Sample records for diepoxybutane cross-links dna

  1. 1,2,3,4-Diepoxybutane-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells


    1,2,3,4-diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins ...

  2. 1,2,3,4-Diepoxybutane-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells.

    Gherezghiher, Teshome B; Ming, Xun; Villalta, Peter W; Campbell, Colin; Tretyakova, Natalia Y


    1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

  3. DNA interstrand cross-linking by a mycotoxic diepoxide.

    Millard, J T; Katz, J L; Goda, J; Frederick, E D; Pierce, S E; Speed, T J; Thamattoor, D M


    The diepoxide mycotoxin (2R, 3R, 8R, 9R)-4,6-decadiyne-2,3:8,9-diepoxy-1,10-diol (repandiol) was both isolated from the mushroom Hydnum repandum and synthesized de novo. Repandiol was found to form interstrand cross-links within a restriction fragment of DNA, linking deoxyguanosines on opposite strands primarily within the 5'-GNC and 5'-GNNC sequences preferred by diepoxyoctane. However, repandiol was a significantly less efficient cross-linker than either of the diepoxyalkanes (diepoxyoctane and diepoxybutane) to which it was compared.

  4. Extreme dryness and DNA-protein cross-links

    Bieger-Dose, A.; Dose, K.; Meffert, R.; Mehler, M.; Risi, S.

    Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32p by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

  5. Swelling properties of cross-linked DNA gels.

    Costa, Diana; Miguel, M Graça; Lindman, Björn


    This work represents our contribution to the field of physical chemistry of DNA gels, and concerns the synthesis and study of novel chemically cross-linked DNA gels. The use of covalent DNA gels is a very promising way to study DNA-cosolute interactions, as well as the dynamic behaviour of DNA and cationic compacting agents, like lipids, surfactants and polycations. Manipulating DNA in new ways, like DNA networks, allows a better understanding and characterization of DNA-cosolute complexes at the molecular level, and also allows us to follow the assembly structures of these complexes. The use of responsive polymer gels for targeted delivery of toxic and/or labile drugs has, during the past few years, shown to be a promising concept. The features found in the proposed system would find applications in a broader field of gel/drug interaction, for the development of controlled release and targeted delivery devices.

  6. Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore.

    Zhang, Xinyue; Price, Nathan E; Fang, Xi; Yang, Zhiyu; Gu, Li-Qun; Gates, Kent S


    Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.

  7. Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    Steen, H; Petersen, J; Mann, M


    Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass....... coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis....... Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component...

  8. Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    Steen, H; Petersen, J; Mann, M


    of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole...

  9. Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay

    de Jong, Steven; Zijlstra, J G; Timmer-Bosscha, H; Mulder, N H; de Vries, Liesbeth


    Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under

  10. Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins.

    Olszewska, Agata; Pohl, Radek; Brázdová, Marie; Fojta, Miroslav; Hocek, Michal


    Nucleotides, 2'-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).

  11. Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

    Zhen, W P; Buchardt, O; Nielsen, Henrik


    A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair...... fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen...... was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA....

  12. DNA interstrand cross-link induced by estrogens as well as their complete and synergic carcinogenesis


    The estrogens show negative activity in Ames test, but estrodiol and diethylstilbestrol in estrogens both are carcinogens based upon animal experiments and epidemiological investigation. It is concluded from the di-region theory, a mechanism conception put forward by one of the present authors, that the carcinogenesis of estrogens is switched on by the covalent cross-link between complementary DNA bases induced by them. We verified for the first time by the DNA alkaline elution method that both estrodiol and diethylstilbestrol cause covalent cross-link between DNA-protein and DNA interstrands after metabolic activation with dosage correlation, but neither the non-carcinogens cholesterol nor pyrene can lead to these sorts of cross-link in the same condition. It has been known that there is a synergetic effect between estrogen and pollution of polycyclic aromatic hydrocarbons. Although non-carcinogenic pyrene alone cannot induce cross-link, its addition with equal molar quantity to estrodiol culture causes synergically the total and DNA interstrand cross-link ratios to be respectively four and three times more than the ones in the cultivation with estrodiol only. It is shown that not only the estrodiol set off the formation of pyrene bi-radicals, but also the pyrene radicals arouse conversely the production of estrodiol bi-radicals.

  13. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi


    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  14. DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabelling.

    Leuratti, C; Jones, N J; Marafante, E; Kostiainen, R; Peltonen, K; Waters, R


    To date only a few studies have been undertaken on DNA adducts formed by epoxybutene (EB) and diepoxybutane (DEB), the two active metabolites of 1,3-butadiene. Our interests have focused on further investigating DNA alkylation by the two epoxides, especially in relation to the development of a method for human biomonitoring. Here, following the reaction of deoxyadenosine monophosphate and poly(dA-dT)(dA-dT) with DEB and subsequent HPLC, we have identified an adenine adduct. MS analyses indicate the structure of an adenine adducted by DEB at the N6 position. A HPLC/32P-postlabelling method was developed for its measurement in DNA samples and the adduct was detected in calf thymus DNA and DNA from Chinese hamster ovary cells exposed to DEB. The 100% labelling efficiency during postlabelling, the amount of the adduct and its elution before the normal nucleotides during HPLC suggest it could be a suitable indicator of BUT exposure.

  15. Dehydration of an ethanol/water azeotrope through alginate-DNA membranes cross-linked with metal ions by pervaporation.

    Uragami, Tadashi; Banno, Masashi; Miyata, Takashi


    To obtain high dehydration membranes for an ethanol/water azeotrope, dried blend membranes prepared from mixtures of sodium alginate (Alg-Na) and sodium deoxyribonucleate (DNA-Na) were cross-linked by immersing in a methanol solution of CaCl2 or MaCl2. In the dehydration of an ethanol/water azeotropic mixture by pervaporation, the effects of immersion time in methanol solution of CaCl2 or MaCl2 on the permeation rate and water/ethanol selectivity through Alg-DNA/Ca(2+) and Alg-DNA/Mg(2+) cross-linked membranes were investigated. Alg-DNA/Mg(2+) cross-linked membrane immersed for 12h in methanol solution of MaCl2 exhibited the highest water/ethanol selectivity. This results from depressed swelling of the membranes by formation of a cross-linked structure. However, excess immersion in solution containing cross-linker led to an increase in the hydrophobicity of cross-linked membrane. Therefore, the water/ethanol selectivity of Alg-DNA/Mg(2+) cross-linked membranes with an excess immersion in cross-linking solution was lowered. The relationship between the structure of Alg-DNA/Ca(2+) and Alg-DNA/Mg(2+) cross-linked membranes and their permeation and separation characteristics during pervaporation of an ethanol/water azeotropic mixture is discussed in detail.

  16. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir


    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked gu...

  17. DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

    Tretyakova, Natalia Y; Groehler, Arnold; Ji, Shaofei


    Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural

  18. Syntheses of DNA Duplexes That Contain a N4C-alkyl-N4C Interstrand Cross-Link

    Miller, Paul S.


    A simple procedure is described for preparing short DNA duplexes that contain a single N4C-alkyl-N4C interstrand cross-link. The synthesis is carried out on an automated DNA synthesizer using standard phosphoramidite chemistry. The cross-link is introduced during the synthesis of the duplex. The method can be used to prepare mg quantities of cross-linked duplexes suitable for physical studies and for the preparation of larger DNA molecules that can be used as substrates to study DNA repair in whole cell extracts and in living cells in culture. PMID:21400705

  19. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir


    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and thymine N3 (T*) atoms (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). In this work we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxoG) and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and tri-oligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction-monitoring mode. The Nim and 8nitroG are the major products formed (~ 0.05% each), and lesser amounts of 8-oxoG (~ 0.02%), and d(G*pT*) and d(G*-T*) enzymatic digestion products (~ 0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both

  20. Generation of guanine-thymidine cross-links in DNA by peroxynitrite/carbon dioxide.

    Yun, Byeong Hwa; Geacintov, Nicholas E; Shafirovich, Vladimir


    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO(3)(•-) and (•)NO(2) radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2'-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and the thymine N3 (T*) atoms (Crean Nucleic Acids Res. 2008, 36, 742-755). In this work, we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5-7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well-known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitro-G), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxo-G), and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled (15)N,(13)C-labeled 2'-deoxy oligoribonucleotides 5'-dGpT and 5'-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and trioligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction monitoring mode. The NIm and 8-nitro-G are the major products formed (∼0.05% each), and lesser amounts of 8-oxo-G (∼0.02%) and d(G*pT*) and d(G*-T*) enzymatic digestion products (∼0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand

  1. DNA interstrand cross-links of an antitumor trinuclear platinum(II) complex: thermodynamic analysis and chemical probing.

    Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor


    The trinuclear platinum compound [{trans-PtCl(NH(3))(2)}(2)(μ-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](4+) (BBR3464) belongs to the polynuclear class of platinum-based anticancer agents. These agents form in DNA long-range (Pt,Pt) interstrand cross-links, whose role in the antitumor effects of BBR3464 predominates. Our results show for the first time that the interstrand cross-links formed by BBR3464 between two guanine bases in opposite strands separated by two base pairs (1,4-interstrand cross-links) exist as two distinct conformers, which are not interconvertible, not only if these cross-links are formed in the 5'-5', but also in the less-usual 3'-3' direction. Analysis of the conformers by differential scanning calorimetry, chemical probes of DNA conformation, and minor groove binder Hoechst 33258 demonstrate that each of the four conformers affects DNA in a distinctly different way and adopts a different conformation. The results also support the thesis that the molecule of antitumor BBR3464 when forming DNA interstrand cross-links may adopt different global structures, including different configurations of the linker chain of BBR3464 in the minor groove of DNA. Our findings suggest that the multiple DNA interstrand cross-links available to BBR3464 may all contribute substantially to its cytotoxicity.

  2. AlkB recognition of a bulky DNA base adduct stabilized by chemical cross-linking


    E.coli AlkB is a direct DNA/RNA repair protein that oxidatively reverses N1 alkylated purines and N3 alkylated pyrimidines to regular bases.Previous crystal structures have revealed N1-methyl adenine(1-meA) recognition by AlkB and a unique base flipping mechanism,but how the AlkB active site can accommodate bulky base adducts is largely unknown.Employing a previously developed chemical cross-linking technique,we crystallized AlkB with a duplex DNA containing a caged thymine base(cagedT).The structure revealed a flexible hairpin lid and a reorganized substrate recognition loop used by AlkB to accommodate cagedT.These observations demonstrate,at the molecular level,how bulky DNA adducts may be recognized and processed by AlkB.

  3. Structure of a DNA glycosylase that unhooks interstrand cross-links

    Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)


    DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

  4. DNA melting properties of the dityrosine cross-linked dimer of Ribonuclease A.

    Dinda, Amit Kumar; Chattaraj, Saparya; Ghosh, Sudeshna; Tripathy, Debi Ranjan; Dasgupta, Swagata


    Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant (Ka) values for complexation of the monomer and the dimer with ct-DNA are (4.95±0.45)×10(4)M(-1) and (1.29±0.05)×10(6)M(-1) respectively. Binding constant (Kb) values for the binding of the monomer and the dimer with ct-DNA were determined using UV-vis spectroscopy and were found to be (4.96±1.67)×10(4)M(-1) and (4.32±0.31)×10(5)M(-1) respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA-dimer indicated that the melting temperature (Tm) for the ct-DNA-dimer complex is lower compared to the ct-DNA-monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA.

  5. Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers.

    Rahman, Khondaker M; James, Colin H; Thurston, David E


    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH(2) functionalities. The PBD dimer SJG-136, which has a C8-O-(CH(2))(3)-O-C8'' central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8-O-(CH(2))(5)-O-C8'), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8'-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA-interaction behaviour of a PBD dimer of particular C8-C8' linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.

  6. Pathways of homologous recombinantion and DNA interstrand cross-link repair : roles of mammalian RAD54 and SNMI

    M.L.G. Dronkert (Mies)


    textabstractThe aim of this thesis is to investigate mammalian DNA interstrand cross-link (ICL) repair. ICLs are formed by a number of agents used in tumor therapy, like mitomycin C and cisplatin. They constitute one of the most toxic damages to DNA, as they inhibit DNA strand separation. However, l

  7. Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C.

    M.L.G. Dronkert (Mies); J. de Wit (Jan); M. Boeve; M.L. Vasconcelos; H. van Steeg (Harry); T.L.R. Tan; J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)


    textabstractDNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to

  8. Carcinogenesis switched on by DNA cross-link between complementary bases aroused by aflatoxin and N-nitroso compounds

    DAI Qianhuan; LU Ping; PENG Shaohua; ZHANG Qingrong


    The di-region theory put forward by Dai Qianhuan, a molecular mechanism of chemical carcinogenesis, concluded that the carcinogenesis induced by most of the environmental carcinogens is switched on by the cross-linking between DNA complementary bases aroused by the bifunctional alkylation of their metabolic intermediates. It was evidenced in this paper with DNA filter elution method that one carcinogenic mycotoxin, aflatoxin G1, four carcinogenic N-nitroso compounds, N-nitrosodiethyl-amine, N-nitrosodibutyl-amine, N-nitrosomorpholine and N-nitrosopyrrolidine, one carcinogenic diazo color, 4-dimethylaminodiazobenzene and one carcinogenic nitrogen-containing heterocyclic compound, quinoline, all induced DNA interstrands cross-linking with dosage correlation after metabolic activation. However, the non-carcinogens in corresponding series for control, aflatoxin B2, N-nitroso-diphenylamine, 4′-bromo-4-dimethylaminodiazobenzene and isoquinoline, cannot induce DNA interstrands cross-linking at all in the same condition. A method for the determination of cross-linking ratio between DNA complementary bases in total DNA interstrands cross-linking, which has no monitoring measure as yet, has been established for the first time based upon a 24 hour repairing experiment. The DNA complementary pair cross-linking ratio induced by a metabolized carcinogen is correlated with its carcinogenic potential. It may be concluded that the mutations including point and frameshift mutagenesis induced by aflatoxin and other carcinogens are switched on by their corresponding cross-linking base pair between complementary bases. Therefore, the di-region theory is a reasonable molecular mechanism for chemical, endogenous and physical carcinogenesis.

  9. Induction of DNA damage and G2 cell cycle arrest by diepoxybutane through the activation of the Chk1-dependent pathway in mouse germ cells.

    Dong, Jianyun; Wang, Zhi; Zou, Peng; Zhang, Guowei; Dong, Xiaomei; Ling, Xi; Zhang, Xi; Liu, Jinyi; Ye, Dongqing; Cao, Jia; Ao, Lin


    1,2:3,4-Diepoxybutane (DEB) is a major carcinogenic metabolite of 1,3-butadiene (BD), which has been shown to cause DNA strand breaks in cells through its potential genotoxicity. The adverse effect of DEB on male reproductive cells in response to DNA damage has not been thoroughly studied, and the related mechanism is yet to be elucidated. Using mouse spermatocyte-derived GC-2 cells, we demonstrated in the present study that DEB caused the proliferation inhibition and marked cell cycle arrest at the G2 phase but not apoptosis. DEB also induced DNA damage as evidenced by γ-H2AX expression, the comet assay, and the cytokinesis-block micronucleus assay. Meanwhile, DEB triggered the Chk1/Cdc25c/Cdc2 signal pathway, which could be abated in the presence of UCN-01 or Chk1 siRNA. GC-2 cells exposed to DEB experienced ROS generation and pretreatment of N-acetyl-l-cysteine, partly attenuated DEB-induced DNA damage, and G2 arrest. Furthermore, measurement of testicular cells showed an increased proportion of tetraploid cells in mice administrated with DEB, alongside the enhanced expression of p-Chk1. Also, the defective reproductive phenotypes, including reduced sperm motility, increased sperm malformation, and histological abnormality of testes, were observed. In conclusion, these results suggest DEB induces DNA damage and G2 cell cycle arrest by activating the Chk1-dependent pathway, while oxidative stress may be associated with eliciting toxicity in male reproductive cells.

  10. Monitoring human lymphocytic DNA-protein cross-links as biomarkers of biologically active doses of chromate.

    Costa, M; Zhitkovich, A; Toniolo, P; Taioli, E; Popov, T; Lukanova, A


    A simple and sensitive assay for DNA-protein cross-links has been used as a biomarker of chromate exposure and early carcinogenic effects. Pilot studies of DNA-protein cross-links in peripheral blood lymphocytes have been conducted with individuals who had higher exposure to chromate, including welders, and with individuals who had lower levels of exposure such as residents living in a chromium-contaminated area in Jersey City, New Jersey. Studies were also conducted in two Bulgarian cities (Jambol and Burgas) with different levels of air pollution and Cr(VI) exposure and in chrome platers in Bulgaria who had high exposure to chromate. DNA-protein cross-links in U.S. welders and in individuals living in Hudson County, New Jersey around chromium-contaminated areas were significantly higher compared to matched controls. Although blood and urinary levels of chromium were not extensively studied in these populations, we were able to obtain these measurements in the Bulgarian population. Chromium levels in red blood cells of controls living in Burgas were in the order of 1 to 2 ppb chromium, and these individuals had the lowest levels of DNA-protein cross-links. However, the chromium levels in Jambol ranged from about 2 to 7 ppb in red blood cells of city residents to about 22 ppb in chrome platers. DNA-protein cross-links were saturated at about 7 to 8 ppb chromium in the red blood cells, and cross-links correlated well only with chromium levels in red blood cells. Urinary chromium levels did not correlate well with either DNA-protein cross-links or chromium levels in with red blood cells.

  11. Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C.

    Palom, Yolanda; Suresh Kumar, Gopinatha; Tang, Li-Qian; Paz, Manuel M; Musser, Steven M; Rockwell, Sara; Tomasz, Maria


    Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other

  12. UV-Induced DNA Interstrand Cross-Linking and Direct Strand Breaks from a New Type of Binitroimidazole Analogue.

    Han, Yanyan; Chen, Wenbing; Kuang, Yunyan; Sun, Huabing; Wang, Zhiqiang; Peng, Xiaohua


    Four novel photoactivated binitroimidazole prodrugs were synthesized. These agents produced DNA interstrand cross-links (ICLs) and direct strand breaks (DSB) upon UV irradiation, whereas no or very few DNA ICLs and DSBs were observed without UV treatment. Although these four molecules (1-4) contain the same binitroimidazole moiety, they bear four different leaving groups, which resulted in their producing different yields of DNA damage. Compound 4, with nitrogen mustard as a leaving group, showed the highest ICL yield. Surprisingly, compounds 1-3, without any alkylating functional group, also induced DNA ICL formation, although they did so with lower yields, which suggested that the binitroimidazole moiety released from UV irradiation of 1-3 is capable of cross-linking DNA. The DNA cross-linked products induced by these compounds were completely destroyed upon 1.0 M piperidine treatment at 90 °C (leading to cleavage at dG sites), which revealed that DNA cross-linking mainly occurred via alkylation of dGs. We proposed a possible mechanism by which alkylating agents were released from these compounds. HRMS and NMR analysis confirmed that free nitrogen mustards were generated by UV irradiation of 4. Suppression of DNA ICL and DSB formation by a radical trap, TEMPO, indicated the involvement of free radicals in the photo reactions of 3 and 4 with DNA. On the basis of these data, we propose that UV irradiation of compounds 1-4 generated a binitroimidazole intermediate that cross-links DNA. The higher ICL yield observed with 4 resulted from the amine effector nitrogen mustard released from UV irradiation.

  13. Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking.

    Dadová, Jitka; Vrábel, Milan; Adámik, Matej; Brázdová, Marie; Pohl, Radek; Fojta, Miroslav; Hocek, Michal


    N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

  14. Quantitative DNA interstrand cross-link formation by coumarin and thymine: structure determination, sequence effect, and fluorescence detection.

    Sun, Huabing; Fan, Heli; Peng, Xiaohua


    The coumarin analogues have been widely utilized in medicine, biology, biochemistry, and material sciences. Here, we report a detailed study on the reactivity of coumarins toward DNA. A series of coumarin analogues were synthesized and incorporated into oligodeoxynucleotides. A photoinduced [2 + 2] cycloaddition occurs between the coumarin moiety and the thymidine upon 350 nm irradiation forming both syn- and anti-cyclobutane adducts (17 and 18), which are photoreversible by 254/350 nm irradiation in DNA. Quantitative DNA interstrand cross-link (ICL) formation was observed with the coumarin moieties containing a flexible two-carbon or longer chain. DNA cross-linking by coumarins shows a kinetic preference when flanked by an A:T base pair as opposed to a G:C pair. An efficient photoinduced electron transfer between coumarin and dG slows down ICL formation. ICL formation quenches the fluorescence of coumarin, which, for the first time, enables fast, easy, and real-time monitoring of DNA cross-linking and photoreversibility via fluorescence spectroscopy. It can be used to detect the transversion mutation between pyrimidines and purines. Overall, this work provides new insights into the biochemical properties and possible toxicity of coumarins. A quantitative, fluorescence-detectable, and photoswitchable DNA cross-linking reaction of the coumarin moieties can potentially serve as mechanistic probes and tools for bioresearch without disrupting native biological environment.

  15. Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair.

    Sengerová, Blanka; Wang, Anderson T; McHugh, Peter J


    DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases.

  16. Cross-linked Polyethylenimine as Potential DNA Vector for Gene Delivery with High Efficiency and Low Cytotoxicity

    Wei DONG; Guang-Hui JIN; Shu-Feng LI; Qi-Ming SUN; Ding-Yuan MA; Zi-Chun HUA


    Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [ 1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

  17. Evaluation of mutagenic effects of formocresol: detection of DNA-protein cross-links and micronucleus in mouse bone marrow.

    Ramos, Maria Emília Santos Pereira; Cavalcanti, Bruno Coêlho; Lotufo, Letícia Veras Costa; de Moraes, Manoel Odorico; Cerqueira, Eneida de Moraes Marcílio; Pessoa, Cláudia


    The genotoxic potential of formocresol was assessed by comet assay on human peripheral blood lymphocytes and in vivo micronucleus in mice. Peripheral blood lymphocytes, obtained from healthy donors, were exposed directly with different dilutions of formocresol for 45 minutes at 37 degrees C. To verify the possibility of formocresol to induce DNA-protein cross-links, treated lymphocytes were incubated with proteinase K. Micronucleus test was performed on male Swiss mice treated with several dilutions of formocresol by single intraperitoneal injection. After treatment, bone marrow was sampled 24 and 48 hours after formocresol administration. Formocresol did not produce detectable DNA damage as evaluated by comet assay. However, after proteinase K exposure, a dose-dependent increase of DNA migration was observed. Formocresol induced a significant increase in micronucleus frequencies at the highest dilution only at 24 hours after administration. Formocresol induced DNA-protein cross-links and an increased frequency of micronucleus.

  18. Screening for DNA Alkylation Mono and Cross-Linked Adducts with a Comprehensive LC-MS(3) Adductomic Approach.

    Stornetta, Alessia; Villalta, Peter W; Hecht, Stephen S; Sturla, Shana J; Balbo, Silvia


    A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by screening for DNA mono and cross-linked adducts in purified DNA and in DNA extracted from cells treated with PR104A, an experimental DNA alkylating nitrogen mustard prodrug currently under investigation for the treatment of leukemia. The results revealed the ability of this new DNA adductomic approach to detect anticipated and unknown PR104A-induced mono and cross-linked DNA adducts in biological samples. This methodology is expected to be a powerful tool for screening for DNA adducts induced by endogenous or exogenous exposures.

  19. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin


    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.

  20. Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.

    Catalano, Michael J; Liu, Shuo; Andersen, Nisana; Yang, Zhiyu; Johnson, Kevin M; Price, Nathan E; Wang, Yinsheng; Gates, Kent S


    A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

  1. Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA interstrand cross-links

    Gruppi, Francesca; Salyard, Tracy L. Johnson; Rizzo, Carmelo J.


    The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5′-CG-3′ and 5′-GC-3′ sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2′-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides. PMID:25431636

  2. Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

    Jost, Petr; Svobodova, Hana; Stetina, Rudolf


    Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs.

  3. Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

    Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M


    Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation......, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA...... interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA...

  4. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K


    a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we...

  5. Induction of DNA-protein cross-links in developing embryos of the purple sea urchin, Strongylocentrotus purpuratus

    Garman, G.D.; Cherr, G.N. [Univ. of California, Davis, CA (United States). Bodega Marine Lab.; Anderson, S.L. [Lawrence Berkeley Lab., Berkeley, CA (United States)


    Exposure to environmental agents during embryonic development may result in DNA-protein cross-linking (DPC), as has been demonstrated for mammalian cell lines. In the latter, formation of DPC`s upon exposure to a wide variety of agents, including some metals, has been observed. To determine whether DPCs could be detected in the sea urchin embryo during development, the authors adapted a mammalian cell assay utilizing potassium-SDS precipitation and a DNA fluorochrome to quantify relative amounts of free and protein-bound DNA. Sea urchin embryos exposed to a known DPC agent, nickel, through gastrulation exhibited a dose-dependent increase in DPCs, as well as an increase in developmental abnormalities. Morphological studies demonstrated that stage-specific exposure to Ni prior to gastrulation resulted in similar levels of abnormal pluteus larval development as compared to embryos exposed through gastrulation. Sea urchin embryos exhibit temporal differences in DNA transcription and gene expression during development, and these could be affected by modifications in DNA-protein interactions. Therefore, the authors are investigating the hypothesis that the similarities in morphological responses observed may relate to susceptibility of a critical stage of development.

  6. Repair of 8-methoxypsoralen induced DNA interstrand cross-links in Tetrahymena thermophila. The effect of inhibitors of macromolecular synthesis

    Nielsen, P E; Køber, L


    The effect of several growth-inhibiting compounds on the repair of 8-methoxypsoralen-UVA-light-induced DNA interstrand cross-links has been studied in the protozoan Tetrahymena thermophila. The repair process was analyzed by the alkaline elution technique and could be divided into 3 phases......: a protein-DNA complexing phase, a DNA-incision phase and finally a DNA-ligation phase. The incision was found to be completely inhibited by novobiocin (50 micrograms/ml), nalidixic acid (150 micrograms/ml), n-butyrate (15 mM) and cycloheximide (1 microgram/ml), while no effect was observed for cytosine-1......-beta-D-arabinofuranoside (10 mM), puromycin (1 mM), hydroxyurea (5 mM) or 3-aminobenzamide (2.5 mM). None of the compounds showed any effect on the protein-DNA complexing step, and the ligation was partly inhibited only by nalidixic acid (150 micrograms/ml). The involvement of topoisomerases...

  7. Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

    Bennetts, L.E.; De Iuliis, G.N.; Nixon, B.; Kime, M.; Zelski, K. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia); McVicar, C.M.; Lewis, S.E. [Obstetrics and Gynaecology, Queen' s University, Belfast (United Kingdom); Aitken, R.J. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia)], E-mail:


    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear ({beta}-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17{beta}-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of

  8. Ouabain, a cardiac glycoside, inhibits the Fanconi anemia/BRCA pathway activated by DNA interstrand cross-linking agents.

    Dong Wha Jun

    Full Text Available Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs, one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC, activates the Fanconi anemia (FA/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na(+/K(+-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members--digitoxin and digoxin--down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca(2+ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.

  9. Selective activation of mitomycin A by thiols to form DNA cross-links and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential.

    Paz, M M; Das, A; Palom, Y; He, Q Y; Tomasz, M


    Mitomycin A (MA) but not mitomycin C (MC) cross-linked linearized (32)P-pBR322 DNA in the presence of dithiothreitol (DTT) or glutathione (GSH), as shown by a sensitive DNA cross-link assay. Incubation of calf-thymus DNA with MA and DTT or mercaptoethanol (MER) resulted in the formation of MA-DNA adducts, which were isolated from nuclease digests of the drug-DNA complexes by HPLC. The adducts were characterized by their UV absorption spectra, electrospray ionization mass spectrometry (ESIMS), and facile conversion from 7-methoxy- to 7-amino-substituted mitosene type adducts upon 10% NH(4)OH treatment, which were identical with known adducts of MC. Both DNA interstrand and intrastrand cross-link adducts, linking two deoxyguanosine residues at N(2), as well as several deoxyguanosine-N(2) monoadducts of MA, were identified. No DNA adducts were formed with MC under the same conditions. A specificity of DNA cross-link formation for the CpG sequence was observed using 12-mer synthetic oligodeoxyribonucleotides as substrates and as DNA sequence models, in analogy to the known CpG sequence specificity of MC-induced DNA cross-links. MA is known to be more cytotoxic by 2-3 orders of magnitude than MC, and this property correlates with redox potentials of MA (-0.19 V) and MA analogues that are higher than those of MC (-0.40 V) and its analogues. It is suggested that the biochemical basis for the higher cytotoxic potency of MA is MA's propensity to be reductively activated by cellular thiols while MC is resistant to thiol activation. This distinction is probably derived from the large difference between the quinone redox potentials of the two drugs.

  10. Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA

    Jang, Y.J.; Stollar, B.D. (Tufts Univ., Boston, MA (USA))


    Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.

  11. DNA cross-link-dependent RAD50/MRE11/NBS1 subnuclear assembly requires the Fanconi anemia C protein.

    Pichierri, Pietro; Averbeck, Dietrich; Rosselli, Filippo


    Fanconi anemia (FA) is a cancer-predisposition syndrome characterized by hypersensitivity to interstrand-cross-link (ICL) inducers. FA hypersensitivity to ICL has been correlated with alterations in homologous recombination, non-homologous end-joining, telomere maintenance, DNA-damage assessment and checkpoint regulation, processes in which the components of the RAD50/MRE11/NBS1 (RMN) complex are involved. To better characterize the mechanisms by which ICL are processed in human cells and to gain insight into their toxicity in FA, we examined (i). the RMN complex assembling in response to the ICL inducers mitomycin C (MMC) and photoactivated 8-methoxypsoralen and (ii). the proficiency of FA cells to perform RMN activation in response to ICL inducers. We show here that ICL activates the assembly of the RMN proteins into subnuclear foci, and that their formation proceeds independently of ICL incision, a step mainly dependent on XP-F/ERCC1 heterodimer activity. Interestingly, FA cells were unable to form RMN foci in response to either ICL inducer. Analysis by pulsed-field gel electrophoresis and single-cell gel electrophoresis of MMC-treated cells showed that FA cells from complementation group C (FA-C cells, defective in the FANCC gene) form double-strand breaks and unhook MMC-induced ICL similarly to FANCC wild-type cells. These observations imply that the absence of RMN assembly in FA-C cells is not simply due to the absence of DNA ends produced as intermediates of ICL processing, and indicates a direct role for FANCC in RMN focus assembly in response to ICL inducers. Moreover, we show that the formation of foci, including BRCA1 and/or RAD51 proteins, is significantly delayed in FA cells. These alterations in the assembly of DNA-repair proteins in FA provide an interpretation for the DNA-damage processing anomalies observed in FA cells and for the genetic instability and the cancer predisposition of the syndrome.

  12. Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system.

    Hockensmith, J W; Kubasek, W L; Vorachek, W R; von Hippel, P H


    Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser

  13. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    Fengquin, X; Nielsen, Henrik; Zhen, W;


    between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally...

  14. High-density functionalization and cross-linking of DNA: "click" and "bis-click" cycloadditions performed on alkynylated oligonucleotides with fluorogenic anthracene azides.

    Pujari, Suresh S; Ingale, Sachin A; Seela, Frank


    High density functionalization of DNA with ethynyl and octadiynyl side chains followed by CuAAC "click labeling" with 9-azidomethylanthracene was performed. Alkynyl DNA was also cross-linked with fluorogenic 9,10-bis-azidomethylanthracene employing the "bis-click" reaction. By this means the fluorescence of the anthracene moiety was imparted to the virtually nonfluorescent DNA. Phosphoramidites of 8-aza-7-deaza-2'-deoxyadenosine with short and long linker arms in a steric nondemanding 7-position were utilized in solid phase oligodeoxynucleotide synthesis. High density alkynylated DNA-without anthracene residues-was found to be of comparable stability with both long and short linker arms. High density anthracene functionalized DNA became less stable with the short linker compared to that with the long linker connectivity. Interstrand cross-linked homodimers constructed from alkynylated oligonucleotides with fluorogenic 9,10-bis-azidomethylanthracene were hybridized with complementary strands to form double helices. They are more stable when the linker was located at a terminus than in a central position. Short linker anthracene adducts were destabilizing compared to long linker adducts. The fluorogenic anthracene residues not only have a significant effect on the duplex stability, but also impart fluorescence to the species. Fluorescence of cross-linked double helices with long linker connectivity was quenched when the cross-link was in a terminal position and was dequenched when the linker was connecting the two double helices at the center of the molecule. The fluorescence of the anthracene cross-linked double helices was strongly increased (dequenched) when the correct base pair was formed, while no change occurred upon mismatch formation.

  15. Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

    Krisnamurthy Mahalakshmi


    Full Text Available Abstract Background Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs can be repaired through direct joining of broken ends (non homologous end joining, NHEJ or through recombination with the non broken sister chromosome (homologous recombination, HR. Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. Results A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD, but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. Conclusion Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC, most likely

  16. Mass spectrometric proteomics reveals that nuclear protein positive cofactor PC4 selectively binds to cross-linked DNA by a trans-platinum anticancer complex.

    Du, Zhifeng; Luo, Qun; Yang, Liping; Bing, Tao; Li, Xianchan; Guo, Wei; Wu, Kui; Zhao, Yao; Xiong, Shaoxiang; Shangguan, Dihua; Wang, Fuyi


    An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a trans-platinum anticancer complex, trans-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by trans-PtTz.

  17. Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

    Hawkins, Clare L; Pattison, David I; Davies, Michael J


    Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines

  18. Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

    Wickramaratne, Susith; Boldry, Emily J; Buehler, Charles; Wang, Yen-Chih; Distefano, Mark D; Tretyakova, Natalia Y


    DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.

  19. Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

    Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi; Watson, Adam T.; Jo, Aera; Etheridge, Thomas J.; Yuan, Fenghua; Zhang, Yanbin; Kim, YoungChang; Carr, Anthony M.; Cho, Yunje


    Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

  20. The Fanconi anemia/BRCA pathway is involved in DNA interstrand cross-link repair of adriamycin-resistant leukemia cells.

    Yao, Chenjiao; Du, Wei; Chen, Haibing; Xiao, Sheng; Huang, Lihua; Chen, Fangping


    The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.

  1. Chemoprotective Effect of Taurine on Potassium Bromate-Induced DNA Damage, DNA-Protein Cross-Linking and Oxidative Stress in Rat Intestine

    Ahmad, Mir Kaisar; Khan, Aijaz Ahmed; Ali, Shaikh Nisar; Mahmood, Riaz


    Potassium bromate (KBrO3) is widely used as a food additive and is a major water disinfection by-product. It induces multiple organ toxicity in humans and experimental animals and is a probable human carcinogen. The present study reports the protective effect of dietary antioxidant taurine on KBrO3-induced damage to the rat intestine. Animals were randomly divided into four groups: control, KBrO3 alone, taurine alone and taurine+ KBrO3. Administration of KBrO3 alone led to decrease in the activities of intestinal brush border membrane enzymes while those of antioxidant defence and carbohydrate metabolism were also severely altered. There was increase in DNA damage and DNA-protein cross-linking. Treatment with taurine, prior to administration of KBrO3, resulted in significant attenuation in all these parameters but the administration of taurine alone had no effect. Histological studies supported these biochemical results showing extensive intestinal damage in KBrO3-treated animals and greatly reduced tissue injury in the taurine+ KBrO3 group. These results show that taurine ameliorates bromate induced tissue toxicity and oxidative damage by improving the antioxidant defence, tissue integrity and energy metabolism. Taurine can, therefore, be potentially used as a therapeutic/protective agent against toxicity of KBrO3 and related compounds. PMID:25748174

  2. Chemoprotective effect of taurine on potassium bromate-induced DNA damage, DNA-protein cross-linking and oxidative stress in rat intestine.

    Mir Kaisar Ahmad

    Full Text Available Potassium bromate (KBrO3 is widely used as a food additive and is a major water disinfection by-product. It induces multiple organ toxicity in humans and experimental animals and is a probable human carcinogen. The present study reports the protective effect of dietary antioxidant taurine on KBrO3-induced damage to the rat intestine. Animals were randomly divided into four groups: control, KBrO3 alone, taurine alone and taurine+ KBrO3. Administration of KBrO3 alone led to decrease in the activities of intestinal brush border membrane enzymes while those of antioxidant defence and carbohydrate metabolism were also severely altered. There was increase in DNA damage and DNA-protein cross-linking. Treatment with taurine, prior to administration of KBrO3, resulted in significant attenuation in all these parameters but the administration of taurine alone had no effect. Histological studies supported these biochemical results showing extensive intestinal damage in KBrO3-treated animals and greatly reduced tissue injury in the taurine+ KBrO3 group. These results show that taurine ameliorates bromate induced tissue toxicity and oxidative damage by improving the antioxidant defence, tissue integrity and energy metabolism. Taurine can, therefore, be potentially used as a therapeutic/protective agent against toxicity of KBrO3 and related compounds.

  3. The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks.

    Niedernhofer, Laura J; Odijk, Hanny; Budzowska, Magda; van Drunen, Ellen; Maas, Alex; Theil, Arjan F; de Wit, Jan; Jaspers, N G J; Beverloo, H Berna; Hoeijmakers, Jan H J; Kanaar, Roland


    Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

  4. Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG cross-links.

    Debadeep Bhattacharyya

    Full Text Available The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP and oxaliplatin (OX are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR spectroscopy and molecular dynamics (MD simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.

  5. DNA-induced inter-particle cross-linking during expanded bed adsorption chromatography - Impact on future support design

    Theodossiou, Irini; Thomas, Owen R. T.


    We have investigated the effects of adsorbent size, ionic capacity and surface immobilised polymers on dynamic capacity and changes occurring to beds of anion-exchangers during the binding of DNA. During application of low concentrations of "3-20 kilobase" calf thymus DNA feeds to expanded beds o...... exhibited a three-fold higher tendency to interact with neighbouring particles in the presence of DNA than that of the dextran DEAE support. The implications of these findings on the design of future expanded bed materials for separation of both proteins and nucleic acids are discussed....

  6. Evidence for different mechanisms of ‘unhooking’ for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples

    Spanswick Victoria J


    Full Text Available Abstract Background DNA interstrand cross-links (ICLs are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma and solid tumours (ovarian cancer that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the ‘unhooking’ step is common to all ICLs. Methods Using a modification of the single cell gel electrophoresis (Comet assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. Results Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The

  7. Isolation and characterization of electrophiles from 2-haloethylnitrosoureas forming cytotoxic DNA cross-links and cyclic nucleotide adducts and the analysis of base site-selectivity by ab initio calculations.

    Lown, J W; Koganty, R R; Bhat, U G; Chauhan, S M; Sapse, A M; Allen, E B


    E- and Z-2-haloethyldiazotates--electrophilic species hitherto suggested as intermediates in the reactions of 2-haloethylnitrosoureas (HENUs) under physiological conditions--were synthesized and characterized by 1H-, 15N- and 13C-NMR (nuclear magnetic resonance). They were stabilized and solubilized in organic solvents as their 18-crown-6 ether complexes. Characterization of the Z-2-fluoroethyldiazotate by 19F- and 13C-NMR, and comparison with the Z-2-chloroethyl compound, confirmed facile cyclization to the 1,2,3-oxadiazoline and subsequent decomposition to nitrogen and ethylene oxide. The E-2-haloethyldiazotates form DNA interstrand cross-links at a rate, and to an extent, and with a DNA base dependence, which parallels the behaviour of the parent HENUs, while the Z isomers alkylate DNA but show minimal cross-linking. Both E-and Z-(2'-chloroethyl)thioethyldiazotates, neither of which can undergo cyclization, cross-link DNA efficiently. Self-consistent-field (SCF) ab initio calculations provided optimized geometries, atomic charges and LUMO (Lowest Unoccupied Molecular Orbital) atom contributions for the E- and Z-2-haloethyldiazohydroxides. The HSAB (Hard and Soft Acids and Bases) theory, in conjunction with HOMO (Highest Occupied Molecular Orbital) values on key DNA base sites, accounted for the observed site-selectivity in the formation of identified cross-links produced by 1,3-bis-(2-chloroethyl)-1-nitrosourea. Independent chemical studies on cytosine derivatives corroborated the predicted site selectivity of attack by electrophiles and the formation of ethanocytidine cyclic adducts.

  8. Simulation modeling of the tissue disposition of formaldehyde to predict nasal DNA-protein cross-links in Fischer 344 rats, rhesus monkeys, and humans.

    Conolly, R B; Lilly, P D; Kimbell, J S


    Formaldehyde inhalation causes formation of DNA-protein cross-links (DPX) in the nasal mucosa of Fischer 344 (F344) rats and rhesus monkeys. DPX are considered to be part of the mechanism by which cytotoxic and carcinogenic effects of formaldehyde in laboratory animals are exerted, and DPX data have been used as a measure of tissue dose in cancer risk assessments for formaldehyde. Accurate prediction of DPX concentrations in humans is therefore desirable. The goal of this work was to increase confidence in the prediction of human DPX by refining earlier models of formaldehyde disposition and DPX kinetics in the nasal mucosa. Anatomically accurate, computational fluid dynamics models of the nasal airways of F344 rats, rhesus monkeys, and humans were used to predict the regional flux of formaldehyde to the respiratory and olfactory mucosa. A previously developed model of the tissue disposition of formaldehyde and of DPX kinetics was implemented in the graphical simulation tool SIMULINK and linked to the regional flux predictions. Statistical optimization was used to identify parameter values, and good simulations of the data were obtained. The parameter estimates for rats and monkeys were used to guide allometric scale-up to the human case. The relative levels of nasal mucosal DPX in rats, rhesus monkeys, and humans for a given inhaled concentration of formaldehyde were predicted by the model to vary with concentration. This modeling approach reduces uncertainty in the prediction of human nasal mucosal DPX resulting from formaldehyde inhalation.

  9. Influence of glutathione and glutathione S-transferases on DNA interstrand cross-link formation by 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the active anticancer moiety generated by laromustine.

    Penketh, Philip G; Patridge, Eric; Shyam, Krishnamurthy; Baumann, Raymond P; Zhu, Rui; Ishiguro, Kimiko; Sartorelli, Alan C


    Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 ∼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N(3)-cytosinyl),-2-(N(1)-guaninyl)ethane DNA interstrand cross-links via N(1),O(6)-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.

  10. Oxidation of Guanine by Carbonate Radicals Derived from Photolysis of Carbonatotetramminecobalt(III) Complexes and the pH Dependence of Intrastrand DNA Cross-links Mediated by Guanine Radical Reactions

    Crean, Conor; Lee, Young Ae; Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir


    The carbonate radical anion CO3•− is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO3•− radicals is known to yield spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), and a novel intrastrand cross-linked product, 5’-d(CCATCG*CT*ACC) between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et ...

  11. Collagen cross linking: Current perspectives

    Srinivas K Rao


    Full Text Available Keratoconus is a common ectatic disorder occurring in more than 1 in 1,000 individuals. The condition typically starts in adolescence and early adulthood. It is a disease with an uncertain cause and its progression is unpredictable, but in extreme cases, vision deteriorates and can require corneal transplant surgery. Corneal collagen cross-linking (CCL with riboflavin (C3R is a recent treatment option that can enhance the rigidity of the cornea and prevent disease progression. Since its inception, the procedure has evolved with newer instrumentation, surgical techniques, and is also now performed for expanded indications other than keratoconus. With increasing experience, newer guidelines regarding optimization of patient selection, the spectrum of complications and their management, and combination procedures are being described. This article in conjunction with the others in this issue, will try and explore the uses of collagen cross-linking (CXL in its current form.

  12. Electrospinning formaldehyde cross-linked zein solutions

    In order to develop zein fibers with improved physical properties and solvent resistance, formaldehyde was used as the cross-linking reagent before spinning. The cross-linking reaction was carried out in either acetic acid or ethanolic-HCl where the amount of cross-linking reagent was between 1 and...

  13. Conformation-dependent Formation of the G[8-5]U Intrastrand Cross-link in 5-Bromouracil-containing G-quadruplex DNA Induced by UVA Irradiation

    Lin, Guangxin; ZHANG Jing; Zeng, Yu; Luo, Hai; Wang, Yinsheng


    G-quadruplex motifs are known to be present in telomeres of human and other organisms. Recent bioinformatic studies also revealed the widespread existence of these motifs in promoter regions of human genes. Treatment of cultured cells with 5-bromo-2’-deoxyuridine (BrdU) is known to result in the substitution of DNA thymidine with BrdU; such replacement has been shown to sensitize cells to killing induced by UV light. Our previous studies revealed that the exposure of BrdU-carrying duplex DNA ...

  14. Evaluation of DNA damage by alkaline elution technique after inhalation exposure of rats and mice to 1,3-butadiene.

    Vangala, R R; Laib, R J; Bolt, H M


    The alkaline filter elution technique was used to evaluate single strand breaks (SSB), DNA-DNA (DDCL) and DNA-protein cross-links (DPCL) in liver and lung of male rats (Sprague-Dawley) and male mice (B6C3F1) after exposure to 2000 ppm 1,3-butadiene (BD) for 7 days (7 h/day and/or to 100, 250, 500, 1000) 2000 ppm BD for 7 h. SSB were detected in liver DNA of both species at 2000 ppm. Cross-links are more pronounced in mouse lung than in mouse liver. Elution rates of lung DNA from mice exposed for 7 h to different concentrations of BD revealed an increase in cross-links between 250 and 500 ppm, and a further increase in cross-links up to 2000 ppm. No such signs of genotoxicity could be observed for the lung of rats. Our data support the involvement of reactive metabolites (epoxybutene and especially diepoxybutane) in butadiene-induced carcinogenesis in the mouse but not to that extent in the rat.

  15. Corneal collagen cross-linking

    Jankov II Mirko


    Full Text Available Corneal collagen cross-linking (CXL with riboflavin and ultraviolet-A (UVA is a new technique of corneal tissue strengthening by using riboflavin as a photosensitizer and UVA to increase the formation of intra- and interfibrillar covalent bonds by photosensitized oxidation. Keratocyte apoptosis in the anterior segment of the corneal stroma all the way down to a depth of about 300 microns has been described and a demarcation line between the treated and untreated cornea has been clearly shown. It is important to ensure that the cytotoxic threshold for the endothelium has not been exceeded by strictly respecting the minimal corneal thickness. Confocal microscopy studies show that repopulation of keratocytes is already visible 1 month after the treatment, reaching its pre-operative quantity and quality in terms of functional morphology within 6 months after the treatment. The major indication for the use of CXL is to inhibit the progression of corneal ectasias, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photoablation. This treatment has also been used to treat infectious corneal ulcers with apparent favorable results. Combination with other treatments, such as intracorneal ring segment implantation, limited topography-guided photoablation and conductive keratoplasty have been used with different levels of success.

  16. Chemical cross-linking of Chlamydia trachomatis

    Birkelund, Svend; Lundemose, AG; Christiansen, Gunna


    Purified elementary bodies (EBs) of Chlamydia trachomatis serovar L2 were analyzed by chemical cross-linking with disuccinimidyl selenodipropionate. The effect of the cross-linking was analyzed by immunoblotting sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated components which...

  17. Oxidation of guanine by carbonate radicals derived from photolysis of carbonatotetramminecobalt(III) complexes and the pH dependence of intrastrand DNA cross-links mediated by guanine radical reactions.

    Crean, Conor; Lee, Young Ae; Yun, Byeong Hwa; Geacintov, Nicholas E; Shafirovich, Vladimir


    The carbonate radical anion CO(3)(*-) is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO(3)(*-) radicals is known to yield spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) products, and also a novel intrastrand cross-linked product: 5'-d(CCATCG*CT*ACC), featuring a linkage between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et al., Nucleic Acids Res. 2008, 36, 742-755). Involvement of the T-N3 (pK(a) of N3-H is 9.67) suggests that the formation of 5'-d(CCATCG*CT*ACC) might be pH-dependent. This hypothesis was tested by generating CO(3)(*-) radicals through the photodissociation of carbonatotetramminecobalt(III) complexes by steady-state UV irradiation, which allowed for studies of product yields in the pH 5.0-10.0 range. The yield of 5'-d(CCATCG*CT*ACC) at pH 10.0 is approximately 45 times greater than at pH 5.0; this is consistent with the proposed mechanism, which requires N3(H) thymine proton dissociation followed by nucleophilic addition to the C8 guanine radical.

  18. Characterization of the deoxyguanosine-lysine cross-link of methylglyoxal.

    Petrova, Katya V; Millsap, Amy D; Stec, Donald F; Rizzo, Carmelo J


    Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA-protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and Nα-acetyllysine (AcLys) and determined the structure of the cross-link to be the N2-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement.

  19. The effect of cross-link distributions in axially-ordered, cross-linked networks

    Bennett, C. Brad; Kruczek, James; Rabson, D. A.; Matthews, W. Garrett; Pandit, Sagar A.


    Cross-linking between the constituent chains of biopolymers has a marked effect on their materials’ properties. In certain of these materials, such as fibrillar collagen, increases in cross-linking lead to an increase in the melting temperature. Fibrillar collagen is an axially-ordered network of cross-linked polymer chains exhibiting a broadened denaturation transition, which has been explained in terms of the successive denaturation with temperature of multiple species. We model axially-ordered, cross-linked materials as stiff chains with distinct arrangements of cross-link-forming sites. Simulations suggest that systems composed of chains with identical arrangements of cross-link-forming sites exhibit critical behavior. In contrast, systems composed of non-identical chains undergo a crossover. This model suggests that the arrangement of cross-link-forming sites may contribute to the broadening of the denaturation transition in fibrillar collagen.

  20. Cross-linking chemistry of squid beak.

    Miserez, Ali; Rubin, Daniel; Waite, J Herbert


    In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.

  1. Cross-linked structure of network evolution

    Bassett, Danielle S.; Wymbs, Nicholas F.; Porter, Mason A.; Mucha, Peter J.; Grafton, Scott T.


    We study the temporal co-variation of network co-evolution via the cross-link structure of networks, for which we take advantage of the formalism of hypergraphs to map cross-link structures back to network nodes. We investigate two sets of temporal network data in detail. In a network of coupled nonlinear oscillators, hyperedges that consist of network edges with temporally co-varying weights uncover the driving co-evolution patterns of edge weight dynamics both within and between oscillator communities. In the human brain, networks that represent temporal changes in brain activity during learning exhibit early co-evolution that then settles down with practice. Subsequent decreases in hyperedge size are consistent with emergence of an autonomous subgraph whose dynamics no longer depends on other parts of the network. Our results on real and synthetic networks give a poignant demonstration of the ability of cross-link structure to uncover unexpected co-evolution attributes in both real and synthetic dynamical systems. This, in turn, illustrates the utility of analyzing cross-links for investigating the structure of temporal networks.

  2. Cross-linked structure of network evolution

    Bassett, Danielle S., E-mail: [Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (United States); Department of Physics, University of California, Santa Barbara, California 93106 (United States); Sage Center for the Study of the Mind, University of California, Santa Barbara, California 93106 (United States); Wymbs, Nicholas F.; Grafton, Scott T. [Department of Psychology and UCSB Brain Imaging Center, University of California, Santa Barbara, California 93106 (United States); Porter, Mason A. [Oxford Centre for Industrial and Applied Mathematics, Mathematical Institute, University of Oxford, Oxford OX2 6GG (United Kingdom); CABDyN Complexity Centre, University of Oxford, Oxford, OX1 1HP (United Kingdom); Mucha, Peter J. [Carolina Center for Interdisciplinary Applied Mathematics, Department of Mathematics, University of North Carolina, Chapel Hill, North Carolina 27599 (United States); Department of Applied Physical Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 (United States)


    We study the temporal co-variation of network co-evolution via the cross-link structure of networks, for which we take advantage of the formalism of hypergraphs to map cross-link structures back to network nodes. We investigate two sets of temporal network data in detail. In a network of coupled nonlinear oscillators, hyperedges that consist of network edges with temporally co-varying weights uncover the driving co-evolution patterns of edge weight dynamics both within and between oscillator communities. In the human brain, networks that represent temporal changes in brain activity during learning exhibit early co-evolution that then settles down with practice. Subsequent decreases in hyperedge size are consistent with emergence of an autonomous subgraph whose dynamics no longer depends on other parts of the network. Our results on real and synthetic networks give a poignant demonstration of the ability of cross-link structure to uncover unexpected co-evolution attributes in both real and synthetic dynamical systems. This, in turn, illustrates the utility of analyzing cross-links for investigating the structure of temporal networks.

  3. Method of preparing cross-linked enzyme particles

    Mateo, C.; Van Langen, L.M.; Van Rantwijk, F.


    The invention relates to a method of preparing cross-linked enzyme particles using a cross-linking agent. According to the invention, the enzyme particles are formed and subsequently cross-linked using a cross-linking agent having at least n reactive groups where N>=3 and a molecular weight of

  4. Method of preparing cross-linked enzyme particles

    Mateo, C.; Van Langen, L.M.; Van Rantwijk, F.


    The invention relates to a method of preparing cross-linked enzyme particles using a cross-linking agent. According to the invention, the enzyme particles are formed and subsequently cross-linked using a cross-linking agent having at least n reactive groups where N>=3 and a molecular weight of >2,00

  5. The cross linking of EPDM and NBR rubber

    Samardžija-Jovanović Suzana


    Full Text Available In the process of macromolecule cross linking, the choice of type and quantity of the components and the experimental conditions are important to obtain the new cross linked materials with better mechanical and chemical characteristics. The cross linking method depends on the rubber type and structure. Intermolecular cross linking results in the formation elastomer network. The basis of the cross linking process, between ethylene propylene diene rubber (EPDM and acrylonitrile butadiene rubber (NBR, is a chemical reaction. Fillers and other additives are present in different mass ratios in the material. The exploitation properties of the cross linked materials depend on the quantity of additive in the cross linked systems.

  6. [Corneal collagen cross-linking for keratoconus].

    Zotov, V V; Pashtaev, N P; Pozdeeva, N A


    Over the last decade, corneal collagen cross-linking (CXL) has become a conventional treatment method for progressive keratoconus. Laboratory studies have shown that CXL increases the diameter of collagen fibers and also the number of intra- and interfibrillar cross-links, thus, increasing biomechanical strength of the irradiated cornea. As confirmed by a series of clinical and randomized controlled trials, CXL is able to slow down and, perhaps, to stop the progression of keratoconus. In most post-CXL patients visual acuity improves, while keratometric readings, spherical equivalent, and higher order aberrations reduce. Although published results prove CXL effective in the treatment of progressive keratoconus, its late consequences are yet unknown. This article reviews the stages of CXL development and results of published experimental and clinical studies. Prospects for CXL modifications that do not require epithelial debridement are discussed.

  7. Characterization of Cross-Linked Lipase Aggregates

    Prabhavathi Devi, Bethala Lakshmi Anu; Guo, Zheng; Xu, Xuebing


    Commercially available microbial lipases from different sources were immobilized as cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linkers. These CLEAs were assayed based on esterification between lauric acid and n-propanol in solvent-free systems...... change upon CLEA formation. This work presents a characterization of CLEAs based on an esterification activity assay, which is useful for exploring the synthetic application potential of CLEA technology with favorable perspectives....

  8. Thermal Analyse sof Cross-Linked Polyethylene

    Radek Polansky


    Full Text Available The paper summarizes results obtained during the structural analyses measurements (Differential Scanning Calorimetry DSC, Thermogravimetry TG, Thermomechanical analysis TMA and Fourier transform infrared spectroscopy FT-IR. The samples of cross-linked polyethylene cable insulation were tested via these analyses. The DSC and TG were carried out using simultaneous thermal analyzer TA Instruments SDT Q600 with connection of Fourier transform infrared spectrometer Nicolet 380. Thermomechanical analysis was carried out by TMA Q400EM TA Instruments apparatus.

  9. Collagen cross-linking in thin corneas

    Prema Padmanabhan


    Full Text Available Collagen cross-linking (CXL has become the standard of care for progressive keratoconus, after numerous clinical studies have established its efficacy and safety in suitably selected eyes. The standard protocol is applicable in eyes which have a minimum corneal thickness of 400 μm after epithelial debridement. This prerequisite was stipulated to protect the corneal endothelium and intraocular tissues from the deleterious effect of ultraviolet-A (UVA radiation. However, patients with keratoconus often present with corneal thickness of less than 400 μm and could have otherwise benefited from this procedure. A few modifications of the standard procedure have been suggested to benefit these patients without a compromise in safety. Transepithelial cross-linking, pachymetry-guided epithelial debridement before cross-linking, and the use of hypoosmolar riboflavin are some of the techniques that have been attempted. Although clinical data is limited at the present time, these techniques are worth considering in patients with thin corneas. Further studies are needed to scientifically establish their efficacy and safety.

  10. Cross-linking for microbial keratitis

    Jayesh Vazirani


    Full Text Available The success of collagen cross-linking as a clinical modality to modify the clinical course in keratoconus seems to have fueled the search for alternative applications for this treatment. Current clinical data on its efficacy is limited and laboratory data seems to indicate that it performs poorly against resistant strains of bacteria and against slow growing organisms. However, the biological plausibility of crosslinking and the lack of effective strategies in managing infections with these organisms continue to focus attention on this potential treatment. Well-conducted experimental and clinical studies with controls are required to answer the questions of its efficacy in future.

  11. The Cross - linking reaction of HEC

    XIONG Jian; YE Jun; XUAN Zhiyong; XIE Guohui


    @@ Cellulose ethers are important components for light industries such as food and papermaking industries. Its modification research is a frontline science to widen their uses and realize their industrialization. The target of modification is to make high production value, low input and meet the needs better in industries. O-(2-hydroxyethl) cellulose (HEC) is one of the best-known cellulose ether derivatives. It is mainly used as thickeners, dispersants, adhesives, extenders, and films because of its water solubility and gel-forming properties. The present research,by means of cross-linking, we study the influence on HEC about rheological behavior.This will provide a feasible scheme for cellulose ethers modification.

  12. The Cross - linking reaction of HEC

    XIONG; Jian


    Cellulose ethers are important components for light industries such as food and papermaking industries. Its modification research is a frontline science to widen their uses and realize their industrialization. The target of modification is to make high production value, low input and meet the needs better in industries.  O-(2-hydroxyethl) cellulose (HEC) is one of the best-known cellulose ether derivatives. It is mainly used as thickeners, dispersants, adhesives, extenders, and films because of its water solubility and gel-forming properties. The present research,by means of cross-linking, we study the influence on HEC about rheological behavior.This will provide a feasible scheme for cellulose ethers modification.……

  13. Cross-link guided molecular modeling with ROSETTA.

    Abdullah Kahraman

    Full Text Available Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods.

  14. Grafted Cross-Linked Polyolefin Substrates for Peptide Synthesis and Assays


    suited for use in solid-phase biosystems, notably bioassays, such as immunoassays, DNA hybridization assays or PCR amplification. The grafted chains may bear substituents which are such that the polymer-grafted cross-linked polyolefin substrate is swellable by water or aqueous media, in other words...

  15. Cross Linked Metal Particles for Low Noise Bolometer Materials


    SECURITY CLASSIFICATION OF: This final report summarizes WSU’s progress from 4/2/2015 to 09/30/2016 on the project, "Cross-linked Metal Particles ...2016 Final Report: Cross-linked Metal Particles for Low-noise Bolometer Materials The views, opinions and/or findings contained in this report are...peer-reviewed journals: Number of Papers published in non peer-reviewed journals: Final Report: Cross-linked Metal Particles for Low-noise Bolometer

  16. Cross-linked polyethylenimine–tripolyphosphate nanoparticles for gene delivery

    Huang XZ


    Full Text Available Xianzhang Huang,1 Sujing Shen,2 Zhanfeng Zhang,1 Junhua Zhuang1 1Department of Laboratory Science, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2Department of Laboratory Science, Guangdong Second Provincial Traditional Chinese Medicine Hospital, Guangzhou, People’s Republic of China Abstract: The high transfection efficiency of polyethylenimine (PEI makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP. We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site–enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin-labeled small interfering ribonucleic acids (siRNAs was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene

  17. Radiation cross-linked polyolefin-insulated wire

    Sano, K.; Ishitani, H.

    Because radiation cross-linked polyolefin has excellent mechanical heat resistance, its application limit can be expanded extremely by improving the resistance against heat oxidation and flame. This paper is concerning a halogen free radiation cross-linked polyolefin-insulated wire having excellent heat resistance and flameretardant property, which is used for appliances.

  18. Research Progress in Corneal Cross-linking Agents

    Na Li; Xiujun Peng; Zhengjun Fan


    Corneal collagen cross-linking with UVA-riboflavin is cur-rently the only method for preventing the progression of kera-toconus from the pathological perspective. Topical application of a direct cross-linking agent is now attracting widespread at-tention in clinical settings..This article reviews the research progress in the application of indirect or direct cross-linking agents (e.g., riboflavin, glucose, ribose, glutaraldehyde, formaldehyde,.glyceraldehyde,.short chain aliphatic β-nitro alcohol, and genipin) in the treatment of corneal diseases and analyzes the cross-linking efficacy,.toxicity,.and merits and disadvantages of each cross-linking agent,.providing clinical information for further studies.

  19. Elasticity of cross-linked semiflexible biopolymers under tension.

    von der Heydt, Alice; Wilkin, Daniel; Benetatos, Panayotis; Zippelius, Annette


    Aiming at the mechanical properties of cross-linked biopolymers, we set up and analyze a model of two weakly bending wormlike chains subjected to a tensile force, with regularly spaced inter-chain bonds (cross-links) represented by harmonic springs. Within this model, we compute the force-extension curve and the differential stiffness exactly and discuss several limiting cases. Cross-links effectively stiffen the chain pair by reducing thermal fluctuations transverse to the force and alignment direction. The extra alignment due to cross-links increases both with growing number and with growing strength of the cross-links, and is most prominent for small force f. For large f, the additional, cross-link-induced extension is subdominant except for the case of linking the chains rigidly and continuously along their contour. In this combined limit, we recover asymptotically the elasticity of a weakly bending wormlike chain without constraints, stiffened by a factor of 4. The increase in differential stiffness can be as large as 100% for small f or large numbers of cross-links.

  20. Molecular Structures of Isolevuglandin-Protein Cross-Links.

    Bi, Wenzhao; Jang, Geeng-Fu; Zhang, Lei; Crabb, John W; Laird, James; Linetsky, Mikhail; Salomon, Robert G


    Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS-PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11-17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]LGE2 with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide-pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11-17) tends to spontaneously oligomerize.

  1. Current status of accelerated corneal cross-linking

    Michael Mrochen


    Full Text Available Corneal cross-linking with riboflavin is a technique to stabilize or reduce corneal ectasia, in diseases such as keratoconus and post-laser-assisted in situ keratomileusis (LASIK ectasia. There is an interest by patient as well as clinicians to reduce the overall treatment time. Especially, the introduction of corneal cross-linking in combination with corneal laser surgery demands a shorter treatment time to assure a sufficient patient flow. The principles and techniques of accelerated corneal cross-linking is discussed.

  2. Covalently cross-linked polyetheretherketone proton exchange membrane for DMFC

    Luo, H


    Full Text Available -7 cm2/s) and good electrochemical stability. The results suggested that cross-linked polyetheretherketone membrane is particularly promising to be used as proton exchange membrane for the direct methanol fuel cell application....

  3. Gelation threshold of cross-linked polymer brushes.

    Hoffmann, Max; Lang, Michael; Sommer, Jens-Uwe


    The cross-linking of polymer brushes is studied using the bond-fluctuation model. By mapping the cross-linking process into a two-dimensional (2D) percolation problem within the lattice of grafting points, we investigate the gelation transition in detail. We show that the particular properties of cross-linked polymer brushes can be reduced to the distribution of bonds which are formed between the grafted chains, and we propose scaling arguments to relate the gelation threshold to the chain length and the grafting density. The gelation threshold is lower than the percolation threshold for 2D bond percolation because of the longer range and broad distribution of bonds formed by the cross-linking process. We term this type of percolation problem star percolation. We observe a broad crossover from mean-field to critical percolation behavior by analyzing the cluster size distribution near the gelation threshold.

  4. Photochemical Patterning of Ionically Cross-Linked Hydrogels

    Marion Bruchet


    Full Text Available Iron(III cross-linked alginate hydrogel incorporating sodium lactate undergoes photoinduced degradation, thus serving as a biocompatible positive photoresist suitable for photochemical patterning. Alternatively, surface etching of iron(III cross-linked hydrogel contacting lactic acid solution can be used for controlling the thickness of the photochemical pattering. Due to biocompatibility, both of these approaches appear potentially useful for advanced manipulation with cell cultures including growing cells on the surface or entrapping them within the hydrogel.


    Atul; Superna; Bhimasankar; Vijayleela


    To evaluate the outcome of collagen cross linkage using riboflavin 0.1% and ultraviolet A radiation of a wavelength 370nm . PURPOSE : To determine the effect of collagen cross linking for keratoconus on pachymetry , corneal topography, uncorrected visual acuity, specular count, IOP at 1, 3, 6 months . METHODS : The current study was designed as a prospective interventional trial of corneal collagen cross - linking in subjects w...

  6. Elasticity of cross-linked semiflexible biopolymers under tension

    von der Heydt, Alice; Benetatos, Panayotis; Zippelius, Annette


    Aiming at the mechanical properties of cross-linked biopolymers, we set up and analyze a model of two weakly bending wormlike chains subjected to a tensile force, with regularly spaced inter-chain bonds (cross-links) represented by harmonic springs. Within this model, we compute the force-extension curve and the differential stiffness exactly and discuss several limiting cases. Cross-links effectively stiffen the chain pair by reducing thermal fluctuations transverse to the force and alignment direction. The extra alignment due to cross-links increases both with growing number and with growing strength of the cross-links, and is most prominent for small force f. For large f, the additional, cross-link-induced extension is subdominant except for the case of linking the chains rigidly and continuously along their contour. In this combined limit, we recover asymptotically the elasticity of a weakly bending wormlike chain without constraints, stiffened by a factor four. The increase in differential stiffness can ...

  7. Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel.

    Villanueva, Idalis; Klement, Brenda J; von Deutsch, Daniel; Bryant, Stephanie J


    In designing a tissue engineering strategy for cartilage repair, selection of both the bioreactor, and scaffold is important to the development of a mechanically functional tissue. The hydrodynamic environment associated with many bioreactors enhances nutrient transport, but also introduces fluid shear stress, which may influence cellular response. This study examined the combined effects of hydrogel cross-linking and the hydrodynamic environment on early chondrocyte response. Specifically, chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels having two different cross-linked structures, corresponding to a low and high cross-linking density. Both cross-linked gels yielded high water contents (92% and 79%, respectively) and mesh sizes of 150 and 60 A respectively. Cell-laden PEG hydrogels were cultured in rotating wall vessels (RWV) or under static cultures for up to 5 days. Rotating cultures yielded low fluid shear stresses (production, and matrix deposition for glycosaminoglycans (GAG). In static cultures, gel cross-linking had no effect on DNA content, NO production, or GAG production; although GAG production increased with culture time for both cross-linked gels. In rotating cultures, DNA content increased, NO production decreased, and overall GAG production decreased when compared to static controls for the low cross-linked gels. For the high cross-linked gels, the hydrodynamic environment had no effect on DNA content, but exhibited similar results to the low cross-linked gel for NO production, and matrix production. Our findings demonstrated that at early culture times, when there is limited matrix production, the hydrodynamic environment dramatically influences cell response in a manner dependent on the gel cross-linking, which may impact long-term tissue development.

  8. Collagen/elastin hydrogels cross-linked by squaric acid.

    Skopinska-Wisniewska, J; Kuderko, J; Bajek, A; Maj, M; Sionkowska, A; Ziegler-Borowska, M


    Hydrogels based on collagen and elastin are very valuable materials for medicine and tissue engineering. They are biocompatible; however their mechanical properties and resistance for enzymatic degradation need to be improved by cross-linking. Up to this point many reagents have been tested but more secure reactants are still sought. Squaric acid (SqAc), 3,4-dihydroxy 3-cyclobutene 1,2-dione, is a strong, cyclic acid, which reacts easily with amine groups. The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5%, 10% and 20% of SqAc and neutralized via dialysis against deionized water were tested. Cross-linked, 3-D, transparent hydrogels were created. The cross-linked materials are stiffer and more resistant to enzymatic degradation than those that are unmodified. The pore size, swelling ability and surface polarity are reduced due to 5% and 10% of SqAc addition. At the same time, the cellular response is not significantly affected by the cross-linking. Therefore, squaric acid would be regarded as a safe, effective cross-linking agent.

  9. Computer simulation of randomly cross-linked polymer networks

    Williams, T P


    In this work, Monte Carlo and Stochastic Dynamics computer simulations of mesoscale model randomly cross-linked networks were undertaken. Task parallel implementations of the lattice Monte Carlo Bond Fluctuation model and Kremer-Grest Stochastic Dynamics bead-spring continuum model were designed and used for this purpose. Lattice and continuum precursor melt systems were prepared and then cross-linked to varying degrees. The resultant networks were used to study structural changes during deformation and relaxation dynamics. The effects of a random network topology featuring a polydisperse distribution of strand lengths and an abundance of pendant chain ends, were qualitatively compared to recent published work. A preliminary investigation into the effects of temperature on the structural and dynamical properties was also undertaken. Structural changes during isotropic swelling and uniaxial deformation, revealed a pronounced non-affine deformation dependant on the degree of cross-linking. Fractal heterogeneiti...

  10. FTIR Spectroscopic Studies on Cross Linking of SU-8 Photoresist

    Kalaiselvi, S. M. P.; Tan, T. L.; Rawat, R. S.; Lee, P.; Heussler, S. P.; Breese, M. B. H.


    The usage of chemically-amplified, negative tone SU-8 photoresist is numerous, spanning industrial, scientific and medical fields. Hence, in this study, some preliminary studies were conducted to understand the dosage and heat treatment requirements of the SU-8 photoresist essential for pattern generation using X-ray lithography. In this work, using Synchrotron as the X-ray source, SU-8 photoresist was characterized for X-ray lithography in terms of its process parameters such as X-ray exposure dose, post exposure bake (PEB) time and temperature for various photoresist thicknesses which is considered worthwhile in view of applications of SU-8 for the fabrication of very high aspect ratio micro structures. The process parameters were varied and the resultant cross linking of the molecular chains of the photoresist was accurately monitored using a Fourier Transform Infra-Red (FTIR) spectrometer and the results are discussed. The infrared absorption peak at 914 cm-1 in the spectrum of the SU-8 photoresist was found to be a useful indicator for the completion of cross linking in the SU-8 photoresist. Results show that the cross linking of the SU-8 photoresist is at a higher rate from 0 J/cm3 to 30 J/cm3 after which the peak almost saturates regardless of the PEB time. It is a good evidence for the validation of dosage requirement of SU-8 photoresist for effective completion of cross linking, which in turn is a requirement for efficient fabrication of micro and nano structures. An analogous behavior was also observed between the extent of cross linking and the PEB time and temperature. The rate of cross linking declines after a certain period of PEB time regardless of PEB temperature. The obtained results also show a definite relation between variation of the absorbance area of the peak at 914 cm-1 and the X-ray exposure dose.

  11. Advanced Corneal Cross-Linking System with Fluorescence Dosimetry

    Marc D. Friedman


    Full Text Available Purpose. This paper describes an advanced system that combines corneal cross-linking with riboflavin with fluorescence dosimetry, the ability to measure riboflavin diffusion within the cornea both before and during UVA treatment. Methods and Results. A corneal cross-linking system utilizing a digital micromirror device (DMD was assembled and used to measure diffusion coefficients of 0.1% riboflavin in 20% dextran in porcine eyes. A value of (3.3±0.2×10−7 cm2/s was obtained for the stroma. Diffusion coefficients for the transepithelial formulation of 0.1% riboflavin in 0.44% saline and 0.02% BAK were also measured to be 4.7±0.3×10−8 cm2/s for epithelium only and (4.6±0.4×10−7 cm2/s for stroma only. Riboflavin consumption during a UVA treatment was also demonstrated. Conclusion. A new advanced corneal cross-linking system with fluorescence dosimetry of riboflavin has been demonstrated. It is hoped that this method may play a significant role in determining the underlying mechanisms of corneal cross-linking and assist with the development of additional riboflavin formulations. Moreover, dosimetry may prove valuable in providing a method to account for the biological differences between individuals, potentially informing cornea-specific UVA treatment doses in real time.

  12. Simulation of Fracture Nucleation in Cross-Linked Polymer Networks

    Moller, J. C.; Barr, S. A.; Schultz, E. J.; Breitzman, T. D.; Berry, R. J.


    A novel atomistic simulation method is developed whereby polymer systems can undergo strain-rate-controlled deformation while bond scission is enabled. The aim is to provide insight into the nanoscale origins of fracture. Various highly cross-linked epoxy systems including various resin chain lengths and levels of nonreactive dilution were examined. Consistent with the results of physical experiments, cured resin strength increased and ductility decreased with increasing cross-link density. An analysis of dihedral angle activity shows the locations in the molecular network that are most absorptive of mechanical energy. Bond scission occurred principally at cross-link sites as well as between phenyl rings in the bisphenol moiety. Scissions typically occurred well after yield and were accompanied by steady increases in void size and dihedral angle motion between bisphenol moieties and at cross-link sites. The methods developed here could be more broadly applied to explore and compare the atomistic nature of deformation for various polymers such that mechanical and fracture properties could be tuned in a rational way. This method and its results could become part of a solution system that spans multiple length and time scales and that could more completely represent such mechanical events as fracture.

  13. The Database of Ribosomal Cross-links: an update.

    Baranov, P V; Kubarenko, A V; Gurvich, O L; Shamolina, T A; Brimacombe, R


    The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at or http://www. approximately ag_ribo/ag_brimacombe/drc/

  14. Elasticity of Rigidly Cross-Linked Networks of Athermal Filaments

    Zagar, Goran; Onck, Patrick R.; Van der Giessen, Erik


    Actin filaments assemble into network-like structures and play an important role in various cellular mechanical processes. It is known that the response of actin networks cross-linked by stiff proteins is characterized by two distinct regimes: (i) a linear stress strain response for small deformatio

  15. Molecular mechanisms in deformation of cross-linked hydrogel nanocomposite.

    Mathesan, Santhosh; Rath, Amrita; Ghosh, Pijush


    The self-folding behavior in response to external stimuli observed in hydrogels is potentially used in biomedical applications. However, the use of hydrogels is limited because of its reduced mechanical properties. These properties are enhanced when the hydrogels are cross-linked and reinforced with nanoparticles. In this work, molecular dynamics (MD) simulation is applied to perform uniaxial tension and pull out tests to understand the mechanism contributing towards the enhanced mechanical properties. Also, nanomechanical characterization is performed using quasi static nanoindentation experiments to determine the Young's modulus of hydrogels in the presence of nanoparticles. The stress-strain responses for chitosan (CS), chitosan reinforced with hydroxyapatite (HAP) and cross-linked chitosan are obtained from uniaxial tension test. It is observed that the Young's modulus and maximum stress increase as the HAP content increases and also with cross-linking process. Load displacement plot from pullout test is compared for uncross-linked and cross-linked chitosan chains on hydroxyapatite surface. MD simulation reveals that the variation in the dihedral conformation of chitosan chains and the evolution of internal structural variables are associated with mechanical properties. Additional results reveal that the formation of hydrogen bonds and electrostatic interactions is responsible for the above variations in different systems.

  16. Porous Cross-Linked Polyimide-Urea Networks

    Meador, Mary Ann B. (Inventor); Nguyen, Baochau N. (Inventor)


    Porous cross-linked polyimide-urea networks are provided. The networks comprise a subunit comprising two anhydride end-capped polyamic acid oligomers in direct connection via a urea linkage. The oligomers (a) each comprise a repeating unit of a dianhydride and a diamine and a terminal anhydride group and (b) are formulated with 2 to 15 of the repeating units. The subunit was formed by reaction of the diamine and a diisocyanate to form a diamine-urea linkage-diamine group, followed by reaction of the diamine-urea linkage-diamine group with the dianhydride and the diamine to form the subunit. The subunit has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups. The subunit has been chemically imidized to yield the porous cross-linked polyimide-urea network. Also provided are wet gels, aerogels, and thin films comprising the networks, and methods of making the networks.

  17. Femtosecond laser collagen cross-linking without traditional photosensitizers

    Guo, Yizang; Wang, Chao; Celi, Nicola; Vukelic, Sinisa


    Collagen cross-linking in cornea has the capability of enhancing its mechanical properties and thereby providing an alternative treatment for eye diseases such as keratoconus. Currently, riboflavin assisted UVA light irradiation is a method of choice for cross-link induction in eyes. However, ultrafast pulsed laser interactions may be a powerful alternative enabling in-depth treatment while simultaneously diminishing harmful side effects such as, keratocyte apoptosis. In this study, femtosecond laser is utilized for treatment of bovine cornea slices. It is hypothesized that nonlinear absorption of femtosecond laser pulses plays a major role in the maturation of immature cross-links and the promotion of their growth. Targeted irradiation with tightly focused laser pulses allows for the absence of a photosensitizing agent. Inflation test was conducted on half treated porcine cornea to identify the changes of mechanical properties due to laser treatment. Raman spectroscopy was utilized to study subtle changes in the chemical composition of treated cornea. The effects of treatment are analyzed by observing shifts in Amide I and Amide III bands, which suggest deformation of the collagen structure in cornea due to presence of newly formed cross-links.

  18. Scleral lens tolerance after corneal cross-linking for keratoconus

    Visser, Esther Simone; Soeters, Nienke; Tahzib, Nayyirih G.


    Purpose. Subjective and objective evaluation of scleral lens tolerance and fitting before and after corneal cross-linking (CXL) for progressive keratoconus. Methods. In this prospective cohort, evaluations were made of 18 unilateral eyes in patients who underwent CXL and had been wearing scleral len

  19. Lactoferrin binding to transglutaminase cross-linked casein micelles

    Anema, S.G.; de Kruif, C.G.


    Casein micelles in skim milk were either untreated (untreated milk) or were cross-linked using transglutaminase (TGA-milk). Added lactoferrin (LF) bound to the casein micelles and followed Langmuir adsorption isotherms. The adsorption level was the same in both milks and decreased the micellar zeta

  20. Polyimide Aerogels with Three-Dimensional Cross-Linked Structure

    Meador, Mary Ann B. (Inventor)


    A method for creating a three dimensional cross-linked polyimide structure includes dissolving a diamine, a dianhydride, and a triamine in a solvent, imidizing a polyamic acid gel by heating the gel, extracting the gel in a second solvent, supercritically drying the gel, and removing the solvent to create a polyimide aerogel.

  1. 21 CFR 177.2420 - Polyester resins, cross-linked.


    .... Pentaerythritol. Polyoxypropylene ethers of 4,4′-isopropylide-nediphenol (containing an average of 2-7.5 moles of...-pentanediol. (3) Cross-linking agents: Butyl acrylate. Butyl methacrylate. Ethyl acrylate. Ethylhexyl acrylate... the production of the resins or added thereto to impart desired technical or physical...

  2. Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts

    Sheldon, R.A.


    The key to obtaining an optimum performance of an enzyme is often a question of devising an effective method for its immobilization. In the present review, we describe a novel, versatile and effective methodology for enzyme immobilization as CLEAs (cross-linked enzyme aggregates). The method is

  3. Hyper-cross-linked, hybrid membranes via interfacial polymerization

    Raaijmakers, M.J.T.


    Hyper-cross-linked, hybrid membranes consist of covalent networks of alternating organic and inorganic, or biological groups. This thesis reports on the preparation of such hybrid networks via interfacial polymerization. The structure-property relationships of the hybrid networks depend strongly on

  4. Oxidation increases mucin polymer cross-links to stiffen airway mucus gels.

    Yuan, Shaopeng; Hollinger, Martin; Lachowicz-Scroggins, Marrah E; Kerr, Sheena C; Dunican, Eleanor M; Daniel, Brian M; Ghosh, Sudakshina; Erzurum, Serpel C; Willard, Belinda; Hazen, Stanley L; Huang, Xiaozhu; Carrington, Stephen D; Oscarson, Stefan; Fahy, John V


    Airway mucus in cystic fibrosis (CF) is highly elastic, but the mechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

  5. Transient Anisocoria after Corneal Collagen Cross-Linking

    Kymionis, George D.; Grentzelos, Michael A.; Stojanovic, Nela; Paraskevopoulos, Theodore A.; Detorakis, Efstathios T.


    Purpose. To report a case with transient anisocoria after corneal collagen cross-linking (CXL). Methods. Case report. Results. A 24-year-old male underwent corneal collagen cross-linking (CXL) in his right eye for keratoconus. At the end of the procedure, the pupil of the treated eye was irregular and dilated, while the pupil of the fellow eye was round, regular, and reactive (anisocoria). The following day, pupils were round, regular, and reactive in both eyes. Conclusion. Anisocoria may be a transient and innocuous complication after CXL. A possible cause for this complication might be the anesthetic drops used before and during the surgical procedure or/and the ultraviolet A irradiation during the treatment. PMID:25276451

  6. Transient Anisocoria after Corneal Collagen Cross-Linking

    George D. Kymionis


    Full Text Available Purpose. To report a case with transient anisocoria after corneal collagen cross-linking (CXL. Methods. Case report. Results. A 24-year-old male underwent corneal collagen cross-linking (CXL in his right eye for keratoconus. At the end of the procedure, the pupil of the treated eye was irregular and dilated, while the pupil of the fellow eye was round, regular, and reactive (anisocoria. The following day, pupils were round, regular, and reactive in both eyes. Conclusion. Anisocoria may be a transient and innocuous complication after CXL. A possible cause for this complication might be the anesthetic drops used before and during the surgical procedure or/and the ultraviolet A irradiation during the treatment.

  7. Cytokines and growth factors cross-link heparan sulfate

    Migliorini, Elisa; Thakar, Dhruv; Kühnle, Jens; Sadir, Rabia; Dyer, Douglas P.; Li, Yong; Sun, Changye; Volkman, Brian F.; Handel, Tracy M.; Coche-Guerente, Liliane; Fernig, David G.; Lortat-Jacob, Hugues; Richter, Ralf P.


    The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors. PMID:26269427

  8. Electrochemical Characterization of Ultrathin Cross-Linked Metal Nanoparticle Films.

    Han, Chu; Percival, Stephen J; Zhang, Bo


    Here we report the preparation, characterization, and electrochemical study of conductive, ultrathin films of cross-linked metal nanoparticles (NPs). Nanoporous films ranging from 40 to 200 nm in thickness composed of gold and platinum NPs of ∼5 nm were fabricated via a powerful layer-by-layer spin coating process. This process allows preparation of uniform NP films as large as 2 × 2 cm(2) with precise control over thickness, structure, and electrochemical and electrocatalytic properties. Gold, platinum, and bimetallic NP films were fabricated and characterized using cyclic voltammetry, scanning electron microscopy, and conductance measurements. Their electrocatalytic activity toward the oxygen reduction reaction (ORR) was investigated. Our results show that the electrochemical activity of such NP films is initially hindered by the presence of dense thiolate cross-linking ligands. Both electrochemical cycling and oxygen plasma cleaning are effective means in restoring their electrochemical activity. Gold NP films have higher electric conductivity than platinum possibly due to more uniform film structure and closer particle-particle distance. The electrochemical and electrocatalytic performance of platinum NP films can be greatly enhanced by the incorporation of gold NPs. This work focuses on electrochemical characterization of cross-linked NP films and demonstrates several unique properties. These include quick and easy preparation, ultrathin and uniform film thickness, tunable structure and composition, and transferability to many other substrates.

  9. Macrophage response to cross-linked and conventional UHMWPE.

    Sethi, Rajiv K; Neavyn, Mark J; Rubash, Harry E; Shanbhag, Arun S


    To prevent wear debris-induced osteolysis and aseptic loosening, cross-linked ultra-high molecular weight polyethylene's (UHMWPE) with improved wear resistance have been developed. Hip simulator studies have demonstrated very low wear rates with these new materials leading to their widespread clinical use. However, the biocompatibility of this material is not known. We studied the macrophage response to cross-linked UHMWPE (XLPE) and compared it to conventional UHMWPE (CPE) as well as other clinically used orthopaedic materials such as titanium-alloy (TiAlV) and cobalt-chrome alloy (CoCr). Human peripheral blood monocytes and murine macrophages, as surrogates for cells mediating peri-implant inflammation, were cultured onto custom designed lipped disks fabricated from the test materials to isolate cells. Culture supernatants were collected at 24 and 48h and analyzed for cytokines such as IL-1alpha, IL-1beta, TNF-alpha and IL-6. Total RNA was extracted from adherent cells and gene expression was analyzed using qualitative RT-PCR. In both in vitro models, macrophages cultured on cross-linked and conventional polyethylene released similar levels of cytokines, which were also similar to levels on control tissue culture dishes. Macrophages cultured on TiAlV and CoCr-alloy released significantly higher levels of cytokines. Human monocytes from all donors varied in the magnitude of cytokines released when cultured on identical surfaces. The variability in individual donor responses to TiAlV and CoCr surfaces may reflect how individuals respond differently to similar stimuli and perhaps reveal a predisposed sensitivity to particular materials.

  10. Newer protocols and future in collagen cross-linking

    Arthur B Cummings


    Full Text Available Corneal Cross-Linking (CXL is an established surgical procedure for the treatment of corneal disorders such as corneal ectasia and keratoconus. This method of treatment stabilises the corneal structure and increases rigidity, reducing the requirement for corneal transplantation. Since its development, many scientific studies have been conducted to investigate ways of improving the procedure. Biomechanical stability of the cornea after exposure to UV-A light, and the effect of shortening procedure time has been some of the many topics explored

  11. Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.

    Leitner, Alexander; Walzthoeni, Thomas; Kahraman, Abdullah; Herzog, Franz; Rinner, Oliver; Beck, Martin; Aebersold, Ruedi


    Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.

  12. Cross-linked comb-shaped anion exchange membranes with high base stability

    Li, NW; Wang, LZ; Hickner, M


    A unique one-step cross-linking strategy that connects quaternary ammonium centers using Grubbs II-catalyzed olefin metathesis was developed. The cross-linked anion exchange membranes showed swelling ratios of less than 10% and hydroxide conductivities of 18 to 40 mS cm(- 1). Cross-linking improved the membranes' stability to hydroxide degradation compared to their non-cross-linked analogues.

  13. Cross-linking e segmento de anel corneano intraestromal

    Adimara da Candelaria Renesto


    Full Text Available O cross-linking corneano é um procedimento usado para a estabilização mecânica e aumento da rigidez corneana em pacientes com ceratocone (reduzindo a possibilidade de progressão, e também em processos inflamatórios de afinamento corneano. Os segmentos de anéis corneanos intraestromais têm como princípio o aplanamento central da córnea. Inicialmente utilizados para correção de baixa miopia, a principal indicação atual é em pacientes com ceratocone, para melhorar a acuidade visual não corrigida, a acuidade visual corrigida e permitir uma melhor tolerância ao uso de lentes de contato como também retardar a necessidade de um transplante de córnea. O objetivo deste artigo é revisar algumas publicações relacionadas ao cross-linking corneano e à inserção do segmento de anel intraestromal, apresentando suas indicações, resultados e complicações relatadas até o momento.

  14. Fiber optic immunosensor for cross-linked fibrin concentration

    Moskowitz, Samuel E.


    Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

  15. Phosphate uptake studies of cross-linked chitosan bead materials.

    Mahaninia, Mohammad H; Wilson, Lee D


    A systematic experimental study is reported that provides a molecular based understanding of cross-linked chitosan beads and their adsorption properties in aqueous solution containing phosphate dianion (HPO4(2-)) species. Synthetically modified chitosan using epichlorohydrin and glutaraldehyde cross-linkers result in surface modified beads with variable hydrophile-lipophile character and tunable HPO4(2-) uptake properties. The kinetic and thermodynamic adsorption properties of cross-linked chitosan beads with HPO4(2-) species were studied in aqueous solution. Complementary structure and physicochemical characterization of chitosan beads via potentiometry, Raman spectroscopy, DSC, and dye adsorption measurements was carried out to establish structure-property relationships. The maximum uptake (Qm) of bead systems with HPO4(2-) at equilibrium was 52.1mgg(-1); whereas, kinetic uptake results for chitosan bead/phosphate systems are relatively rapid (0.111-0.113min(-1)) with an intraparticle diffusion rate-limiting step. The adsorption process follows a multi-step pathway involving inner- and outer-sphere complexes with significant changes in hydration. Phosphate uptake strongly depends on the composition and type of cross-linker used for preparation of chitosan beads. The adsorption isotherms and structural characterization of bead systems illustrate the role of surface charge, hydrophile-lipophile balance, adsorption site accessibility, and hydration properties of the chitosan bead surface. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Adsorption of methyl orange onto protonated cross-linked chitosan

    Ruihua Huang


    Full Text Available The adsorption of methyl orange (MO from aqueous solutions on protonated cross-linked chitosan was studied in a batch system. The results showed that the adsorption of MO onto protonated cross-linked chitosan was affected significantly by initial MO concentration, adsorbent dosage, adsorption temperature, and contact time. The pH value of solution had a minor impact on the adsorption of MO in a pH range of 1.0–9.1. The equilibrium isotherms at different temperatures (293, 303, and 313 K and pH values (4.5, 6.7, and 9.1 were investigated. Langmuir model was able to describe these Equilibrium data fitted perfectly. The maximum monolayer adsorption capacities obtained from the Langmuir model were 89.29, 130.9, and 180.2 mg/g at 293, 303, and 313 K, respectively. Adsorption kinetics at different concentrations (100, 200 and 300 mg/L and pH values (4.5, 6.7 and 9.1 were also studied. The kinetics was correlated well with the pseudo second-order model.

  17. Supermacroporous chemically cross-linked poly(aspartic acid) hydrogels.

    Gyarmati, Benjámin; Mészár, E Zsuzsanna; Kiss, Lóránd; Deli, Mária A; László, Krisztina; Szilágyi, András


    Chemically cross-linked poly(aspartic acid) (PASP) gels were prepared by a solid-liquid phase separation technique, cryogelation, to achieve a supermacroporous interconnected pore structure. The precursor polymer of PASP, polysuccinimide (PSI) was cross-linked below the freezing point of the solvent and the forming crystals acted as templates for the pores. Dimethyl sulfoxide was chosen as solvent instead of the more commonly used water. Thus larger temperatures could be utilized for the preparation and the drawback of increase in specific volume of water upon freezing could be eliminated. The morphology of the hydrogels was characterized by scanning electron microscopy and interconnectivity of the pores was proven by the small flow resistance of the gels. Compression tests also confirmed the interconnected porous structure and the complete re-swelling and shape recovery of the supermacroporous PASP hydrogels. The prepared hydrogels are of interest for several biomedical applications as scaffolding materials because of their cytocompatibility, controllable morphology and pH-responsive character.

  18. Microbial Keratitis After Collagen Cross-linking Treatment

    Banu Torun Acar


    Full Text Available A 33-year-old woman presented with pain, redness, and diminution of vision that occurred 2 days after collagen cross-linking had been performed for keratoconus in the right eye. Culture results from the patient's contact lens and corneal scrapings were positive for Staphylococcus epidermidis. According to the results of antibiotic susceptibility testing, the patient was treated with hourly topical fortified vancomycin and exocin. Before collagen cross-linking, the best-corrected visual acuity (BCVA was 4/10, the manifest refraction was -7.00 -1.755 3°. Four months after the procedure, the BCVA was 4/10, the manifest refraction was -5.50 -1.75 10°. Slit-lamp examination revealed a mild residual haze in the upper midperipheral cornea, and stromal opacities had disappeared. Collagen crosslinking is less invasive compared to other methods for treatment of keratoconus, but epithelial debridement and bandage contact lens wearing may lead to the development of bacterial keratitis. (Turk J Oph thal mol 2012; 42: 300-2

  19. Covalently Cross-Linked Arabinoxylans Films for Debaryomyces hansenii Entrapment

    Ramsés González-Estrada


    Full Text Available In the present study, wheat water extractable arabinoxylans (WEAX were isolated and characterized, and their capability to form covalently cross-linked films in presence of Debaryomyces hansenii was evaluated. WEAX presented an arabinose to xylose ratio of 0.60, a ferulic acid and diferulic acid content of 2.1 and 0.04 µg∙mg−1 WEAX, respectively and a Fourier Transform Infra-Red (FT-IR spectrum typical of WEAX. The intrinsic viscosity and viscosimetric molecular weight values for WEAX were 3.6 dL∙g−1 and 440 kDa, respectively. The gelation of WEAX (1% w/v with and without D. hansenii (1 × 107 CFU∙cm−2 was rheologically investigated by small amplitude oscillatory shear. The entrapment of D. hansenii decreased gel elasticity from 1.4 to 0.3 Pa, probably by affecting the physical interactions between WEAX chains. Covalently cross-linked WEAX films containing D. hansenii were prepared by casting. Scanning electron microscopy images show that WEAX films containing D. hansenii were porous and consisted of granular-like and fibre microstructures. Average tensile strength, elongation at break and Young’s modulus values dropped when D. hansenii was present in the film. Covalently cross-lined WEAX containing D. hansenii could be a suitable as a functional entrapping film.

  20. One-step electrospinning of cross-linked chitosan fibers.

    Schiffman, Jessica D; Schauer, Caroline L


    Chitin is a nitrogen-rich polysaccharide that is abundant in crustaceans, mollusks, insects, and fungi and is the second most abundant organic material found in nature next to cellulose. Chitosan, the N-deacetylated derivative of chitin, is environmentally friendly, nontoxic, biodegradable, and antibacterial. Fibrous mats are typically used in industries for filter media, catalysis, and sensors. Decreasing fiber diameters within these mats causes many beneficial effects such as increased specific surface area to volume ratios. When the intrinsically beneficial effects of chitosan are combined with the enhanced properties of nanofibrous mats, applications arise in a wide range of fields, including medical, packaging, agricultural, and automotive. This is particularly important as innovative technologies that focus around bio-based materials are currently of high urgency, as they can decrease dependencies on fossil fuels. We have demonstrated that Schiff base cross-linked chitosan fibrous mats can be produced utilizing a one-step electrospinning process that is 25 times faster and, therefore, more economical than a previously reported two-step vapor-cross-linking method. These fibrous mats are insoluble in acidic, basic, and aqueous solutions for 72 h. Additionally, this improved production method results in a decreased average fiber diameter, which measures 128 +/- 40 nm. Chemical and structural analyses were conducted utilizing Fourier transform infrared spectroscopy, solubility studies, and scanning electron microscopy.

  1. Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins.

    Pantelis Georgiades

    Full Text Available Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.

  2. Thermally Cross-Linked Anion Exchange Membranes from Solvent Processable Isoprene Containing Ionomers


    capacities (IECs). Solution cast membranes were thermally cross- linked to form anion exchange membranes. Cross-linking was achieved by taking advantage...distribution is unlimited. Thermally Cross-Linked Anion Exchange Membranes from Solvent Processable Isoprene Containing Ionomers The views...Box 12211 Research Triangle Park, NC 27709-2211 Anion Exchnage Membrane, Polymer synthesis, Morphology, Anion Conductivity REPORT DOCUMENTATION PAGE

  3. Small Strain Topological Effects of Biopolymer Networks with Rigid Cross-Links

    Zagar, G.; Onck, P. R.; Van der Giessen, E.; Garikipati, K; Arruda, EM


    Networks of cross-linked filamentous biopolymers form topological structures characterized by L, T and X cross-link types of connectivity 2, 3 and 4, respectively. The distribution of cross-links over these three types proofs to be very important for the initial elastic shear stiffness of isotropic

  4. Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

    Olde Damink, L.H.H.; Dijkstra, P.J.; Luyn, van M.J.A.; Wachem, van P.B.; Nieuwenhuis, P.; Feijen, J.


    A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). Cross-linking using EDC involves the activation of carboxylic acid groups to give O-acylisourea groups, which form cross-links

  5. Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH).

    Helwak, Aleksandra; Tollervey, David


    RNA-RNA interactions have critical roles in many cellular processes, but studying them is difficult and laborious. Here we describe an experimental procedure, termed cross-linking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein is UV-cross-linked in cell cultures to stabilize RNA interactions, and it is purified under denaturing conditions. RNAs associated with the bait protein are partially truncated, and the ends of RNA duplexes are ligated together. After linker addition, cDNA library preparation and high-throughput sequencing, the ligated duplexes give rise to chimeric cDNAs, which unambiguously identify RNA-RNA interaction sites independent of bioinformatic predictions. This protocol is optimized for studying miRNA targets bound by Argonaute (AGO) proteins, but it should be easily adapted for other RNA-binding proteins and classes of RNA. The protocol requires ∼5 d to complete, excluding the time required for high-throughput sequencing and bioinformatic analyses.

  6. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena


    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  7. Cross-linked hyaluronic acid in pressure ulcer prevention.

    Beniamino, P; Vadalà, M; Laurino, C


    Long-term bedridden patients are at high risk of acquring pressure ulcers (PUs). In this group of patients, prevention is necessary to cut the health costs, improve quality of life and reduce the mortality. Here, we evaluated the effectiveness of a cross-linked hyaluronic acid (HA) as plastic bulking-agent filling and remodelling the deep dermis and subcutaneous space of the skin areas exposed to the risk of necrosis. Our work hypothesis has been to inflate a sub-dermal elastic cushion, filled with a natural ECM component, with the aim to induce a stronger tissue background resistant to the ulcerative process. All the patients had an increased risk of PUs, at the sacral, ileum or heel skin. Patients were being nursed accordingly to the standard orthopaedic ward management with a pressure relieveing air mattress. The standard protocol consisted in body mobilisation every 3 hours, 24 hours a day and accurate cleaning of the skin with liquid soap and water without any towel friction and without adding any cream or lotion for the skin protection. Our filling protocol enclosed: accurate disinfection of the skin to be injected with povidone-iodine solution, followed by a local anaesthesia with 28G 13 mm needle, injecting 1.5 ml of 1% xylocaine. Then slow, deep, subcutaneous injection of cross-linked HA was performed with a 18G long needle, in order to deliver a homogeneous, soft gel layer underneath and around the whitish erythematous skin edges at risk of ulceration. Patients' tolerability of the compound and adverse events were also recorded. There were 15 patients (78-94 years old) who participated in the study. All tolerated the procedure very well and no serious side effects were declared. No skin pressure ulceration was detected in the four weeks follow-up Conclusion: We have demonstrated the safety and tolerability of a cross-linked HA subdermal injection in PUs prevention. The compound stratifies in a soft, elastic, interstitial bulk into the deep dermis, thus

  8. Biocatalytic cross-linking of pectic polysaccharides for designed food functionality

    Zaidel, Dayang Norulfairuz Abang; Meyer, Anne S.


    Recent research has demonstrated how cross-linking of pectic polysaccharides to obtain gel formation can be promoted by enzymatic catalysis reactions, and provide opportunities for functional upgrading of pectic polysaccharides present in agro-industrial sidestreams. This review highlights...... the mechanisms of formation of functional pectic polysaccharide cross-links, including covalent cross-links (notably phenolic esters and uronyl ester linkages) and non-covalent, ionic cross-links (which involve calcium and borate ester links). The treatise examines how such cross-links can be designed via...

  9. Stiffening of semiflexible biopolymers and cross-linked networks

    Van Dillen, T; Van der Giessen, E


    We study the mechanical stiffening behavior in two-dimensional (2D) cross-linked networks of semiflexible biopolymer filaments under simple shear. Filamental constituents immersed in a fluid undergo thermally excited bending motions. Pulling out these undulations results in an increase in the axial stiffness. We analyze this stiffening behavior of 2D semiflexible filaments in detail: we first investigate the average, {static} force-extension relation by considering the initially present undulated configuration that is pulled straight under a tensile force, and compare this result with the average response in which undulation dynamics is allowed during pulling, as derived earlier by MacKintosh and coworkers. We will show that the resulting mechanical behavior is rather similar, but with the axial stiffness being a factor 2 to 4 larger in the dynamic model. Furthermore, we study the stretching contribution in case of extensible filaments and show that, for 2D filaments, the mechanical response is dominated by {...

  10. Conventional Versus Cross-Linked Polyethylene for Total Hip Arthroplasty.

    Surace, Michele F; Monestier, Luca; Vulcano, Ettore; Harwin, Steven F; Cherubino, Paolo


    The clinical and radiographic outcomes of 88 patients who underwent primary total hip arthroplasty with either conventional polyethylene or cross-linked polyethylene (XLPE) from the same manufacturer were compared. There were no significant differences between the 2 subpopulations regarding average age, gender, side affected, or prosthetic stem and cup size. The average follow-up was 104 months (range, 55 to 131 months). To the authors' knowledge, this is the longest follow-up for this particular insert. Clinical and radiographic evaluations were performed at 1, 3, 6, and 12 months and then annually. Results showed that XLPE has a significantly greater wear reduction than that of standard polyethylene in primary total hip arthroplasty. At the longest available follow-up for these specific inserts, XLPE proved to be effective in reducing wear.

  11. Studies on N-vinylformamide cross-linked copolymers

    Świder, Joanna; Tąta, Agnieszka; Sokołowska, Katarzyna; Witek, Ewa; Proniewicz, Edyta


    Copolymers of N-vinylformamide (NVF) cross-linked with three multifunctional monomers, including divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), and N,N‧-methylenebisacrylamide (MBA) were synthetized by a three-dimensional free radical polymerization in inverse suspension using 2,2‧-azobis(2-methylpropionamide) dihydrochloride (AIBA) as an initiator. Methyl silicon oil was used as the continuous phase during the polymerization processes. Fourier-transform adsorption infrared (FT-IR) spectra revealed the presence of silicone oil traces and suggested that silicone oil strongly interacted with the copolymers surface. Purification procedure allowed to completely remove the silicon oil traces from P(NVF-co-DVB) only. The morphology and the structure of the investigated copolymers were examined by optical microscopy, FT-IR, and FT-Raman (Fourier-transform Raman spectroscopy) methods.

  12. Grass Cell Walls: A Story of Cross-Linking

    Hatfield, Ronald D.; Rancour, David M.; Marita, Jane M.


    Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p

  13. Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

    Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B.


    Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium. PMID:24395229

  14. The wear of cross-linked polyethylene against itself.

    Joyce, T J; Ash, H E; Unsworth, A


    Cross-linked polyethylene (XLPE) may have an application as a material for an all-plastic surface replacement finger joint. It is inexpensive, biocompatible and can be injection-moulded into the complex shapes that are found on the ends of the finger bones. Further, the cross-linking of polyethylene has significantly improved its mechanical properties. Therefore, the opportunity exists for an all-XLPE joint, and so the wear characteristics of XLPE sliding against itself have been investigated. Wear tests were carried out on both reciprocating pin-on-plate machines and a finger function simulator. The reciprocating pin-on-plate machines had pins loaded at 10 N and 40 N. All pin-on-plate tests show wear factors from the plates very much greater than those of the pins. After 349 km of sliding, a mean wear factor of 0.46 x 10(-6) mm3/N m was found for the plates compared with 0.021 x 10(-6) mm3/N m for the pins. A fatigue mechanism may be causing this phenomenon of greater plate wear. Tests using the finger function simulator give an average wear rate of 0.22 x 10(-6) mm3/N m after 368 km. This sliding distance is equivalent to 12.5 years of use in vivo. The wear factors found were comparable with those of ultra-high molecular weight polyethylene (UHMWPE) against a metallic counterface and, therefore, as the loads across the finger joint are much less than those across the knee or the hip, it is probable that an all-XLPE finger joint will be viable from a wear point of view.

  15. Anisotropic Thermal Conductivity Measurements on Cross-Linked Polybutadienes in Uniaxial Elongation

    Venerus, David C.; Kolev, Dimitre


    Cross-linked elastomers have numerous applications including automobiles, sporting goods, and biomedical devices. During both their processing and application, these materials experience large mechanical stresses and thermal gradients. In this study, we investigate the mechanical and thermal transport behavior of cross-linked polybutadienes. These materials have been prepared by cross linking well-entangled polybutadienes using an organic peroxide cross-linking agent at low concentration. Samples obtained after nearly complete conversion of the cross-linking agent, which can be characterized as lightly cross-linked (i.e., more than 10 entanglements per cross-link), were subjected to a series of large strain, uniaxial deformations. Measurements of the tensile stress and two components of the thermal conductivity tensor will be reported as a function of elongation. These data are also used to examine the stress-thermal rule in which the stress and thermal conductivity tensors are linearly related.

  16. Yield and Failure Behavior Investigated for Cross-Linked Phenolic Resins Using Molecular Dynamics

    Monk, Joshua D.; Lawson, John W.


    Molecular dynamics simulations were conducted to fundamentally evaluate the yield and failure behavior of cross-linked phenolic resins at temperatures below the glass transition. Yield stress was investigated at various temperatures, strain rates, and degrees of cross-linking. The onset of non-linear behavior in the cross-linked phenolic structures was caused by localized irreversible molecular rearrangements through the rotation of methylene linkers followed by the formation or annihilation of neighboring hydrogen bonds. The yield stress results, with respect to temperature and strain rate, could be fit by existing models used to describe yield behavior of amorphous glasses. The degree of cross-linking only indirectly influences the maximum yield stress through its influence on glass transition temperature (Tg), however there is a strong relationship between the degree of cross-linking and the failure mechanism. Low cross-linked samples were able to separate through void formation, whereas the highly cross-linked structures exhibited bond scission.

  17. Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation

    Lai, Jui-Yang, E-mail:


    In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50 min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150 min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation. - Highlights: • We report the development of photo-cross-linked AM as a limbal stem cell niche. • Cross-linked structure of tissue materials was controlled by UV irradiation time. • Biostability and matrix permeability of AM depended on cross-linking density. • All the studied photo-cross-linked AM showed good in vitro biocompatibility.

  18. Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue mouse and rat fibroblasts.

    Erexson, G L; Tindall, K R


    We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue(R) mouse (BBM1) and Big Blue(R) rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H(2)O(2). Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H(2)O(2). GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 microM DEB plus 0, 2 or 20 units of GSH-Px with and without 2x10(8) species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.

  19. A core cross-linked polymeric micellar platium(IV) prodrug with enhanced anticancer efficiency.

    Hou, Jie; Shang, Jincai; Jiao, Chengbin; Jiang, Peiyue; Xiao, Huijie; Luo, Lan; Liu, Tongjun


    A core cross-linked polymeric micellar cisplatin(IV) conjugate prodrug is prepared by attaching the cisplatin(IV) to mPEG-b-PLL biodegradable copolymers to form micellar nanoparticles that can disintegrate to release the active anticancer agent cisplatin(II) in a mild reducing environment. Moreover, in vitro studies show that this cisplatin(IV) conjugate prodrug displays enhanced cytotoxicity against HepG2 cancer cells compared with cisplatin(II). Further studies demonstrate that the high cellular uptake and platinum-DNA adduct of this cisplatin(IV) conjugate prodrug can induce more cancer-cell apoptosis than cisplatin(II), which is responsible for its enhanced anticancer activity.

  20. Differential Enzymatic 16O/18O Labelling for the Detection of Cross-Linked Nucleic Acid-Protein Heteroconjugates.

    Flett, Fiona J; Sachsenberg, Timo; Kohlbacher, Oliver; Mackay, C Logan; Interthal, Heidrun


    Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterisation of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential 16O/18O labelling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labelled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates which were subjected to the labelling reaction and analysed by high resolution FT-ICR mass spectrometry.

  1. Riboflavin-ultraviolet a corneal cross-linking for keratoconus

    El-Raggal Tamer


    Full Text Available Purpose: To evaluate the safety, efficacy of riboflavin-ultraviolet A irradiation (UVA corneal cross-linking and present refractive changes induced by the treatment in cases of keratoconus. Materials and Methods: The study includes 15 eyes of 9 patients with keratoconus with an average keratometric (K reading less than 54 D and minimal corneal thickness greater than 420 microns. The corneal epithelium was removed manually within the central 8.5 mm diameter area and the cornea was soaked with riboflavin eye drops (0.1% in 20% dextran t-500 for 30 minutes followed by exposure to UVA radiation (365 nm, 3 mW/cm 2 for 30 minutes. During the follow-up period, uncorrected visual acuity (UCVA, best spectacle-corrected visual acuity (BSCVA, manifest refraction, slit lamp examination and topographic changes were recorded at the first week, first month, 3 and 6 months. Results: There was statistically significant improvement of UCVA from a preoperative mean of 0.11 ± 0.07 (range 0.05-0.3 to a postoperative mean of 0.15 ± 0.06 (range 0.1-0.3 (P < 0.05. None of the eyes lost lines of preoperative UCVA but 1 eye lost 1 line of preoperative BSCVA. The preoperative mean K of 49.97 ± 2.81 D (range 47.20-51.75 changed to 48.34 ± 2.64 D (range 45.75-50.40. This decrease in K readings was statistically significant (P < 0.05. All eyes developed minimal faint stromal haze that cleared in 14 eyes within 1 month. In only 1 eye, this resulted in a very faint corneal scar. Other sight threatening complications were not encountered in this series. Progression of the original disease was not seen in any of the treated eyes within 6 months of follow-up. Conclusion: Riboflavin-UVA corneal cross-linking is a safe and promising method for keratoconus. Larger studies with longer follow up are recommended.

  2. Energy harvesting from vibration with cross-linked polypropylene piezoelectrets

    Xiaoqing Zhang


    Full Text Available Piezoelectret films are prepared by modification of the microstructure of polypropylene foam sheets cross-linked by electronic irradiation (IXPP, followed by proper corona charging. Young’s modulus, relative permittivity, and electromechanical coupling coefficient of the fabricated films, determined by dielectric resonance spectra, are about 0.7 MPa, 1.6, and 0.08, respectively. Dynamic piezoelectric d33 coefficients up to 650 pC/N at 200 Hz are achieved. The figure of merit (FOM, d33 ⋅ g33 for a more typical d33 value of 400 pC/N is about 11.2 GPa−1. Vibration-based energy harvesting with one-layer and two-layer stacks of these films is investigated at various frequencies and load resistances. At an optimum load resistance of 9 MΩ and a resonance frequency of 800 Hz, a maximum output power of 120 μW, referred to the acceleration g due to gravity, is obtained for an energy harvester consisting of a one-layer IXPP film with an area of 3.14 cm2 and a seismic mass of 33.7 g. The output power can be further improved by using two-layer stacks of IXPP films in electric series. IXPP energy harvesters could be used to energize low-power electronic devices, such as wireless sensors and LED lights.

  3. Cross-linking da cornea: protocolo padrão

    Marcony R. Santhiago

    Full Text Available RESUMO O objetivo desta revisão é de determinar as indicações e eficácia da cirurgia que promove novas ligações covalentes entre as fibras de colágeno da córnea, conhecida como Cross-Linking (CXL, assim como esclarecer seus objetivos. O ceratocone é uma doença ectasica da córnea, bilateral, assimétrica, que, principalmente, cursa com encurvamento e afinamentos progressivo, e se inicia em geral na segunda década de vida. O uso primário do CXL tem sido na interrupção da progressão do Ceratocone. Apesar do conhecido encurvamento no estroma da córnea ocorrer nesses pacientes, a fisiopatologia por trás do ceratocone ainda é desconhecida e parece ser multifatorial. Pela evidencia literária disponível até o momento, o CXL da córnea esta, portanto indicado nos pacientes com doença em progressão. Concluímos que existe evidencia suficiente para afirmar que o CXL da córnea é eficaz na estabilização da doença ectásica da cornea.

  4. Cross-linking of polytetrafluoroethylene during room-temperature irradiation

    Pugmire, David L [Los Alamos National Laboratory; Wetteland, Chris J [Los Alamos National Laboratory; Duncan, Wanda S [Los Alamos National Laboratory; Lakis, Rollin E [Los Alamos National Laboratory; Schwartz, Daniel S [Los Alamos National Laboratory


    Exposure of polytetrafluoroethylene (PTFE) to {alpha}-radiation was investigated to detennine the physical and chemical effects, as well as to compare and contrast the damage mechanisms with other radiation types ({beta}, {gamma}, or thermal neutron). A number of techniques were used to investigate the chemical and physical changes in PTFE after exposure to {alpha}-radiation. These techniques include: Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and fluorescence spectroscopy. Similar to other radiation types at low doses, the primary damage mechanism for the exposure of PTFE to {alpha}-radiation appears to be chain scission. Increased doses result in a change-over of the damage mechanism to cross-linking. This result is not observed for any radiation type other than {alpha} when irradiation is performed at room temperature. Finally, at high doses, PTFE undergoes mass-loss (via smallfluorocarbon species evolution) and defluorination. The amount and type of damage versus sample depth was also investigated. Other types of radiation yield damage at depths on the order of mm to cm into PTFE due to low linear energy transfer (LET) and the correspondingly large penetration depths. By contrast, the {alpha}-radiation employed in this study was shown to only induce damage to a depth of approximately 26 {mu}m, except at very high doses.

  5. Synthesis and Properties of Cross-Linked Polyamide Aerogels

    Williams, Jarrod C.; Meador, Mary Ann; McCorkle, Linda


    We report the first synthesis of cross-linked polyamide aerogels through step growth polymerization using a combination of diamines, diacid chloride and triacid chloride. Polyamide oligomers endcapped with amines are prepared as stable solutions in N-methylpyrrolidinone from several different diamine precursors and 1,3-benzenedicarbonyl dichloride. Addition of 1,3,5-benzenetricarbonyl trichloride yields gels which form in under five minutes according to the scheme shown. Solvent exchange of the gels into ethanol, followed by drying using supercritical CO2 extraction gives colorless aerogels with densities around 0.1 to 0.2 gcm3. Thicker monolithes of the polyamide aerogels are stiff and strong, while thin films of certain formulations are highly flexible, durable, and even translucent. These materials may have use as insulation for deployable space structures, rovers, habitats or extravehicular activity suits as well as in many terrestrial applications. Strucure property relationships of the aerogels, including surface area, mechanical properties, and thermal conductivity will be discussed.

  6. Energy harvesting from vibration with cross-linked polypropylene piezoelectrets

    Zhang, Xiaoqing; Wu, Liming; Sessler, Gerhard M.


    Piezoelectret films are prepared by modification of the microstructure of polypropylene foam sheets cross-linked by electronic irradiation (IXPP), followed by proper corona charging. Young's modulus, relative permittivity, and electromechanical coupling coefficient of the fabricated films, determined by dielectric resonance spectra, are about 0.7 MPa, 1.6, and 0.08, respectively. Dynamic piezoelectric d33 coefficients up to 650 pC/N at 200 Hz are achieved. The figure of merit (FOM, d33 ṡ g33) for a more typical d33 value of 400 pC/N is about 11.2 GPa-1. Vibration-based energy harvesting with one-layer and two-layer stacks of these films is investigated at various frequencies and load resistances. At an optimum load resistance of 9 MΩ and a resonance frequency of 800 Hz, a maximum output power of 120 μW, referred to the acceleration g due to gravity, is obtained for an energy harvester consisting of a one-layer IXPP film with an area of 3.14 cm2 and a seismic mass of 33.7 g. The output power can be further improved by using two-layer stacks of IXPP films in electric series. IXPP energy harvesters could be used to energize low-power electronic devices, such as wireless sensors and LED lights.

  7. Energy harvesting from vibration with cross-linked polypropylene piezoelectrets

    Zhang, Xiaoqing [Shanghai Key Laboratory of Special Artificial Microstructure Materials and Technology & School of Physics Science and Engineering, Tongji University, Shanghai 200092 (China); Institute for Telecommunications Technology, Merckstrasse 25, 64283 Darmstadt (Germany); Wu, Liming [Shanghai Key Laboratory of Special Artificial Microstructure Materials and Technology & School of Physics Science and Engineering, Tongji University, Shanghai 200092 (China); Sessler, Gerhard M., E-mail: [Institute for Telecommunications Technology, Merckstrasse 25, 64283 Darmstadt (Germany)


    Piezoelectret films are prepared by modification of the microstructure of polypropylene foam sheets cross-linked by electronic irradiation (IXPP), followed by proper corona charging. Young’s modulus, relative permittivity, and electromechanical coupling coefficient of the fabricated films, determined by dielectric resonance spectra, are about 0.7 MPa, 1.6, and 0.08, respectively. Dynamic piezoelectric d{sub 33} coefficients up to 650 pC/N at 200 Hz are achieved. The figure of merit (FOM, d{sub 33} ⋅ g{sub 33}) for a more typical d{sub 33} value of 400 pC/N is about 11.2 GPa{sup −1}. Vibration-based energy harvesting with one-layer and two-layer stacks of these films is investigated at various frequencies and load resistances. At an optimum load resistance of 9 MΩ and a resonance frequency of 800 Hz, a maximum output power of 120 μW, referred to the acceleration g due to gravity, is obtained for an energy harvester consisting of a one-layer IXPP film with an area of 3.14 cm{sup 2} and a seismic mass of 33.7 g. The output power can be further improved by using two-layer stacks of IXPP films in electric series. IXPP energy harvesters could be used to energize low-power electronic devices, such as wireless sensors and LED lights.

  8. Subclinical inflammatory response after accelerated corneal cross-linking

    Hassan Hashemi; Nahid Ashraf; Ebrahim Jafarzadehpur; Alireza Hedayatfar; Soheila Asgari


    Background: To evaluate the inlfammatory response after accelerated collagen cross-linking (CXL) in eyes with keratoconus. Methods: Consecutive eyes with keratoconus undergoing CXL surgery were included in this non-randomized interventional study. Aqueous lfare was measured pre- and post-operatively with a laser lfare photometer at 1 week, 1, 3 and 6 months after CXL. Results: Sixty eyes of 60 patients were entered into the study. Before CXL, the mean lfare value was 4.5 photons per millisecond (ph/ms). The lfare values observed at week 1 (7.1 ph/ms; P=0.008), month 1 (6.5 ph/ms; P=0.04), month 3 (6.7 ph/ms; P=0.004) and month 6 (6.7 ph/ms; P=0.004) were signiifcantly higher compared to baseline. Flare values were not signiifcantly different from week 1 up to 6 months after CXL (P=0.930). No statistically significant correlation was detected between the amount of inlfammation and keratometric indices. Conclusions: Accelerated CXL in patients with keratoconus may cause a subclinical inflammatory response which is evident as slight but rather long-lasting rise of aqueous lfare.

  9. Pyridinium cross-links in heritable disorders of collagen

    Pasquali, M.; Still, M.J.; Dembure, P.P. [Emory Univ., Atlanta, GA (United States)] [and others


    Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited disorders of collagen that is characterized by skin fragility, skin hyperextensibility, and joint hypermobility. EDS type VI is caused by impaired collagen lysyl hydroxylase (procollagen-lysine, 2-oxoglutarate 5-dioxygenase; E.C., the ascorbate-dependent enzyme that hydroxylates lysyl residues on collagen neopeptides. Different alterations in the gene for collagen lysyl hydroxylase have been reported in families with EDS type VI. In EDS type VI, impairment of collagen lysyl hydroxylase results in a low hydroxylysine content in mature collagen. Hydroxylysine is a precursor of the stable, covalent, intermolecular cross-links of collagen, pyridinoline (Pyr), and deoxypyridinoline (Dpyr). Elsewhere we reported in preliminary form that patients with EDS type VI had a distinctive alteration in the urinary excretion of Pyr and Dpyr. In the present study, we confirm that the increased Dpyr/Pyr ratio is specific for EDS type VI and is not observed in other inherited or acquired collagen disorders. In addition, we find that skin from patients with EDS type VI has reduced Pyr and increased Dpyr, which could account for the organ pathology. 19 refs., 1 tab.

  10. Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry

    Steen, Hanno; Jensen, Ole Nørregaard


    Photochemical cross-linking is a commonly used method for studying the molecular details of protein-nucleic acid interactions. Photochemical cross-linking aids in defining nucleic acid binding sites of proteins via subsequent identification of cross-linked protein domains and amino acid residues....... Mass spectrometry (MS) has emerged as a sensitive and efficient analytical technique for determination of such cross-linking sites in proteins. The present review of the field describes a number of MS-based approaches for the characterization of cross-linked protein-nucleic acid complexes...... and for sequencing of peptide-nucleic acid heteroconjugates. The combination of photochemical cross-linking and MS provides a fast screening method to gain insights into the overall structure and formation of protein-oligonucleotide complexes. Because the analytical methods are continuously refined and protein...

  11. Fabrication of patterned calcium cross-linked alginate hydrogel films and coatings through reductive cation exchange.

    Bruchet, Marion; Melman, Artem


    Calcium cross-linked alginate hydrogels are widely used in targeted drug delivery, tissue engineering, wound treatment, and other biomedical applications. We developed a method for preparing homogeneous alginate hydrogels cross-linked with Ca(2+) cations using reductive cation exchange in homogeneous iron(III) cross-linked alginate hydrogels. Treatment of iron(III) cross-linked alginate hydrogels with calcium salts and sodium ascorbate results in reduction of iron(III) cations to iron(II) that are instantaneously replaced with Ca(2+) cations, producing homogeneous ionically cross-linking hydrogels. Alternatively, the cation exchange can be performed by photochemical reduction in the presence of calcium chloride using a sacrificial photoreductant. This approach allows fabrication of patterned calcium alginate hydrogels through photochemical patterning of iron(III) cross-linked alginate hydrogel followed by the photochemical reductive exchange of iron cations to calcium.

  12. An Open Data Format for Visualization and Analysis of Cross-Linked Mass Spectrometry Results.

    Hoopmann, Michael R; Mendoza, Luis; Deutsch, Eric W; Shteynberg, David; Moritz, Robert L


    Protein-protein interactions are an important element in the understanding of protein function, and chemical cross-linking shotgun mass spectrometry is rapidly becoming a routine approach to identify these specific interfaces and topographical interactions. Protein cross-link data analysis is aided by dozens of algorithm choices, but hindered by a lack of a common format for representing results. Consequently, interoperability between algorithms and pipelines utilizing chemical cross-linking remains a challenge. pepXML is an open, widely-used format for representing spectral search algorithm results that has facilitated information exchange and pipeline development for typical shotgun mass spectrometry analyses. We describe an extension of this format to incorporate cross-linking spectral search results. We demonstrate application of the extension by representing results of multiple cross-linking search algorithms. In addition, we demonstrate adapting existing pepXML-supporting software pipelines to analyze protein cross-linking results formatted in pepXML. Graphical Abstract ᅟ.

  13. Tailoring the properties of cholecyst-derived extracellular matrix using carbodiimide cross-linking.

    Burugapalli, Krishna


    Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N\\'-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR-FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59 degrees C for non-cross-linked CEM to 78 degrees C for the highest EDC cross-linking concentration, while IR peak area ratios for the free -NH(2) group at 3290 cm(-1) to that of the amide I band at 1635 cm(-1) decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol\\/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC\\/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.

  14. In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

    Heinz, Andrea; Ruttkies, Christoph K H; Jahreis, Günther; Schräder, Christoph U; Wichapong, Kanin; Sippl, Wolfgang; Keeley, Fred W; Neubert, Reinhard H H; Schmelzer, Christian E H


    Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Radiation cross-linking in ultra-high molecular weight polyethylene for orthopaedic applications

    Oral, Ebru; Muratoglu, Orhun K.


    The motivation for radiation cross-linking of ultra-high molecular weight polyethylene (UHMWPE) is to increase its wear resistance to be used as bearing surfaces for total joint arthroplasty. However, radiation also leaves behind long-lived residual free radicals in this polymer, the reactions of which can detrimentally affect mechanical properties. In this review, we focus on the radiation cross-linking and oxidative stability of first and second generation highly cross-linked UHMWPEs develo...

  16. Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts

    Bennett, K L; Kussmann, M; Björk, P;


    The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro...... revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross...

  17. Effect of Cross-Linking and Enzymatic Hydrolysis Composite Modification on the Properties of Rice Starches

    Gao-Qiang Liu


    Full Text Available Native rice starch lacks the versatility necessary to function adequately under rigorous industrial processing, so modified starches are needed to meet the functional properties required in food products. This work investigated the impact of enzymatic hydrolysis and cross-linking composite modification on the properties of rice starches. Rice starch was cross-linked with epichlorohydrin (EPI with different concentrations (0.5%, 0.7%, 0.9% w/w, on a dry starch basis, affording cross-linked rice starches with the three different levels of cross-linking that were named R1, R2, and R3, respectively. The cross-linked rice starches were hydrolyzed by α-amylase and native, hydrolyzed, and hydrolyzed cross-linked rice starches were comparatively studied. It was found that hydrolyzed cross-linked rice starches showed a lower the degree of amylase hydrolysis compared with hydrolyzed rice starch. The higher the degree of cross-linking, the higher the capacity to resist enzyme hydrolysis. Hydrolyzed cross-linked rice starches further increased the adsorptive capacities of starches for liquids and decreased the trend of retrogradation, and it also strengthened the capacity to resist shear compared to native and hydrolyzed rice starches.

  18. Structural and spectral characteristics of the cross-linked dimer derived from electrooxidation of cyclic 1,N2-propanoguanosine.

    Murakami, Hiroya; Esaka, Yukihiro; Uno, Bunji


    The acetaldehyde-derived cyclic propano adduct of 2'-deoxyguanosine was easily oxidized electrochemically into the cross-linked dimer as an oxidative product. The structural and spectroscopic characteristics of the dimer were investigated by MS, (1)H and (13)C-NMR, UV, and DFT calculations. The dimer formation was inferred from a molecular ionic peak of m/z 705 ([(2M-2H)+H](+), M being the molecular weight of the monomer) on the ESI-MS spectra and the chemical formula as C(28)H(36)N(10)O(12) provided by the high-resolution ESI-MS results. The C2-N5 linkage between the two monomers in the dimer was deduced from the (1)H- and (13)C-NMR spectral results. In addition, the correlations in the 2-dimensional NMR spectra (DQF-COSY and HMBC) were consistently explained by the structure of the C2-N5 cross-linked dimer. UV spectral measurements also support the C2-N5 linking in the dimer formation. The formation of the cross-link dimer as an oxidative lesion of the acetaldehyde-derived cyclic propano adduct of guanosine is expected to interfere with DNA replication and to contribute to acetaldehyde-mediated genotoxicity.

  19. Alkaline battery containing a separator of a cross-linked copolymer of vinyl alcohol and unsaturated carboxylic acid

    Hsu, L. C.; Philipp, W. H.; Sheibley, D. W.; Gonzalez-Sanabria, O. D. (Inventor)


    A battery separator for an alkaline battery is described. The separator comprises a cross linked copolymer of vinyl alcohol units and unsaturated carboxylic acid units. The cross linked copolymer is insoluble in water, has excellent zincate diffusion and oxygen gas barrier properties and a low electrical resistivity. Cross linking with a polyaldehyde cross linking agent is preferred.




    Full Text Available To evaluate the outcome of collagen cross linkage using riboflavin 0.1% and ultraviolet A radiation of a wavelength 370nm . PURPOSE : To determine the effect of collagen cross linking for keratoconus on pachymetry , corneal topography, uncorrected visual acuity, specular count, IOP at 1, 3, 6 months . METHODS : The current study was designed as a prospective interventional trial of corneal collagen cross - linking in subjects with progressive keratoconus between a period of J anuary 2013 to J uly 2014 including 50 eyes of 30 patients. This study rece ived approval from Ethics committee. Informed written consent was obtained from all patients prior to treatment. RESULTS: Mean follow up period of 6 months. There was statistically significant decline in effective k readings from mean pre op (51.7D to pos t op value (49.65D . Pachymetry showed initial increase post operatively followed by reduction in corneal thickness in 3 month s follow up , followed by gradual increase in thickness over a period of 6 months though it was not equivalent to pre - operative values. paired t test p value was 0.00 1 in 1 n 3 month and 0. 043 in 6 month, concluding significant reduction in early post op period with im provement over time . Specular count reduced from pre - op (2673.80 levels to post - op (2654.60 levels ,the reduction in specular count was not statistically significant P value (0.014 The uncorrected visual acuity showed decrease in refractive error with in crease in visual acuity gradually over 6 months, log MAR scale visual acuity was used for statistical analysis with P value (0.001 which was statistically significant .visual acuity improved by 1.5 lines in snellens equivalence or from 0.5 to 0.4 in log MA R char y ± 20cells/mm 3 , P value(0. 001 . IOP statically show ed no significant change in pre - operative and post operatively , paired t test p value was 0. 44 showing it was not significant . CONCLUSIONS : 1. Visual acuity improved in

  1. Encapsulation of volatiles by homogenized partially-cross linked alginates.

    Inguva, Pavan K; Ooi, Shing Ming; Desai, Parind M; Heng, Paul W S


    Cross-linked calcium alginate gels are too viscous to be efficaciously incorporated into spray dried formulations. Thus, viscosity reduction is essential to ensure the processability of calcium alginate gels to be sprayed. Viscosity reduction by high pressure homogenization can open new formulation possibilities. Presently, testing of microcapsule integrity is also limited because either single particle tests neglect collective particle behaviours in bulk or bulk testing methods are often associated with single compressions which may not fully characterize individual particle strengths. The aim of this study was sub-divided into three objectives. First objective was to evaluate the impact of high pressure homogenization on gel viscosity. Second objective was to explore the use of the homogenized gels with modified starch for microencapsulation by spray drying. The final objective was to develop a stamping system as microcapsule strength tester that can assess microcapsules in bulk and evaluate the impact of multiple compressions. Collectively, this study would lead towards developing a pressure-activated patch of microcapsules with encapsulated volatiles and the method to assess the patch efficacy. The alginate gels largely experienced an exponential decay in viscosity when homogenized. Furthermore, the homogenized gels were successfully incorporated in spray drying formulations for microencapsulation. The custom-designed microcapsule strength tester was successfully used and shown to possess the required sensitivity to discern batches of microcapsules containing volatiles to have different release profiles. Addition of homogenized gels strengthened the microcapsules only at high wall to core ratios with low mass-load alginate gels. High mass-load gels weaken the microcapsules, exhibiting a higher release at low stamping pressures and wrinkling on the microcapsules surface.

  2. Integrated Cryogenic Satellite Communications Cross-Link Receiver Experiment

    Romanofsky, R. R.; Bhasin, K. B.; Downey, A. N.; Jackson, C. J.; Silver, A. H.; Javadi, H. H. S.


    An experiment has been devised which will validate, in space, a miniature, high-performance receiver. The receiver blends three complementary technologies; high temperature superconductivity (HTS), pseudomorphic high electron mobility transistor (PHEMT) monolithic microwave integrated circuits (MMIC), and a miniature pulse tube cryogenic cooler. Specifically, an HTS band pass filter, InP MMIC low noise amplifier, HTS-sapphire resonator stabilized local oscillator (LO), and a miniature pulse tube cooler will be integrated into a complete 20 GHz receiver downconverter. This cooled downconverter will be interfaced with customized signal processing electronics and integrated onto the space shuttle's 'HitchHiker' carrier. A pseudorandom data sequence will be transmitted to the receiver, which is in low Earth orbit (LEO), via the Advanced Communication Technology Satellite (ACTS) on a 20 GHz carrier. The modulation format is QPSK and the data rate is 2.048 Mbps. The bit error rate (BER) will be measured in situ. The receiver is also equipped with a radiometer mode so that experiment success is not totally contingent upon the BER measurement. In this mode, the receiver uses the Earth and deep space as a hot and cold calibration source, respectively. The experiment closely simulates an actual cross-link scenario. Since the receiver performance depends on channel conditions, its true characteristics would be masked in a terrestrial measurement by atmospheric absorption and background radiation. Furthermore, the receiver's performance depends on its physical temperature, which is a sensitive function of platform environment, thermal design, and cryocooler performance. This empirical data is important for building confidence in the technology.

  3. The theory and art of corneal cross-linking

    Rebecca McQuaid


    Full Text Available Before the discovery of corneal cross-linking (CXL, patients with keratoconus would have had to undergo corneal transplantation, or wear rigid gas permeable lenses (RGPs that would temporarily flatten the cone, thereby improving the vision. The RGP contact lens (CL would not however alter the corneal stability and if the keratoconus was progressive, the continued steepening of the cone would occur under the RGP CL. To date, the Siena Eye has been the largest study to investigate long term effects of standard CXL. Three hundred and sixty-three eyes were treated and monitored over 4 years, producing reliable long-term results proving long-term stability of the cornea by halting the progression of keratoconus, and proving the safety of the procedure. Traditionally, CXL requires epithelial removal prior to corneal soakage of a dextran-based 0.1% riboflavin solution, followed by exposure of ultraviolet-A (UV-A light for 30 min with an intensity of 3 mW/cm2. A series of in vitro investigations on human and porcine corneas examined the best treatment parameters for standard CXL, such as riboflavin concentration, intensity, wavelength of UV-A light, and duration of treatment. Photochemically, CXL is achieved by the generation of chemical bonds within the corneal stroma through localized photopolymerization, strengthening the cornea whilst minimizing exposure to the surrounding structures of the eye. In vitro studies have shown that CXL has an effect on the biomechanical properties of the cornea, with an increased corneal rigidity of approximately 70%. This is a result of the creation of new chemical bonds within the stroma.

  4. Transglutaminases: widespread cross-linking enzymes in plants.

    Serafini-Fracassini, Donatella; Del Duca, Stefano


    Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied. The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive. It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction. The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still incompletely defined physiological roles. At present, it is not possible to classify this enzyme family in plants owing to

  5. Feruloylated arabinoxylans are oxidatively cross-linked by extracellular maize peroxidase but not by horseradish peroxidase.

    Burr, Sally J; Fry, Stephen C


    Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

  6. Analytical characterisation of glutardialdehyde cross-linking products in gelatine-gum arabic complex coacervates

    Fuguet, Elisabet [Advanced Measurement and Imaging, Unilever Food and Health Research Institute, Olivier van Noortlaan 120, 3133 AT Vlaardingen (Netherlands)], E-mail:; Platerink, Chris van [Advanced Measurement and Imaging, Unilever Food and Health Research Institute, Olivier van Noortlaan 120, 3133 AT Vlaardingen (Netherlands); Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands); Janssen, Hans-Gerd [Advanced Measurement and Imaging, Unilever Food and Health Research Institute, Olivier van Noortlaan 120, 3133 AT Vlaardingen (Netherlands); van' t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam (Netherlands)


    Encapsulates having shells of cross-linked mixtures of proteins and polysaccharides are widely used in the food and pharmaceutical industry for controlled release of actives and flavour compounds. In order to be able to predict the behaviour and the release characteristics of the microcapsules, a better understanding of the nature and extent of the cross-linking reaction is needed. Several analytical techniques were applied for the characterisation of glutardialdehyde (GDA) cross-linked encapsulates made of gelatine and gum arabic. To allow the use of sensitive, high-resolution methods such as chromatography and mass spectrometry, the sample first had to be hydrolysed. In this way, a mixture of amino acids, small peptides and the cross-link moieties was obtained. High-resolution liquid chromatography coupled to high-resolution mass spectrometry (HPLC-MS) was applied to detect possible cross-link markers through a comparison of HPLC-MS mass-chromatograms obtained for cross-linked and non-cross-linked coacervates. HPLC-MS/MS was used to identify the species responsible for the differences. Cross-linking occurred between GDA molecules and lysine and hydroxylysine {epsilon}-amino groups, and up to eight cross-link products of different nature could be identified. They included pyridinium ions and Schiff bases, and also unreacted GDA condensation products. Next, based on the insight gained in the possible chemical structures present in the cross-link markers, methods for selective labelling of these functionalities were employed to allow easier detection of related reaction products. Both liquid chromatography (LC) and gas chromatography (GC) were used in these experiments. Unfortunately, these approaches failed to detect new cross-link markers, most likely as a result of the low levels at which these are present.

  7. Feruloylated Arabinoxylans Are Oxidatively Cross-Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase

    Sally J. Burr; Stephen C. Fry


    Covalent cross-linking of soluble extraceUular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H_2O_2/peroxidase, O_2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extra-cellular, feruloylated [pentosyl-~3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H_2O_2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H_2O_2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [~3H]arabinoxylans in vitro only if H_20_2 was also added, indicating a peroxiclase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present, Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [~3H]arabino-xylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.




    Cross-linked, hydrophobically associating homo- and copolymers were synthesized by free-radical cyclo(co)polymerization of alkylmethyldiallylammonium bromide monomers with a small amount of N,N'-methylenebisacrylamide in aqueous solution using ammonium persulfate as the initiator. The cross-linked h

  9. Rheological properties of dispersions of enzymatically cross-linked apo-α-lactalbumin

    Saricay, Yunus; Wierenga, Peter A.; Vries, de Renko


    The enzymatic cross-linking of apo-α-lactalbumin (α-LA) with horseradish peroxidase (HRP) leads to the formation of hydrophilic protein aggregates with controlled size and architecture. We explore the rheological properties of dispersions of these HRP-cross-linked α-LA aggregates with a hydrodyna

  10. Covalently Cross-Linked Sulfone Polybenzimidazole Membranes with Poly(Vinylbenzyl Chloride) for Fuel Cell Applications

    Yang, Jingshuai; Aili, David; Li, Qingfeng;


    Covalently cross-linked polymer membranes were fabricated from poly(aryl sulfone benzimidazole) (SO(2) PBI) and poly(vinylbenzyl chloride) (PVBCl) as electrolytes for high-temperature proton-exchange-membrane fuel cells. The cross-linking imparted organo insolubility and chemical stability against...


    A facile method utilizing microwave irradiation is described that accomplishes the cross-linking reaction of PVA with metallic and bimetallic systems. Nanocomposites of PVA-cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-Pt, Pt-Fe, Cu...


    A facile microwave irradiation approach that results in a cross-linking reaction of poly (vinyl alcohol) (PVA) with metallic and bimetallic systems is described. Nanocomposites of PVA cross-linked metallic systems such as Pt, Cu, and In and bimetallic systems such as Pt-In, Ag-P...

  13. Effects of Supercritical CO 2 Conditioning on Cross-Linked Polyimide Membranes

    Kratochvil, Adam M.


    The effects of supercritical CO2 (scCO2) conditioning on high-performance cross-linked polyimide membranes is examined through gas permeation and sorption experiments. Under supercritical conditions, the cross-linked polymers do not exhibit a structural reorganization of the polymer matrix that was observed in the non-cross-linkable, free acid polymer. Pure gas permeation isotherms and mixed gas permeabilities and selectivities show the cross-linked polymers to be much more stable to scCO2 conditioning than the free acid polymer. In fact, following scCO2 conditioning, the mixed gas CO2 permeabilities of the cross-linked polymers increased while the CO2/CH4 separation factors remained relatively unchanged. This response highlights the stability and high performance of these cross-linked membranes in aggressive environments. In addition, this response reveals the potential for the preconditioning of cross-linked polymer membranes to enhance productivity without sacrificing efficiency in practical applications which, in effect, provides another tool to \\'tune\\' membrane properties for a given separation. Finally, the dual mode model accurately describes the sorption and dilation characteristics of the cross-linked polymers. The changes in the dual mode sorption model parameters before and after the scCO2 exposure also provide insights into the alterations in the different glassy samples due to the cross-linking and scCO2 exposure. © 2010 American Chemical Society.

  14. Self-assembly made durable: water-repellent materials formed by cross-linking fullerene derivatives.

    Wang, Jiaobing; Shen, Yanfei; Kessel, Stefanie; Fernandes, Paulo; Yoshida, Kaname; Yagai, Shiki; Kurth, Dirk G; Möhwald, Helmuth; Nakanishi, Takashi


    Fullerene flakes: A diacetylene-functionalized fullerene derivative self-organizes into flakelike microparticles (see picture). Both the diacetylene and C(60) moieties can be effectively cross-linked, which leads to supramolecular materials with remarkable resistivity to solvent, heat, and mechanical stress. Moreover, the surface of the cross-linked flakelike objects is highly durable and water-repellent.

  15. Quantification of carboxyl groups in carbodiimide cross-linked collagen sponges

    Everaerts, Frank; Torrianni, Mark; Hendriks, Marc; Feijen, Jan


    Glutaraldehyde (GA) fixation of bioprosthetic tissue is a well adapted technique, with commercial products on the market for almost 40 years. Amine groups present in tissue react with GA to form different types of cross-links. An estimation of the degree of cross-linking of the tissue can be obtaine

  16. Infrared microspectroscopic determination of collagen cross-links in articular cartilage

    Rieppo, Lassi; Kokkonen, Harri T.; Kulmala, Katariina A. M.; Kovanen, Vuokko; Lammi, Mikko J.; Töyräs, Juha; Saarakkala, Simo


    Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n=27) were treated with threose to increase the collagen cross-linking while the other half (n=27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r=0.84 (p<0.001), r=0.87 (p<0.001) and r=0.92 (p<0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

  17. Glucose-mediated cross-linking of collagen in rat tendon and skin

    Mentink, CJAL; Hendriks, M; Levels, AAG; Wolffenbuttel, BHR


    Back-ground: Cross-linking of macromolecules like collagen plays an important role in the development of complications in diabetes and ageing. One of the underlying mechanisms of this cross-linking is the formation of advanced glycation endproducts (AGEs). Methods: In this study, we assessed the use

  18. Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid

    Oudgenoeg, G.; Hilhorst, R.; Piersma, S.R.; Boeriu, C.G.; Gruppen, H.; Hessing, M.; Voragen, A.G.J.; Laane, C.


    The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase

  19. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat.

    Rothamel, D.; Schwarz, F.; Sager, M.; Herten, M. van; Sculean, A.; Becker, J.M.


    The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendE

  20. Investigation of anisotropic thermal transport in cross-linked polymers

    Simavilla, David Nieto

    Thermal transport in lightly cross-linked polyisoprene and polybutadine subjected to uniaxial elongation is investigated experimentally. We employ two experimental techniques to assess the effect that deformation has on this class of materials. The first technique, which is based on Forced Rayleigh Scattering (FRS), allows us to measure the two independent components of the thermal diffusivity tensor as a function of deformation. These measurements along with independent measurements of the tensile stress and birefringence are used to evaluate the stress-thermal and stress-optic rules. The stress-thermal rule is found to be valid for the entire range of elongations applied. In contrast, the stress-optic rule fails for moderate to large stretch ratios. This suggests that the degree of anisotropy in thermal conductivity depends on both orientation and tension in polymer chain segments. The second technique, which is based on infrared thermography (IRT), allows us to measure anisotropy in thermal conductivity and strain induced changes in heat capacity. We validate this method measurements of anisotropic thermal conductivity by comparing them with those obtained using FRS. We find excellent agreement between the two techniques. Uncertainty in the infrared thermography method measurements is estimated to be about 2-5 %. The accuracy of the method and its potential application to non-transparent materials makes it a good alternative to extend current research on anisotropic thermal transport in polymeric materials. A second IRT application allows us to investigate the dependence of heat capacity on deformation. We find that heat capacity increases with stretch ratio in polyisoprene specimens under uniaxial extension. The deviation from the equilibrium value of heat capacity is consistent with an independent set of experiments comparing anisotropy in thermal diffusivity and conductivity employing FRS and IRT techniques. We identify finite extensibility and strain

  1. Assessment of protein function following cross-linking by alpha-dicarbonyls.

    Miller, Antonia G; Gerrard, Juliet A


    Protein cross-linking via the Maillard reaction with alpha-dicarbonyl compounds has been the subject of intense scrutiny in the literature. We report here a study of the impact of this cross-linking on enzyme function. Protein function following glycation was examined by treating ribonuclease A with methylglyoxal, glyoxal, and diacetyl, which cross-linked the enzyme and impaired its activity. The effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5-dimethylpyrazole-1-carboxamidine, on the cross-linking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein cross-linking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported antiaging compounds in vivo.

  2. Characteristic of hyaluronic acid derivative films cross-linked by polyethylene glycol of low water content

    Chen Jinghua; Chen Jingtao; Xu Zheng; Gu Qisheng


    Objective: To test the characteristics of byaluronic acid (HA) derivative cross-linked by polyethylene glycol films of low water content. Methods: The cross-linked HA film with 200 μm thickness was got at atmospheric pressure at 25℃ for 5 d. After dried, cross-linked films of 10 mm×10 mm were weighed and immersed in phosphate buffered saline (PBS pH 7.45) at 37℃ for 24 h. Then the solution fraction and water content were estimated. Meanwhile, cross-linked HA derivative films were immersed in phosphate buffered saline (PBS: pH 7.45) at 37℃ for determined time and then implanted subcutaneously in the back of white rats to test in vitro or in vivo degradation characteristic. Results and Conclusion: HA hydrogel cross-linked by polyethylene glycol with water content is as low as 60% and this kind of HA derivative has a slow degradation rate.


    Sawant, Akshada; Kothandapani, Anbarasi; Zhitkovich, Anatoly; Sobol, Robert W.; Patrick, Steve M.


    Mismatch repair (MMR) deficiency gives rise to cisplatin resistance and can lead to poor prognosis in cancers. Various models have been proposed to explain this low level of resistance caused due to loss of MMR proteins. We have shown that MMR proteins are required to maintain cisplatin interstrand cross-links (ICLs) on the DNA leading to increased cellular sensitivity. In our previous studies, we have shown that BER processing of the cisplatin ICLs is mutagenic. Polymerase β (Polβ) can generate mismatches which leads to the activation and the recruitment of mismatch repair proteins. In this paper, we distinguished between the requirement of different downstream MMR proteins for maintaining cisplatin sensitivity. We show that the MutSα (MSH2-MSH6) heterocomplex is required to maintain cisplatin sensitivity, whereas the Mutsβ complex has no effect. These results can be correlated with the increased repair of cisplatin ICLs and ICL induced DNA double strand breaks (DSBs) in the resistant cells. Moreover, we show that MLH1 proficient cells displayed a cisplatin sensitive phenotype when compared with the MLH1 deficient cells and the ATPase activity of MLH1 is essential to mediate this effect. Based on these results, we propose that MutSα as well as the downstream MMR pathway proteins are essential to maintain a cisplatin sensitive phenotype as a consequence of processing Polβ induced mismatches at sites flanking cisplatin ICLs. PMID:26519826

  4. Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

    LU; Xiuling; ZHENG; Chunyang; XU; Yuhong; SU; Zhiguo


    Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the twoα subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pI of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.

  5. Corneal changes following collagen cross linking and simultaneous topography guided photoablation with collagen cross linking for keratoconus

    Prema Padmanabhan


    Full Text Available Purpose: To compare the outcome of Collagen cross-linking (CXL with that following topography-guided customized ablation treatment (T-CAT with simultaneous CXL in eyes with progressive keratoconus. Materials and Methods: This was a prospective, non-randomized single centre study of 66 eyes with progressive keratoconus. Of these, 40 eyes underwent CXL and 26 eyes underwent T-CAT + CXL. The refractive, topographic, tomographic and aberrometric changes measured at baseline, 1, 3 and 6 months post-operatively were compared between both groups. Results: After a mean follow-up of 7.7 ± 1.3 months, the mean retinoscopic cylinder decreased by 1.02 ± 3.16 D in the CXL group ( P = 0.1 and 2.87 ± 3.22 D in the T-CAT + CXL group ( P = 0.04. The Best corrected visual acuity increased by 2 lines or more in 10% of eyes in the CXL group and in 23.3% of eyes in the T-CAT + CXL group. The mean steepest-K reduced by 0.40 ± 3.71 D ( P = 0.77 in the CXL group and by 2.91 ± 2.01D ( P = 0.03 in the T-CAT + CXL group. The sag factor and surface asymmetry index showed no significant change in the CXL group but reduced by 3.59 ± 5.94 D ( P = 0.01 and 0.72 ± 1.18 ( P = 0.02 respectively in the T-CAT + CXL group. There was a significant increase in the highest posterior corneal elevation in both groups (9.57 ± 14.93 μ in the CXL group and 7.85 ± 9.25 μ in the T-CAT + CXL group, P ≤ 0.001 for both. There was significantly greater reduction of mean coma ( P < 0.001 and mean higher-order aberrations ( P = 0.01 following T-CAT + CXL compared to CXL. Conclusions: CAT + CXL is an effective approach to confer biomechanical stability and to improve the corneal contour in eyes with keratoconus and results in better refractive, topographic and aberrometric outcomes than CXL alone.

  6. Chemistry and physical properties of melt-processed and solution-cross-linked corn zein.

    Sessa, David J; Mohamed, Abdellatif; Byars, Jeffrey A


    Corn zein was cross-linked with glutaraldehyde (GDA) using glacial acetic acid (HAc) as catalyst. The objectives are to evaluate the swelling characteristics of GDA cross-linked zein gels in water, ethanol, and their combinations. Similar formulations, upon solvent evaporation, form films. The mechanical properties of the films are compared to compression molded tensile bars from GDA melt-processed zein as a second objective. Chemistry of the cross-linking reaction was based on the aldehyde binding characteristics defined by use of fluorescence spectroscopy; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to demonstrate the cross-linking reaction; FTIR to observe absorption differences of the cross-linked product; differential scanning calorimetry, dynamic mechanical analysis and thermogravimetric analysis to assess thermal properties; and the use of Instron Universal Testing Machine to evaluate mechanical properties. A reaction mechanism for acid catalyzed GDA cross-linking of zein is proposed. Thermal and mechanical properties of tensile bars cut from either film or formed by compression molding were similar, where both showed increased tensile strengths, ductility and stiffness when compared with unmodified controls. Samples that were reacted with 8% GDA by weight based on weight of zein from either process retained their integrity when tensile bars from each were subjected to boiling water for 10 min or soaking in either water or HAc for 24 h. The melt-processed, cross-linked zein is a more environmentally friendly method that would eliminate the need for HAc recovery.

  7. Modified gum arabic cross-linked gelatin scaffold for biomedical applications

    Sarika, P.R. [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547 (India); Cinthya, Kuriakose [Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, Kerala 695 012 (India); Jayakrishnan, A. [Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036 (India); Anilkumar, P.R., E-mail: [Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, Kerala 695 012 (India); James, Nirmala Rachel, E-mail: [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547 (India)


    The present work deals with development of modified gum arabic cross-linked gelatin scaffold for cell culture. A new biocompatible scaffold was developed by cross-linking gelatin (Gel) with gum arabic, a polysaccharide. Gum arabic was subjected to periodate oxidation to obtain gum arabic aldehyde (GAA). GAA was reacted with gelatin under appropriate pH to prepare the cross-linked hydrogel. Cross-linking occurred due to Schiff's base reaction between aldehyde groups of oxidized gum arabic and amino groups of gelatin. The scaffold prepared from the hydrogel was characterized by swelling properties, degree of cross-linking, in vitro degradation and scanning electron microscopy (SEM). Cytocompatibility evaluation using L-929 and HepG2 cells confirmed non-cytotoxic and non-adherent nature of the scaffold. These properties are essential for generating multicellular spheroids and hence the scaffold is proposed to be a suitable candidate for spheroid cell culture. - Highlights: • Gum arabic cross-linked gelatin scaffold was developed for tissue engineering. • Cross-linking was achieved by Schiff's base reaction. • The scaffold is non-cytotoxic and non adherent to fibroblast and hepatocytes. • The scaffolds are potential candidates for spheroid cell culture.

  8. Understanding chemical reactivity for homo- and heterobifunctional protein cross-linking agents.

    Chen, Fan; Nielsen, Simone; Zenobi, Renato


    Chemical cross-linking, combined with mass spectrometry, has been applied to map three-dimensional protein structures and protein-protein interactions. Proper choice of the cross-linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross-linkers with proteins are quite sparse. In this study, we investigated chemical cross-linking from the aspects of the protein structures and the cross-linking reagents involved, by using two structurally well-known proteins, glyceraldehyde 3-phosohate dehydrogenase and ribonuclease S. Chemical cross-linking reactivity was compared using a series of homo- and hetero-bifunctional cross-linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydrosuccinimide and succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross-linking efficiency. Moreover, the reactive groups of the chemical cross-linker also play an important role; a higher cross-linking reaction efficiency was found for maleimides compared to 2-pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N-hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Shell and core cross-linked poly(L-lysine)/poly(acrylic acid) complex micelles.

    Hsieh, Yi-Hsuan; Hsiao, Yung-Tse; Jan, Jeng-Shiung


    We report the versatility of polyion complex (PIC) micelles for the preparation of shell and core cross-linked (SCL and CCL) micelles with their surface properties determined by the constituent polymer composition and cross-linking agent. The negatively and positively charged PIC micelles with their molecular structure and properties depending on the mixing weight percentage and polymer molecular weight were first prepared by mixing the negatively and positively charged polyions, poly(acrylic acid) (PAA) and poly(L-lysine) (PLL). The feasibility of preparing SCL micelles was demonstrated by cross-linking the shell of the negatively and positively charged micelles using cystamine and genipin, respectively. The core of the micelles can be cross-linked by silica deposition to stabilize the assemblies. The shell and/or core cross-linked micelles exhibited excellent colloid stability upon changing solution pH. The drug release from the drug-loaded SCL micelles revealed that the controllable permeability of the SCL micelles can be achieved by tuning the cross-linking degree and the SCL micelles exhibited noticeable pH-responsive behavior with accelerated release under acidic conditions. With the versatility of cross-linking strategies, it is possible to prepare a variety of SCL and CCL micelles from PIC micelles.

  10. Novel antimicrobial superporous cross-linked chitosan/pyromellitimide benzoyl thiourea hydrogels.

    Mohamed, Nadia A; Abd El-Ghany, Nahed A; Fahmy, Mona M


    In this work, chitosan (CS) was cross-linked with different amounts of pyromellitimide benzoyl thiourea moieties. The structure of the cross-linked CS was confirmed by elemental analyses, FTIR and (1)H- NMR spectroscopy. The cross-linking process proceeds via reacting of the amino groups of CS with the isothiocyanate groups of the N,N'-bis [4-(isothiocyanate carbonyl)phenyl] pyromellitimide cross-linker. The amount of the cross-linker was varied with respect to CS to produce four new pyromellitimide benzoyl thiourea cross-linked CS (PIBTU-CS) hydrogels designated as PIBTU-CS-1, PIBTU-CS-2, PIBTU-CS-3, and PIBTU-CS-4 of increasing cross-linking degree percent of 11, 22, 44 and 88%, respectively. The scanning electron microscopy observation indicates the extremely porous structure of the hydrogels. XRD results showed that the crystallinity of CS was decreased upon cross-linking. The four hydrogels exhibit a higher antibacterial activity on Bacillus subtilis and Streptococcus pneumoniae as Gram positive bacteria and against Escherichia coli as Gram negative bacteria and higher antifungal activity on Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum than that of the parent CS as shown from their higher inhibition zone diameters and their lower MIC values. The swell ability of the hydrogel as well as their antimicrobial activity increased with increasing cross-linking density.

  11. Grafted, cross-linked carbon black as a double-layer capacitor electrode material

    Richner, R.; Mueller, S.; Wokaun, A.


    Isocyanate prepolymers readily react with oxidic functional groups on carbon black. On carbon black grafted with diisocyanates, reactive isocyanate groups are available for cross-linking to a polyurethane system. This cross-linked carbon black was considered as a new active material for electrochemical electrodes. Active material for electric double-layer capacitor electrodes was produced which had values of specific capacitance of up to 200 F/g. Cross-linking efficiencies of up to 58 % of the polymers utilised were achieved. (author)

  12. Characterization of solid UV cross-linked PEGDA for biological applications

    Castro, David


    This paper reports on solid UV cross-linked Poly(ethylene)-glycol-diacrylate (PEGDA) as a material for microfluidic devices for biological applications. We have evaluated biocompatibility of PEGDA through two separate means: 1) by examining cell viability and attachment on cross-linked PEGDA surfaces for cell culture applications, and 2) by determining if cross-linked PEGDA inhibits the polymerase chain reaction (PCR) processes for on-chip PCR. Through these studies a correlation has been found between degree of curing and cell viability, attachment, as well as on PCR outcome.

  13. Permanent Set of Cross-Linking Networks: Comparison of Theory with Molecular Dynamics Simulations

    Rottach, Dana R.; Curro, John G.; Budzien, Joanne


    The permanent set of cross-linking networks is studied by molecular dynamics. The uniaxial stress for a bead-spring polymer network is investigated as a function of strain and cross-link density history, where cross-links are introduced in unstrained and strained networks. The permanent set...... is found from the strain of the network after it returns to the state-of-ease where the stress is zero. The permanent set simulations are compared with theory using the independent network hypothesis, together with the various theoretical rubber elasticity theories: affine, phantom, constrained junction...

  14. A genetic anomaly of oriented collagen biosynthesis and cross-linking: Keratoconus.

    Bourges, J L; Robert, A M; Robert, L


    Oriented collagen biosynthesis is one of the major mechanisms involved in tissue and organ formation during development. Corneal biogenesis is one example. Defects in this process lead to anomalies in tissue structure and function. The transparency of cornea and its achievement are a good example as well as its pathological modifications. Keratoconus is one example of this type of pathologies, involving also inappropriate cross-linking of collagen fibers. Among the tentatives to correct this anomaly, the riboflavin-potentiated UV-cross-linking (CXL) of keratoconus corneas appears clinically satisfactory, although none of the experiments and clinical results published prove effective cross-linking. The published results are reviewed in this article.

  15. Permanent Set of Cross-Linking Networks: Comparison of Theory with Molecular Dynamics Simulations

    Rottach, Dana R.; Curro, John G.; Budzien, Joanne;


    is found from the strain of the network after it returns to the state-of-ease where the stress is zero. The permanent set simulations are compared with theory using the independent network hypothesis, together with the various theoretical rubber elasticity theories: affine, phantom, constrained junction......The permanent set of cross-linking networks is studied by molecular dynamics. The uniaxial stress for a bead-spring polymer network is investigated as a function of strain and cross-link density history, where cross-links are introduced in unstrained and strained networks. The permanent set...

  16. Langevin dynamcis simulations of driven polymer translocation into a cross-linked gel.

    Sean, David; Slater, Gary W


    We investigate the dynamics of driving a polyelectrolyte such as DNA through a nanopore and into a cross-linked gel. Placing the gel on the trans-side of the nanopore can increase the translocation time while not negatively affecting the capture rates. Thus, this setup combines the mechanics of gel electrophoresis with nanopore translocation. However, contrary to typical gel electrophoresis scenarios, the effect of the field is localized in the immediate vicinity of the nanopore and becomes negligible inside the gel matrix. Thus, we investigate the process by which a semiflexible polymer can be pushed into a gel matrix via a localized field and we describe how the dynamics of gel penetration depends upon the field intensity, polymer stiffness, and gel pore size. Our simulation results show that a semiflexible polymer enters the gel region with two distinct mechanisms depending upon the ratio between the bending length scale and the gel pore size. In both regimes, the gel fibers cause a net increase in the mean translocation time. Interestingly, the translocation rate is found to be constant (a potentially useful feature for many applications) during the predominant part of the translocation process when the polymer is stiff over a length scale comparable to the gel pore size.

  17. Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins.

    Chen, Rong; Wang, Jian-Bo; Zhang, Xian-Qing; Ren, Jing; Zeng, Cheng-Ming


    Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS-PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.

  18. Reaction of 1,2,3,4-Diepoxybutane with 2′-Deoxyguanosine: Initial Products and Their Stabilities and Decomposition Patterns under Physiological Conditions

    Zhang, Xin-Yu; Elfarra, Adnan A.


    1,2,3,4-Diepoxybutane (DEB), an in vivo metabolite of 1,3-butadiene (BD), is a carcinogen and a potent mutagen. Previously, DEB was shown to react with 2′-deoxyguanosine (dG) under physiological conditions to produce seven major nucleoside adducts resulting from alkylation at the N1- (P8 and P9), N7- (P5 and P5′) and both the N1- and N2-positions of dG to form six-membered (P4-1 and P4-2) and seven-membered fused ring systems (P6), respectively (Zhang and Elfarra, Chem. Res. Toxicol. 2003, 16...

  19. Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

    Richard, S; Tamas, C; Sell, D R; Monnier, V M


    Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

  20. Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer

    Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H.; Nevosad, Andreas; Teichert, Christian; S. White, Matthew.; S. Sariciftci, Niyazi.; Scharber, Markus C.


    We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium–tin–oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor–acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process. PMID:24817837

  1. Stabilized sulfonated aromatic polymers by in situ solvothermal cross-linking

    Maria Luisa eDi Vona


    Full Text Available The cross-link reaction via sulfone bridges of sulfonated polyetheretherketone (SPEEK by thermal treatment at 180 °C in presence of dimethylsulfoxide (DMSO is discussed. The modifications of properties subsequent to the cross-linking are presented. The mechanical strength as well as the hydrolytic stability increased with the thermal treatment time, i.e., with the degree of cross-linking. The proton conductivity was determined as function of temperature, IEC, degree of cross-linking and hydration number. The memory effect, which is the membrane ability to remember the water uptake reached at high temperature also at lower temperature, is exploited in order to achieve high values of conductivity. Membranes swelled at 110 °C can reach a conductivity of 0.14 S/cm at 80°C with a hydration number ( of 73.

  2. An Investigation on Rheology of Peroxide Cross-linking of Low Density Polyethylene

    Ghasemi, Ismaeil; Rasmussen, Henrik K.; Szabo, Peter


    One of the most important post-reactor modifications of polyethylene is cross-linking. It improves some properties of polyethylene such as environmental stress cracking resistance, chemical and abrasion resistance, and service temperature. In this study, the effect of peroxide cross-linking on th......One of the most important post-reactor modifications of polyethylene is cross-linking. It improves some properties of polyethylene such as environmental stress cracking resistance, chemical and abrasion resistance, and service temperature. In this study, the effect of peroxide cross......-linking on the rheological behaviour of low density polyethylene was investigated by using a combination of creep test and differential scanning calorimeter (DSC) in isotherm condition. The used peroxide was di-cumyl peroxide and its concentration was 2 wt%. The experiments were carried out at 150,160, and 170 degrees C...


    A.G. Gurin


    Full Text Available A possibility of applying a pulse-periodic high-current induction electron accelerators to radiation polyethylene cross-linking is considered in the article. A comparative analysis with other devices used for irradiation is made.

  4. Molecular Dynamics Simulations of Polymer Networks Undergoing Sequential Cross-Linking and Scission Reactions

    Rottach, Dana R.; Curro, John G.; Budzien, Joanne;


    The effects of sequential cross-linking and scission of polymer networks formed in two states of strain are investigated using molecular dynamics simulations. Two-stage networks are studied in which a network formed in the unstrained state (stage 1) undergoes additional cross-linking in a uniaxia......The effects of sequential cross-linking and scission of polymer networks formed in two states of strain are investigated using molecular dynamics simulations. Two-stage networks are studied in which a network formed in the unstrained state (stage 1) undergoes additional cross...... good agreement with the predictions of Flory and Fricker. It was found that the fractional stress reduction upon removal of the first-stage cross-links could be accurately calculated from the slip tube model of Rubinstein and Panyukov modified to use the theoretical transfer functions of Fricker.  ...

  5. Comparative study of PBI Cross Linked Utilizing Agents of Varying Steric Configurations

    Kirkebcek, Andreas; Aili, David; Li, Qingfeng;


    The high thermal and chemical stability of poly[2,2'-(m-phenylene)-5,5' bibenzimidazole] (PBI) accounts for its wise spread use in high temperature polymer electrolyte membrane fuel cells (HT- PEMFC). By doping the membrane with phosphoric acid (PA) ionic conductivity is obtained. Thus conductivity...... ionic or covalent cross linking. When considering such, little attention is devoted to explore the effect of the sterical configuration of the cross linking agent. In this contribution three different cross linking agents are utilized to evaluate how these affects final membrane properties....... is dependent on the amount of PA present within the membrane. However mechanical properties are reduced are significantly reduced due to the plasticizing effect shown by PA [1]. This effect is due to PBI chain displacement. This effect can be lessened by use of cross linking [2-4]. This can be obtained using...

  6. Riboflavin cross-linking of collagen porous scaffolds for periodontal regeneration

    Seciu A-M.


    Full Text Available Periodontitis treatment using occlusive membranes presented variable efficacy due to their rapid degradation in the complex biologic environment of the damaged periodontium. The aim of this study was to prepare novel composites based on collagen, chondroitin sulfate and fibronectin, and to establish the optimal parameters for their photochemical cross-linking using riboflavin and UV exposure. The degree of cross-linking, biodegradability and density of all scaffold variants were investigated. Their cytotoxicity was evaluated in a culture of gingival fibroblasts by MTT assay and light microscopy. The results indicated that the higher the cross-linking degree, the lower was the scaffold biodegradation. Cell culture studies showed that composite scaffolds were favorable for cellular survival. In conclusion, the cross-linking method using riboflavin and UV exposure resulted in stable and biocompatible collagen-based composite scaffolds that could be used for periodontitis treatment.

  7. Hydrogen peroxide and ferulic acid-mediated oxidative cross-linking ...



    Dec 15, 2009 ... Oxidative cross-linked casein mediated by hydrogen peroxide and ferulic acid was prepared at ... functional properties of food proteins treated (Motoki and. Seguro, 1998 ..... Team of Northeast Agricultural University (No.

  8. CrossWork: Software-assisted identification of cross-linked peptides

    Rasmussen, Morten; Refsgaard, Jan; Peng, Li;


    The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major...... obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross......-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. Cross...

  9. A Review of Collagen Cross-Linking in Cornea and Sclera

    Xiao Zhang


    Full Text Available Riboflavin/UVA cross-linking is a technique introduced in the past decades for the treatment of keratoconus, keratectasia, and infectious keratitis. Its efficacy and safety have been investigated with clinical and laboratory studies since its first clinical application by Wollensak for the treatment of keratoconus. Although its complications are encountered during clinical practice, such as infection inducing risk, minimal invasion merits a further investigation on its future application in clinical practice. Recently, collagen cross-linking in sclera shows a promising prospect. In present study, we summarized the representative studies describing the clinical and laboratory application of collagen cross-linking published in past decades and provided our opinion on the positive and negative results of cross-linking in the treatment of ophthalmic disorders.

  10. New insights into the pros and cons of cross-linking decellularized bioartificial organs.

    Hussein, Kamal H; Park, Kyung-Mee; Lee, Yun-Suk; Woo, Jae-Seok; Kang, Byung-Jae; Choi, Ki-Young; Kang, Kyung-Sun; Woo, Heung-Myong


    ABSTRACTDecellularization is an attractive method for scaffold designing in regenerative medicine. The resulting extracellular matrix (ECM) consists of structural proteins such as collagen and elastin, growth factors, and glycosaminoglycans, which can direct site-appropriate remodeling after in vivo implantation. Mainly, collagen and elastin of ECM are exposed to the enzymatic biodegradation in the host. To control the biodegradation process, treatment of decellularized tissue by a cross-linking agent is required. Cross-linking also reduces antigenicity and increases the storage properties. Cross-linkers should be nontoxic, with the ability to preserve the ECM components, especially glycosaminoglycans and associated growth factors for retention of scaffold bioactivity. In this review, we describe the different cross-linking agents and methods of evaluation of cross-linking efficiency.

  11. Glutaraldehyde vapor cross-linked nanofibrous PVA mat with in situ formed silver nanoparticles.

    Destaye, Addisu Getachew; Lin, Cheng-Keng; Lee, Cheng-Kang


    Polyvinyl alcohol (PVA) nanofibrous mat can be easily prepared via electrospinning its aqueous solution. However, the obtained nanofibrous mat is instantaneously dissolved in water. Therefore, rendering the environmentally friendly nanofibrous mat water insoluble by cross-linking mechanism is of great interest. The electrospun PVA nanofibrous mat with an average fiber diameter of ca. 400 nm could be effectively cross-linked by glutaraldehyde vapor at room temperature. The cross-linking not only resulted in a water-insoluble nanofibrous mat but also generated an excess amount of unreacted aldehyde functional groups that could reduce silver salts into silver nanoparticles. The in situ formed silver nanoparticles along the fibrous surface showed excellent antimicrobial activity against Escherichia coli. The vapor cross-linked nanofibrous mat shows a high potential to be used for efficiently capturing and killing pathogenic bacteria.

  12. Plasticizer migration from cross-linked flexible PVC. 1. Effects on tribology

    Pannico, M.; Persico, P.; Ambrogi, V.; Carfagna, C.


    Utilization of soft PVC is restricted by plasticizer migration that can affect material properties, as well as its toxicity. Modifying the chemical structure of PVC is one of the most effective tool to reduce the diffusion of plasticizer. In this work, a soft cross-linked PVC was obtained using a difunctional amine, namely isophoron diamine (IPDA) as the cross-linking agent. The gel content (wt %) was evaluated by weighting the insoluble portion obtained through solvent extraction technique. Thermogravimetric analysis (TGA) revealed that cross-linking reactions promote thermal degradation phenomena in the polymer matrix. Tribological properties of soft uncross-linked, cross-linked and rigid PVC were determined. Soft formulations were held in contact for 32 days with rigid PVC sheets. Plasticizer migration towards the interface causes an increase of dynamic friction compared to that of the reference rigid PVC.

  13. The level of cross-linking and the structure of anisotropic magnetorheological elastomers

    Borin, Dmitry, E-mail: [Institute of Fluid Mechanics, Technische Universitaet Dresden, Dresden 01062 (Germany); Guenther, David [Institute of Fluid Mechanics, Technische Universitaet Dresden, Dresden 01062 (Germany); Hintze, Christian; Heinrich, Gert [Leibniz Institute of Polymer Research, Dresden 01069 (Germany); Odenbach, Stefan [Institute of Fluid Mechanics, Technische Universitaet Dresden, Dresden 01062 (Germany)


    The influence of the concentration of the magnetic powder on the level of cross-linking of magnetorheological elastomers (MREs) has been studied. Afterwards the structural characterisation of manufactured MREs has been performed by using non-destructive method, specifically the computed tomography. The correlation between internal structures of MREs and the developing of its cross-linking level during the curing was found. It was shown that changes in the concentration of the powder significantly affect morphologies of the sample.

  14. Novel thermoplastic vulcanizates (TPVs based on silicone rubber and polyamide exploring peroxide cross-linking

    K. Naskar


    Full Text Available Novel thermoplastic vulcanizates (TPVs based on silicone rubber (PDMS and polyamide (PA12 have been prepared by dynamic vulcanization process. The effect of dynamic vulcanization and influence of various types of peroxides as cross-linking agents were studied in detail. All the TPVs were prepared at a ratio of 50/50 wt% of silicone rubber and polyamide. Three structurally different peroxides, namely dicumyl peroxide (DCP, 3,3,5,7,7-pentamethyl 1,2,4-trioxepane (PMTO and cumyl hydroperoxide (CHP were taken for investigation. Though DCP was the best option for curing the silicone rubber, at high temperature it suffers from scorch safety. An inhibitor 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO was added with DCP to stabilize the radicals in order to increase the scorch time. Though CHP (hydroperoxide had higher half life time than DCP at higher temperature, it has no significant effect on cross-linking of silicone rubber. PMTO showed prolonged scorch safety and better cross-linking efficiency rather than the other two. TPVs of DCP and PMTO were made up to 11 minutes of mixing. Increased values of tensile strength and elongation at break of PMTO cross-linked TPV indicate the superiority of PMTO. Scanning electron micrographs correlate with mechanical properties of the TPVs. High storage modulus (E' and lower loss tangent value of the PMTO cross-linked TPV indicate the higher degree of cross-linking which is also well supported by the overall cross-link density value. Thus PMTO was found to be the superior peroxide for cross-linking of silicone rubber at high temperature.

  15. Sorption of substituted indoles on highly cross-linked polystyrene from water-acetonitrile solutions

    Shafigulin, R. V.; Myakishev, A. A.; Il'Ina, E. A.; Il'in, M. M.; Davankov, V. A.; Bulanova, A. V.


    The sorption of first synthesized indole derivatives by highly cross-linked polystyrenes from water-acetonitrile solutions was studied by high-performance liquid chromatography. The retention factors and differences in the Gibbs energy of adsorption from infinite diluted solutions were calculated, and the applicability of the Snyder-Soczewinski and Scott-Kucera models for describing the chromatographic retention of sorbates on a polymer network of highly cross-linked polystyrene was shown.

  16. Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices

    Perera, Handunge Kumudu Irani; Handuwalage, Charith Sandaruwan


    Background Protein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method. Methods Methanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °...

  17. Computational exploration of polymer nanocomposite mechanical property modification via cross-linking topology

    Lacevic, N; Gee, R; Saab, A; Maxwell, R


    Molecular dynamics simulations have been performed in order to study the effects of nanoscale filler cross-linking topologies and loading levels on the mechanical properties of a model elastomeric nanocomposite. The model system considered here is constructed from octa-functional polyhedral oligomeric silsesquioxane (POSS) dispersed in a poly(dimethylsiloxane) (PDMS) matrix. Shear moduli, G, have been computed for pure and for filled and unfilled PDMS as a function of cross-linking density, POSS fill loading level, and polymer network topology. The results reported here show that G increases as the cross-linking (covalent bonds formed between the POSS and the PDMS network) density increases. Further, G is found to have a strong dependence on cross-linking topology. The increase in shear modulus, G, for POSS filled PDMS is significantly higher than that for unfilled PDMS cross-linked with standard molecular species, suggesting an enhanced reinforcement mechanism for POSS. In contrast, in blended systems (POSS/PDMS mixture with no cross-linking) G was not observed to significantly increase with POSS loading. Finally, we find intriguing differences in the structural arrangement of bond strains between the cross-linked and the blended systems. In the unfilled PDMS the distribution of highly strained bonds appears to be random, while in the POSS filled system, the strained bonds form a net-like distribution that spans the network. Such a distribution may form a structural network 'holding' the composite together and resulting in increases in G compared to an unfilled, cross-linked system. These results are of importance for engineering of new POSS-based multifunctional materials with tailor-made mechanical properties.

  18. Computational exploration of polymer nanocomposite mechanical property modification via cross-linking topology.

    Lacevic, Naida; Gee, Richard H; Saab, Andrew; Maxwell, Robert


    Molecular dynamics simulations have been performed in order to study the effects of nanoscale filler cross-linking topologies and loading levels on the mechanical properties of a model elastomeric nanocomposite. The model system considered here is constructed from octafunctional polyhedral oligomeric silsesquioxane (POSS) dispersed in a poly(dimethylsiloxane) (PDMS) matrix. Shear moduli, G, have been computed for pure and for filled and unfilled PDMS as a function of cross-linking density, POSS fill loading level, and polymer network topology. The results reported here show that G increases as the cross-linking (covalent bonds formed between the POSS and the PDMS network) density increases. Further, G is found to have a strong dependence on cross-linking topology. The increase in shear modulus, G, for POSS filled PDMS is significantly higher than that for unfilled PDMS cross-linked with standard molecular species, suggesting an enhanced reinforcement mechanism for POSS. In contrast, in blended systems (POSS/PDMS mixture with no cross-linking) G was not observed to significantly increase with POSS loading. Finally, we find intriguing differences in the structural arrangement of bond strains between the cross-linked and the blended systems. In the unfilled PDMS the distribution of highly strained bonds appears to be random, while in the POSS filled system, the strained bonds form a netlike distribution that spans the network. Such a distribution may form a structural network "holding" the composite together and resulting in increases in G compared to an unfilled, cross-linked system. These results are of importance for engineering of new POSS-based multifunctional materials with tailor-made mechanical properties.

  19. Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins.

    Vincent, S H; Holeman, B; Cully, B C; Muller-Eberhard, U


    Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

  20. Characterization of the receptor for endothelial cell growth factor (ECGF) by affinity cross-linking

    Friesel, R.; Burgess, W.H.; Mehlman, T.; Maciag, T.


    The authors have demonstrated high affinity receptors for ECGF on endothelial cells by covalent cross-linking of (/sup 125/I)-ECGF with disuccinimidyl suberate and observe a single chain cross-linked polypeptide species with an apparent M/sub r/ of 170K. The M/sub r/ 170K species represents (/sup 125/I)-ECGF bound to its receptor since (i) excess unlabeled ECGF inhibits the cross-linking of (/sup 125/I)-ECGF, (ii) labeling of the M/sub r/170K species does not take place in the absence of cross-linker, (iii) cells previously shown to be refractory to ECGF and lack ECGF receptors do not yield a cross-linked species, (iv) the cross-linked species can be immunoprecipitated with anti-ECGF antibodies, and (v) preincubation of cells with ECGF at 37/sup 0/C significantly reduces cross-linking while incubation at 4/sup 0/C does not. These data demonstrate that ECGF induced cell proliferation occurs through the occupancy of a specific cell surface polypeptide receptor with an apparent M/sub r/ of 150K, and suggests that internalization of the receptor-ligand complex may be relevant to ECGF-induced signal transduction.

  1. Directing the oligomer size distribution of peroxidase-mediated cross-linked bovine alpha-lactalbumin.

    Heijnis, Walter H; Wierenga, Peter A; van Berkel, Willem J H; Gruppen, Harry


    Enzymatic protein cross-linking is a powerful tool to change protein functionality. For optimal functionality in gel formation, the size of the cross-linked proteins needs to be controlled, prior to heating. In the current study, we addressed the optimization of the horseradish peroxidase-mediated cross-linking of calcium-depleted bovine alpha-lactalbumin. To characterize the formed products, the molecular weight distribution of the cross-linked protein was determined by size exclusion chromatography. At low ionic strength, more dimers of alpha-lactalbumin are formed than at high ionic strength, while the same conversion of monomers is observed. Similarly, at pH 5.9 more higher oligomers are formed than at pH 6.8. This is proposed to be caused by local changes in apo alpha-lactalbumin conformation as indicated by circular dichroism spectroscopy. A gradual supply of hydrogen peroxide improves the yield of cross-linked products and increases the proportion of higher oligomers. In conclusion, this study shows that the size distribution of peroxidase-mediated cross-linked alpha-lactalbumin can be directed toward the protein oligomers desired.

  2. Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications.

    Steffensen, Charlotte L; Andersen, Mogens L; Degn, Peter E; Nielsen, Jacob H


    Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.

  3. Smooth muscle cell phenotype modulation and contraction on native and cross-linked polyelectrolyte multilayers.

    Moussallem, Maroun D; Olenych, Scott G; Scott, Shannon L; Keller, Thomas C S; Schlenoff, Joseph B


    Smooth muscle cells convert between a motile, proliferative "synthetic" phenotype and a sessile, "contractile" phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young's modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle alpha-actin (smActin) that are characteristic of the smooth muscle "contractile" phenotype. Consistent with those cells being "contractile", A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca(2+) ionophore.

  4. Cross-linked chitosan improves the mechanical properties of calcium phosphate-chitosan cement.

    Aryaei, Ashkan; Liu, Jason; Jayatissa, Ahalapitiya H; Jayasuriya, A Champa


    Calcium phosphate (CaP) cements are highly applicable and valuable materials for filling bone defects by minimally invasive procedures. The chitosan (CS) biopolymer is also considered as one of the promising biomaterial candidates in bone tissue engineering. In the present study, some key features of CaP-CS were significantly improved by developing a novel CaP-CS composite. For this purpose, CS was the first cross-linked with tripolyphosphate (TPP) and then mixed with CaP matrix. A group of CaP-CS samples without cross-linking was also prepared. Samples were fabricated and tested based on the known standards. Additionally, the effect of different powder (P) to liquid (L) ratios was also investigated. Both cross-linked and uncross-linked CaP-CS samples showed excellent washout resistance. The most significant effects were observed on Young's modulus and compressive strength in wet condition as well as surface hardness. In dry conditions, the Young's modulus of cross-linked samples was slightly improved. Based on the presented results, cross-linking does not have a significant effect on porosity. As expected, by increasing the P/L ratio of a sample, ductility and injectability were decreased. However, in the most cases, mechanical properties were enhanced. The results have shown that cross-linking can improve the mechanical properties of CaP-CS and hence it can be used for bone tissue engineering applications.

  5. Cross-linked survey analysis is an approach for separating cause and effect in survey research.

    Redelmeier, Donald A; Thiruchelvam, Deva; Lustig, Andrew J


    We developed a new research approach, called cross-linked survey analysis, to explore how an acute exposure might lead to changes in survey responses. The goal was to identify associations between exposures and outcomes while reducing some ambiguities related to interpreting cause and effect in survey responses from a population-based community questionnaire. Cross-linked survey analysis differs from a cross-sectional, longitudinal, and panel survey analysis by individualizing the timeline to the unique history of each respondent. Cross-linked survey analysis, unlike a repeated-measures self-matching design, does not track changes in a repeated survey question given to the same respondent at multiple time points. Pilot data from three analyses (n = 1,177 respondents) illustrate how a cross-linked survey analysis can control for population shifts, temporal trends, and reverse causality. Accompanying graphs provide an intuitive display to readers, summarize results, and show differences in response distributions. Population-based individual-level linkages also reduce selection bias and increase statistical power compared with a single-center cross-sectional survey. Cross-linked survey analysis has limitations related to unmeasured confounding, pragmatics, survivor bias, statistical models, and the underlying artifacts in survey responses. We suggest that a cross-linked survey analysis may help in epidemiology science using survey data. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. A novel strategy for preparing mechanically robust ionically cross-linked alginate hydrogels

    Jejurikar, Aparna; Lawrie, Gwen; Groendahl, Lisbeth [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Martin, Darren, E-mail: [Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland 4072 (Australia)


    The properties of alginate films modified using two cross-linker ions (Ca{sup 2+} and Ba{sup 2+}), comparing two separate cross-linking techniques (the traditional immersion (IM) method and a new strategy in a pressure-assisted diffusion (PD) method), are evaluated. This was achieved through measuring metal ion content, water uptake and film stability in an ionic solution ([Ca{sup 2+}] = 2 mM). Characterization of the internal structure and mechanical properties of hydrated films were established by cryogenic scanning electron microscopy and tensile testing, respectively. It was found that gels formed by the PD technique possessed greater stability and did not exhibit any delamination after 21 day immersion as compared to gels formed by the IM technique. The Ba{sup 2+} cross-linked gels possessed significantly higher cross-linking density as reflected in lower water content, a more dense internal structure and higher Young's modulus compared to Ca{sup 2+} cross-linked gels. For the Ca{sup 2+} cross-linked gels, a large improvement in the mechanical properties was observed in gels produced by the PD technique and this was attributed to thicker pore walls observed within the hydrogel structure. In contrast, for the Ba{sup 2+} cross-linked gels, the PD technique resulted in gels that had lower tensile strength and strain energy density and this was attributed to phase separation and larger macropores in this gel.

  7. Digestibility of β-lactoglobulin following cross-linking by Trametes versicolor laccase and apple polyphenols



    Full Text Available β-Lactoglobulin (BLG is an important nutrient of dairy products and an important allergen in cow’s milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS. The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG, with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin–pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders.

  8. Exogenous collagen cross-linking recovers tendon functional integrity in an experimental model of partial tear.

    Fessel, Gion; Wernli, Jeremy; Li, Yufei; Gerber, Christian; Snedeker, Jess G


    We investigated the hypothesis that exogenous collagen cross-linking can augment intact regions of tendon to mitigate mechanical propagation of partial tears. We first screened the low toxicity collagen cross-linkers genipin, methylglyoxal and ultra-violet (UV) light for their ability to augment tendon stiffness and failure load in rat tail tendon fascicles (RTTF). We then investigated cross-linking effects in load bearing equine superficial digital flexor tendons (SDFT). Data indicated that all three cross-linking agents augmented RTTF mechanical properties but reduced native viscoelasticity. In contrast to effects observed in fascicles, methylglyoxal treatment of SDFT detrimentally affected tendon mechanical integrity, and in the case of UV did not alter tendon mechanics. As in the RTTF experiments, genipin cross-linking of SDFT resulted in increased stiffness, higher failure loads and reduced viscoelasticity. Based on this result we assessed the efficacy of genipin in arresting tendon tear propagation in cyclic loading to failure. Genipin cross-linking secondary to a mid-substance biopsy-punch significantly reduced tissue strains, increased elastic modulus and increased resistance to fatigue failure. We conclude that genipin cross-linking of injured tendons holds potential for arresting tendon tear progression, and that implications of the treatment on matrix remodeling in living tendons should now be investigated.

  9. A Comparative Study of the Characteristics of Cross-Linked, Oxidized and Dual-Modified Rice Starches

    Feng-Xiang Yu


    Full Text Available Rice starch was cross-linked with epichlorohydrin (0.3%, w/w, on a dry starch basis and oxidized with sodium hypochlorite (2.5% w/w, respectively. Two dual-modified rice starch samples (oxidized cross-linked rice starch and cross-linked oxidized rice starch were obtained by the oxidation of cross-linked rice starch and the cross-linking of oxidized rice starch at the same level of reagents. The physicochemical properties of native rice starch, cross-linked rice starch and oxidized rice starch were also studied parallel with those of the two dual-modified rice starch samples using rapid visco analysis (RVA, differential scanning calorimetry (DSC, dynamic rheometry and scanning electron microscopy (SEM. It was found that the levels of cross-linking and oxidation used in this study did not cause any significant changes in the morphology of rice starch granules. Cross-linked oxidized starch showed lower swelling power (SP and solubility, and higher paste clarity in comparison with native starch. Cross-linked oxidized rice starch also had the lowest tendency of retrogradation and highest ability to resistant to shear compared with native, cross-linked, oxidized and oxidized cross-linked rice starches. These results suggest that the undesirable properties in native, cross-linked and oxidized rice starch samples could be overcome through dual-modification.

  10. Click Cross-Linking-Improved Waterborne Polymers for Environment-Friendly Coatings and Adhesives.

    Hu, Jianqing; Peng, Kaimei; Guo, Jinshan; Shan, Dingying; Kim, Gloria B; Li, Qiyao; Gerhard, Ethan; Zhu, Liang; Tu, Weiping; Lv, Weizhong; Hickner, Michael A; Yang, Jian


    Waterborne polymers, including waterborne polyurethanes (WPU), polyester dispersions (PED), and polyacrylate emulsions (PAE), are employed as environmentally friendly water-based coatings and adhesives. An efficient, fast, stable, and safe cross-linking strategy is always desirable to impart waterborne polymers with improved mechanical properties and water/solvent/thermal and abrasion resistance. For the first time, click chemistry was introduced into waterborne polymer systems as a cross-linking strategy. Click cross-linking rendered waterborne polymer films with significantly improved tensile strength, hardness, adhesion strength, and water/solvent resistance compared to traditional waterborne polymer films. For example, click cross-linked WPU (WPU-click) has dramatically improved the mechanical strength (tensile strength increased from 0.43 to 6.47 MPa, and Young's modulus increased from 3 to 40 MPa), hardness (increased from 59 to 73.1 MPa), and water resistance (water absorption percentage dropped from 200% to less than 20%); click cross-linked PED (PED-click) film also possessed more than 3 times higher tensile strength (∼28 MPa) than that of normal PED (∼8 MPa). The adhesion strength of click cross-linked PAE (PAE-click) to polypropylene (PP) was also improved (from 3 to 5.5 MPa). In addition, extra click groups can be preserved after click cross-linking for further functionalization of the waterborne polymeric coatings/adhesives. In this work, we have demonstrated that click modification could serve as a convenient and powerful approach to significantly improve the performance of a variety of traditional coatings and adhesives.

  11. Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat.

    Rothamel, Daniel; Schwarz, Frank; Sager, Martin; Herten, Monika; Sculean, Anton; Becker, Jürgen


    The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendExtend (BME) (glutaraldehyde cross-linked bovine type I collagen), (4) Ossix (OS) (enzymatic-cross-linked bovine type I collagen), (5) TutoDent (TD) (non-cross-linked bovine type I collagen, and (6-8) VN(1-3) (chemical cross-linked porcine type I and III collagens). Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. After 2, 4, 8, 16, and 24 weeks of healing, the rats were sacrificed and explanted specimens were prepared for histologic and histometric analysis. The following parameters were evaluated: biodegradation over time, vascularization, tissue integration, and foreign body reaction. Highest vascularization and tissue integration was noted for BG followed by BM, BME, and VN(1); TD, VN(2), and VN(3) showed prolongated, while OS exhibited no vascularization. Subsequently, biodegradation of BG, BM, BME and VN(1) was faster than TD, VN(2), and VN(3). OS showed only a minute amount of superficial biodegradation 24 weeks following implantation. Biodegradation of TD, BM, BME, VN(2), and VN(3) was associated with the presence of inflammatory cells. Within the limits of the present study, it was concluded that cross-linking of bovine and porcine-derived collagen types I and III was associated with (i) prolonged biodegradation, (ii) decreased tissue integration and vascularization, and (iii) in case of TD, BM, BME, VN(2), and VN(3) foreign body reactions.

  12. Vitamin E-diffused highly cross-linked UHMWPE particles induce less osteolysis compared to highly cross-linked virgin UHMWPE particles in vivo.

    Bichara, David A; Malchau, Erik; Sillesen, Nanna H; Cakmak, Selami; Nielsen, G Petur; Muratoglu, Orhun K


    Recent in vitro findings suggest that UHMWPE wear particles containing vitamin E (VE) may have reduced biologic activity and decreased osteolytic potential. We hypothesized that particles from VE-stabilized, radiation cross-linked UHMWPE would cause less osteolysis in a murine calvarial bone model when compared to virgin gamma irradiated cross-linked UHMWPE. Groups received equal amount of particulate debris overlaying the calvarium for 10 days. Calvarial bone was examined using high resolution micro-CT and histomorphometric analyses. There was a statistically significant difference between virgin (12.2%±8%) and VE-UHMWPE (3%±1.4%) groups in regards to bone resorption (P=0.005) and inflammatory fibrous tissue overlaying the calvaria (0.48 vs. 0.20, PUHMWPE particles have reduced osteolytic potential in vivo when compared to virgin UHMWPE.

  13. Protein structure prediction guided by cross-linking restraints - A systematic evaluation of the impact of the cross-linking spacer length

    Hofmann, Tommy; Meiler, Jens; Kalkhof, Stefan


    Recent development of high-resolution mass spectrometry (MS) instruments enables chemical cross-linking (XL) to become a high-throughput method for obtaining structural information about proteins. Restraints derived from XL-MS experiments have been used successfully for structure refinement and protein-protein docking. However, one formidable question is under which circumstances XL-MS data might be sufficient to determine a protein's tertiary structure de novo? Answering this question will not only include understanding the impact of XL-MS data on sampling and scoring within a de novo protein structure prediction algorithm, it must also determine an optimal cross-linker type and length for protein structure determination. While a longer cross-linker will yield more restraints, the value of each restraint for protein structure prediction decreases as the restraint is consistent with a larger conformational space. In this study, the number of cross-links and their discriminative power was systematically analyz...

  14. Vitamin E-diffused highly cross-linked UHMWPE particles induce less osteolysis compared to highly cross-linked virgin UHMWPE particles in vivo

    Bichara, David A; Malchau, Erik; Sillesen, Nanna H


    when compared to virgin gamma irradiated cross-linked UHMWPE. Groups received equal amount of particulate debris overlaying the calvarium for 10 days. Calvarial bone was examined using high resolution micro-CT and histomorphometric analyses. There was a statistically significant difference between...... virgin (12.2%±8%) and VE-UHMWPE (3%±1.4%) groups in regards to bone resorption (P=0.005) and inflammatory fibrous tissue overlaying the calvaria (0.48 vs. 0.20, P

  15. Preparation of cross-linked hen-egg white lysozyme crystals free of cracks

    Yan, Er-Kai; Lu, Qin-Qin; Zhang, Chen-Yan; Liu, Ya-Li; He, Jin; Chen, Da; Wang, Bo; Zhou, Ren-Bin; Wu, Ping; Yin, Da-Chuan


    Cross-linked protein crystals (CLPCs) are very useful materials in applications such as biosensors, catalysis, and X-ray crystallography. Hence, preparation of CLPCs is an important research direction. During the preparation of CLPCs, an often encountered problem is that cracks may appear in the crystals, which may finally lead to shattering of the crystals into small pieces and cause problem in practical applications. To avoid cross-link induced cracking, it is necessary to study the cracking phenomenon in the preparation process. In this paper, we present an investigation on how to avoid cracking during preparation of CLPCs. An orthogonal experiment was designed to study the phenomenon of cross-link induced cracking of hen-egg white lysozyme (HEWL) crystals against five parameters (temperature, solution pH, crystal growth time, glutaraldehyde concentration, and cross-linking time). The experimental results showed that, the solution pH and crystal growth time can significantly affect cross-link induced cracking. The possible mechanism was studied, and optimized conditions for obtaining crack-free CLPCs were obtained and experimentally verified. PMID:27703210

  16. Supercritical CO2 Foaming of Radiation Cross-Linked Isotactic Polypropylene in the Presence of TAIC

    Chen-Guang Yang


    Full Text Available Since the maximum foaming temperature window is only about 4 °C for supercritical CO2 (scCO2 foaming of pristine polypropylene, it is important to raise the melt strength of polypropylene in order to more easily achieve scCO2 foaming. In this work, radiation cross-linked isotactic polypropylene, assisted by the addition of a polyfunctional monomer (triallylisocyanurate, TAIC, was employed in the scCO2 foaming process in order to understand the benefits of radiation cross-linking. Due to significantly enhanced melt strength and the decreased degree of crystallinity caused by cross-linking, the scCO2 foaming behavior of polypropylene was dramatically changed. The cell size distribution, cell diameter, cell density, volume expansion ratio, and foaming rate of radiation-cross-linked polypropylene under different foaming conditions were analyzed and compared. It was found that radiation cross-linking favors the foamability and formation of well-defined cell structures. The optimal absorbed dose with the addition of 2 wt % TAIC was 30 kGy. Additionally, the foaming temperature window was expanded to about 8 °C, making the handling of scCO2 foaming of isotactic polypropylene much easier.

  17. Incorporation of Decanethiol-Passivated Gold Nanoparticles into Cross-Linked Poly(Dimethylsiloxane Films

    Motohiro Tagaya


    Full Text Available Cross-linking degree of a poly(dimethylsiloxane (PDMS film was controlled, and the incorporation of hydrophobic decanethiol-passivated gold (Au nanoparticles into the film was investigated. FT-IR spectra indicated that the hydrosilylation reaction between a vinyl group and a hydrosilyl group occurred with the cross-linking. The swelling degree of the film in toluene changed with a cross-linker concentration, indicating the control of the cross-linking degree of PDMS film. By EDX analysis, the amount of incorporated Au nanoparticles increased with decreasing a cross-linker concentration, indicating the enlarged free volume of the film. The Au nanoparticle-PDMS composite film containing a cross-linker at 6 wt% showed brown color attributed to plasmon resonance of Au nanoparticles, suggesting the Au nanoparticles in the film at monodispersion state. The UV-visible absorbance of the composite film decreased without spectralshift by swelling with toluene, and the changes were reversible. The aggregation among Au nanoparticles in the composite film after calcination also depended on the cross-linking degree. Thus, the control of cross-linking degree of PDMS film successfully leaded to a simple way of fabricating the Au nanoparticle-PDMS composite film at the mono-dispersion state.

  18. Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking.

    van Montfort, Bart A; Schuurman-Wolters, Gea K; Wind, Joyce; Broos, Jaap; Robillard, George T; Poolman, Bert


    A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.

  19. Effects of partial hydrolysis and subsequent cross-linking on wheat gluten physicochemical properties and structure.

    Wang, Kaiqiang; Luo, Shuizhong; Cai, Jing; Sun, Qiaoqiao; Zhao, Yanyan; Zhong, Xiyang; Jiang, Shaotong; Zheng, Zhi


    The rheological behavior and thermal properties of wheat gluten following partial hydrolysis using Alcalase and subsequent microbial transglutaminase (MTGase) cross-linking were investigated. The wheat gluten storage modulus (G') and thermal denaturation temperature (Tg) were significantly increased from 2.26 kPa and 54.43°C to 7.76 kPa and 57.69°C, respectively, by the combined action of partial hydrolysis (DH 0.187%) and cross-linking. The free SH content, surface hydrophobicity, and secondary structure analysis suggested that an appropriate degree of Alcalase-based hydrolysis allowed the compact wheat gluten structure to unfold, increasing the β-sheet content and surface hydrophobicity. This improved its molecular flexibility and exposed additional glutamine sites for MTGase cross-linking. SEM images showed that a compact 3D network formed, while SDS-PAGE profiles revealed that excessive hydrolysis resulted in high-molecular-weight subunits degrading to smaller peptides, unsuitable for cross-linking. It was also demonstrated that the combination of Alcalase-based partial hydrolysis with MTGase cross-linking might be an effective method for modifying wheat gluten rheological behavior and thermal properties.

  20. A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

    Chavez, Juan D.; Eng, Jimmy K.; Schweppe, Devin K.; Cilia, Michelle; Rivera, Keith; Zhong, Xuefei; Wu, Xia; Allen, Terrence; Khurgel, Moshe; Kumar, Akhilesh; Lampropoulos, Athanasios; Larsson, Mårten; Maity, Shuvadeep; Morozov, Yaroslav; Pathmasiri, Wimal; Perez-Neut, Mathew; Pineyro-Ruiz, Coriness; Polina, Elizabeth; Post, Stephanie; Rider, Mark; Tokmina-Roszyk, Dorota; Tyson, Katherine; Vieira Parrine Sant'Ana, Debora; Bruce, James E.


    Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions. PMID:27997545

  1. Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

    Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo


    An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration.

  2. Synthesis, Characterization, and Antibacterial Activity of Cross-Linked Chitosan-Glutaraldehyde

    Long-Biao Guo


    Full Text Available This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR band at 1559 cm−1, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug.

  3. Ice templated and cross-linked xylan/nanocrystalline cellulose hydrogels.

    Köhnke, Tobias; Elder, Thomas; Theliander, Hans; Ragauskas, Arthur J


    Structured xylan-based hydrogels, reinforced with cellulose nanocrystals (CNCs), have successfully been prepared from water suspensions by cross-linking during freeze-casting. In order to induce cross-linking during the solidification/sublimation operation, xylan was first oxidized using sodium periodate to introduce dialdehydes. The oxidized xylan was then mixed with CNCs after which the suspension was frozen unidirectionally in order to control the ice crystal formation and by that the pore morphology of the material. Finally the ice crystal templates were removed by freeze-drying. During the freeze-casting process hemiacetal bonds are formed between the aldehyde groups and hydroxyl groups, either on other xylan molecules or on CNCs, which cross-links the system. The proposed cross-linking reaction was confirmed by using cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy. The pore morphology of the obtained materials was analyzed by scanning electron microscopy (SEM). The materials were also tested for compressive strength properties, both in dry and water swollen state. All together this study describes a novel combined freeze-casting/cross-linking process which enables fabrication of nanoreinforced biopolymer-based hydrogels with controlled porosity and 3-D architecture.

  4. Cross-Linking Poly(lactic acid) Film Surface by Neutral Hyperthermal Hydrogen Molecule Bombardment.

    Du, Wangli; Shao, Hong; He, Zhoukun; Tang, Changyu; Liu, Yu; Shen, Tao; Zhu, Yan; Lau, Woon-ming; Hui, David


    Constructing a dense cross-linking layer on a polymer film surface is a good way to improve the water resistance of poly(lactic acid) (PLA). However, conventional plasma treatments have failed to achieve the aim as a result of the unavoidable surface damage arising from the charged species caused by the uncontrolled high energy coming from colliding ions and electrons. In this work, we report a modified plasma method called hyperthermal hydrogen-induced cross-linking (HHIC) technology to construct a dense cross-linking layer on PLA film surfaces. This method produces energy-controlled neutral hyperthermal hydrogen, which selectively cleaves C-H bonds by molecule collision from the PLA film without breaking other bonds (e.g., C-C bonds in the polymer backbone), and results in subsequent cross-linking of the carbon radicals generated from the organic molecules. The formation of a dense cross-linking layer can serve as a barrier layer to significantly improve both the hydrophobicity and water vapor barrier property of the PLA film. Because of the advantage of selective cleavage of C-H bonds by HHIC treatment, the original physical properties (e.g., mechanical strength and light transmittance) of the PLA films are well-preserved.

  5. Studies on Cross-linking of succinic acid with chitosan/collagen

    Tapas Mitra


    Full Text Available The present study summarizes the cross-linking property of succinic acid with chitosan /collagen. In detail, the chemistry behind the cross-linking and the improvement in mechanical and thermal properties of the cross-linked material were discussed with suitable instruments and bioinformatics tools. The concentration of succinic acid with reference to the chosen polymers was optimized. A 3D scaffold prepared using an optimized concentration of succinic acid (0.2% (w/v with chitosan (1.0% (w/v and similarly with collagen (0.5% (w/v, was subjected to surface morphology, FT-IR analysis, tensile strength assessment, thermal stability and biocompatibility. Results revealed, cross-linking with succinic acid impart appreciable mechanical strength to the scaffold material. In silico analysis suggested the prevalence of non-covalent interactions, which played a crucial role in improving the mechanical and thermal properties of the cross-linked scaffold. The resultant 3D scaffold may find application as wound dressing material, as an implant in clinical applications and as a tissue engineering material.

  6. Mechanical and biocompatible characterization of a cross-linked collagen-hyaluronic acid wound dressing.

    Kirk, James F; Ritter, Gregg; Finger, Isaac; Sankar, Dhyana; Reddy, Joseph D; Talton, James D; Nataraj, Chandra; Narisawa, Sonoko; Millán, José Luis; Cobb, Ronald R


    Collagen scaffolds have been widely employed as a dermal equivalent to induce fibroblast infiltrations and dermal regeneration in the treatment of chronic wounds and diabetic foot ulcers. Cross-linking methods have been developed to address the disadvantages of the rapid degradation associated with collagen-based scaffolds. To eliminate the potential drawbacks associated with glutaraldehyde cross-linking, methods using a water soluble carbodiimide have been developed. In the present study, the glycosaminoglycan (GAG) hyaluronic acid (HA), was covalently attached to an equine tendon derived collagen scaffold using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to create ntSPONGE The HA was shown to be homogeneously distributed throughout the collagen matrix. In vitro analyses of the scaffold indicated that the cross-linking enhanced the biological stability by decreasing the enzymatic degradation and increasing the thermal denaturation temperature. The material was shown to support the attachment and proliferation of mouse L929 fibroblast cells. In addition, the cross-linking decreased the resorption rate of the collagen as measured in an intramuscular implant model in rabbits. The material was also shown to be biocompatible in a variety of in vitro and in vivo assays. These results indicate that this cross-linked collagen-HA scaffold, ntSPONGE has the potential for use in chronic wound healing.

  7. Novel Technique of Transepithelial Corneal Cross-Linking Using Iontophoresis in Progressive Keratoconus

    Raffa, Paolo; Rosati, Marianna


    In this work, the authors presented the techniques and the preliminary results at 6 months of a randomized controlled trial (NCT02117999) comparing a novel transepithelial corneal cross-linking protocol using iontophoresis with the Dresden protocol for the treatment of progressive keratoconus. At 6 months, there was a significant average improvement with an average flattening of the maximum simulated keratometry reading of 0.72 ± 1.20 D (P = 0.01); in addition, corrected distance visual acuity improved significantly (P = 0.08) and spherical equivalent refraction was significantly less myopic (P = 0.02) 6 months after transepithelial corneal cross-linking with iontophoresis. The novel protocol using iontophoresis showed comparable results with standard corneal cross-linking to halt progression of keratoconus during 6-month follow-up. Investigation of the long-term RCT outcomes are ongoing to verify the efficacy of this transepithelial corneal cross-linking protocol and to determine if it may be comparable with standard corneal cross-linking in the management of progressive keratoconus. PMID:27597895

  8. Novel Technique of Transepithelial Corneal Cross-Linking Using Iontophoresis in Progressive Keratoconus

    Marco Lombardo


    Full Text Available In this work, the authors presented the techniques and the preliminary results at 6 months of a randomized controlled trial (NCT02117999 comparing a novel transepithelial corneal cross-linking protocol using iontophoresis with the Dresden protocol for the treatment of progressive keratoconus. At 6 months, there was a significant average improvement with an average flattening of the maximum simulated keratometry reading of 0.72±1.20 D (P=0.01; in addition, corrected distance visual acuity improved significantly (P=0.08 and spherical equivalent refraction was significantly less myopic (P=0.02 6 months after transepithelial corneal cross-linking with iontophoresis. The novel protocol using iontophoresis showed comparable results with standard corneal cross-linking to halt progression of keratoconus during 6-month follow-up. Investigation of the long-term RCT outcomes are ongoing to verify the efficacy of this transepithelial corneal cross-linking protocol and to determine if it may be comparable with standard corneal cross-linking in the management of progressive keratoconus.

  9. Cross-linking study on skeletal muscle actin: properties of suberimidate-treated actin.

    Ohara, O; Takahashi, S; Ooi, T; Fujiyoshi, Y


    Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular cross-links in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.

  10. Synthesis of cross-linked magnetic composite microspheres containing carboxyl groups

    Jili ZHAO; Zhaorang HAN; Qiang SONG; Ying WANG; Dan SUN


    Fe3O4 magnetic nano-particles were prepared by a co-precipitation method and were modified using oleic acid. Then, the cross-linked magnetic composite microspheres containing a carboxyl group were prepared by using an improved emulsion polymerization with divinylbenzene (DVB) as the cross-linking agent. The composite microspheres comprised the Fe3O4 magnetic nano-partictes as cores and the copolymer of styrene and acrylic acid as shells. The morphology and structure of the composite microsphere were characterized by FT-IR, transmission electron microscopy (TEM), X-ray diffrac-tion (XRD), X-ray photoelectron spectrum (XPS) and so on. The results show that the composite microspheres were well dispersed in emulsion with uniform sizes and carboxyl groups on their surface. They were cross-linked and stable in 1 mol/L of HCl and DMF.

  11. Photochromic cross-link polymer for color changing and sensing surface

    Fu, Richard; Shi, Jianmin; Forsythe, Eric; Srour, Merric


    Photochromic cross-link polymers were developed using patented ultraviolet (UV) photoinitiator and commercial photochromic dyes. The photochromic dyes have been characterized by measuring absorbance before and after UV activation using UV-visible (Vis) spectrometry with varying activation intensities and wavelengths. Photochromic cross-link polymers were characterized by a dynamic xenon and UV light activation and fading system. The curing processes on cloth were established and tested to obtain effective photochromic responses. Both PulseForge photonic curing and PulseForge plus heat surface curing processes had much better photochromic responses (18% to 19%, 16% to 25%, respectively) than the xenon lamp treatment (8%). The newly developed photochromic cross-link polymer showed remarkable coloration contrasts and fast and comparable coloration and fading rates. Those intelligent, controlled color changing and sensing capabilities will be used on flexible and "drapeable" surfaces, which will incorporate ultra-low power sensors, sensor indicators, and identifiers.

  12. Optimizing end-group cross-linking polymer electrolytes for fuel cell applications

    Kim, Yu Seung [Los Alamos National Laboratory; Lee, Kwan Soo [Los Alamos National Laboratory; Jeong, Myung - Hwan [GIST, KOREA; Lee, Jae - Suk [GIST, KOREA


    This paper demonstrates the optimization of proton conductivity and water uptake for cross-linkable polymer electrolytes through synthesis and characterization of end-group cross-linkable sulfonated poly(arylene ether) copolymers (ESF-BPs). The extent of reaction of cross-linking was controlled by reaction time resulting in a series of polymers with two, independent tunable parameters, degree of sulfonation (DS) and degree of cross-linking (DC). For the polymers presented, cross-linking improved proton conductivity while reducing water uptake, an uncommon trend in polymer electrolytes where water is critical for proton conduction. Other trends relating to changes are reported and the results yield insight into the role of DS and DC and how to optimize electrochemical properties and performance of polymer electrolytes through these tunable parameters. Select polymer electrolytes were tested in fuel cells where performance and durability with accelerated relative humidity cycling were compared with Nafion{reg_sign}.

  13. Cross-linked aromatic cationic polymer electrolytes with enhanced stability for high temperature fuel cell applications

    Ma, Wenjia; Zhao, Chengji; Yang, Jingshuai


    Diamine-cross-linked membranes were prepared from cross-linkable poly(arylene ether ketone) containing pendant cationic quaternary ammonium group (QPAEK) solution by a facile and general thermal curing method using 4,4′-diaminodiphenylmethane with rigid framework and 1,6-diaminohexane with flexible...... framework as cross-linker, respectively. Self-cross-linked cationic polymer electrolytes membranes were also prepared for comparison. The diamines were advantageously distributed within the polymeric matrix and its amine function groups interacted with the benzyl bromide of QPAEK, resulting in a double...... that the diamine-cross-linked membranes using the rigid cross-linker show much improved properties than that using the flexible cross-linker. More properties relating to the feasibility in high temperature proton exchange membrane fuel cell applications were investigated in detail....

  14. Genipin Cross-Linked Polymeric Alginate-Chitosan Microcapsules for Oral Delivery: In-Vitro Analysis

    Hongmei Chen


    Full Text Available We have previously reported the preparation of the genipin cross-linked alginate-chitosan (GCAC microcapsules composed of an alginate core with a genipin cross-linked chitosan membrane. This paper is the further investigation on their structural and physical characteristics. Results showed that the GCAC microcapsules had a smooth and dense surface and a networked interior. Cross-linking by genipin substantially reduced swelling and physical disintegration of microcapsules induced by nongelling ions and calcium sequestrants. Strong resistance to mechanical shear forces and enzymatic degradation was observed. Furthermore, the GCAC membranes were permeable to bovine serum albumin and maintained a molecular weight cutoff at 70 KD, analogous to the widely studied alginate-chitosan, and alginate-poly-L-lysine-alginate microcapsules. The release features and the tolerance of the GCAC microcapsules in the stimulated gastrointestinal environment were also investigated. This GCAC microcapsule formulation offers significant potential as a delivery vehicle for many biomedical applications.

  15. Cross-linked PAN-based thin-film composite membranes for non-aqueous nanofiltration

    Pérez-Manríquez, Liliana


    A new approach on the development of cross-linked PAN based thin film composite (TFC) membranes for non-aqueous application is presented in this work. Polypropylene backed neat PAN membranes fabricated by phase inversion process were cross-linked with hydrazine to get excellent solvent stability toward dimethylformamide (DMF). By interfacial polymerization a selective polyamide active layer was coated over the cross-linked PAN using N,N′-diamino piperazine (DAP) and trimesoyl chloride (TMC) as monomers. Permeation and molecular weight cut off (MWCO) experiments using various dyes were done to evaluate the performance of the membranes. Membranes developed by such method show excellent solvent stability toward DMF with a permeance of 1.7 L/m2 h bar and a molecular weight cut-off of less than 600 Da.

  16. Light-triggered cross-linking of alginates with caged Ca2+.

    Cui, Jiaxi; Wang, Miao; Zheng, Yijun; Rodríguez Muñiz, Gemma Maria; del Campo, Aránzazu


    A strategy to light-trigger ionic cross-linking of alginates by incorporating a photosensitive Ca2+ cage (nitr-T) is presented. Upon irradiation, free Ca2+ was released, and this caused gelation of the alginate solution. Addition of Ca2+ "on-demand" allowed us to obtain homogeneous alginate (ALG) gels using concentrated initial ALG solutions (10%), not possible with other ionic gelation approaches. The cross-linking degree and derived mechanical properties of the hydrogel were modulated by the exposure dose. The light-mediated cross-linked alginate hydrogel displayed a significant improvement in the mechanical properties and homogeneity when compared to mixtures of alginate and soluble Ca2+ at comparable concentrations.

  17. Synthesis of acrylic and allylic bifunctional cross-linking monomers derived from PET waste

    Cruz-Aguilar, A.; Herrera-González, A. M.; Vázquez-García, R. A.; Navarro-Rodríguez, D.; Coreño, J.


    An acrylic and two novel allylic monomers synthesized from bis (hydroxyethyl) terephthalate, BHET, are reported. This was obtained by glycolysis of post-consumer PET with boiling ethylene glycol. The bifunctional monomer bis(2-(acryloyloxy)ethyl) terephthalate was obtained from acryloyl chloride, while the allylic monomers 2-(((allyloxi)carbonyl)oxy) ethyl (2-hydroxyethyl) terephthalate and bis(2-(((allyloxi)carbonyl)oxy)ethyl) terephthalate, from allyl chloroformate. Cross-linking was studied in bulk polymerization using two different thermal initiators. Monomers were analyzed by means of 1H NMR and the cross-linked polymers by infrared spectroscopy. Gel content higher than 90% was obtained for the acrylic monomer. In the case of the mixture of the allylic monomers, the cross-linked polymer was 80 % using BPO initiator, being this mixture 24 times less reactive than the acrylic monomer.

  18. Effect of cross-linking degree on selected properties of retrograded starch adipate.

    Kapelko, M; Zięba, T; Michalski, A; Gryszkin, A


    The aim of this study was to determine the effects of the concentration of paste used to produce retrograded starch, and esterification degree, on selected properties of the resultant distarch adipate. Starch paste was prepared from native potato starch (1, 4, 10, 18 or 30 g/100g), frozen, defrosted and dried. Thus produced preparations of retrograded starch were cross-linked with various doses of a cross-linking agent (0.125, 0.25, 0.5, 1.0 or 2.0 ml per 100g of starch). Properties of the produced adipates depended on both the concentration of paste used to produce retrograded starch and the degree of substitution with adipic acid residues. Solubility in water and swelling power of the cross-linked preparations of retrograded starch, as well as pasting temperature and viscosity of produced pastes, all decreased along with the increasing degree of substitution with adipic acid residues.

  19. Cheese whey protein recovery by ultrafiltration through transglutaminase (TG) catalysis whey protein cross-linking.

    Wen-Qiong, Wang; Lan-Wei, Zhang; Xue, Han; Yi, Lu


    In whey ultrafiltration (UF) production, two main problems are whey protein recovery and membrane fouling. In this study, membrane coupling protein transglutaminase (TG) catalysis protein cross-linking was investigated under different conditions to find out the best treatment. We found that the optimal conditions for protein recovery involved catalyzing whey protein cross-linking with TG (40U/g whey proteins) at 40°C for 60min at pH 5.0. Under these conditions, the recovery rate was increased 15-20%, lactose rejection rate was decreased by 10%, and relative permeate flux was increase 30-40% compared to the sample without enzyme treatment (control). It was noticeable that the total resistance and cake resistance were decreased after enzyme catalysis. This was mainly due to the increased particle size and decreased zeta potential. Therefore, membrane coupling enzyme catalysis protein cross-linking is a potential means for further use.

  20. Solution processed organic light-emitting diodes using the plasma cross-linking technology

    He, Kongduo; Liu, Yang; Gong, Junyi; Zeng, Pan; Kong, Xun; Yang, Xilu; Yang, Cheng; Yu, Yan; Liang, Rongqing; Ou, Qiongrong


    Solution processed multilayer organic light-emitting diodes (OLEDs) present challenges, especially regarding dissolution of the first layer during deposition of a second layer. In this work, we first demonstrated a plasma cross-linking technology to produce a solution processed OLED. The surfaces of organic films can be cross-linked after mixed acetylene and Ar plasma treatment for several tens of seconds and resist corrosion of organic solvent. The film thickness and surface morphology of emissive layers (EMLs) with plasma treatment and subsequently spin-rinsed with chlorobenzene are nearly unchanged. The solution processed triple-layer OLED is successfully fabricated and the current efficiency increases 50% than that of the double-layer OLED. Fluorescent characteristics of EMLs are also observed to investigate factors influencing the efficiency of the triple-layer OLED. Plasma cross-linking technology may open up a new pathway towards fabrication of all-solution processed multilayer OLEDs and other soft electronic devices.

  1. Improvement on Physical Properties of Pullulan Films by Novel Cross-Linking Strategy.

    Chen, Chieh-Ting; Chen, Kuan-I; Chiang, Hsin-Han; Chen, Yu-Kuo; Cheng, Kuan-Chen


    Pullulan based films possess several advantages, including high transparency, low toxicity, good biodegradability, good mechanical properties, and low oxygen permeability, are preferable for food packaging. The application of pullulan films on food packaging, however, has inherent disadvantage of high water solubility. In this study, glutaraldehyde and glycerol were used as the cross-linking reagent and the plasticizer respectively to improve water resistance and physical properties of the pullulan films. Effects of cross-linking degree on physical properties, including water absorptions, swelling behaviors, water vapor permeability and tensile strengths of films were evaluated. FTIR results demonstrated that the pullulan films were successfully cross-linked by glutaraldehyde. The tensile strength of pullulan films could be enhanced significantly (P glycerol as a plasticizer enhanced the extensibility of films as well as the hydrophilicity, resulting in higher water vapor permeability.

  2. Study on Chemical Cross-linking Modification of Hyaluronan and the Biocompatibility of its Derivatives

    HU Guo-ying; LIU Xin; GU Han-qing


    Objective: Prepare cross-linked HA gels with higher mechanical stability,lower degradation velocity and desirable biocompatibility,so as to extend the usage of HA.Method: 1.Test molecular weight of HA (MrHA) by viscosimetry;2.Prepare cross-linked HA gels by DVS,GTA,DEC;3.Discuss the cross-linking and degradation procedure;4,evaluate the biocompatibility of the best HA gels.Results: The mechanical stability and durability to degradation of HA-DVS gels are superior to those of other gels,and when HA :DVS = 40:1 (g/g),at 35℃ and in 0.2M NaOH solution,the HA-DVS gel shows the best mechanical stability,and its cytotoxicity reaches class I,hemolysis ratio is lower than 5 %.Conclusion:Our HADVS gel can be used to prepare biologic scaffolds.

  3. Comparison of Wear and Oxidation in Retrieved Conventional and Highly Cross-Linked UHMWPE Tibial Inserts.

    Currier, Barbara H; Currier, John H; Franklin, Katherine J; Mayor, Michael B; Reinitz, Steven D; Van Citters, Douglas W


    Two groups of retrieved tibial inserts from one manufacturer's knee system were analyzed to evaluate the effect of a highly cross-linked bearing surface on wear and in vivo oxidation. The two groups ((1) conventional gamma-inert sterilized and (2) highly cross-linked, coupled with the same rough (Ra=0.25) Ti-6Al-4V tray) were matched with statistically similar in vivo duration and patient variables. The retrieved inserts were analyzed for ketone oxidation and wear in the form of dimensional change. The difference in oxidation rate between highly cross-linked and conventional gamma-inert sterilized inserts did not reach statistical significance. Observations suggest that the majority of wear can be accounted for by the backside interface with the rough Ti-6Al-4V tray; however, wear measured by thickness-change rate was statistically indistinguishable between the two bearing materials.

  4. Designing of superporous cross-linked hydrogels containing acrylic-based polymer network

    Ray Debajyoti


    Full Text Available Biodegradable cross-linked polymer, 2-hydroxyethyl methacrylate-co-acrylic acid was synthesized by free radical polymerization technique using N,N"-methylene-bis-acrylamide as cross-linker and benzoyl peroxide as reaction initiator. FT-IR, 1 H-NMR, scanning electron microscopy (SEM, and thermogravimetric analysis (TGA studies of the copolymer along with homopolymers were carried out. FT-IR studies showed no interactions on copolymerization. SEM studies of the copolymer were carried out and mean particle size was found to be 50 µm. TGA analysis indicated an increase in thermal stability by cross-linking the polymer network. Swelling behavior of the copolymer showed more swelling by increasing pH of the medum and the prepared polymer was found to be biodegradable. The prepared cross-linked polymer system holds good for further drug delivery studies in connection to its super swelling and biodegradability.

  5. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering

    Mathapati, Santosh; Bishi, Dillip Kumar [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India); Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Guhathakurta, Soma [Departmet of Engineering Design, Indian Institute of Technology Madras, Chennai (India); Cherian, Kotturathu Mammen [Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Venugopal, Jayarama Reddy; Ramakrishna, Seeram [Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Verma, Rama Shanker, E-mail: [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India)


    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p > 0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP–G–CA–ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP–G–CA–ET). DBP–G–CA–ET exhibited a significant (p > 0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p < 0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. - Highlights: ► We improved the quality of patch biomaterial for cardiovascular surgical procedures. ► Bovine pericardium was

  6. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase.

    Truong, Van-Den; Clare, Debra A; Catignani, George L; Swaisgood, Harold E


    Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.

  7. Solvent Composition is Critical for Carbodiimide Cross-Linking of Hyaluronic Acid as an Ophthalmic Biomaterial

    Jui-Yang Lai


    Full Text Available Hyaluronic acid (HA is one of the most important ophthalmic biomaterials, while also being used for tissue engineering and drug delivery. Although chemical cross-linking is an effective way to improve the material performance, it may as a consequence be detrimental to the living cells/tissues. Given that the cross-linking efficiency is mediated by the solvent composition during the chemical modification, this study aims to explore the stability and biocompatibility of carbodiimide cross-linked HA in relation to material processing conditions by varying the acetone/water volume ratio (from 70:30 to 95:5 at a constant 1-ethyl-3-(3-dimethyl aminopropyl carbodiimide (EDC concentration of 100 mM. Our results indicated that after the EDC treatment in the presence of an acetone/water mixture (85:15, v/v, the HA hydrogel membranes have the lowest equilibrium water content, the highest stress at break and the greatest resistance to hyaluronidase digestion. Live/Dead assays and pro-inflammatory cytokine expression analyses showed that the cross-linked HA hydrogel membranes, irrespective of the solvent composition, are compatible with human RPE cell lines without causing toxicity and inflammation. However, it should be noted that the test samples prepared by the cross-linking in the presence of acetone/water mixtures containing 70, 75, and 95 vol % of acetone slightly inhibit the metabolic activity of viable ARPE-19 cultures, probably due to the alteration in the ionic interaction between the medium nutrients and polysaccharide biomaterials. In summary, the water content, mechanical strength and RPE cell proliferative capacity strongly depends on the solvent composition for carbodiimide cross-linking of HA materials.

  8. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

    Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.; Briggaman, R. A.


    Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data

  9. 1,3-Diphenylethenylcarbazolyl-Based Monomer for Cross-Linked Hole Transporting Layers

    Maryte Daskeviciene


    Full Text Available A new cross-linkable monomer containing 1,3-diphenylethenylcarbazolyl-based hole-transporting moieties and four reactive epoxy groups, was prepared by a multistep synthesis route from 1,3-bis(2,2-diphenylethenyl-9H-carbazol-2-ol and its application for the in situ formation of cross-linked hole transporting layers was investigated. A high concentration of flexible aliphatic epoxy chains ensures good solubility and makes this compound an attractive cross-linking agent. The synthesized compounds were characterized by various techniques, including differential scanning calorimetry, xerographic time of flight, and electron photoemission in air methods.

  10. Dynamically vulcanized biobased polylactide/natural rubber blend material with continuous cross-linked rubber phase.

    Chen, Yukun; Yuan, Daosheng; Xu, Chuanhui


    We prepared a biobased material, dynamically vulcanized polylactide (PLA)/natural rubber (NR) blend in which the cross-linked NR phase owned a continuous network-like dispersion. This finding breaks the traditional concept of a sea-island morphology formed after dynamic vulcanization of the blends. The scan electron microscopy and dissolution/swell experiments provided the direct proof of the continuous cross-linked NR phase. This new biobased PLA/NR blend material with the novel structure is reported for the first time in the field of dynamic vulcanization and shows promise for development for various functional applications.

  11. Increasing Thermal Stability of Gelatin by UV-Induced Cross-Linking with Glucose

    Evan M. Masutani


    Full Text Available The effects of ultraviolet (254 nm radiation on a hydrated gelatin-glucose matrix were investigated for the development of a physiologically thermostable substrate for potential use in cell scaffold production. Experiments conducted with a differential scanning calorimeter indicate that ultraviolet irradiation of gelatin-glucose hydrogels dramatically increases thermal stability such that no melting is observed at temperatures of at least 90°C. The addition of glucose significantly increases the yield of cross-linked product, suggesting that glucose has a role in cross-link formation. Comparisons of lyophilized samples using scanning electron microscopy show that irradiated materials have visibly different densities.

  12. A novel DTPA cross-linking of hyaluronic acid and metal complexation thereof.

    Buffa, Radovan; Běťák, Jiří; Kettou, Sofiane; Hermannová, Martina; Pospíšilová, Lucie; Velebný, Vladimír


    Macromolecular conjugates of a natural polysaccharide, hyaluronic acid, with diethylenetriaminepentaacetic acid (DTPA)-metal complexes were synthesized and characterized by FTIR, NMR, SEC-MALLS and ICP analysis. Several parameters of the cross-linking reaction as molecular weight of starting HA, temperature, equivalent of DTPA bis-anhydride, concentration of HA, presence of transacylation catalyst DMAP and reaction time were studied. The mechanism for the reaction was suggested and relationship between the molecular weight assigned by SEC-MALLS, reaction parameters and rheological properties of the final cross-linked products were investigated. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Preparation and in vitro evaluation of chitosan matrices cross-linked by formaldehyde vapors.

    Rao, B S; Murthy, K V


    Rifampicin-chitosan matrices were prepared by a chemical cross-linking method to develop a sustained-release form. The effects of cross-linking agent (formaldehyde) on the drug release rate and release kinetics were investigated in this study. Moreover, the kinetics of rifampicin released from chitosan matrices exposed to formaldehyde vapors for predetermined time intervals were analyzed using Ritger and Peppas exponential equation. The in vitro release kinetics exhibited a non-Fickian transport model. Increasing the exposure time to formaldehyde vapors decreased the release rate of rifampicin from chitosan matrices as a result of formation of greater structural strength and tighter texture.

  14. Reinforced films based on cross-linked water-soluble sulfonated carbon nanotubes with sulfonated polystyrene.

    Dai, Ying; Haiping, Hong; Guiver, Michael; Welsh, Jeffry S


    Reinforced films based on sulfonated polystyrene cross-linked with water-soluble sulfonated carbon nanotubes were fabricated using a free-standing film-making method. Transmission and scanning electron microscopy (TEM and SEM), and X-ray photoelectron spectroscopy (XPS) were used to verify the cross-linking reaction. The mechanical properties of these films demonstrated that the tensile strength increases with an increase in the sulfonated nanotube concentration. At 5 wt% nanotube loading, the tensile strength increased 84% compared with polymer containing no nanotube loading. The relationships between structure and mechanical properties are discussed and a possible direction for making ultra thin and ultra lightweight film is proposed.

  15. Kinetics of enzyme-catalyzed cross-linking of feruloylated arabinan from sugar beet

    Abang Zaidel, Dayang Norulfairuz; Arnous, Anis; Holck, Jesper


    the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular...... weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased...

  16. In Situ Cross-Linking of Stimuli-Responsive Hemicellulose Microgels during Spray Drying

    Zhao, Weifeng; Nugroho, Robertus Wahyu N.; Odelius, Karin; Edlund, Ulrica; Zhao, Changsheng; Albertsson, Ann-Christine


    Chemical cross-linking during spray drying offers the potential for green fabrication of microgels with a rapid stimuli response and good blood compatibility and provides a platform for stimuli-responsive hemicellulose microgels (SRHMGs). The cross-linking reaction occurs rapidly in situ at elevated temperature during spray drying, enabling the production of microgels in a large scale within a few minutes. The SRHMGs with an average size range of ∼1–4 μm contain O-acetyl-galactoglucomannan as...

  17. Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids▿

    Dierkes, Lindsay E.; Peebles, Craig L.; Firek, Brian A.; Hendrix, Roger W.; Duda, Robert L.


    The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution. PMID:19091865

  18. Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.

    Petrotchenko, Evgeniy V; Serpa, Jason J; Hardie, Darryl B; Berjanskii, Mark; Suriyamongkol, Bow P; Wishart, David S; Borchers, Christoph H


    Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including

  19. Induction of DNA–protein cross-links by ionizing radiation and their elimination from the genome

    Nakano, Toshiaki; Mitsusada, Yusuke [Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526 (Japan); Salem, Amir M.H. [Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526 (Japan); Department of Pathology, Medical Research Division, National Research Centre, El-Bohouth St., Dokki, Giza 12311 (Egypt); Shoulkamy, Mahmoud I. [Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526 (Japan); Department of Zoology, Biological Science Building, Faculty of Science, Minia University, Minia 61519 (Egypt); Sugimoto, Tatsuya [Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526 (Japan); Hirayama, Ryoichi; Uzawa, Akiko [Research Center for Charged Particle Therapy, National Institute of Radiological Sciences (NIRS), Chiba 263-8555 (Japan); Furusawa, Yoshiya [Development and Support Center, National Institute of Radiological Sciences (NIRS), Chiba 263-8555 (Japan); Ide, Hiroshi, E-mail: [Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526 (Japan)


    Highlights: • Normoxic and hypoxic mouse tumors were irradiated with X-rays and C-ion beams. • DNA–protein cross-links (DPCs) and DNA double-strand breaks (DSBs) were analyzed. • C-ion beams produced more DPCs than did X-rays in normoxic and hypoxic tumor cells. • DPCs were eliminated from the genome much more slowly than DSBs. • Persisting DPCs may have deleterious effects on cells in conjunction with DSBs. - Abstract: Ionizing radiation produces various types of DNA lesions, such as base damage, single-strand breaks, double-strand breaks (DSBs), and DNA–protein cross-links (DPCs). Of these, DSBs are the most critical lesions underlying the lethal effects of ionizing radiation. With DPCs, proteins covalently trapped in DNA constitute strong roadblocks to replication and transcription machineries, and hence can be lethal to cells. The formation of DPCs by ionizing radiation is promoted in the absence of oxygen, whereas that of DSBs is retarded. Accordingly, the contribution of DPCs to the lethal events in irradiated cells may not be negligible for hypoxic cells, such as those present in tumors. However, the role of DPCs in the lethal effects of ionizing radiation remains largely equivocal. In the present study, normoxic and hypoxic mouse tumors were irradiated with X-rays [low linear energy transfer (LET) radiation] and carbon (C)-ion beams (high LET radiation), and the resulting induction of DPCs and DSBs and their removal from the genome were analyzed. X-rays and C-ion beams produced more DPCs in hypoxic tumors than in normoxic tumors. Interestingly, the yield of DPCs was slightly but statistically significantly greater (1.3- to 1.5-fold) for C-ion beams than for X-rays. Both X-rays and C-ion beams generated two types of DPC that differed according to their rate of removal from the genome. This was also the case for DSBs. The half-lives of the rapidly removed components of DPCs and DSBs were similar (<1 h), but those of the slowly removed components

  20. The Effect of Glutaraldehyde Cross-Linking on the Enzyme Activity of Immobilized &beta-Galactosidase on Chitosan Bead

    He Chen


    Full Text Available The effect of glutaraldehyde solution concentration, cross-linking time, cross-linking pH and cross-linking temperature on the enzyme activity of the immobilized &beta-galactosidase on Chitosan beads were studied. The enzyme activity was measured after immobilized by detecting the absorbance value at 420 nm.The results were as follows: the immobilized enzyme activity reached the maximum when the concentration of glutaraldehyde solution was 0.3%.The immobilized enzyme had optimal cross-linking time of 3h, optimal cross-linking pH of 6.5, optimal cross-linking temperature of 25°C, under these conditions, the immobilized enzyme activity reached 101, 96, 90 U/g, respectively.

  1. Physicochemical properties and micro-structural characteristics in starch from kudzu root as affected by cross-linking.

    Chen, Boru; Dang, Leping; Zhang, Xiao; Fang, Wenzhi; Hou, Mengna; Liu, Tiankuo; Wang, Zhanzhong


    Kudzu starch was cross-linked with sodium trimetaphosphate (STMP) at different temperatures, time and of STMP concentrations in this work. The cross-linked starches (CLSs) were fractionated further into cross-linked amylose and amylopectin in order to compare the effect of cross-linking on the microstructure. According to scanning electron microscope (SEM), CLSs displayed the resemble appearance of spherical and polygonal shapes like NS. X-ray diffraction (XRD) revealed that amylose of native starch (A), NS and CLS displayed a combination of A-type and B-type structure, while that was not found in amylose of cross-linked starch (CLA). The deconvoluted fourier transform infrared (FT-IR) indicated that crystal structure of kudzu starch was losing with the proceeding of cross-linking reaction. The CLSs exhibited a higher retrogradation and freeze-thaw stability than NS. This was accompanied by a significant decrease in sedimentation, transparency, swelling power and solubility.

  2. DNA extraction from formalin-fixed material.

    Campos, Paula F; Gilbert, Thomas M P


    The principal challenges facing PCR-based analyses of DNA extracted from formalin-fixed materials are fragmentation of the DNA and cross-linked protein-DNA complexes. Here, we present an efficient protocol to extract DNA from formalin-fixed or paraffin-embedded tissues (FFPE). In this protocol, protein-DNA cross-links are reversed using heat and alkali treatment, yielding significantly longer fragments and larger amounts of PCR-amplifiable DNA than standard DNA extraction protocols.

  3. Design of phosphated cross-linked microspheres of bael fruit gum as a biodegradable carrier.

    Mahammed, Nawaz; Gowda, D V; Deshpande, Rohan D; Thirumaleshwar, Shailesh


    Present work was aimed at designing of phosphated cross-linked microspheres of bael fruit gum (BFG) by emulsification method using sodium-tri-meta phosphate as a cross-linking agent for treatment of colon cancer using 5-fluorouracil as model drug. Stirring speed was found to be 1,000 rpm for about 5 h to be optimal to obtain reproducible microspheres. It was found that there is an increase in particle size as polymer concentration is increased whereas a reduction in particle size was observed as there is increase in stirring speed. Cross-linked BFG microspheres were successfully prepared by emulsification method. Optimum surfactant concentration was found to be 2 % w/w. Scanning electron microscopy studies showed that the drug-loaded microspheres were non-aggregated and in spherical shape. Differential scanning calorimetry and Fourier transform infrared-spectroscopy studies showed that drug and excipients are compatible. Release studies showed that drug release was more profound in cecal medium induced with enzymes causing degradation of the cross linked BFG than that of the release showed in simulated intestinal fluid. Stability studies showed that there were no significant changes in the drug content and physical appearance of microspheres.

  4. Elevated carboxy terminal cross linked telopeptide of type I collagen in alcoholic cirrhosis

    Møller, S; Hansen, M; Hillingsø, Jens


    BACKGROUND: The carboxy terminal cross linked telopeptide of type I collagen (ICTP) has been put forward as a marker of bone resorption. Patients with alcoholic liver disease may have osteodystrophy. AIMS: To assess circulating and regional concentrations of ICTP in relation to liver dysfunction...

  5. TGFβ affects collagen cross-linking independent of chondrocyte phenotype but strongly depending on physical environment

    Bastiaansen-Jenniskens, Y.M.; Koevoet, W.; Bart, A.C.W. de; Zuurmond, A.-M.; Bank, R.A.; Verhaar, J.A.N.; Groot, J. de; Osch, G.J.V.M. van


    Transforming growth factor beta (TGFβ) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGFβ, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of

  6. Ionically cross-linked hyaluronic acid: wetting, lubrication, and viscoelasticity of a modified adhesion barrier gel

    Katherine Vorvolakos


    Full Text Available Katherine Vorvolakos1, Irada S Isayeva1, Hoan-My Do Luu1, Dinesh V Patwardhan1, Steven K Pollack21Division of Chemistry and Material Science, 2Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, USAAbstract: Hyaluronic acid (HA, in linear or cross-linked form, is a common component of cosmetics, personal care products, combination medical products, and medical devices. In all cases, the ability of the HA solution or gel to wet surfaces and/or disrupt and lubricate interfaces is a limiting feature of its mechanism of action. We synthesized ferric ion–cross-linked networks of HA based on an adhesion barrier, varied the degree of cross-linking, and performed wetting goniometry, viscometry, and dynamic mechanical analysis. As cross-linking increases, so do contact angle, viscosity, storage modulus, and loss modulus; thus, wetting and lubrication are compromised. These findings have implications in medical device materials, such as adhesion barriers and mucosal drug delivery vehicles.Keywords: hyaluron, adhesion barrier, wetting, contact angle, viscosity, lubrication, elasticity, viscoelastic, hydrogel, ferric

  7. ROMP-based thermosetting polymers from modified castor oil with various cross-linking agents

    Ding, Rui

    Polymers derived from bio-renewable resources are finding an increase in global demand. In addition, polymers with distinctive functionalities are required in certain advanced fields, such as aerospace and civil engineering. In an attempt to meet both these needs, the goal of this work aims to develop a range of bio-based thermosetting matrix polymers for potential applications in multifunctional composites. Ring-opening metathesis polymerization (ROMP), which recently has been explored as a powerful method in polymer chemistry, was employed as a unique pathway to polymerize agricultural oil-based reactants. Specifically, a novel norbornyl-functionalized castor oil alcohol (NCA) was investigated to polymerize different cross-linking agents using ROMP. The effects of incorporating dicyclopentadiene (DCPD) and a norbornene-based crosslinker (CL) were systematically evaluated with respect to curing behavior and thermal mechanical properties of the polymers. Isothermal differential scanning calorimetry (DSC) was used to investigate the conversion during cure. Dynamic DSC scans at multiple heating rates revealed conversion-dependent activation energy by Ozawa-Flynn-Wall analysis. The glass transition temperature, storage modulus, and loss modulus for NCA/DCPD and NCA/CL copolymers with different cross-linking agent loading were compared using dynamic mechanical analysis. Cross-link density was examined to explain the very different dynamic mechanical behavior. Mechanical stress-strain curves were developed through tensile test, and thermal stability of the cross-linked polymers was evaluated by thermogravimetric analysis to further investigate the structure-property relationships in these systems.

  8. Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs)

    Sheldon, R.A.


    Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational

  9. Temperature dependence of creep compliance of highly cross-linked epoxy: A molecular simulation study

    Khabaz, Fardin, E-mail:; Khare, Ketan S., E-mail:; Khare, Rajesh, E-mail: [Department of Chemical Engineering, Texas Tech University, Lubbock, TX 79409 (United States)


    We have used molecular dynamics (MD) simulations to study the effect of temperature on the creep compliance of neat cross-linked epoxy. Experimental studies of mechanical behavior of cross-linked epoxy in literature commonly report creep compliance values, whereas molecular simulations of these systems have primarily focused on the Young’s modulus. In this work, in order to obtain a more direct comparison between experiments and simulations, atomistically detailed models of the cross-linked epoxy are used to study their creep compliance as a function of temperature using MD simulations. The creep tests are performed by applying a constant tensile stress and monitoring the resulting strain in the system. Our results show that simulated values of creep compliance increase with an increase in both time and temperature. We believe that such calculations of the creep compliance, along with the use of time temperature superposition, hold great promise in connecting the molecular insight obtained from molecular simulation at small length- and time-scales with the experimental behavior of such materials. To the best of our knowledge, this work is the first reported effort that investigates the creep compliance behavior of cross-linked epoxy using MD simulations.

  10. Mocaf cross-linking with gluten to improve the quality of mocaf dough

    Raharja, Sapta; Udin, Faqih; Suparno, Ono; Febrianti, Faricha Helfi; Nuraisyah, Ani


    Crosslink between mocaf and gluten is conducted to increase the using of mocaf which has very big potential in Indonesia. The effort of cross-linking between mocaf and gluten is to get mocaf flour with better dough quality. This study aims to produce a cross-linked mocaf-gluten flour and to evaluate the influence of heating temperature (X1) and the addition of gluten concentration (X2) using completely randomized design factorial (RAFL). The cross-linking is carried out in alkaline solution with 10%, 20%, and 30% gluten addition and heating temperature at 50, 55, and 60 °C. The result showed that mocaf - gluten flour with the treatment of 30% gluten addition at 55 °C had the largest amount of protein and baking expansion (i.e 19.77% and 2.78 mL/g). Swelling power of the flour was increasing along with the increasing of water absorbing capacity of the mocaf - gluten flour. Birefringence properties of mocaf - gluten flour tended to be reduced as the increasing heating temperature. FTIR analysis of mocaf - gluten flour showed that there was peak strengthening of the infrared spectrum of the C - N bond at 1167-1159 cm-1 which was presumably resulted from the gluten addition and the cross-linking properties.

  11. A minimal model for stabilization of biomolecules by hydrocarbon cross-linking

    Hamacher, K.; Hübsch, A.; McCammon, J. A.


    Programmed cell death regulating protein motifs play an essential role in the development of an organism, its immune response, and disease-related cellular mechanisms. Among those motifs the BH3 domain of the BCL-2 family is found to be of crucial importance. Recent experiments showed how the isolated, otherwise unstructured BH3 peptide can be modified by a hydrocarbon linkage to regain function. We parametrized a reduced, dynamic model for the stability effects of such covalent cross-linking and confirmed that the model reproduces the reinforcement of the structural stability of the BH3 motif by cross-linking. We show that an analytically solvable model for thermostability around the native state is not capable of reproducing the stabilization effect. This points to the crucial importance of the peptide dynamics and the fluctuations neglected in the analytic model for the cross-linking system to function properly. This conclusion is supported by a thorough analysis of a simulated Gō model. The resulting model is suitable for rational design of generic cross-linking systems in silicio.

  12. Sensitizing potential of enzymatically cross-linked peanut proteins in a mouse model of peanut allergy.

    Smit, Joost|info:eu-repo/dai/nl/250600706


    SCOPE: The cross-linking of proteins by enzymes to form high-molecular-weight protein, aggregates can be used to tailor the technological or physiological functionality of food products. Aggregation of dietary proteins by food processing may promote allergic sensitization, but the effects of enzymat

  13. Design and Preparation of Cross-Linked Polystyrene Nanoparticles for Elastomer Reinforcement

    Ming Lu


    Full Text Available Cross-linked polystyrene (PS particles in a latex form were synthesized by free radical emulsion polymerization. The nano-PS-filled elastomer composites were prepared by the energy-saving latex compounding method. Results showed that the PS particles took a spherical shape in the size of 40–60 nm with a narrow size distribution, and the glass-transition temperature of the PS nanoparticles increased with the cross-linking density. The outcomes from the mechanical properties demonstrated that when filled into styrene-butadiene rubber (SBR, nitrile-butadiene rubber (NBR, and natural rubber (NR, the cross-linked PS nano-particles exhibited excellent reinforcing capabilities in all the three matrices, and the best in the SBR matrix. In comparison with that of the carbon black filled composites, another distinguished advantage of the cross-linked PS particles filled elastomer composites was found to be light weight in density, which could help to save tremendous amount of energy when put into end products.

  14. Polymers and Cross-Linking: A CORE Experiment to Help Students Think on the Submicroscopic Level

    Bruce, Mitchell R. M.; Bruce, Alice E.; Avargil, Shirly; Amar, Francois G.; Wemyss, Thomas M.; Flood, Virginia J.


    The Polymers and Cross-Linking experiment is presented via a new three phase learning cycle: CORE (Chemical Observations, Representations, Experimentation), which is designed to model productive chemical inquiry and to promote a deeper understanding about the chemistry operating at the submicroscopic level. The experiment is built on two familiar…

  15. 21 CFR 177.2710 - Styrene-divinylbenzene resins, cross-linked.


    ... SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: POLYMERS Substances..., preparing, treating, packaging, transporting, or holding food, in accordance with the following prescribed... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Styrene-divinylbenzene resins, cross-linked....

  16. Substituent distribution within cross-linked and hydroxypropylated sweet potato starch and potato starch

    Zhao, J.; Schols, H.A.; Chen Zenghong,; Jin Zhengyu,; Buwalda, P.L.; Gruppen, H.


    Revealing the substituents distribution within starch can help to understand the changes of starch properties after modification. The distribution of substituents over cross-linked and hydroxypropylated sweet potato starch was investigated and compared with modified potato starch. The starches were

  17. EVA交联机理的研究%The Research of EVA Cross-linking Mechanism

    王川艳; 苑会林


    研究了过氧化二异丙苯(DCP)对乙烯-醋酸乙烯共聚物(EVA)交联的影响,分析交联过程的反应机理.对交联的EVA样品进行紫外光辐照,分析在该过程中所发生的反应,并研究加入紫外线吸收剂2-羟基-4-正辛氧基-二苯甲酮(UV-531)后,对交联EVA样品在紫外光辐照下交联的影响.%The effect of peroxide (DCP) on Ethylene-Vinyl Acetate (EVA) cross-linking were studied, the reaction mechanism of EVA cross-linking were analyzed. Upon the exposure of cross-linked EVA samples to ultraviolet light, the reaction during this process were analyzed, and also, after adding the ultraviolet absorber (UV-531) , the influences to cross-linked EVA samples during the ultraviolet radiation process were researched.

  18. Dynamic Heterogeneity in Highly Cross-linked Epoxy in the Vicinity of Glass Transition

    Lin, Po-Han; Khare, Rajesh


    Cross-linked epoxy has been widely used in aerospace and electronics industries. The highly cross-linked nature of these systems leads to different chain dynamics as compared to the linear polymeric systems. In this work, we have used molecular dynamics (MD) simulations to study the dynamic heterogeneity in cross-linked epoxy near the glass transition temperature. Well-relaxed atomistic models of cross-linked epoxy were first created by employing the simulated annealing polymerization approach. The specific epoxy system studied consisted of diglycidyl ether of bisphenol-A (DGEBA) as the epoxy monomer and trimethylene glycol di-p-aminobenzoate (TMAB) as the cross-linker. The glass transition temperature of these model structures was determined from MD simulation by monitoring their volume-temperature behaviour in a stepwise cooling run. The chain dynamics of these systems were characterized by their local translational and orientational mobility. Furthermore, dynamic heterogeneity was studied by analyzing the spatial distribution of the mobile and immobile atoms in the system near the glass transition temperature.

  19. Preparation of Nanocellulose Reinforced Chitosan Films, Cross-Linked by Adipic Acid.

    Falamarzpour, Pouria; Behzad, Tayebeh; Zamani, Akram


    Adipic acid, an abundant and nontoxic compound, was used to dissolve and cross-link chitosan. After the preparation of chitosan films through casting technique, the in situ amidation reaction was performed at 80-100 °C as verified by Fourier transform infrared (FT-IR). The reaction was accompanied by the release of water which was employed to investigate the reaction kinetics. Accordingly, the reaction rate followed the first-order model and Arrhenius equation, and the activation energy was calculated to be 18 kJ/mol. Furthermore, the mechanical properties of the chitosan films were comprehensively studied. First, optimal curing conditions (84 °C, 93 min) were introduced through a central composite design. In order to evaluate the effects of adipic acid, the mechanical properties of physically cross-linked (uncured), chemically cross-linked (cured), and uncross-linked (prepared by acetic acid) films were compared. The use of adipic acid improved the tensile strength of uncured and chemically cross-linked films more than 60% and 113%, respectively. Finally, the effect of cellulose nanofibrils (CNFs) on the mechanical performance of cured films, in the presence of glycerol as a plasticizer, was investigated. The plasticized chitosan films reinforced by 5 wt % CNFs showed superior properties as a promising material for the development of chitosan-based biomaterials.

  20. Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent

    Alexandre Arsenault


    Full Text Available Laccase from Coriolopsis Polyzona was insolubilized as cross-linked enzyme aggregates (CLEAs for the first time with chitosan as the cross-linking agent. Concentrations between 0.01 and 1.867 g/L of chitosan were used and between 0.05 and 600 mM of 1-ethyl-3-(3-dimethylaminopropylcarbodiimide hydrochloride. The laccase was precipitated using ammonium sulphate and cross-linked simultaneously. Specific activity and thermal stability of these biocatalysts were measured. Activities of up to 737 U/g were obtained when 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS was used as a substrate. Moreover, the stability of these biocatalysts was improved with regards to thermal degradation compared to free laccase when exposed to denaturing conditions of high temperature and low pH. The CLEAs stability against chemical denaturants was also tested but no significant improvement was detected. The total amount of ABTS to be oxidized during thermal degradation by CLEAs and free laccase was calculated and the insolubilized enzymes were reported to oxidize more substrate than free laccase. The formation conditions were analyzed by response surface methodology in order to determine an optimal environment for the production of efficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24 hours of formation at pH 3 and at 4°C without agitation, the CLEAs exhibit the best specific activity.

  1. Standard and hypoosmolar corneal cross-linking in various pachymetry groups

    Soeters, Nienke; Tahzib, Nayyirih G


    PURPOSE: To investigate the influence of corneal thickness on the outcome of corneal cross-linking (CXL) for progressive keratoconus. METHODS: In this cohort study, 72 unilateral eyes were treated by CXL and divided into three groups according to central corneal thickness (CCT) measured by ultrasoun

  2. The peptidoglycan of Mycobacterium abscessus is predominantly cross-linked by L,D-transpeptidases.

    Lavollay, Marie; Fourgeaud, Martine; Herrmann, Jean-Louis; Dubost, Lionel; Marie, Arul; Gutmann, Laurent; Arthur, Michel; Mainardi, Jean-Luc


    Few therapeutic alternatives remain for the treatment of infections due to multiresistant Mycobacterium abscessus. Here we show that the peptidoglycans of the "rough" and "smooth" morphotypes contain predominantly 3→3 cross-links generated by l,d-transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.

  3. Cross-linking Electrospun Polydioxanone-Soluble Elastin Blends: Material Characterization

    Michael J. McClure


    Full Text Available The purpose of this study was to establish whether material properties of elastin co-electrospun with polydioxanone (PDO would change over time in both the uncross-linked state and the cross-linked state. First, uncross-linked scaffolds were placed in phosphate buffered saline (PBS for three separate time periods: 15 minutes, 1 hour, and 24 hours, and subsequently tested using uniaxial materials testing. Several cross-linking reagents were then investigated to verify their ability to crosslink elastin: 1-ethyl-3-(dimethylaminopropyl-carbodiimide (EDC, ethylene glycol diglycidyl ether (EGDE, and genipin. Uniaxial tensile testing was performed on scaffolds cross-linked with EDC and genipin, yielding results that warranted further investigation for PDO-elastin blends. Material properties of the cross-linked scaffolds were then found within range of both pig femoral artery and human femoral artery. These results demonstrate PDO-elastin blends could potentially be favorable as vascular grafts, thus warranting future in vitro and in vivo studies.

  4. Is dialdehyde starch a valuable cross-linking agent for collagen/elastin based materials?

    Skopinska-Wisniewska, J; Wegrzynowska-Drzymalska, K; Bajek, A; Maj, M; Sionkowska, A


    Collagen and elastin are the main structural proteins in mammal bodies. They provide mechanical support, strength, and elasticity to various organs and tissues, e.g. skin, tendons, arteries, and bones. They are readily available, biodegradable, biocompatible and they stimulate cell growth. The physicochemical properties of collagen and elastin-based materials can be modified by cross-linking. Glutaraldehyde is one of the most efficient cross-linking agents. However, the unreacted molecules can be released from the material and cause cytotoxic reactions. Thus, the aim of our work was to investigate the influence of a safer, macromolecular cross-linking agent--dialdehyde starch (DAS). The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5 and 10% of DAS and neutralized via dialysis against deionized water were tested. The homogenous, transparent, stiff hydrogels were obtained. The DAS addition causes the formation of intermolecular cross-linking bonds but does not affect the secondary structure of the proteins. As a result, the thermal stability, mechanical strength, and, surprisingly, swelling ability increased. At the same time, the surface properties test and in vitro study show that the materials are attractive for 3T3 cells. Moreover, the materials containing 10% of DAS are more resistant to enzymatic degradation.

  5. Corneal Cross-Linking for Pediatric Keratoconus : Long-Term Results

    Godefrooij, Daniel A; Soeters, Nienke; Imhof, Saskia M; Wisse, Robert P L


    PURPOSE: To assess the efficacy and safety of cross-linking in pediatric patients with keratoconus and to provide a systematic literature overview regarding this subject. METHODS: In this prospective cohort, 54 eyes of 36 pediatric patients with keratoconus underwent standard epithelium-off cross-li

  6. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan


    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI protein

  7. Preparation of Nanocellulose Reinforced Chitosan Films, Cross-Linked by Adipic Acid

    Pouria Falamarzpour


    Full Text Available Adipic acid, an abundant and nontoxic compound, was used to dissolve and cross-link chitosan. After the preparation of chitosan films through casting technique, the in situ amidation reaction was performed at 80–100 °C as verified by Fourier transform infrared (FT-IR. The reaction was accompanied by the release of water which was employed to investigate the reaction kinetics. Accordingly, the reaction rate followed the first-order model and Arrhenius equation, and the activation energy was calculated to be 18 kJ/mol. Furthermore, the mechanical properties of the chitosan films were comprehensively studied. First, optimal curing conditions (84 °C, 93 min were introduced through a central composite design. In order to evaluate the effects of adipic acid, the mechanical properties of physically cross-linked (uncured, chemically cross-linked (cured, and uncross-linked (prepared by acetic acid films were compared. The use of adipic acid improved the tensile strength of uncured and chemically cross-linked films more than 60% and 113%, respectively. Finally, the effect of cellulose nanofibrils (CNFs on the mechanical performance of cured films, in the presence of glycerol as a plasticizer, was investigated. The plasticized chitosan films reinforced by 5 wt % CNFs showed superior properties as a promising material for the development of chitosan-based biomaterials.

  8. Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with β-casein

    Boeriu, C.G.; Oudgenoeg, G.; Spekking, W.T.J.; Berendsen, L.B.J.M.; Vancon, L.; Boumans, H.; Gruppen, H.; Berkel, W.J.H. van; Laane, C.; Voragen, A.G.J.


    Heterologous conjugates of wheat arabinoxylan and β-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of β-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the β-casein and the feruloylated arabinoxylan was

  9. Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications

    Sell, Scott A; Garg, Koyal; McClure, Michael J; Bowlin, Gary L [Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23284-3067 (United States); Francis, Michael P [Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298-0709 (United States); Simpson, David G [Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23298-0709 (United States)], E-mail:


    The purpose of this study was to enhance the mechanical properties and slow the degradation of an electrospun fibrinogen scaffold, while maintaining the scaffold's high level of bioactivity. Three different cross-linkers were used to achieve this goal: glutaraldehyde vapour, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and genipin in ethanol. Scaffolds with a fibrinogen concentration of 120 mg ml{sup -1} were electrospun and cross-linked with one of the aforementioned cross-linkers. Mechanical properties were determined through uniaxial tensile testing performed on scaffolds incubated under standard culture conditions for 1 day, 7 days and 14 days. Cross-linked scaffolds were seeded with human foreskin fibroblasts (BJ-GFP-hTERT) and cultured for 7, 14 and 21 days, with histology and scanning electron microscopy performed upon completion of the time course. Mechanical testing revealed significantly increased peak stress and modulus values for the EDC and genipin cross-linked scaffolds, with significantly slowed degradation. However, cross-linking with EDC and genipin was shown to have some negative effect on the bioactivity of the scaffolds as cell migration throughout the thickness of the scaffold was slowed.

  10. Mature enzymatic collagen cross-links, hydroxylysylpyridinoline and lysylpyridinoline, in the aging human vitreous

    Ponsioen, T.L.; van Deemter, M.; Bank, R.A.; Snabel, J.M.; Zijlstra, G.S.; van der Worp, R.J.; Hooymans, J.M.M.; Los, L.I.


    Purpose. The vitreous body of the human eye undergoes progressive morphologic changes with aging. Since the enzymatic collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are known to be important for the integrity of the collagen matrix, the presence in the vitreous on agin

  11. Mature Enzymatic Collagen Cross-Links, Hydroxylysylpyridinoline and Lysylpyridinoline, in the Aging Human Vitreous

    Ponsioen, Theodorus L.; van Deemter, Marielle; Bank, Rudolf A.; Snabel, Johanna M.; Zijlstra, Gerrit S.; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.


    PURPOSE. The vitreous body of the human eye undergoes progressive morphologic changes with aging. Since the enzymatic collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are known to be important for the integrity of the collagen matrix, the presence in the vitreous on agin

  12. Formation and Distribution of Space-Charge in Cross-Linked Polyethylene

    Zhang, Ye-Wen; Li, Ji-Xiao; Zheng, Fei-Hu; Peng, Zong-Ren; Wu, Chang-Shun; Xia, Zhong-Fu


    The formation and distribution of space-charge in a cross-linked polyethylene (XLPE) sample are investigated by means of pressure wave propagation, infrared spectroscopy and electrostatic force microscopy (EFM). The related mechanism of space-charge distribution and the structure of XLPE are discussed. The EFM images show that quite large quantitative space-charges locate at the surface of spherulites.

  13. Crown ether activation of cross-linked subtilisin Carlsberg crystals in organic solvents

    Unen, van Dirk-Jan; Sakodinskaya, Inna K.; Engbersen, Johan F.J.; Reinhoudt, David N.


    The activity of cross-linked subtilisin Carlsberg crystals in the catalysis of peptide bond formation can be significantly enhanced by pretreatment of the enzyme crystals with crown ethers. Soaking of the enzyme crystals in a solution of crown ether in acetonitrile followed by evaporation of the sol

  14. Phosphoric acid doped polysulfone membranes with aminopyridine pendant groups and imidazole cross-links

    Hink, Steffen; Elsøe, Katrine; Cleemann, Lars Nilausen;


    Udel polysulfone based membranes with 4-aminopyridine pendant groups and cross-linking imidazole units are synthesized in a simple two step reaction. The ratio of 4-aminopyridine and imidazole is varied and the materials are extensively characterized. The average phosphoric acid uptake (in 85 wt...

  15. Cross-Linked Nanoporous Materials from Reactive and Multifunctional Block Polymers

    Seo, Myungeun; Amendt, Mark A.; Hillmyer, Marc A. (UMM)


    Polylactide-b-poly(styrene-co-2-hydroxyethylmethacrylate) (PLA-b-P(S-co-HEMA)) and polylactide-b-poly(styrene-co-2-hydroxyethylacrylate) (PLA-b-P(S-co-HEA)) were synthesized by combination of ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization. {sup 1}H nuclear magnetic resonance spectroscopy and size exclusion chromatography data indicated that the polymerizations were controlled and that hydroxyl groups were successfully incorporated into the block polymers. The polymers were reacted with 4,4{prime}-methylenebis(phenyl isocyanate) (MDI) to form the corresponding cross-linked materials. The materials were annealed at 150 C to complete the coupling reaction. Robust nanoporous materials were obtained from the cross-linked polymers by treatment with aqueous base to hydrolyze the PLA phase. Small-angle X-ray scattering study combined with scanning electron microscopy showed that MDI-cross-linked PLA-b-P(S-co-HEMA)/PLA-b-P(S-co-HEA) can adopt lamellar, hexagonally perforated lamellar, and hexagonally packed cylindrical morphologies after annealing. In particular, the HPL morphology was found to evolve from lamellae due to increase in volume fraction of PS phase as MDI reacted with hydroxyl groups. The reaction also kinetically trapped the morphology by cross-linking. Bicontinuous morphologies were also observed when dibutyltin dilaurate was added to accelerate reaction between the polymer and MDI.

  16. Effect of dual modification with hydroxypropylation and cross-linking on physicochemical properties of taro starch.

    Hazarika, Bidyut Jyoti; Sit, Nandan


    Dual modification of taro starch by hydroxypropylation and cross-linking was carried out and the properties of the modified starches were investigated. Two different levels of hydroxypropylation (5 and 10%) and cross-linking (0.05 and 0.10%) were used in different sequences. The amylose contents of the starch decreased due to single and dual modification. For the dual-modified starches, the swelling, solubility and clarity was found to increase with level of hydroxypropylation and decrease with level of cross-linking. The freeze-thaw stability of the dual-modified starches was also affected by the sequence of modification. The viscosities of the cross-linked and dual-modified starches were more than native and hydroxypropylated starches. The firmness of the dual-modified starches was also higher than native and single modified starches. The dual-modified starches have benefits of both type of modifications and could be used for specific purposes e.g. food products requiring high viscosity as well as freeze-thaw stability.

  17. Vitamin E diffused highly cross-linked polyethylene in total hip arthroplasty at five years

    Nebergall, Audrey K; Greene, M. E.; Laursen, M B


    AIMS: The objective of this five-year prospective, blinded, randomised controlled trial (RCT) was to compare femoral head penetration into a vitamin E diffused highly cross-linked polyethylene (HXLPE) liner with penetration into a medium cross-linked polyethylene control liner using radiostereome......AIMS: The objective of this five-year prospective, blinded, randomised controlled trial (RCT) was to compare femoral head penetration into a vitamin E diffused highly cross-linked polyethylene (HXLPE) liner with penetration into a medium cross-linked polyethylene control liner using...... radiostereometric analysis. PATIENTS AND METHODS: Patients scheduled for total hip arthroplasty (THA) were randomised to receive either the study E1 (32 patients) or the control ArComXL polyethylene (35 patients). The median age (range) of the overall cohort was 66 years (40 to 76). RESULTS: The five-year median...... (interquartile range) proximal femoral head penetration into the E1 was -0.05 mm (-0.13 to -0.02) and 0.07 mm (-0.03 to 0.16) for ArComXL. At three and five years, the penetration was significantly greater in the ArComXL group compared with the E1 group (p = 0.029 and p = 0.019, respectively). All patient...

  18. Synthesis, Optimization, Property, Characterization, and Application of Dialdehyde Cross-Linking Guar Gum

    Tang Hongbo


    Full Text Available Dialdehyde cross-linking guar gum (DCLGG, as a novel material, was synthesized using phosphorus oxychloride as a cross-linking reagent, sodium periodate as an oxidant, and ethanol as a solvent through keeping the original particle form of guar gum. The process parameters such as the reaction temperature, reaction time, pH, amount of sodium periodate, and amount of ethanol were optimized by the response surface methodology in order to obtain the regression model of the oxidization. The covalent binding of L-asparagine onto the surfaces of DCLGG was further investigated. The results showed that the best technological conditions for preparing DCLGG were as follows: reaction temperature = 40°C, reaction time = 3.0 h, pH = 4.0, and amount of ethanol = 74.5%. The swelling power of DCLGG was intermediate between cross-linking guar gum and dialdehyde guar gum. The cross-linking and dialdehyde oxidization reduced the viscosity of GG. The cross-liking reduced the melting enthalpy of GG. However, the oxidization increased melting enthalpy of ACLGG. The thermal stability of GG was increased by cross-linking or oxidization. The variation of the onset decomposition temperature and end decomposition temperature of GG was not consistent with thermal stability of GG. L-asparagine could be chemically bound well by DCLGG through forming Schiff base under the weak acidity. The maximum adsorption capacity of L-asparagine on DCLGG with aldehyde content of 56.2% reached 21.9 mg/g.

  19. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering.

    Mathapati, Santosh; Bishi, Dillip Kumar; Guhathakurta, Soma; Cherian, Kotturathu Mammen; Venugopal, Jayarama Reddy; Ramakrishna, Seeram; Verma, Rama Shanker


    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p>0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP-G-CA-ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP-G-CA-ET). DBP-G-CA-ET exhibited a significant (p>0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (ppericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Wear measurement of highly cross-linked UHMWPE using a 7Be tracer implantation technique.

    Wimmer, Markus A; Laurent, Michel P; Dwiwedi, Yasha; Gallardo, Luis A; Chipps, Kelly A; Blackmon, Jeffery C; Kozub, Raymond L; Bardayan, Daniel W; Gross, Carl J; Stracener, Daniel W; Smith, Michael S; Nesaraja, Caroline D; Erikson, Luke; Patel, Nidhi; Rehm, Karl E; Ahmad, Irshad; Greene, John P; Greife, Uwe


    The very low wear rates achieved with the current highly cross-linked ultrahigh molecular weight polyethylenes (UHMWPE) used in joint prostheses have proven to be difficult to measure accurately by gravimetry. Tracer methods are therefore being explored. The purpose of this study was to perform a proof-of-concept experiment on the use of the radioactive tracer beryllium-7 ((7)Be) for the determination of in vitro wear in a highly cross-linked orthopedic UHMWPE. Three cross-linked and four conventional UHMWPE pins made from compression-molded GUR 1050, were activated with 10(9) to 10(10) (7)Be nuclei using a new implantation setup that produced a homogenous distribution of implanted nuclei up to 8.5 μm below the surface. The pins were tested for wear in a six-station pin-on-flat apparatus for up to 7.1 million cycles (178 km). A Germanium gamma detector was employed to determine activity loss of the UHMWPE pins at preset intervals during the wear test. The wear of the cross-linked UHMWPE pins was readily detected and estimated to be 17 ± 3 μg per million cycles. The conventional-to-cross-linked ratio of the wear rates was 13.1 ± 0.8, in the expected range for these materials. Oxidative degradation damage from implantation was negligible; however, a weak dependence of wear on implantation dose was observed limiting the number of radioactive tracer atoms that can be introduced. Future applications of this tracer technology may include the analysis of location-specific wear, such as loss of material in the post or backside of a tibial insert.

  1. Light-Sensitive Ruthenium Complex-Loaded Cross-linked Polymeric Nanoassemblies for the Treatment of Cancer.

    Dickerson, M; Howerton, B; Bae, Y; Glazer, E


    This work focuses on improving the efficacy of photoactivatable Ru complexes for photodynamic therapy by employing cross-linked nanoassemblies (CNAs) as a delivery approach. The effects of complex photoactivation, hydrophobicity, and solution ionic strength and pH on complex loading and release from CNAs were analyzed. The cell cytotoxicity of CNA formulations was similar to free Ru complexes despite reduced or eliminated DNA interactions. The release rate and the amount of each Ru complex released (%) varied inversely with complex hydrophobicity, while the effect of solution ionic strength was dependent on complex hydrophobicity. Premature release of two photoactivatable prodrugs prior to irradiation was believed to account for higher activity in cells studies compared to DNA interaction studies; however, for photostable (1)O2 generator-loaded CNAs this cannot explain the high cytotoxicity and lack of DNA interactions because release was incomplete after 48 hrs. The cause remains unclear, but among other possibilities, accelerated release in a cell culture environment may be responsible.

  2. Oegylated and cross-linking carbazole dendrons and dendrimers: Synthesis, characterization, assembly and thin film fabrication

    Felipe, Mary Jane Legaspi


    Dendrimers and dendrons (fractional dendrimers) are macromolecular structures that have well-defined molecular weights and precise number of functional groups. Tailoring these structures has provided designer molecules that can be used for various applications including drug delivery, sensors, and anti-biofouling surfaces. Overall, this dissertation provides novel protocols for the understanding of molecular design, synthesis, and structure-property relationship of OEGylated and conjugated carbazole dendrons and dendrimers. In this design, the use of oligo(ethylene glycol) (OEG) allows for the fabrication of biocompatible materials and imparts hydrophilicity on the structure while the carbazole functionality allows the cross-linking of these designer molecules. Such fine-tuning of macromolecular structures leading to the fabrication of anti-biofouling thin films, nanostructuring at the air-water interface, and assembly into supramolecular superstructures are considered in this dissertation. Chapter 2 details the synthesis, characterization, and electrochemical cross-linking of OEGylated linear dendrons and "Janus-type" dendrimers. Cross-linking the carbazole moieties enables the deposition of these films on Au, indium tin oxide-coated glass, and doped silicon through cyclic voltammetry and provides films with secondary level of organization imparted by the inter- and intra-molecular interaction among the carbazole units. Chapter 3 describes the fabrication of nonspecific protein adsorption resistant surfaces through electrochemical grafting of three different dendrons on SAM carbazole-coated gold substrates. The predictable shape of each dendron and the ability to cross-link the carbazole units have enabled parametrization of OEG conformation and density on these interfaces. Chapter 4 demonstrates the fundamental architectural requirements for obtaining stable films with OEGylated linear dendron molecules providing a new architectural design of nanostructuring

  3. Statistical Inferences from Formaldehyde Dna-Protein Cross-Link Data

    Physiologically-based pharmacokinetic (PBPK) modeling has reached considerable sophistication in its application in the pharmacological and environmental health areas. Yet, mature methodologies for making statistical inferences have not been routinely incorporated in these applic...

  4. Wheat gluten films cross-linked with 1-ethyl-3-(3-demethylaminopropyl) carbodiimide and N-hydroxysuccinimide

    Tropini, V.; Lens, J.P.; Mulder, W.J.; Silvestre, F.


    Wheat gluten films were cast from aqueous dispersions containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linking reagents and glycerol as a plasticizer. Cross-linking was carried out to improve film properties such as water sensitivity and tensile

  5. Simulation and theory of self-assembly and network formation in reversibly cross-linked equilibrium polymers

    Kindt, James T.


    A simulation model of hard spheres capable of reversible assembly into chains, which then may reversibly cross-link into networks, has been studied through grand canonical Monte Carlo simulation. Effects of varying intra- and interchain bond strengths, chain flexibilities, and restrictions on cross-linking angle were investigated. Observations including chain-length distributions and phase separation could be captured in most cases using a simple model theory. The coupling of chain growth to cross-linking was shown to be highly sensitive to the treatment of cross-linking by chain ends. In some systems, ladderlike domains of several cross-links joining two chains were common, resulting from cooperativity in the cross-linking. Extended to account for this phenomenon, the model theory predicts that such cooperativity will suppress phase separation in weakly polymerizing chains and at high cross-link concentration. In the present model, cross-linking stabilizes the isotropic phase with respect to the nematic phase, causing a shift in the isotropic-nematic transition to higher monomer concentration than in simple equilibrium polymers.

  6. Comparative studies of photochemical cross-linking methods for stabilizing the bulk hetero-junction morphology in polymer solar cells

    Carlé, Jon Eggert; Andreasen, Birgitta; Tromholt, Thomas;


    -light illumination to give solvent resistant films and reduced phase separation and growth of PCBM crystallites in polymer:PCBM films. The stability of solar cells based on the cross-linked polymers was tested under various conditions. This study showed that cross-linking can improve morphological stability...

  7. Effect of PDMS cross-linking degree on the permeation performance of PAN/PDMS composite nanofiltration membranes

    Stafie, N.; Stamatialis, Dimitrios; Wessling, Matthias


    This work focuses on the effect of poly(dimethyl siloxane) (PDMS) cross-linking on the permeation performance of the poly(acrylonitrile) (PAN)/PDMS nanofiltration (NF) composite membrane. PDMS membrane of various cross-linking degrees could be obtained by changing the ratio of a vinyl-terminated

  8. Synthesis and properties of reprocessable sulfonated polyimides cross-linked via acid stimulation for use as proton exchange membranes

    Zhang, Boping; Ni, Jiangpeng; Xiang, Xiongzhi; Wang, Lei; Chen, Yongming


    Cross-linked sulfonated polyimides are one of the most promising materials for proton exchange membrane (PEM) applications. However, these cross-linked membranes are difficult to reprocess because they are insoluble. In this study, a series of cross-linkable sulfonated polyimides with flexible pendant alkyl side chains containing trimethoxysilyl groups is successfully synthesized. The cross-linkable polymers are highly soluble in common solvents and can be used to prepare tough and smooth films. Before the cross-linking reaction is complete, the membranes can be reprocessed, and the recovery rate of the prepared films falls within an acceptable range. The cross-linked membranes are obtained rapidly when the cross-linkable membranes are immersed in an acid solution, yielding a cross-linking density of the gel fraction of greater than 90%. The cross-linked membranes exhibit high proton conductivities and tensile strengths under hydrous conditions. Compared with those of pristine membranes, the oxidative and hydrolytic stabilities of the cross-linked membranes are significantly higher. The CSPI-70 membrane shows considerable power density in a direct methanol fuel cell (DMFC) test. All of these results suggest that the prepared cross-linked membranes have great potential for applications in proton exchange membrane fuel cells.

  9. Insights into the swelling process and drug release mechanisms from cross-linked pectin/high amylose starch matrices

    Fernanda M. Carbinatto


    Full Text Available Cross-linked pectin/high amylose mixtures were evaluated as a new excipient for matrix tablets formulations, since the mixing of polymers and cross-linking reaction represent rational tools to reach materials with modulated and specific properties that meet specific therapeutic needs. Objective: In this work the influence of polymer ratio and cross-linking process on the swelling and the mechanism driving the drug release from swellable matrix tablets prepared with this excipient was investigated. Methods: Cross-linked samples were characterized by their micromeritic properties (size and shape, density, angle of repose and flow rate and liquid uptake ability. Matrix tablets were evaluated according their physical properties and the drug release rates and mechanisms were also investigated. Results: Cross-linked samples demonstrated size homogeneity and irregular shape, with liquid uptake ability insensible to pH. Cross-linking process of samples allowed the control of drug release rates and the drug release mechanism was influenced by both polymer ratio and cross-linking process. The drug release of samples with minor proportion of pectin was driven by an anomalous transport and the increase of the pectin proportion contributed to the erosion of the matrix. Conclusion: The cross-linked mixtures of high amylose and pectin showed a suitable excipient for slowing the drug release rates.

  10. Polymer-additive extraction via pressurized fluids and organic solvents of variously cross-linked poly(methylmethacrylates).

    Nazem, N; Taylor, L T


    Variously cross-linked poly(methylmethacrylates) (PMMAs) are synthesized with three additives incorporated at theoretically 1000 microg of the additive per gram of prepared polymer. The additives are Irganox 1010, Irganox 1076, and Irgafos 168. The in-house" synthesized polyacrylates are then subjected to supercritical fluid extraction (SFE) to determine if additive recovery is a function of percent cross-linking. Although considerable work in this regard has been performed with non-cross-linked polyolefins, the literature is lacking regarding polyacrylates. Some additive degradation apparently occurs during the synthesis, as judged by the increased complexity of the extract high-performance liquid chromatographic trace and the low percent recoveries observed especially for the Irganoxes. For low polymer cross-linking (1%), it appears that both PMMA synthetic reproducibility and readily observed polymer swelling during SFE are serious issues that adversely affect additive percent recovery and precision of results. Higher percent cross-linking yields more consistent analytical data than low percent cross-linking, even though the amount of additive extracted in all PMMA samples (regardless of cross-linking percentage) is essentially the same whether the extraction is via SFE or liquid-solid extraction with methylene chloride. Results for comparably cross-linked poly(ethylmethacrylate) and poly(butylmethacrylate) are similar to PMMA.

  11. Corneal cross-linking in a 10-year-old child with stage III keratoconus.

    Abbondanza, Marco; Felice, Valentina De; Abbondanza, Gabriele


    Keratoconus is a corneal dystrophy characterized by progressive thinning, conical shape of the cornea and irregular astigmatism. It is particularly insidious when it occurs in very young patients. We report the case of a 10-year-old child with an aggressive stage III keratoconus, who was suggested to undergo a Penetrating Keratoplasty. We performed Corneal Collagen Cross-linking with epithelium removal instead, which was successful in arresting the rapid progression of keratoconus. Eighteen months later, Kmax had decreased by 1.3 D, astigmatism by 0.8 D, thinnest pachimetry had improved by 69 μm and CDVA was 20/32. This case confirms that Corneal Collagen Cross-linking is a safe and effective procedure for the treatment of keratoconus even in its aggressive forms. We encourage ophthalmic surgeons to favor conservative treatments when dealing with very young patients. © NEPjOPH.

  12. Polymeric Nanocapsule from Silica Nanoparticle@Cross-linked Polymer Nanoparticles via One-Pot Approach

    Shen Ruoping


    Full Text Available Abstract A facile strategy was developed here to prepare cross-linked polymeric nanocapsules (CP nanocapsules with silica nanoparticles as templates. The silica nanoparticle@cross-linked polymer nanoparticles were prepared by the encapsulation of the silica nanoparticles by the one-pot approach via surface-initiated atom transfer radical polymerization of hydroxyethyl acrylate in the presence ofN,N′-methylenebisacrylamide as a cross-linker from the initiator-modified silica nanoparticles. After the silica nanoparticle templates were etched with hydrofluoric acid, the CP nanocapsules with particle size of about 100 nm were obtained. The strategy developed was confirmed with Fourier transform infrared, thermogravimetric analysis and transmission electron microscopy.

  13. Development and Evaluation of Dual Cross-Linked Pulsatile Beads for Chronotherapy of Rheumatoid Arthritis

    Abanesh kumar Bansal


    Full Text Available In the present investigation, pulsatile release beads were prepared by ionic gelation technique. Lornoxicam dual cross-linked beads were prepared by dropping dispersed phase of lornoxicam, pectin, and sodium alginate into the dispersion phase of different concentrations of calcium chloride solution followed by aluminium chloride solution. The formulated beads were further coated by Eudragit L & S 100 in the ratio 1 : 2 w/w in order to achieve desired lag time. In vitro release study showed lag time of 5–8 h before release of lornoxicam from the formulated beads. Thus, formulated dual cross-linked beads when administered at bed time may release lornoxicam when needed most for chronotherapeutics of early morning rheumatoid arthritis attacks in chronic patients.

  14. The cross-linking influence of electromagnetic radiation on water-soluble polyacrylan compositions with biopolymers

    B. Grabowska


    Full Text Available The results of examinations of the cross-linking influence of electromagnetic radiation - in a microwave range – on polyacrylancompositions with biopolymers, are presented in the hereby paper. The cross-linking process of the tested compositions was determined on the basis of the FT-IR spectroscopic methods. It was shown that microwave operations can lead to the formation of new cross-linkedstructures with strong covalent bonds. The adsorption process and formation of active centres in polymer molecules as well as in highsilica sand were found due to microwave radiations. In this process hydroxyl groups (-OH - present in a polymer - and silane groups (Si- O-H - present in a matrix - are mainly taking part. Spectroscopic and strength tests performed for the system: biopolymer binding agent – matrix indicate that the microwave radiation can be applied for hardening moulding sands with biopolymer binders.

  15. Metal cation cross-linked nanocellulose hydrogels as tissue engineering substrates.

    Zander, Nicole E; Dong, Hong; Steele, Joshua; Grant, John T


    The use of cellulose materials for biomedical applications is attractive due to their low cost, biocompatibility, and biodegradability. Specific processing of cellulose to yield nanofibrils further improves mechanical properties and suitability as a tissue engineering substrate due to the similarity to the fibrous structure, porosity, and size-scale of the native extracellular matrix. In order to generate the substrate, nanocellulose hydrogels were fabricated from carboxylated cellulose nanofibrils via hydrogelation using metal salts. Hydrogels cross-linked with Ca(2+) and Fe(3+) were investigated as tissue culture substrates for C3H10T1/2 fibroblast cells. Control substrates as well as those with physically adsorbed and covalently attached fibronectin protein were evaluated with X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR), and enzyme linked immunosorbent assay (ELISA). Significantly more cells were attached to surfaces modified with protein, with the highest number of cells adhered to the calcium cross-linked hydrogels with covalently attached protein.

  16. Biochemical properties of bioplastics made from wheat gliadins cross-linked with cinnamaldehyde.

    Balaguer, M Pau; Gómez-Estaca, Joaquín; Gavara, Rafael; Hernandez-Munoz, Pilar


    The aim of this work has been to study the modification of gliadin films with cinnamaldehyde as a potential cross-linker agent. The molecular weight profile and cross-linking density showed that cinnamaldehyde increased reticulation in the resulting films. The participation of free amino groups of the protein in the newly created entanglements could be a possible mechanism of connection between the polypeptidic chains. The combination of a Schiff base and a Michael addition is a feasible approach to understanding this mechanism. The protein solubility in different media pointed to lower participation by both noncovalent and disulfide bonds in stabilizing the structure of the cross-linked films. The new covalent bonds formed by the cinnamaldehyde treatment hampered water absorption and weight loss, leading to more water-resistant matrices which had not disintegrated after 5 months. The properties of this novel bioplastic could be modified to suit the intended application by using cinnamaldehyde, a naturally occurring compound.

  17. Thermally Conductive-Silicone Composites with Thermally Reversible Cross-links.

    Wertz, J T; Kuczynski, J P; Boday, D J


    Thermally conductive-silicone composites that contain thermally reversible cross-links were prepared by blending diene- and dienophile-functionalized polydimethylsiloxane (PDMS) with an aluminum oxide conductive filler. This class of thermally conductive-silicones are useful as thermal interface materials (TIMs) within Information Technology (IT) hardware applications to allow rework of valuable components. The composites were rendered reworkable via retro Diels-Alder cross-links when temperatures were elevated above 130 °C and required little mechanical force to remove, making them advantageous over other TIM materials. Results show high thermal conductivity (0.4 W/m·K) at low filler loadings (45 wt %) compared to other TIM solutions (>45 wt %). Additionally, the adhesion of the material was found to be ∼7 times greater at lower temperatures (25 °C) and ∼2 times greater at higher temperatures (120 °C) than commercially available TIMs.

  18. pH-sensitive genipin-cross-linked chitosan microspheres for heparin removal.

    Kamiński, Kamil; Zazakowny, Karolina; Szczubiałka, Krzysztof; Nowakowska, Maria


    Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell significantly in water at pH values below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The kinetics of heparin binding was found to be pH dependent and was faster and more efficient at a lower pH. That can be also controlled by the weight of ChGp microspheres used. Rate and efficiency of heparin adsorption at pH 7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrimethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin removal in biomedical applications.

  19. Anisotropic nanoparticles with controllable morphologies from non-cross-linked seeded emulsion polymerization.

    Niu, Qun; Pan, Mingwang; Yuan, Jinfeng; Liu, Xiao; Wang, Xiaomei; Yu, Haifeng


    A simple and elegant approach to fabricate anisotropic P(VC-co-AAEM)/PS nanoparticles with controllable morphologies via emulsifier-free seeded emulsion polymerization is presented. Non-cross-linked P(VC-co-AAEM) seeds with hydrophilic surface are first synthesized through copolymerization of vinyl chloride (VC) and acetoacetoxyethyl methacrylate (AAEM), which are used to prepare P(VC-co-AAEM)/PS NPs with multiple bulges by SEP of styrene. Electron microscopy observation indicates that the content of AAEM in seeds is crucial to control the phase separation and morphology of the composite NPs. Moreover, the thermodynamic immiscibility between PVC and PS is the driving force for the formation of PS bulges onto the P(VC-co-AAEM) seeds. The resultant anisotropic NPs with non-cross-linked feature may promisingly serve as compatibilizers for further polymer processing. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Preparation of bioimprinting cross-linked enzyme aggregates of phenylalanine ammonia lyase and it's partial properties

    Jiandong CUI


    Full Text Available Phenylalanine ammonia lyase (PAL is a key enzyme for production of L-phenylalanine. Currently, PAL is mainly obtained from Rhodotorula PAL However, Rhodotorula PAL exhibits poor stability, which limits its industrial application. In this study, bioimprinting cross-linked enzyme aggregates of PAL (PAL-iCLEAs is developed by combining cross-linked enzyme aggregates technology and imprinted enzyme method. The most optimal imprinting molecule substrate is screened. Moreover, some characteristics of the PAL-iCLEAs are examined. The results show that the most suitable substrates for preparing PAL-iCLEAs is tran-cinnamic acid. The optimal temperature and pH was 50 ℃ and 10.5, respectively. In addition, PAL-iCLEAs shows good reusability, the recovery of PAL activity still remained 32% after reusing 9 times.

  1. Synthesis of Cross-Linked Polymeric Micelle pH Nanosensors

    Ek, Pramod Kumar; Jølck, Rasmus Irming; Andresen, Thomas Lars


    at the micelle shell using CuAAC results in a stabilized micelle pH nanosensor. Compared to the postmicelle modification strategy, the mixed-micellization approach increases the control of the overall composition of the nanosensors.Both approaches provide stable nanosensors with similar pKa profiles and thereby......The design flexibility that polymeric micelles offer in the fabrication of optical nanosensors for ratiometric pH measurements is investigated. pH nanosensors based on polymeric micelles are synthesized either by a mixed-micellization approach or by a postmicelle modification strategy. In the mixed......-micellization approach, self-assembly of functionalized unimers followed by shell cross-linking by copper-catalyzed azide-alkyne cycloaddition (CuAAC) results in stabilized cRGD-functionalized micelle pH nanosensors. In the postmicelle modification strategy, simultaneous cross-linking and fluorophore conjugation...

  2. Mechanical properties and collagen cross-linking of the patellar tendon in old and young men

    Couppé, C; Hansen, P; Kongsgaard, M


    Age-related loss in muscle mass and strength impairs daily life function in the elderly. However, it remains unknown whether tendon properties also deteriorate with age. Cross-linking of collagen molecules provides structural integrity to the tendon fibrils and has been shown to change with age...... in animals but has never been examined in humans in vivo. In this study, we examined the mechanical properties and pyridinoline and pentosidine cross-link and collagen concentrations of the patellar tendon in vivo in old (OM) and young men (YM). Seven OM (67 +/- 3 years, 86 +/- 10 kg) and 10 YM (27 +/- 2...... contractions. Percutaneous tendon biopsies were taken and analyzed for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), pentosidine, and collagen concentrations. We found no significant differences in the dimensions or mechanical properties of the tendon between OM and YM. Collagen concentrations were...

  3. [Use of native and cross-linked collagen membranes for guided tissue and bone regeneration].

    Schwarz, Frank; Sager, Martin; Rothamel, Daniel; Herten, Monika; Sculean, Anton; Becker, Jürgen


    A material which is used as a barrier for GBR/GTR procedures has to satisfy several physicochemical characteristics such as biocompatibility, tissue integration, barrier function, and dimensional stability. Recently, many investigations reported on the use of products derived from type I and type III porcine or bovine collagen. Collagen membranes are predominantly resorbed by enzymatic activity (protease and collagenase). To decrease resorption, various physical and chemical cross-linking techniques have been used. Although nowadays cross-linking of collagen seems to be a commonly used procedure, its impact on physicochemical properties of the membrane is still unknown. The aim of the present literature review is to evaluate the potential use of different collagen membranes for GBR/GTR procedures.

  4. Acute corneal hydrops during pregnancy with spontaneous resolution after corneal cross-linking for keratoconus: a case report.

    Stock, Ricardo Alexandre; Thumé, Thaís; Bonamigo, Elcio Luiz


    Keratoconus may progress to acute corneal hydrops even after cross-linking. In some cases, keratoconus progresses during pregnancy. In this report, we present a case of a patient with increased anterior stromal resistance after cross-linking that would favor nonprogression of keratoconus during pregnancy. We report that cross-linking is likely to have had a protective effect in a white pregnant patient with acute corneal hydrops who showed rapid improvement, as documented by corneal topography. Improvement occurred within 8 days, whereas up to 250 days are reported in the literature. No keratoconus progression occurred in the 20-month follow-up period. Cross-linking failed to prevent the occurrence of acute corneal hydrops after rupture of Descemet's membrane but most likely helped to accelerate the resolution of the condition. Corneal hardening resulting from cross-linking may have also contributed to stabilizing keratoconus during pregnancy.

  5. The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

    D. V. Shepherd


    Full Text Available We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropylcarbodiimide, NHS (N-hydroxysuccinimide combination was considered. Atomic Force Microscopy and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer or water in any of the fibres.

  6. The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

    Shepherd, D. V.; Shepherd, J. H.; Ghose, S.; Kew, S. J.; Cameron, R. E.; Best, S. M.


    We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.

  7. Molecular contacts for chlorosome envelope proteins revealed by cross-linking studies with chlorosomes from Chlorobium tepidum

    Li, Hui; Frigaard, Niels-Ulrik; Bryant, Donald A


    type and mutants lacking a single chlorosome protein were cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and analyzed by gel electrophoresis. Similar cross-linking products were observed when the time and temperature were varied or when EDC...... was replaced with glutaraldehyde. Specific interactions between chlorosome proteins in cross-linked products were identified by immunoblotting with polyclonal antibodies raised against recombinant chlorosome proteins. We confirmed these interactions by demonstrating that these products were missing...... in appropriate mutants. Confirming the location of CsmA in the paracrystalline baseplate, cross-linking showed that CsmA forms dimers, trimers, and homomultimers as large as dodecamers and that CsmA directly interacts with the Fenna-Matthews-Olson protein. Cross-linking further suggests that the precursor form...

  8. Synthesis and Characterisation of Novel Cross Linked Biopolyesters from Olive Oil as Eco-friendly Biodegradable Materials

    J. Shakina


    Full Text Available Novel cross linked biopolyesters were synthesised from naturally available olive oil, oligomeric polytriglyceride fumarate (o-PTF was prepared by glycerolysis of the olive oil followed by condensation with maleic anhydride. The (o-PTF was characterised by spectral (FTIR, UV, NMR and physicochemical properties (specific gravity, viscosity, saponification value, iodine value. The molecular weight of o-PTF was estimated using gel permeation chromatography (GPC. The cross linked biopolyesters were prepared by treating o-PTF of olive with N-vinyl-2-pyrrolidone and vinyl acetate. The cross linked biopolyesters were evaluated for spectral properties (FT IR, cross linked density, thermal properties, mechanical properties and biodegradation. The potential antifungal and anti bacterial activities of the newly prepared biopolyesters were evaluated. The environment degradation was assessed by soil burial test. The outcome of the studies has revealed that the newly prepared cross linked biopolyesters are potential biodegradable material for various consumer application like package materials and agricultural applications.

  9. Blind testing of cross-linking/mass spectrometry hybrid methods in CASP11.

    Schneider, Michael; Belsom, Adam; Rappsilber, Juri; Brock, Oliver


    Hybrid approaches combine computational methods with experimental data. The information contained in the experimental data can be leveraged to probe the structure of proteins otherwise elusive to computational methods. Compared with computational methods, the structures produced by hybrid methods exhibit some degree of experimental validation. In spite of these advantages, most hybrid methods have not yet been validated in blind tests, hampering their development. Here, we describe the first blind test of a specific cross-link based hybrid method in CASP. This blind test was coordinated by the CASP organizers and utilized a novel, high-density cross-linking/mass-spectrometry (CLMS) approach that is able to collect high-density CLMS data in a matter of days. This experimental protocol was developed in the Rappsilber laboratory. This approach exploits the chemistry of a highly reactive, photoactivatable cross-linker to produce an order of magnitude more cross-links than homobifunctional cross-linkers. The Rappsilber laboratory generated experimental CLMS data based on this protocol, submitted the data to the CASP organizers which then released this data to the CASP11 prediction groups in a separate, CLMS assisted modeling experiment. We did not observe a clear improvement of assisted models, presumably because the properties of the CLMS data-uncertainty in cross-link identification and residue-residue assignment, and uneven distribution over the protein-were largely unknown to the prediction groups and their approaches were not yet tailored to this kind of data. We also suggest modifications to the CLMS-CASP experiment and discuss the importance of rigorous blind testing in the development of hybrid methods. Proteins 2016; 84(Suppl 1):152-163. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  10. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre


    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  11. Progress of research on corneal collagen cross-linking for corneal melting

    Ke-Ren Xiao


    Full Text Available Corneal collagen cross-linking(CXLcould increase the mechanical strength, biological stability and halt ectasia progression due to covalent bond formed by photochemical reaction between ultraviolet-A and emulsion of riboflavin between collagen fibers in corneal stroma. Corneal melting is an autoimmune related noninfectious corneal ulcer. The mechanism of corneal melting, major treatment, the basic fundamental of ultraviolet-A riboflavin induced CXL and the clinical researches status and experiment in CXL were summarized in the study.

  12. Histological response to injected gluteraldehyde cross-linked bovine collagen based implant in a rat model

    Cağlar Melda


    Full Text Available Abstract Background The aim of present study is to investigate the short and long term histopathological alterations caused by submucosal injection of gluteraldehyde cross-linked bovine collagen based on an experimental rat model. Methods Sixty Sprague-Dawley rats were assigned into two groups as group I and II each containing 30 rats. 0.1 ml of saline solution and 0.1 ml of gluteraldehyde cross-linked bovine collagen were injected into the submucosa of bladder of first (control and second groups, respectively. Both group I and II were further subdivided into 3 other groups as Group IA, IB, IC and Group IIA, IIB, IIC according to the sacrification period. Group IA and IIA, IB and IIB, IC and IIC rats (10 rats for each group were sacrificed 3, 6, and 12 months after surgical procedure, respectively. Two slides prepared from injection site of the bladder were evaluated completely for each rat by being unaware of the groups and at random by two independent senior pathologists to determine the fibroblast invasion, collagen formation, capillary ingrowth and inflammatory reaction. Additionally, randomized brain sections from each rat were also examined to detect migration of the injection material. The measurements were made using an ocular micrometer at ×10 magnification. The results were assessed using t-tests for paired and independent samples, with p Results Migration to the brain was not detected in any group. Significant histopathological changes in the gluteraldehyde cross-linked bovine collagen injected groups were fibroblast invasion in 93.3%, collagen formation in 73.3%, capillary ingrowth in 46.6%, inflamatory reaction in 20%. Conclusion We emphasize that the usage of gluteraldehyde cross-linked bovine collagen in children appears to be safe for endoscopic treatment of vesicoureteral reflux.

  13. Synthesis and Properties of Lactic Acid-based Cross-linked Poly(ester-amide)

    Yue Ying HE; Cong Ming XIAO


    A novel lactic acid-based cross-linked poly(ester-amide) (LCPEA) was synthesized. The gel fraction of the LCPEA could be modulated by the reaction conditions and it affected the mechanical and thermal properties of the LCPEA. The tensile strength, elastic modulus and bend strength of the LCPEA of 65% gel fraction were 4.65, 136.55 and 39.63 MPa, respectively. The thermal decomposition temperature (50 wt%) of the LCPEA was around 410 ℃.

  14. Identification of dityrosine cross-linked sites in oxidized human serum albumin.

    Annibal, Andrea; Colombo, Graziano; Milzani, Aldo; Dalle-Donne, Isabella; Fedorova, Maria; Hoffmann, Ralf


    Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.

  15. Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.

    Au, Phil Chi Khang; Helliwell, Chris; Wang, Ming-Bo


    RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.

  16. Long-term results of cornea collagen cross-linking with riboflavin for keratoconus

    Vinay Agrawal


    Full Text Available Corneal collagen cross-linking with riboflavin and UVA light (CXL is the only method designed to arrest the progression of keratoconus. Visual improvement generally starts 3 months after treatment. Reduction is coma seen on aberrometry in early postoperative phase is also responsible for the improvement in visual acuity. In the light of currently available data we can thus say that CXL is a safe procedure that is successful in arresting keratoconus.

  17. Comparative study between novel self cross-linking and conventional fluorinated acrylic latex

    Li Jun Chen


    Novel self cross-linking fluorinated acrylic latex (SCLFAL) has been successfully prepared via starved seeded semi-batch emulsion polymerization. The resultant SCLFAL is characterized by Fourier transform infrared (FTTR) spectrometry. Contact angle (CA) and glass transition temperature (Tg) of the film are investigated. Results show that CA and Tg of the film can be improved when the moderate amount of HPMA is introduced into the mixed monomers.

  18. Effects of polymerization on the oxygen carrying and redox properties of diaspirin cross-linked hemoglobin.

    Rogers, M S; Ryan, B B; Cashon, R E; Alayash, A I


    Human hemoglobin site specifically cross-linked with bis(3,5-dibromosalicyl)fumarate results in a low oxygen affinity hemoglobin-based red cell substitute (alpha-DBBF). Polymerization of alpha-DBBF by bis(maleoylglycylamide) polyethylene glycol (BMAA-PEG) yields poly alpha-DBBF which offers the added benefits of reduced renal clearance and increased retention in the vascular circulation. Oxygen equilibrium curves for poly alpha-DBBF are slightly left-shifted (higher O2 affinity) compared to those of alpha-DBBF; with a diminished cooperativity and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide binding), poly alpha-DBBF exhibits a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. The rate of nitric oxide binding to the oxidized form of poly alpha-DBBF shows little or no change compared to the intramolecularly cross-linked derivative. The reduction of cyanomet poly alpha-DBBF by dithionite is several fold faster than that of HbA0 and alpha-DBBF whereas the slow subsequent cyanide dissociation from the ferrous iron remained unchanged among all proteins. The propensity of poly alpha-DBBF for auto-oxidation is slightly enhanced over alpha-DBBF whereas the extent of oxidative modification by hydrogen peroxide is very similar. Polymerization appears to selectively modify ligand interactions and redox kinetics of the tetrameric cross-linked form which reflects a possibly more open heme pocket. The data suggests that changes in oxygenation properties of hemoglobin brought about by a given modification are not necessarily predictive of other functional changes.

  19. A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation.

    MacGregor, Helen J; Kato, Yoji; Marshall, Lindsay J; Nevell, Thomas G; Shute, Janis K


    The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

  20. Polymer Composition and Substrate Influences on the Adhesive Bonding of a Biomimetic, Cross-Linking Polymer

    Matos-Pérez, Cristina R.; White, James D.; Wilker, Jonathan J.


    Hierarchical biological materials such as bone, sea shells, and marine bioadhesives are providing inspiration for the assembly of synthetic molecules into complex structures. The adhesive system of marine mussels has been the focus of much attention in recent years. Several catechol-containing polymers are being developed to mimic the cross-linking of proteins containing 3,4-dihydroxyphenylalanine (DOPA) used by shellfish for sticking to rocks. Many of these biomimetic polymer systems have be...

  1. Sorption of adamantane phenylamide derivatives on hyper-cross-linked polystyrene from water-acetonitrile eluents

    Shafigulin, R. V.; Konstantinov, A. V.; Bulanova, A. V.; Il'in, M. M.; Davankov, V. A.


    Study of the main physicochemical features of the sorption of phenylamide adamantane derivatives on hyper-cross-linked polystyrene from water-acetonitrile solutions shows that both hydrophobic and electronic interactions make a large contribution to retention, especially for a chlorine-containing derivative in which there are π- p and π- d interactions between the outer-shell electrons of the chlorine atom in addition to π- π interactions between aromatic fragments of the sorbate and sorbent.

  2. Fabrication and properties of irradiation-cross-linked poly(vinyl alcohol)/clay aerogel composites.

    Chen, Hong-Bing; Liu, Bo; Huang, Wei; Wang, Jun-Sheng; Zeng, Guang; Wu, Wen-Hao; Schiraldi, David A


    Poly(vinyl alcohol) (PVOH)/clay aerogel composites were fabricated by an environmentally friendly freeze-drying of the aqueous precursor suspensions, followed by cross-linking induced by gamma irradiation without chemical additives. The influences of cross-linking conditions, i.e., absorbed dose and polymer loading as well as density on the aerogel structure and properties, were investigated. The absorbed dose of 30 kGy was found to be the optimum dose for fabricating strong PVOH composites; the compressive modulus of an aerogel prepared from an aqueous suspension containing 2 wt % PVOH/8 wt % clay increased 10-fold, and that containing 1 wt % PVOH/9 wt % clay increased 12 times upon cross-linking with a dose of 30 kGy. Increasing the solids concentration led to an increase in the mechanical strength, in accordance with the changes in microstructure from layered structure to network structure. The increase of absorbed dose also led to decreased porous size of the network structure. Cross-linking and the increase of the PVOH lead to decreased thermal stability. The strengthened PVOH/clay aerogels possess very low flammability, as measured by cone calorimetry, with heat, smoke, and volatile products release value decreasing as increasing clay content. The mechanism of flame retardation in these materials was investigated with weight loss, FTIR, WAXD, and SEM of the burned residues. The proposed mechanism is that with decreasing fuel content (increasing clay content), increased heat and mass transport barriers are developed; simultaneously low levels of thermal conductivity are maintained during the burning.

  3. Corneal Cross-Linking: An Example of Photoinduced Polymerization as a Treatment Modality in Keratoconus.

    Kubrak-Kisza, Magdalena; Kisza, Krystian Jerzy; Misiuk-Hojło, Marta


    The cornea is one of the principal refractive elements in the human eye and plays a crucial role in the process of vision. Keratoconus is the most common corneal dystrophy, found mostly among young adults. It is characterized by a reduced number of collagen cross-links in the corneal stroma, resulting in reduced biomechanical stability and an abnormal shape of the cornea. These changes lead to progressive myopia, corneal thinning, central scarring and irregular astigmatism, causing severely impaired vision. Hard contact lenses, photorefractive keratectomy or intracorneal rings are the most common treatment options for refractive error caused by keratoconus. However, these techniques do not treat the underlying cause of the corneal ectasia and therefore are not able to stop the progression of the disease. Riboflavin photoinduced polymerization of corneal collagen, also known as corneal cross-linking (CXL), has been introduced as the first therapy which, by stabilizing the structure of the cornea, prevents the progression of keratoconus. It stiffens the cornea using the photo-sensitizer riboflavin in combination with ultraviolet irradiation. This is a current review of the CXL procedure as a therapy for keratoconus, which relies on photoinduced polymerization of human tissue. We have focused on its biomechanical and physiological influences on the human cornea and have reviewed the previous and current biochemical theories behind cross-linking reactions in the cornea.

  4. An Update on the Safety and Efficacy of Corneal Collagen Cross-Linking in Pediatric Keratoconus

    El Rami, Hala; Chelala, Elias; Fakhoury, Henry; Cherfan, Carole; Cherfan, George; Jarade, Elias


    Keratoconus is a degenerative disease that affects adolescents and young adults and presents with variable thinning and conical deformation of the corneal apex. The resultant irregular astigmatism can progress to levels that can significantly affect everyday activities and overall quality of life. Therefore, stopping the progression of the disease is an essential part in managing patients with keratoconus. Corneal collagen cross-linking is a minimally invasive procedure that stiffens the anterior corneal stroma by creating strong covalent bonds between collagen fibrils. Over the past decade, many studies have proved its safety and efficacy in halting keratoconus progression in adults. This review of the literature highlights the growing trend towards using this treatment in pediatric keratoconic patients. In children, keratoconus tends to be more severe and fast progression is often encountered requiring closer follow-up intervals. Standard cross-linking shows comparable results in children with a good safety-efficacy profile during follow-up periods of up to three years. Further research is needed to standardize and evaluate transepithelial and accelerated cross-linking protocols as these could be of tremendous help in a population where cooperation and compliance are major issues. PMID:26491663

  5. Analysis of the effective dose of ultraviolet light in corneal cross-linking.

    Zhang, Yong; Wang, Kuan-Chen; Chang, Chao-Kai; Lin, Jui-Teng


    To analyze the efficacy of ultraviolet (UV) light initiating corneal cross-linking (CXL). The time-dependent absorption of UV light due to the depletion of the initiator (riboflavin) was calculated. The effective dose of CXL with corneal surface covered by a thin layer of riboflavin was derived analytically. The cross linking time was calculated by the depletion level of the riboflavin concentration. A comprehensive method was used to derive analytic formulas. The effective dose of CXL was reduced by a factor (R) which was proportional to the thickness (d) and concentrations (C0) of the riboflavin surface layer. Our calculations showed that the conventional dose of 5.4 J/cm(2) had a reduced effective dose of 4.3 and 3.45 J/cm(2), for d was 100 and 200 µm, respectively, and C0=0.1%. The surface cross linking time was calculated to be T*=10.75s, for a depletion level of 0.135 and UV initial intensity of 30 mW/cm(2). The volume T* was exponentially increasing and proportional to exp (bdC0), with b being the steady state absorption coefficient. The effective dose of CXL is reduced by a factor proportional to the thickness and concentrations of the riboflavin surface layer. The wasted dose should be avoided by washing out the extra riboflavin surface layer prior to the UV light exposure.

  6. Riboflavin-UVA-induced corneal collagen cross-linking in pediatric patients.

    Caporossi, Aldo; Mazzotta, Cosimo; Baiocchi, Stefano; Caporossi, Tomaso; Denaro, Rosario; Balestrazzi, Angelo


    Evaluation of stability and functional response after riboflavin-UVA–induced cross-linking in a population of patients younger than 18 years with progressive keratoconus after 36 months of follow-up. Prospective nonrandomized phase II open trial conducted at the Department of Ophthalmology, Siena University, Italy. The "Siena CXL Pediatrics" trial involved 152 patients aged 18 years or younger (10–18 years) with clinical and instrumental evidence of keratoconus progression. The population was divided into 2 groups according to corneal thickness (>450 and 450 μm) and +0.14 and +0.15 Snellen lines, respectively, in the thinner group (corneal thickness <450 μm). Patients in the latter group already showed a better and faster functional recovery than the thicker group at 3-month follow-up. Topographic results showed statistically significant improvement in K readings and asymmetry index values. Coma reduction was also statistically significant. The study demonstrated significant and rapid functional improvement in pediatric patients younger than 18 years with progressive keratoconus, undergoing riboflavin-UVA–induced cross-linking. In pediatric age, a good functional response and keratoconus stability was obtained after corneal cross-linking in a 36-month follow-up.

  7. Fluorescent and cross-linked organic-inorganic hybrid nanoshells for monitoring drug delivery.

    Sun, Lijuan; Liu, Tianhui; Li, Hua; Yang, Liang; Meng, Lingjie; Lu, Qinghua; Long, Jiangang


    Functionalized and monodisperse nanoshells have attracted significant attention owing to their well-defined structure, unique properties, and wide range of potential applications. Here, the synthesis of cross-linked organic-inorganic hybrid nanoshells with strong fluorescence properties was reported via a facile precipitation polymerization of hexachlorocyclotriphosphazene (HCCP) and fluorescein on silica particles used as templates. The resulting poly(cyclotriphosphazene-co-fluorescein) (PCTPF) nanoshells were firm cross-linked shells with ∼2.2 nm mesopores that facilitated the transport of drug molecules. The fluorescent nanoshells also exhibited excellent water dispersibility and biocompatibility; thus, they can be considered as ideal drug vehicles with high doxorubicin storage capacity (26.2 wt %) and excellent sustained release (up to 14 days). Compared to doxorubicin (DOX) alone, the PCTPF nanoshells more efficiently delivered DOX into and killed cancer cells. Moreover, the PCTPF nanoshells also exhibited remarkable fluorescent emission properties and improved photobleaching stability in both suspension and solid state owing to the covalent immobilization of fluorescein in the highly cross-linked organic-inorganic hybrids. The exceptional fluorescent properties enabled the release of DOX as well as the distribution of nanoshells and DOX to be monitored.

  8. Preparation and Characterization of a pH-Responsive Core Cross-linked Polymer Micelle

    Kousaka, Shouta; Sugahara, Makoto; Endo, Tatsuya; Yusa, Shin-ichi, E-mail: [Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280 (Japan)


    Poly(ethylene glycol)-b-poly(2-(diethylamino) ethyl methacrylate-co-2-cinnamoyl-oxyethyl acrylate) (PEG-b-P(DEA/CEA)) was prepared by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. pH-responsive association behaviour of PEG-b-P(DEA/CEA) in 0.1 M NaCl was characterized by dynamic light scattering (DLS). As solution pH is increased from an acidic pH, the hydrodynamic radius (R{sub h}) increases, indicative of the polymer micelle formation. The formation of a micelle was also supported by static light scattering (SLS) data. The cinnamoyl groups in the core of the polymer micelle undergo photodimerization, yielding cross-links between polymer chains. The core of the polymer micelle was fixed, which was confirmed by DLS, SLS, and small angle X-ray scattering (SAXS) techniques. When pH is decreased to 3, R{sub h} of the core cross-linked (CCL) polymer micelle slightly increases due to the protonation of the DEA unit in the cross-linked core. The reversible pH-induced swelling and shrinking behaviour can be observed.

  9. Preparation of novel ferrocene-based shell cross-linked thermoresponsive hybrid micelles with antitumor efficacy.

    Wei, Hua; Quan, Chang-Yun; Chang, Cong; Zhang, Xian-Zheng; Zhuo, Ren-Xi


    The shell cross-linked (SCL) thermoresponsive hybrid poly(N-isopropylacrylamide-co-aminoethyl methacrylate)-b-polymethyl methacrylate (P(NIPAAm-co-AMA)-b-PMMA) micelle consisting of a cross-linked thermoresponsive hybrid shell and a hydrophobic core domain was fabricated via a two-step process: micellization of P(NIPAAm-co-AMA)-b-PMMA in aqueous solution followed by cross-linking of the hydrophilic shell layer via the amidation reaction between the amine groups of AMA units and the carboxylic acid functions of 1,1'-ferrocenedicarboxylic acid. The SCL micelle showed reversible dispersion/aggregation in response to the temperature cycles through the lower critical solution temperature (LCST) of the thermoresponsive hybrid shell at around 36 degrees C, observed by turbidity measurements and dynamic light scattering (DLS). Besides the usage as an inorganic difunctional cross-linker, the inorganic ferrocene segment further endowed the SCL hybrid micelle with the antitumor efficacy, namely, the resulting SCL micelle exhibited a remarkable cytotoxic effect for HeLa cells with a very low IC50. The results showed that the SCL hybrid micelle developed in this study could be potentially used as an antitumor agent, which is unique compared to the conventional tumor therapy by using the antitumor drug loaded in the micellar core.

  10. EGDMA-cross-linked polystyrene resin: An efficient support for gel phase peptide synthesis

    P K Ajikumar; K S Devaky


    This article illustrates the application of a 2% ethyleneglycol dimethacrylate-cross-linked polystyrene support (EGDMA-PS) in manual solid phase peptide synthesis. This copolymer has been characterised and optimised for peptide synthesis by performing the synthesis of a few model peptides and two biologically important peptides. EGDMA-cross-linked polystyrene support was prepared by the suspension polymerisation of the monomers EGDMA and styrene. EGDMA-PS resin undergoes facile swelling in a variety of solvents, both polar and nonpolar, used in peptide synthesis. The polymer was functionalised by Friedel-Crafts chloromethylation reaction. Peptides were assembled on a 2% cross-linked chloromethyl polymer support of capacity 1.63 mmol Cl/g. The biological peptides synthesised are an 11-residue peptide ATP binding site of the CDC2 kinase and a difficult sequence-a nineresidue peptide 34-42 corresponding to a portion of the hydrophobic terminus of the-amyloid protein 1-42. After synthesis, the peptides were cleaved from the support by treating with neat TFA. Purity of the peptides obtained in good yield was checked by TLC and HPLC methods and found to be fairly high.

  11. Core Cross-linked Star Polymers for Temperature/pH Controlled Delivery of 5-Fluorouracil

    Elizabeth Sánchez-Bustos


    Full Text Available RAFT polymerization with cross-linking was used to prepare core cross-linked star polymers bearing temperature sensitive arms. The arms consisted of a diblock copolymer containing N-isopropylacrylamide (NIPAAm and 4-methacryloyloxy benzoic acid (4MBA in the temperature sensitive block and poly(hexyl acrylate forming the second hydrophobic block, while ethyleneglycol dimethacrylate was used to form the core. The acid comonomer provides pH sensitivity to the arms and also increases the transition temperature of polyNIPAAm to values in the range of 40 to 46°C. Light scattering and atomic force microscopy studies suggest that loose core star polymers were obtained. The star polymers were loaded with 5-fluorouracil (5-FU, an anticancer agent, in values of up to 30 w/w%. In vitro release experiments were performed at different temperatures and pH values, as well as with heating and cooling temperature cycles. Faster drug release was obtained at 42°C or pH 6, compared to normal physiological conditions (37°C, pH 7.4. The drug carriers prepared acted as nanopumps changing the release kinetics of 5-FU when temperatures cycles were applied, in contrast with release rates at a constant temperature. The prepared core cross-linked star polymers represent advanced drug delivery vehicles optimized for 5-FU with potential application in cancer treatment.

  12. Molecular dynamics simulations of highly cross-linked polymer networks: prediction of thermal and mechanical properties

    Shenogina, Natalia; Tsige, Mesfin; Mukhopadhyay, Sharmila; Patnaik, Soumya


    We use all-atom molecular dynamics (MD) simulations to predict the mechanical and thermal properties of thermosetting polymers. Atomistic simulation is a promising tool which can provide detailed structure-property relationships of densely cross-linked polymer networks. In this work we study the thermo-mechanical properties of thermosetting polymers based on amine curing agents and epoxy resins and have focused on the DGEBA/DETDA epoxy system. At first we describe the modeling approach to construction of realistic all-atom models of densely cross-linked polymer matrices. Subsequently, a series of atomistic simulations was carried out to examine the simulation cell size effect as well as the role of cross-linking density and chain length of the resin strands on thermo-mechanical properties at different temperatures. Two different methods were used to deform the polymer networks. Both static and dynamic approaches to calculating the mechanical properties were considered and the thermo-mechanical properties obtained from our simulations were found in reasonable agreement with experimental values.

  13. Application of cross-linked and hydrolyzed arabinoxylans in baking of model rye bread.

    Buksa, Krzysztof; Nowotna, Anna; Ziobro, Rafał


    The role of water extractable arabinoxylan with varying molar mass and structure (cross-linked vs. hydrolyzed) in the structure formation of rye bread was examined using a model bread. Instead of the normal flour, the dough contained starch, arabinoxylan and protein, which were isolated from rye wholemeal. It was observed that the applied mixes of these constituents result in a product closely resembling typical rye bread, even if arabinoxylan was modified (by cross-linking or hydrolysis). The levels of arabinoxylan required for bread preparation depended on its modification and mix composition. At 3% protein, the maximum applicable level of poorly soluble cross-linked arabinoxylan was 3%, as higher amounts of this preparation resulted in an extensively viscous dough and diminished bread volume. On the other hand highly soluble, hydrolyzed arabinoxylan could be used at a higher level (6%) together with larger amounts of rye protein (3% or 6%). Further addition of arabinoxylan leads to excessive water absorption, resulting in a decreased viscosity of the dough during baking and insufficient gas retention. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Wood Sawdust/Natural Rubber Ecocomposites Cross-Linked by Electron Beam Irradiation

    Elena Manaila


    Full Text Available The obtaining and characterization of some polymeric eco-composites based on wood sawdust and natural rubber is presented. The natural rubber was cross-linked using the electron beam irradiation. The irradiation doses were of 75, 150, 300 and 600 kGy and the concentrations of wood sawdust were of 10 and 20 phr, respectively. As a result of wood sawdust adding, the physical and mechanical properties such as hardness, modulus at 100% elongation and tensile strength, showed significant improvements. The presence of wood sawdust fibers has a reinforcing effect on natural rubber, similar or better than of mineral fillers. An increase in the irradiation dose leads to the increasing of cross-link density, which is reflected in the improvement of hardness, modulus at 100% elongation and tensile strength of blends. The cross-linking rates, appreciated using the Flory-Rehner equation, have increased with the amount of wood sawdust in blends and with the irradiation dose. Even if the gel fraction values have varied irregularly with the amount of wood sawdust and irradiation dose it was over 90% for all blends, except for the samples without wood sawdust irradiated with 75 kGy. The water uptake increased with increasing of fiber content and decreased with the irradiation dose.

  15. Cross-linked polystyrene sulfonic acid and polyethylene glycol as a low-fouling material.

    Alghunaim, Abdullah; Zhang Newby, Bi-min


    A negatively charged hydrophilic low fouling film was prepared by thermally cross-linking a blend consisting of polystyrene sulfonic acid (PSS) and polyethylene glycol (PEG). The film was found to be stable by dip-washing. The fouling resistance of this material toward bacterial (Escherichia coli) and colloidal (polystyrene particles) attachment, non-specific protein (fibronectin) adsorption and cell (3T3 NIH) adhesion was evaluated and was compared with glass slides modified with polyethylene glycol (PEG) brushes, oxidized 3-mercaptopropyltrimethoxysilane (sulfonic acid, SA), and n-octadecyltrichlorosilane (OTS). The extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory and thermodynamic models based on surface energy were used to explain the interaction behaviors of E. coli/polystyrene particles-substrate and protein-substrate interactions, respectively. The cross-linked PSS-PEG film was found to be slightly better than SA and PEG toward resisting non-specific protein adsorption, and showed comparable low attachment results as those of PEG toward particle, bacterial and NIH-3T3 cells adhesion. The low-fouling performance of PSS-PEG, a cross-linked film by a simple thermal curing process, could allow this material to be used for applications in aqueous environments, where most low fouling hydrophilic polymers, such as PSS or PEG, could not be easily retained.

  16. Light-harvesting cross-linked polymers for efficient heterogeneous photocatalysis.

    Wang, Cheng; Xie, Zhigang; deKrafft, Kathryn E; Lin, Wenbin


    Nonporous, phosphorescent cross-linked polymers (Ru-CP and Ir-CP) were synthesized via Pd-catalyzed Sonogashira cross-coupling reactions between tetra(p-ethynylphenyl)methane and dibrominated Ru(bpy)(3)(2+) or Ir(ppy)(2)(bpy)(+), respectively. The resultant particulate cross-linked polymer (CP) materials have very high catalyst loadings (76.3 wt % for Ru-CP and 71.6 wt % for Ir-CP), and are nonporous with negligibly small surface areas (2.9 m(2)/g for Ru-CP and 2.7 m(2)/g for Ir-CP). Despite their nonporous nature, the insoluble CP materials serve as highly active and recyclable heterogeneous photocatalysts for a range of organic transformations such as aza-Henry reaction, aerobic amine coupling, and dehalogenation of benzyl bromoacetate. An efficient light-harvesting mechanism, which involves collection of photons by exciting the (3)MLCT states of the phosphors and migration of the excited states to the particle surface, is proposed to account for the very high catalytic activities of these nonporous CPs. Steady-state and time-resolved emission data, as well as the reduced catalytic activity of Os(bpy)(3)(2+)-doped Ru-CPs supports efficient excited state migration for the CP frameworks. This work uncovers a new strategy in designing highly efficient photocatalysts based on light-harvesting cross-linked polymers.

  17. Cross-linked carbon nanotubes buckygel actuators: an in-depth study

    Gendron, David; Bubak, Grzegorz; Ceseracciu, Luca; Ansaldo, Alberto; Ricci, Davide


    Recently, materials that can convert electrical energy into mechanical work have drawn great attention. Applications in robotics, tactile or optical displays and microelectrochemical systems are currently investigated. Likewise, interest in actuators devices is increasing toward applications where low voltage and low weight properties are required. One way to achieve such prerequisites is to combine the mechanical and electronic properties of carbon nanotubes (CNTs) with the stability and conductivity of ionic liquids. Indeed, the CNTs can be dispersed in ionic liquids to form hybrid composites also named bucky gels, thanks to the non-covalent (π-π stacking and cation-π) interactions. In our previous studies, we demonstrated an improvement in actuator performance whilst using cross-linked CNTs. Indeed, our preliminary results showed an increase in the capacitance together with a faster response of the actuator. At the time, these results were explained by an actuation mechanism model. Herein, we designed new experiments in order to allow us to get a deeper insight in the effect the crosslinking process on the carbon nanotubes properties. Thus, we present a set of electromechanical and electrochemical data that shed light on the chemical modification of the CNTs, the different cross-linking strategies and also on the uses of cross-linked CNTS polymer blends. Finally, corresponding bucky gels actuators performances will also be discussed.

  18. An atomistic model for cross-linked HNBR elastomers used in seals

    Molinari, Nicola; Sutton, Adrian; Stevens, John; Mostofi, Arash


    Hydrogenated nitrile butadiene rubber (HNBR) is one of the most common elastomeric materials used for seals in the oil and gas industry. These seals sometimes suffer ``explosive decompression,'' a costly problem in which gases permeate a seal at the elevated temperatures and pressures pertaining in oil and gas wells, leading to rupture when the seal is brought back to the surface. The experimental evidence that HNBR and its unsaturated parent NBR have markedly different swelling properties suggests that cross-linking may occur during hydrogenation of NBR to produce HNBR. We have developed a code compatible with the LAMMPS molecular dynamics package to generate fully atomistic HNBR configurations by hydrogenating initial NBR structures. This can be done with any desired degree of cross-linking. The code uses a model of atomic interactions based on the OPLS-AA force-field. We present calculations of the dependence of a number of bulk properties on the degree of cross-linking. Using our atomistic representations of HNBR and NBR, we hope to develop a better molecular understanding of the mechanisms that result in explosive decompression.

  19. Capacity of cognitive radio under imperfect secondary and cross link channel state information

    Sboui, Lokman


    In this paper, we study the ergodic capacity of secondary user channel in a spectrum sharing scenario in which the secondary transmitter is instantaneously aware of estimated versions of the cross link (between the secondary transmitter and the primary receiver) and the secondary link Channel State Information (CSI). The secondary link optimal power profile along with the ergodic capacity are derived for a class of fading channels, under an average power constraint and an instantaneous interference outage constraint. We also show that our framework is rather general as it encompasses several previously studied spectrum sharing settings as special cases. In order to gain some insights on the capacity behavior, numerical results are shown for independent Rayleigh fading channels where it is found for instance, that at low SNR regime, only the secondary channel estimation matters and that the cross link CSI has no effect on the ergodic capacity; whereas at high SNR regime, the capacity is rather driven by the cross link CSI. © 2011 IEEE.

  20. Scheimpflug imaged corneal changes on anterior and posterior surfaces after collagen cross-linking

    Ziad Hassan


    Full Text Available AIM: To compare the anterior and posterior corneal parameters before and after collagen cross-linking therapy for keratoconus.METHODS: Collagen cross-linking was performed in 31 eyes of 31 keratoconus patients (mean age 30.6±8.9y. Prior to treatment and an average 7mo after therapy, Scheimpflug analysis was performed using Pentacam HR. In addition to corneal thickness assessments, corneal radius, elevation, and aberrometric measurements were performed both on anterior and posterior corneal surfaces. Data obtained before and after surgery were statistically analyzed.RESULTS: In terms of horizontal and vertical corneal radius, and central corneal thickness no deviations were observed an average 7mo after operation. Corneal higher order aberration showed no difference neither on anterior nor on posterior corneal surfaces. During follow-up period, no significant deviation was detected regarding elevation values obtained by measurement in mm units between the 3.0-8.0 mm-zones.CONCLUSION: Corneal stabilization could be observed in terms of anterior and posterior corneal surfaces, elevation and higher order aberration values 7mo after collagen cross-linking therapy for keratoconus.

  1. Synthesis of cross-linked carbon nanotube mats and their applications

    Ventura, Darryl N.


    Carbon nanotubes (CNTs) possess excellent tensile strength and electron transport properties that make them a promising component in future materials and technologies. The covalent cross-linking of carbon nanotubes is one avenue of producing thin, flexible mats that can be used in a variety of applications. Here we describe the cross-linking of functionalized CNTs through the Michael addition and imine formation reaction schemes. Our method of cross-linking is an improvement over traditional techniques such as laser irradiation because it is not detrimental to the nanotube structure. Our method also does not rely on weak van der Waals interactions that are sometimes used in the synthesis of nanotube mats. Furthermore, our filtration-from-suspension procedure can be readily scaled up for industrial use and does not require high pressure or high temperature reaction chambers. Such CNT-based composites can be used in a wide variety of applications. We demonstrate their use in a hydrogen sensor and a dye-sensitized solar cell.

  2. Evaluation of ultrasonic atomization as a new approach to prepare ionically cross-linked chitosan microparticles.

    Albertini, Beatrice; Passerini, Nadia; Rodriguez, Lorenzo


    Ultrasonic atomization was evaluated as a new approach for the preparation of ionically cross-linked controlled-release chitosan microparticles loaded with theophylline as the model drug, using tripolyphosphate (TPP) as counter-ion. It was possible to nebulize both 2% and 3% (w/v) chitosan solutions as a function of their viscosity, usually not processed by employing the conventional nebulizer. The results of the chitosan molecular characterization using the SEC-MALS analysis revealed that ultrasonic atomization caused a certain depolymerization, probably due to the main chain scission of the 1,4-glycosidic bond; however, Fourier transform-infrared spectroscopy revealed the absence of other chemical modifications. The ultrasonic atomization allowed preparation of TPP cross-linked chitosan microparticles mostly ranging between 50 and 200 mum. As regards manufacturing parameters, the linking time and washing medium were found to affect the properties of the microparticles, while the stirring rate of the TPP solution did not show any influence. The evaluation of the formulation variables revealed that chitosan concentration strongly affected both the feasibility of the ultrasonic atomization and the drug release. All the microparticles showed an encapsulation efficiency of > 50 % and, after an initial burst effect, a controlled release of drug for 48 h. In conclusion, the ultrasonic atomization could be proposed as a robust and innovative single-step procedure with scale-up potential to successfully prepare ionically cross-linked chitosan microparticles.

  3. Optimizing performance in cross-linking negative-tone molecular resists

    Lawson, Richard A.; Narcross, Hannah; Sharp, Brandon; Chun, Jun Sung; Neisser, Mark; Tolbert, Laren M.; Henderson, Clifford L.


    Many different types of non-traditional resist designs have shown promise for future generations of patterning, but there is a greater need for understanding and developing additives and ancillary materials for these novel resists compared to traditional polymeric positive tone systems which are quite mature. With the goal of meeting some of these needs, we carried out multiple different studies of negative tone molecular resists based on epoxide cross-linking. We have developed methods for controlling cross-linking in these materials using photo-decomposable nucleophiles (PDNs) which have shown resolution improvements in one resist from 26 nm down to 18 nm so far. Aqueous base developed systems have been made by introducing phenol groups to these resists. Although the first molecule designed successfully patterns in aqueous base, its performance is limited compared to organic solvent development. A series of di-functional epoxide molecular resists have been made and generally show much worse sensitivity than a fourfunctional epoxide resist. Underlayers (ULs) have been designed specifically to leverage the high reactivity of epoxides to create ULs that can cross-link to the resist. These ULs improve the adhesion of some of these molecular resists, but also show significant sensitivity improvements compared to imaging on bare silicon. Sensitivity in one resist was improved by 5-12 mJ/cm2 simply by using the UL.

  4. Electrospinning of Cross-Linked Magnetic Chitosan Nanofibers for Protein Release.

    Nicknejad, Ehsan Tayerani; Ghoreishi, Seyyed Mohammad; Habibi, Neda


    A poly(vinylalcohol) (PVA) electrospun/magnetic/chitosan nanocomposite fibrous cross-linked network was fabricated using in situ cross-linking electrospinning technique and used for bovine serum albumin (BSA) loading and release applications. Sodium tripolyphosphate (TPP) and glutaraldehyde (GA) were used as cross-linkers which modified magnetic-Fe3O4 chitosan as Fe3O4/CS/TPP and Fe3O4/CS/GA, respectively. BSA was used as a model protein drugs which was encapsulated to form Fe3O4/CS/TPP/BSA and Fe3O4/CS/GA/BSA nanoparticles. The composites were electrospun with PVA to form nanofibers. Nanofibers were characterized by field emission scanning electron microscopy (FESEM) and Fourier transform infrared spectroscopy (FTIR). The characterization results suggest that Fe3O4 nanoparticles with average size of 45 nm were successfully bound on the surface of chitosan. The cross-linked nanofibers were found to contain uniformly dispersed Fe3O4 nanoparticles. The size and morphology of the nanofibers network was controlled by varying the cross-linker type. FTIR data show that these two polymers have intermolecular interactions. The sample with TPP cross-linker showed an enhancement of the controlled release properties of BSA during 30-h experimental investigation. Graphical Abstract ᅟ.

  5. Reasons for revision of first-generation highly cross-linked polyethylenes.

    Kurtz, Steven M; Medel, Francisco J; MacDonald, Daniel W; Parvizi, Javad; Kraay, Matthew J; Rimnac, Clare M


    Over a 10-year period, we prospectively evaluated the reasons for revision of contemporary and highly cross-linked polyethylene formulations in amulticenter retrieval program. Two hundred twelve consecutive retrievals were classified as conventional gamma inert sterilized (n = 37), annealed (Cross fire,[Stryker Orthopedics, Mahwah, NJ] n = 72), or remelted (Longevity [Zimmer ,Warsaw, Ind], XLPE[Smith and Nephew, Memphis, Tenn], Durasul [Zimmer,Warsaw, Ind] n = 103) liners. The most frequent reasons for revision were loosening (35%), instability(28%), and infection (21%) and were not related to polyethylene formulation (P = .17). Annealed and remelted liners had comparable linear penetration rates(0.03 and 0.04 mm/y, respectively, on average), and these were significantly lower than the rate in conventional retrievals (0.11 mm/y, P ≤ .0005). This retrieval study including first-generation highly cross linked liners demonstrated lower wear than conventional polyethylene. Although loosening remained as the most prevalent reason for revision, we could not demonstrate a relationship between wear and loosening.The long-term clinical performance of first-generation highly cross-linked liners remains promising based on the midterm outcomes of the components documented in this study [corrected].

  6. Crosslink density, oxidation and chain scission in retrieved, highly cross-linked UHMWPE tibial bearings.

    Reinitz, Steven D; Currier, Barbara H; Levine, Rayna A; Van Citters, Douglas W


    Irradiated, thermally stabilized, highly cross-linked UHMWPE bearings have demonstrated superior wear performance and improved in vitro oxidation resistance compared with terminally gamma-sterilized bearings, yet retrieval analysis reveals unanticipated in vivo oxidation in these materials despite fewer or no measurable free radicals. There has been little evidence to date that the oxidation mechanism in thermally stabilized materials is the same as that in conventional materials, and so it is unknown whether oxidation in these materials is leading to chain scission and a degradation of mechanical properties, molecular weight, and crosslink density. The aim of this study was to determine whether measured in vivo oxidation in retrieved, highly cross-linked tibial bearings corresponds with a decreasing crosslink density. Analysis of three tibial bearing materials revealed that crosslink density decreased following in vivo duration, and that the change in crosslink density was strongly correlated with oxidation. The results suggest that oxidation in highly cross-linked materials is causing chain scissions that may, in time, impact the material properties. If in vivo oxidation continues over longer durations, there is potential for a clinically significant degradation of mechanical properties.

  7. Cross-linked multilayer-dye films deposited onto silica surfaces with high affinity for pepsin

    Bucatariu, Florin; Ghiorghita, Claudiu-Augustin; Cocarta, Ana-Irina; Dragan, Ecaterina Stela


    Cross-linked thin films based on pH-responsive polymers with a specific ligand inside the organic layer are useful materials in separation processes or in fabrication of controlled delivery systems. Herein, we report the step-by-step deposition of polymer multilayers based on poly(ethyleneimine) (PEI), poly(acrylic acid) (PAA) and poly(sodium methacrylate) (PMAA) followed by the Congo red (CR) immobilization onto composite Daisogel silica microparticles and silicon wafers. The non-crosslinked composites were not stable in extreme basic medium (pH = 13), while thermal and chemical cross-linked samples with CR inside were stable over a wide range of pH. The interaction properties of different proteins [pepsin (PEP), lysozyme, trypsin, bovine serum albumin] with modified solid surfaces were followed by potentiometric titrations, UV and AFM measurements. Only the PEP macromolecules were sorbed onto the Daisogel composite microparticles with CR inside the cross-linked multilayer. The maximum sorbed amount was nearly 200 mg PEP/g Daisogel//(PEI/PAA)4.5 + CR. This high sorbed amount was in accordance with the AFM images, the average high and roughness increased drastically after the sorption of PEP.

  8. Rheological characterization of cataplasm bases composed of cross-linked partially neutralized polyacrylate hydrogel.

    Wang, Jian; Zhang, Hongqin; An, Dianyun; Yu, Jian; Li, Wei; Shen, Teng; Wang, Jianxin


    Viscoelasticity is a useful parameter for characterizing the intrinsic properties of the cross-linked polyacrylate hydrogel used in cataplasm bases. The aim of this study was to investigate the effects of various formulation parameters on the rheological characteristics of polyacrylate hydrogel. The hydrogel layers were formed using a partially neutralized polyacrylate (Viscomate(™)), which contained acrylic acid and sodium acrylate in different copolymerization ratios, as the cross-linked gel framework. Dihydroxyaluminum aminoacetate (DAAA), which produces aluminum ions, was used as the cross-linking agent. Rheological analyses were performed using a "stress amplitude sweep" and a "frequency sweep". The results showed that greater amounts of acrylic acid in the structure of Viscomate as well as higher concentrations of DAAA and Viscomate led to an increase in the elastic modulus (G'). However, greater amounts of acrylic acid in the structure of Viscomate and higher concentrations of DAAA had an opposite on the viscous modulus (G″); this might be owing to higher steric hindrance. The results of this study can serve as guidelines for the optimization of formulations for cataplasms.

  9. Fracture mechanics of collagen fibrils: influence of natural cross-links.

    Svensson, Rene B; Mulder, Hindrik; Kovanen, Vuokko; Magnusson, S Peter


    Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human collagen fibrils. There was an initial rise in modulus followed by a plateau with reduced modulus, which was finally followed by an even greater increase in stress and modulus before failure. The RTTs also displayed the initial increase and plateau phase, but the third region was virtually absent and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish the underlying mechanisms.

  10. Azide-based cross-linking of polymers of intrinsic microporosity (PIMs) for condensable gas separation

    Du, Naiying


    Cross-linked polymers of intrinsic microporosity (PIM)s for gas separation membranes, were prepared by a nitrene reaction from a representative PIM in the presence of two different diazide cross-linkers. The reaction temperature was optimized using TGA. The homogenous membranes were cast from THF solutions of different ratios of PIM to azides. The resulting cross-linked structures of the PIMs membranes were formed at 175 °C after 7.5 h and confirmed by TGA, XPS, FT-IR spectroscopy and gel content analysis. These resulting cross-linked polymeric membranes showed excellent gas separation performance and can be used for O 2/N 2 and CO 2/N 2 gas pairs, as well as for condensable gases, such as CO 2/CH 4, propylene/propane separation. Most importantly, and differently from typical gas separation membranes derived from glassy polymers, the crosslinked PIMs showed no obvious CO 2 plasticization up to 20 atm pressure of pure CO 2 and CO 2/CH 4 mixtures. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.

    Choi, Yongki; Moody, Issa S; Sims, Patrick C; Hunt, Steven R; Corso, Brad L; Seitz, David E; Blaszczak, Larry C; Blaszcazk, Larry C; Collins, Philip G; Weiss, Gregory A


    The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.

  12. Preliminary clinical and radiographic results of large ceramic heads on highly cross-linked polyethylene.

    Meftah, Morteza; Ebrahimpour, Prouskeh Bruce; He, Chuan; Ranawat, Amar S; Ranawat, Chitranjan S


    Data are limited regarding large ceramic femoral heads with highly cross-linked polyethylene. We hypothesized that large ceramic head articulation with highly cross-linked polyethylene is safe with a low wear rate, comparable to metal-on-highly cross-linked polyethylene.The study group comprised 63 patients (72 hips) who had undergone total hip replacement (THR) with ceramic-on-highly cross-linked polyethylene between April 2006 and March 2007 with a minimum 2-year follow-up. Postoperative Western Ontario and Mc-Master Universities Arthritis Index (WOMAC) and Hospital for Special Surgery (HSS) scores were used for clinical assessment. Six-week and 2-year radiographs were analyzed by 2 independent observers using Roman 1.70 software. Twenty-six patients (29 hips) had 32-mm and 37 patients (43 hips) had 36-mm Biolox delta ceramic femoral heads (Ceramtec, Plochingen, Germany). Mean patient age was 60.9 ± 8.9 years, and mean follow-up was 2.9 ± 0.5 years. Mean postoperative WOMAC and HSS hip scores were 30.4 and 36.6, respectively. Mean wear at 1 and 2 years postoperatively was 0.06 ± 0.28 and 0.006 ± 0.12 mm/yr for all hips, respectively. Mean wear at 1 and 2 years postoperatively for the 32-mm femoral head was 0.063 ± 0.278 and 0.007 ± 0.126 mm/yr, respectively, and for the 36-mm femoral head was 0.057 ± 0.292 and 0.006 ± 0.118 mm/yr, respectively. No patient had any clinical complications, such as reoperation, infection, fractures, or radiographic evidence of osteolysis or loosening. The early results of THR with large ceramic heads demonstrate high safety and efficacy. Our data with 2-year follow-up show low wear rates, similar to published data for metal-on-highly cross-linked polyethylene.

  13. Graft copolymerization onto polybutadiene: Cross-linking and thermal degradation of vinyl polymers and copolymers

    Jiang, Dayue (David)

    This work consists of three parts. In Part I, the graft copolymerization of methyl methacrylate, methyl acrylate, methacylic acid and acrylic acid onto polybutadiene and its copolymers by benzoyl peroxide, BPO, or 2, 2'azobis(2-methylpropionitrile), AIBN, initiation were explored. The results show that these monomers can be grafted onto butadiene region of butadiene-containing polymers. The extent of both graft copolymerization and homopolymerization are dependent on the time and temperature of the reaction and the concentration of all of the reactants. One must specify the monomer, initiator and solvent for the efficient graft copolymerization. The methyl methacrylate adds directly to the radical sites which are formed on the backbone by the interaction of the polymer and the primary radical form the initiator, while for the other three monomers, the graft copolymerization occurs by addition of macro-radical to the double bonds. In Part II, the cross-linking of polybutadiene, butadiene-styrene copolymers, and polystyrene by irradiation, thermal and chemical processes, and Friedel-Crafts chemistry and the effect of cross-linking on the thermal stability were investigated. The proof of cross-linking of the polymer comes from the insolubility of the product after the cross-linking reaction and is characterized by gel content and swelling ratio. The results show that the thermal stability of the polymer can be improved by cross-linking. In Part III, the thermal degradation of three vinyl polymers, poly(vinylsulfonic acid) and its sodium salt and poly(vinylphosphonic acid) were studied by combination technique: TGA/FTIR. The results show that TGA/FTIR combined with analysis of residues provides an excellent opportunity to understand the degradation pathway of the compounds. The observation of foaming indicates that the char which is formed contains carbon as well as the inorganic salts which have been observed. The carbon is in a partially graphitized form. The salts

  14. Nondestructive fluorescence-based quantification of threose-induced collagen cross-linking in bovine articular cartilage

    Kinnunen, Jussi; Kokkonen, Harri T.; Kovanen, Vuokko; Hauta-Kasari, Markku; Vahimaa, Pasi; Lammi, Mikko J.; Töyräs, Juha; Jurvelin, Jukka S.


    Extensive collagen cross-linking affects the mechanical competence of articular cartilage: it can make the cartilage stiffer and more brittle. The concentrations of the best known cross-links, pyridinoline and pentosidine, can be accurately determined by destructive high-performance liquid chromatography (HPLC). We explore a nondestructive evaluation of cross-linking by using the intrinsic fluorescence of the intact cartilage. Articular cartilage samples from bovine knee joints were incubated in threose solution for 40 and 100 h to increase the collagen cross-linking. Control samples without threose were also prepared. Excitation-emission matrices at wavelengths of 220 to 950 nm were acquired from the samples, and the pentosidine and pyridinoline cross-links and the collagen concentrations were determined using HPLC. After the threose treatment, pentosidine and lysyl pyridinole (LP) concentrations increased. The intrinsic fluorescence, excited below 350 nm, decreased and was related to pentosidine [r=-0.90, 240/325 nm (excitation/emission)] or LP (r=-0.85, 235/285 nm) concentrations. Due to overlapping, the changes in emission could not be linked specifically to the recorded cross-links. However, the fluorescence signal enabled a nondestructive optical estimate of changes in the pentosidine and LP cross-linking of intact articular cartilage.

  15. Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation.

    Veríssimo, D M; Leitão, R F C; Ribeiro, R A; Figueiró, S D; Sombra, A S B; Góes, J C; Brito, G A C


    The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.

  16. Relationship between cross-linking conditions of ethylene vinyl acetate and potential induced degradation for crystalline silicon photovoltaic modules

    Jonai, Sachiko; Hara, Kohjiro; Tsutsui, Yuji; Nakahama, Hidenari; Masuda, Atsushi


    In this study, we investigated the relationship in crystalline silicon (c-Si) photovoltaic (PV) modules between the cross-linking level of copolymer of ethylene and vinyl acetate (EVA) as the encapsulant and the degree of degradation due to potential induced degradation (PID) phenomenon. We used three methods for the determination of cross-linking level of EVA: xylene method, which is one of the solvent extraction methods (SEM), curing degree by differential scanning calorimetry (DSC), and viscoelastic properties by dynamic mechanical analysis (DMA). The results indicate that degradation of PV modules by PID test depends on the cross-linking level of EVA. The PV modules encapsulated by EVA with higher cross-linking level show lower degradation degree due to PID phenomenon. Also we showed that EVA with higher cross-linking level tended to be higher volume resistivity. This tendency is similar to that for electrical resistance value during the PID test. The PID test was also done by changing thickness of EVA between front cover glass and c-Si with the same cross-linking level. The PV modules encapsulated by thicker EVA between front cover glass and c-Si cell show lower degradation by PID. From these results, the PV modules encapsulated by EVA with higher cross-linking level, higher volume resistivity and increased thickness would be tolerant of PID phenomenon.

  17. Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone

    Willems, Nop M.B.K., E-mail: [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Langenbach, Geerling E.J. [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Stoop, Reinout [Dept. of Metabolic Health Research, TNO, P.O. Box 2215, 2301 CE Leiden (Netherlands); Toonder, Jaap M.J. den [Dept. of Mechanical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Mulder, Lars [Dept. of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Zentner, Andrej [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Everts, Vincent [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands)


    The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2 M ribose at 37 °C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness. - Highlights: • The assessment of effects of glycation in bone using HPLC, microCT, and nanoindentation • Ribose incubation: 300‐fold increase in the number of pentosidine cross-links • 300‐fold increase in the number of pentosidine cross-links: no changes in bone tissue stiffness.

  18. Sub- T g Cross-Linking of a Polyimide Membrane for Enhanced CO 2 Plasticization Resistance for Natural Gas Separation

    Qiu, Wulin


    Decarboxylation-induced thermal cross-linking occurs at elevated temperatures (∼15 °C above glass transition temperature) for 6FDA-DAM:DABA polyimides, which can stabilize membranes against swelling and plasticization in aggressive feed streams. Despite this advantage, such a high temperature might result in collapse of substructure and transition layers in the asymmetric structure of a hollow fibers based on such a material. In this work, the thermal cross-linking of the 6FDA-DAM:DABA at temperatures much below the glass transition temperature (∼387 °C by DSC) was demonstrated. This sub-Tg cross-linking capability enables extension to asymmetric structures useful for large scale membranes. The resulting polymer membranes were characterized by swelling in known solvents for the un-cross-linked materials, TGA analysis, and permeation tests of aggressive gas feed stream at higher pressure. The annealing temperature and time clearly influence the degree of cross-linking of the membranes, and results in a slight difference in selectivity for membranes under various cross-linking conditions. Results indicate that the sub-Tg thermal cross-linking of 6FDA-DAM:DABA dense film membrane can be carried out completely even at a temperature as low as 330 °C. Permeabilities were tested for the polyimide membranes using both pure gases (He, O2, N2, CH4, CO2) and mixed gases (CO2/CH4). The selectivity of the cross-linked membrane can be maintained even under very aggressive CO2 operating conditions that are not possible without cross-linking. Moreover, the plasticization resistance was demonstrated up to 700 psia for pure CO 2 gas or 1000 psia for 50% CO2 mixed gas feeds. © 2011 American Chemical Society.


    Oral, Ebru; Beckos, Christine Godleski; Malhi, Arnaz S.; Muratoglu, Orhun K.


    Vitamin E-stabilized, highly cross-linked ultrahigh molecular weight polyethylene (UHMWPE) is a promising oxidation and wear resistant UHMWPE with improved mechanical strength in comparison with the first generation, irradiated and melted UHMWPE. One approach of incorporating vitamin E in UHMWPE is through blending of vitamin E in UHMWPE powder followed by consolidation and radiation crosslinking. However radiation crosslinking efficiency of UHMWPE decreases in the presence of vitamin E. Therefore an optimum vitamin E concentration and radiation dose level needs to be determined to achieve a cross-link density comparable to 100-kGy irradiated and melted UHMWPE, which has shown excellent wear properties in vivo. We investigated the cross-link density and mechanical properties of vitamin E-blended UHMWPEs as a function of vitamin E concentration in the blend and gamma irradiation doses up to 200 kGy. We found that 0.3 wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 65 kGy for virgin UHMWPE and 1.0 wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 25 kGy for virgin UHMWPE even when the former were irradiated to a radiation dose of 200 kGy. In addition, higher plasticity at vitamin E concentrations at and above 0.3 wt% indicated that increased chain scissioning may be prevalent. Since the wear resistance of this irradiated UHMWPE would be expected to be low, vitamin E concentrations equal to or above 0.3 wt% are not recommended for subsequent irradiation to achieve a wear resistant cross-linked UHMWPE. The long–term oxidative stability of irradiated blends with low vitamin E concentrations has yet to be studied to determine an optimum between cross-link density and long-term oxidative stability. PMID:18514813

  20. Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils.

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J; Buehler, Markus J


    Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength.

  1. Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

    Zheng, Qiuling; Zhang, Hao; Wu, Shiyong; Chen, Hao


    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca2+ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology.

  2. Affinity of anticancer drug, daunomycin, to core histones in solution:comparison of free and cross-linked proteins



    Aim: The interaction of anthracyclinc anticancer drugs with chromatin, nuclco-somes and historic H1 has been extensively studied. In the present study, for the first time, we have investigated the binding of anthracycline antibiotic, daunomycin,to free and cross-linked thymus core histones (CL-core) in solution and in the absence of DNA. Methods: Fluorescence, UV/Vis spectroscopy and equilibrium dialysis techniques were used. Results: The UV spectroscopy results show that daunomycin induces hypochromicity in the absorption spectra of the core histones.Fluorescence emission intensity is decreased upon daunomycin binding and the process is concentration dependent. The equilibrium dialysis shows that the bind-ing is positive cooperative with the binding sites as Scatchard plot and Hill Coef-ficient confirm it. Conclusion: The results suggest that daunomycin shows much higher affinity to core histories free in solution than to CL-core, implying that the binding is most likely due to the accessibility of these proteins to the environment.It is suggested that daunomycin binds strongly to open state of histones, such as in tumor cells, rather than to their compact structure seen in normal chromatin.

  3. Epoxy Cross-Linked Collagen and Collagen-Laminin Peptide Hydrogels as Corneal Substitutes

    May Griffith


    Full Text Available A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE, was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl carbodiimide (EDC/N-hydroxysuccinimide (NHS as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF42·XH2O was used to catalyze the formation of secondary amine bonds. To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers.

  4. Collagen cross-linking using rose bengal and green light to increase corneal stiffness.

    Cherfan, Daniel; Verter, E Eri; Melki, Samir; Gisel, Thomas E; Doyle, Francis J; Scarcelli, Giuliano; Yun, Seok Hyun; Redmond, Robert W; Kochevar, Irene E


    Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application. Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment. RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability. RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

  5. Scleral wound healing with cross-link technique using riboflavin and ultraviolet A on rabbit eyes.

    Damasceno, Nadyr A; Miguel, Nadia C; Ventura, Marcelo Palis; Burnier, Miguel; Avila, Marcos P; Damasceno, Eduardo F


    The aim of study was to evaluate the cross-link using riboflavin and ultraviolet A (UVA) for improving scleral wound healing. This was an experimental study involving four New Zealand rabbits (eight eyes). Therapy procedure was chosen for the right eye and control procedure for the left one. UVA irradiation of 365 nm with a surface irradiance of 3 mW/cm(2) and a photosensitizer of riboflavin drops were applied for 30 minutes on the right eye at 2 mm from the limbus. Sclerotomy incision was performed at 2 mm from the limbus in both right (on the cross-link-treated area) and left eye. Then, 30 days after surgery, a morphological analysis and histological staining with hematoxylin-eosin and picrosirius red were performed, and the sclerotomy cicatrization of right and left eyes was compared. The variables investigated were as follows: sclerotomy incision pictures and measurements were made using the ImageJ Software. Scleral thickness was measured (employing the anterior optical coherence tomography and the digital caliper). Collagen fiber density stained with picrosirius red staining was measured using the Image Pro Plus software. The morphological analysis showed that in all samples, the right eye presented sclerotomy closure, and in two eyes, among them, there were no visible edges of the sclerotomies incision. The left eye presented sclerotomy closure and incision edges. The Image Pro demonstrated a higher density of collagen fibers in the right eye when compared to the one. The statistical analysis was significant when compared to the collagen fiber density in the treated eyes with the control eyes. The cross-link procedure resulted in a better sclerotomy wound healing.

  6. Corneal collagen cross-linking to stop corneal ectasia exacerbated by radial keratotomy.

    Mazzotta, Cosimo; Baiocchi, Stefano; Denaro, Rosario; Tosi, Gian Marco; Caporossi, Tomaso


    To assess the efficacy of riboflavin ultraviolet A (UV-A) corneal collagen cross-linking in the management of keratoconic corneal ectasia exacerbated by radial keratotomy (RK). A patient with progressive corneal ectasia and hyperopic shift, occurring 10 years after RK performed in the left eye, was treated with riboflavin UV-A corneal collagen cross-linking according to the Siena protocol: Pilocarpin 0.1% drop (1 hour before), lidocaine 4% drops 15 minutes before, mechanical scraping of epithelium (9-mm-diameter area), preirradiation stromal soaking for 10 minutes in riboflavin 0.1%-dextrane 20% (Ricrolin; Sooft Italy) applied every 2 minutes, and 30 minutes of total exposure (6 steps of 5 minutes) to solid-state UV-A illuminator (Caporossi, Baiocchi, Mazzotta Vega X linker; CSO Opthalmics, Florence, Italy), energy delivered 3 mW/cm, and irradiated area 9 mm in diameter. After the operation, uncorrected visual acuity and best spectacle-corrected visual acuity improved from 0.2 to 0.6 and from 0.3 to 0.8 Snellen lines, respectively, in a 12-month follow-up. Improved topographical K readings and corneal symmetry index were also recorded starting from the first postoperative month and continuing thereafter. No adverse effects were recorded after treatment. Riboflavin UV-A-induced corneal cross-linking seems to be a promising surgical option in the management of unstable corneal ectasia exacerbated by RK, particularly in eyes with preexisting keratoconus. A large cohort and longer follow-up are needed to determine its long-term efficacy in this clinical setting.

  7. Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds.

    Balakrishnan, Biji; Jayakrishnan, A


    The injectable polymer scaffolds which are biocompatible and biodegradable are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural proteins and polysaccharides are ideal scaffolds for tissue engineering since they resemble the extracellular matrices of the tissue comprised of various amino acids and sugar-based macromolecules. Here, we report a new class of hydrogels derived from oxidized alginate and gelatin. We show that periodate-oxidized sodium alginate having appropriate molecular weight and degree of oxidation rapidly cross-links proteins such as gelatin in the presence of small concentrations of sodium tetraborate (borax) to give injectable systems for tissue engineering, drug delivery and other medical applications. The rapid gelation in the presence of borax is attributed to the slightly alkaline pH of the medium as well as the ability of borax to complex with hydroxyl groups of polysaccharides. The effect of degree of oxidation and concentration of alginate dialdehyde, gelatin and borax on the speed of gelation was examined. As a general rule, the gelling time decreased with increase in concentration of oxidized alginate, gelatin and borax and increase in the degree of oxidation of alginate. Cross-linking parameters of the gel matrix were studied by swelling measurements and trinitrobenzene sulphonic acid (TNBS) assay. In general, the degree of cross-linking was found to increase with increase in the degree of oxidation of alginate, whereas the swelling ratio and the degree of swelling decreased. The gel was found to be biocompatible and biodegradable. The potential of the system as an injectable drug delivery vehicle and as a tissue-engineering scaffold is demonstrated by using primaquine as a model drug and by encapsulation of hepatocytes inside the gel matrix, respectively.

  8. Porous chitosan scaffold cross-linked by chemical and natural procedure applied to investigate cell regeneration

    Yao, Chih-Kai [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Liao, Jiunn-Der, E-mail: [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Center of Micro/Nano Science and Technology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Chung, Chia-Wei; Sung, Wei-I. [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Chang, Nai-Jen [Institute of Biomedical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China)


    Highlights: Black-Right-Pointing-Pointer Polymeric scaffolds, made from chitosan-based films fixed by chemical (citrate) or natural method (genipin), were developed. Black-Right-Pointing-Pointer Nano-indentation with a constant harmonic frequency was applied on porous scaffolds to explore their surface mechanics. Black-Right-Pointing-Pointer The relationship between surface mechanical property and cell-surface interactions of scaffold materials was demonstrated. Black-Right-Pointing-Pointer Porous scaffolds cross-linked by genipin showed adequate cell affinity, non-toxicity, and suitable mechanical properties. - Abstract: Porous chitosan scaffold is used for tissue engineering and drug delivery, but is limited as a scaffold material due to its mechanical weakness, which restrains cell adhesion on the surface. In this study, a chemical reagent (citrate) and a natural reagent (genipin) are used as cross-linkers for the formation of chitosan-based films. Nanoindentation technique with a continuous stiffness measurement system is particularly applied on the porous scaffold surface to examine the characteristic modulus and nanohardness of a porous scaffold surface. The characteristic modulus of a genipin-cross-linked chitosan surface is Almost-Equal-To 2.325 GPa, which is significantly higher than that of an uncross-linked one ( Almost-Equal-To 1.292 GPa). The cell-scaffold surface interaction is assessed. The cell morphology and results of an MTS assay of 3T3-fibroblast cells of a genipin-cross-linked chitosan surface indicate that the enhancement of mechanical properties induced cell adhesion and proliferation on the modified porous scaffold surface. The pore size and mechanical properties of porous chitosan film can be tuned for specific applications such as tissue regeneration.

  9. Development of casein microgels from cross-linking of casein micelles by genipin.

    Silva, Naaman F Nogueira; Saint-Jalmes, Arnaud; de Carvalho, Antônio F; Gaucheron, Frédéric


    Casein micelles are porous colloidal particles, constituted of casein molecules, water, and minerals. The vulnerability of the supramolecular structure of casein micelles face to changes in the environmental conditions restrains their applications in other domains besides food. Thus, redesigning casein micelles is a challenge to create new functionalities for these biosourced particles. The objective of this work was to create stable casein microgels from casein micelles using a natural cross-linker, named genipin. Suspensions of purified casein micelles (25 g L(-1)) were mixed with genipin solutions to have final concentrations of 5, 10, and 20 mM genipin. Covalently linked casein microgels were formed via cross-linking of lysyl and arginyl residues of casein molecules. The reacted products exhibited blue color. The cross-linking reaction induced gradual changes on the colloidal properties of the particles. The casein microgels were smaller and more negatively charged and presented smoother surfaces than casein micelles. These results were explained based on the cross-linking of free NH2 present in an external layer of κ-casein. Light scattering and rheological measurements showed that the reaction between genipin and casein molecules was intramicellar, as one single population of particles was observed and the values of viscosity (and, consequently, the volume fraction of the particles) were reduced. Contrary to the casein micelles, the casein microgels were resistant to the presence of dissociating agents, e.g., citrate (calcium chelating) and urea, but swelled as a consequence of internal electrostatic repulsion and the disruption of hydrophobic interactions between protein chains. The casein microgels did not dissociate at the air-solution interface and formed solid-like interfaces rather than a viscoelastic gel. The potential use of casein microgels as adaptable nanocarriers is proposed in the article.

  10. T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

    Varga, F.; Rumpler, M.; Zoehrer, R.; Turecek, C.; Spitzer, S.; Thaler, R; Paschalis, E.P.; Klaushofer, K.


    Thyroid hormones (T3, T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process...

  11. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts

    Chu, Chenyu; Deng, Jia [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Xiang, Lin; Wu, Yingying [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Wei, Xiawei [State Key Laboratory of Biotherapy and Laboratory for Aging Research, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041 (China); Qu, Yili [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Man, Yi, E-mail: [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)


    Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

  12. Evaluation of cross-linked chitosan microparticles containing acyclovir obtained by spray-drying

    Stulzer, Hellen Karine [Laboratorio Quitech, Departamento de Quimica, Universidade Federal de Santa Catarina (Brazil); Laboratorio de Controle de Qualidade, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina (Brazil); Laboratorio de Controle de Qualidade, Departamento de Ciencias Farmaceuticas, Universidade Estadual de Ponta Grossa (Brazil)], E-mail:; Tagliari, Monika Piazzon [Laboratorio de Controle de Qualidade, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina (Brazil); Parize, Alexandre Luis [Laboratorio Quitech, Departamento de Quimica, Universidade Federal de Santa Catarina (Brazil); Silva, Marcos Antonio Segatto [Laboratorio de Controle de Qualidade, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina (Brazil); Laranjeira, Mauro Cesar Marghetti [Laboratorio Quitech, Departamento de Quimica, Universidade Federal de Santa Catarina (Brazil)


    The aim of this study was to obtain microparticles containing acyclovir (ACV) and chitosan cross-linked with tripolyphosphate using the spray-drying technique. The resultant system was evaluated through loading efficiency, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), in vitro release and stability studies. The results obtained indicated that the polymer/ACV ratio influenced the final properties of the microparticles, with higher ratios giving the best encapsulation efficiency, dissolution profiles and stability. The DSC and XRPD analyses indicated that the ACV was transformed into amorphous form during the spray-drying process.

  13. Charge regulation and energy dissipation while compressing and sliding a cross-linked chitosan hydrogel layer

    Liu, Chao; Thormann, Esben; Tyrode, Eric


    Interactions between a silica surface and a surface coated with a grafted cross-linked hydrogel made from chitosan/PAA multilayers are investigated, utilizing colloidal probe atomic force microscopy. Attractive double-layer forces are found to dominate the long-range interaction over a broad range...... of pH and ionic strength conditions. The deduced potential at the hydrogel/aqueous interface is found to be very low. This situation is maintained in the whole pH-range investigated, even though the degree of protonation of chitosan changes significantly. This demonstrates that pH-variations change...

  14. Scleral wound healing with cross-link technique using riboflavin and ultraviolet A on rabbit eyes

    Damasceno NA


    Full Text Available Nadyr A Damasceno,1 Nadia C Miguel,2 Marcelo Palis Ventura,3 Miguel Burnier Jr,4 Marcos P Avila,5 Eduardo F Damasceno3 1Ophthalmology Department, Hospital Naval Marcílio Dias, 2Laboratory of Neurohistology and Cell Ultrastructure, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 3Ophthalmology Department, Universidade Federal Fluminense, Niterói, Brazil; 4Ophthalmology Department, McGill University, Montreal, QC, Canada; 5Ophthalmology Department, Universidade Federal de Goiás, Goiania, Brazil Purpose: The aim of study was to evaluate the cross-link using riboflavin and ultraviolet A (UVA for improving scleral wound healing.Materials and methods: This was an experimental study involving four New Zealand rabbits (eight eyes. Therapy procedure was chosen for the right eye and control procedure for the left one. UVA irradiation of 365 nm with a surface irradiance of 3 mW/cm2 and a photosensitizer of riboflavin drops were applied for 30 minutes on the right eye at 2 mm from the limbus. Sclerotomy incision was performed at 2 mm from the limbus in both right (on the cross-link-treated area and left eye. Then, 30 days after surgery, a morphological analysis and histological staining with hematoxylin–eosin and picrosirius red were performed, and the sclerotomy cicatrization of right and left eyes was compared. The variables investigated were as follows: sclerotomy incision pictures and measurements were made using the ImageJ Software. Scleral thickness was measured (employing the anterior optical coherence tomography and the digital caliper. Collagen fiber density stained with picrosirius red staining was measured using the Image Pro Plus software.Results: The morphological analysis showed that in all samples, the right eye presented sclerotomy closure, and in two eyes, among them, there were no visible edges of the sclerotomies incision. The left eye presented sclerotomy closure and incision edges

  15. Towards aging mechanisms of cross-linked polyethylene (XLPE) cable insulation materials in nuclear power plants

    Liu, Shuaishuai; Fifield, Leonard S.; Bowler, Nicola


    Cross-linked polyethylene (XLPE) cable insulation material undergoes simultaneous, accelerated thermal and gamma-radiation aging to simulate the long-term aging environment within nuclear power plants (NPPs). A variety of materials characterization tests, including scanning electron microscopy, thermo-gravimetric analysis, differential scanning calorimetry, oxidation induction time, gel-fraction and dielectric properties measurement, are conducted on pristine and differently aged XLPE samples. A preliminary model of one possible aging mechanism of XLPE cable insulation material under gamma radiation at elevated temperature of 115 °C is suggested.

  16. Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

    Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt


    as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple...... of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

  17. Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

    Doerig Christian


    Full Text Available Abstract Background Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1, which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. Methods Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC were subjected to in vitro modification by tissue transglutaminase-2 (TG2 to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyllysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. Results This study identified a substrate motif for (TG2 and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyllysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo


    You-xiang Wang; Ying Zhu; Jia-cong Shen


    Enhanced stability of polyplexes in physiological condition was an important prerequisite for successful systemic gene delivery. Herein novel method was reported to develop stable gene vector by nanotechnology. Thiolated polyplexes were constructed and then cross-linked with gold nanoparticles (AuNPs) by gold-thiol interactions. TEM pictures showed that AuNPs were attached to the shell of spherical polyplexes. The hybrid gene vector was stable enough in physiological condition and maintained efficient transfection, which showed great potential in gene delivery research and application.

  19. Progress of corneal collagen cross-linking combined with refractive surgery

    Na Li


    Full Text Available As a photochemical reaction that can stiffen the cornea, corneal collagen cross-linking (CXL is the only promising method of preventing the progress of keratectasia, such as keratoconus and secondary ectasia following refractive surgery. The aim of CXL is to stabilize the underlying condition, with a small chance of visual improvement. Combining CXL with refractive surgery targeting both stabilization and reshaping of the corneal tissue for visual function improvement is a good treatment option. This review aims to provide a comprehensive and unbiased summary of the published research regarding combined CXL and refractive surgery, including measures and results, to help elucidate the future direction of CXL.

  20. Inactivation of Matrix-bound MMPs by Cross-linking Agents in Acid Etched Dentin

    Scheffel, Débora Lopes Salles; Hebling, Josimeri; Scheffel, Régis Henke; Agee, Kelly A.; Turco, Gianluca; de Souza Costa, Carlos Alberto; Pashley, David H.


    Objectives Published TEM analysis of in vivo resin-dentin bonds shows that in 44 months almost 70% of collagen fibrils from the hybrid layer disappear. Matrix metalloproteinases (MMPs) play an important role in that process and are thought to be the main factor responsible for the solubitization of dentin collagen. Therefore, this study aimed to evaluate the inactivation of matrix-bound MMPs by carbodiimide (EDC) or proanthocyanidin (PA) both cross-linking agents, or the MMP-inhibitor, chlorhexidine (CHX), on acid-etched dentin using a simplified MMP assay method. Methods Dentin beams (1×1×6mm) were obtained from mid-coronal dentin of sound third molars and randomly divided into 6 groups (G) according to the dentin treatment: G1: Deionized water (control), G2: 0.1M EDC, G3: 0.5M EDC, G4: 0.5M EDC+35% HEMA, G5: 5% Proanthocyanidin (PA) and G6: 2% CHX. The beams were etched for 15s with 37% phosphoric acid, rinsed and then immersed for 60s in one of the treatment solutions. The total MMP activity of dentin was analyzed for 1 h by colorimetric assay (Sensolyte). Data were submitted to Wilcoxon non-parametric test and Mann-Whitney tests (p>0.05). Results All experimental cross-linking solutions significantly reduced MMP activity compared to control, except 0.1M EDC (53.6% ±16.1). No difference was observed between cross-linking agents and 2% CHX 0.5M EDC + 35% HEMA (92.3% ±8.0) was similar to 0.5M EDC (89.1% ±6.4), 5% PA (100.8% ±10.9) and 2% CHX (83.4% ±10.9). Conclusion Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity. Mixing 0.5M EDC and 35% HEMA did not influence EDC inhibitor potential. PMID:23786610

  1. Corneal cross-linking in a child with osteogenesis imperfecta syndrome and keratoconus

    Sergio Kwitko


    Full Text Available Cross-linking (CXL is a well-established procedure in children with keratoconus (KC, but cases of CXL and osteogenesis imperfecta (OI have not been published in the literature, despite the association between physiopathology of these diseases. This is the first case, to the best of our knowledge, of a young girl with both OI and KC that underwent a CXL treatment. In this case, CXL was performed at 6-years-old prior to an expected progression, without complications and probably stopped further keratoconus progression.

  2. Investigation of aging effects in cross-linked polyethylene insulated cable using terahertz waves

    Yan, Zhijin; Shi, Wei; Hou, Lei; Xu, Ming; Yang, Lei; Dong, Chengang; Li, Shengtao


    A method to detect the degree of aging in cross-linked polyethylene (XLPE) insulated cables, using terahertz (THz) waves, is reported in this letter. With increased aging time, the real permittivity decreases in the frequency range from 0.7 to 2.5 THz. The imaginary permittivity and absorption coefficient of unaged XLPE sample are larger than those of thermally aged XLPE insulated cables. These findings indicate that the aging degree of XLPE can be obtained by measuring its permittivity and absorption coefficient by THz waves.

  3. Viscoelastic Model of Cross-Linked Polyethylene Including Effects of Temperature and Crystallinity

    Olasz, L.; Gudmundson, P.


    Characterization of the mechanical behavior of cross-linked polyethylene (XLPE) commonly used in high voltage cable insulation was performed by an extensive set of isothermal uniaxial tensile relaxation tests. Tensile relaxation experiments were complemented by pressure-volume-temperature experiments as well as density and crystallinity measurements. Based on the experimental results, a viscoelastic power law model with four parameters was formulated, incorporating temperature and crystallinity dependence. It was found that a master curve can be developed by both horizontal and vertical shifting of the relaxation curves. The model was evaluated by making comparisons of the predicted stress responses with the measured responses in relaxation tests with transient temperature histories.

  4. The Effect of the Semiconductive Screen on Space Charge Suppression in Cross-Linked Polyethylene

    Li, Lin; Han, Bai; Song, Wei; Wang, Xuan; Lei, Qing-Quan


    The space charge distributions of cross-linked polyethylene (XLPE) with Borouge's Borlink™ semiconductive screen type LE0550 and LE0595 from a pulsed electro-acoustic method are obtained. The contact interface morphology at the semiconductive screen and the structure of XLPE near the interface are characterized. The dielectric spectrum and the conductivity current of XLPE with the different semiconductive electrodes are compared. The semiconductive screen changes the structure and the dielectric characteristic of XLPE near the contact interface, which may be the main reason for space charge suppression in XLPE with Borouge's type LE0550 semiconductive screen.

  5. Cross-Linked Liquid Crystalline Systems From Rigid Polymer Networks to Elastomers

    Broer, Dirk


    With rapidly expanding interest in liquid crystalline polymers and elastomers among the liquid crystal community, researchers are currently exploring the wide range of possible application areas for these unique materials, including optical elements on displays, tunable lasers, strain gauges, micro-structures, and artificial muscles. Written by respected scientists from academia and industry around the world, who are not only active in the field but also well-known in more traditional areas of research, "Cross-Linked Liquid Crystalline Systems: From Rigid Polymer Networks to Elastomers&qu

  6. Effect of Cross-linking Agent on Barbituric Acid-initiated Resin


    To improve the tensile bond strength of dentin bonding agents, the adhesion between dentin and MMA resin was investigated by applying initiator systems containing 1-cyclohexyl-5-ethyl barbituric acid (CEB), copper acetyl acetonate (CAA), quaternary ammonium chloride (QAC), cupric ion, with the addition of TEGDMA, and pretreatment solution for dentin, and an HEMA primer. The effects of a cross-linking agent on barbituric acid-initiated dentin bonding resin were examined by measuring degree of conversion, molecular weight, hardness of bonding resin, and tensile bond strengths.

  7. Triosidines: novel Maillard reaction products and cross-links from the reaction of triose sugars with lysine and arginine residues

    Tessier, Frederic J; Monnier, Vincent M; Sayre, Lawrence M; Kornfield, Julia A


    The role of the highly reactive triose sugars glyceraldehyde and glyceraldehyde-3-phosphate in protein cross-linking and other amino acid modifications during the Maillard reaction was investigated...

  8. The molecular mechanism of photodynamic therapy to fibrosis: Regulation on the pyridinoline cross-link formation in collagen

    ZHANG JuCheng; LIU Wei; YI ZhongZhou; CHEN Rui; LI Ying; MIN Yong


    Fibrotic processes in tissues are characterised by an excessive accumulation of collagen containing increased levels of pyridinoline cross-links,which has been taken as an important criterion to diagnose the fibrosis.Hypocrellin B (HB) is a natural photosensitizer with excellent photodynamic activity but very low dark-toxicity.In the current work,main purpose was to clarify if photodynamic action of HB could be applied to regulating the level of pyridinoline cross-link in collagen.To use gelatin as a model collagen,it was observed that the pyridinoline cross-link formation was effectively degraded by HB photodynamic action in the gelatin,suggesting the level of pyridinoline cross-link formation in collagen may be photodynamically regulated to a normal level.Furthermore,mechanism of the photodynamic action of HB was studied.

  9. Cross-linked supramolecular polymer metallogels constructed via a self-sorting strategy and their multiple stimulus-response behaviors.

    Wang, Xu-Qing; Wang, Wei; Yin, Guang-Qiang; Wang, Yu-Xuan; Zhang, Chang-Wei; Shi, Jia-Meng; Yu, Yihua; Yang, Hai-Bo


    Novel cross-linked supramolecular polymer metallogels were successfully constructed from four components via a self-sorting strategy, and feature interesting multiple stimulus-response behaviors under various external stimuli, including halide, base, and competitive guests.

  10. Structure and kinetics of chemically cross-linked protein gels from small-angle X-ray scattering

    Kaieda, Shuji; Halle, Bertil


    Glutaraldehyde (GA) reacts with amino groups in proteins, forming intermolecular cross-links that, at sufficiently high protein concentration, can transform a protein solution into a gel. Although GA has been used as a cross-linking reagent for decades, neither the cross-linking chemistry nor the microstructure of the resulting protein gel have been clearly established. Here we use small-angle X-ray scattering (SAXS) to characterise the microstructure and structural kinetics of gels formed by cross-linking of pancreatic trypsin inhibitor, myoglobin or intestinal fatty acid-binding protein. By comparing the scattering from gels and dilute solutions, we extract the structure factor and the pair correlation function of the gel. The protein gels are spatially heterogeneous, with dense clusters linked by sparse networks. Within the clusters, adjacent protein molecules are almost in contact, but the protein concentration in the cluster is much lower than in a crystal. At the $\\sim$ 1 nm SAXS resolution, the native ...

  11. Gingival Crevicular Fluid Calprotectin, Osteocalcin and Cross-Linked N-Terminal Telopeptid Levels in Health and Different Periodontal Diseases

    Sema Becerik


    Full Text Available Aim: The aim of the present study was to investigate gingival crevicular fluid (GCF calprotectin, osteocalcin and cross-linked N-terminal telopeptide (NTx levels in health along with different periodontal diseases.

  12. Glutaraldehyde Cross-Linking of TendonMechanical Effects at the Level of the Tendon Fascicle and Fibril

    Hansen, P.; Svensson, R.B.; Aagaard, P.;


    allowed for a detailed characterization of the effect of cross-linking in rat-tail tendon. The cross-link inducing agent glutaraldehyde augmented the tensile strength of tendon fascicles. Stress at failure increased from 8 MPa to 39 MPa. The mechanical effects of glutaraldehyde at the tendon fibril level......Conclusive insight into the microscopic principles that govern the strength of tendon and related connective tissues is lacking and the importance of collagen cross-linking has not been firmly established. The combined application of whole-tissue mechanical testing and atomic force spectroscopy...... were examined by atomic force microscopy. Peak forces increased from 1379 to 2622 pN while an extended Hertz fit of force-indentation data showed a 24 fold increase in Young's modulus on indentation. The effect of glutaraldehyde cross-linking on the tensile properties of a single collagen fibril...

  13. Dynamic constitutional frameworks for DNA biomimetic recognition.

    Catana, Romina; Barboiu, Mihail; Moleavin, Ioana; Clima, Lilia; Rotaru, Alexandru; Ursu, Elena-Laura; Pinteala, Mariana


    Linear and cross-linked dynamic constitutional frameworks generated from reversibly interacting linear PEG/core constituents and cationic sites shed light on the dominant coiling versus linear DNA binding behaviours, closer to the histone DNA binding wrapping mechanism.

  14. Stromal haze after combined riboflavin-UVA corneal collagen cross-linking in keratoconus: in vivo confocal microscopic evaluation.

    Mazzotta, Cosimo; Balestrazzi, Angelo; Baiocchi, Stefano; Traversi, Claudio; Caporossi, Aldo


    The technique of corneal collagen cross-linking consists of photopolymerization of stromal fibres by the combined action of a photosensitizing substance (riboflavin or vitamin B2) and ultraviolet light from a solid state UVA source. Photopolymerization increases the rigidity of corneal collagen and its resistance to keratectasia. In this report we present two cases, studied through in vivo confocal microscopy, with stage III keratoconus that developed stromal haze after the cross-linking treatment.

  15. Quantitative assessment of fibrinogen cross-linking by epsilon aminocaproic acid in patients with end-stage liver disease.

    Quach, Thien; Tippens, Melissa; Szlam, Fania; Van Dyke, Rebecca; Levy, Jerrold H; Csete, Marie


    Analysis of the effectiveness of antifibrinolytic therapy for liver transplant recipients is hampered by lack of quantitative assays for assessing drug effects. We adapted chemical engineering tools used in polymerization studies to quantify fibrinogen cross-linking by plasma from liver transplant patients obtained before and after epsilon aminocaproic acid (EACA) therapy. A target fluorescein isothiocyanate-fibrinogen (FITC-fibrinogen) molecule was constructed; it fluoresces in a quantifiable pattern when in solution, and undergoes cross-linking in the presence of plasmin inhibitors. Cross-linking quenches the fluorescent signal, and the quenching is a quantifiable endpoint. Thus fluorescence from this reporter molecule can be used to assess functional improvement in fibrinogen cross-linking as a result of antifibrinolytic therapies, and it is sensitive to picomolar amounts of plasmin inhibitors and activators. Cross-linking of FITC-fibrinogen by patient plasma, before and after EACA therapy, was assessed using fluorescence spectrometry. Fluorescence patterns from FITC-fibrinogen indicated no significant cross-linking of the target fibrinogen as a consequence of EACA in posttreatment plasma. When the fibrinogen-FITC target was assayed without plasma in the presence of EACA at concentrations that bracket therapeutic levels (100 and 400 microg/ml), significant fluorescence quenching (target FITC-fibrinogen cross-linking) was achieved. These results suggest that fibrinogen-FITC fluorescence is sensitive enough to detect EACA activity in clinically relevant ranges, but that EACA given in usual doses is insufficient to promote fibrinogen cross-linking in patients with end-stage liver disease.

  16. Oxidized pectin cross-linked carboxymethyl chitosan: a new class of hydrogels.

    Fan, Lihong; Sun, Yi; Xie, Weiguo; Zheng, Hua; Liu, Shuhua


    Oxidation of pectin was performed with sodium periodate to prepare pectin dialdehyde (PD). In this study we used the cross-linking reaction of the active aldehyde of PD and the amino of carboxymethyl chitosan (CMC) to prepare the hydrogels. By controlling the proportion of pectin dialdehyde and CMC we made different kinds of hydrogels. We systematically studied the characters of the hydrogels using Fourier transform infrared spectroscopy analysis of the pectin dialdehyde, CMC and the hydrogels, and also X-ray diffractometry and scanning electron microscopy analysis of the instrument of the hydrogels. Equilibrium swelling showed that the gels retained about 88-93% water. The water vapor transmission rate (WVTR) and the evaporation of water from gels showed that such hydrogels were optimal for maintaining a moist environment conducive for wound healing. Examination of the hemolytic potential showed that the hydrogels were nonhemolytic in nature. The hydrogels were non-toxic and blood-compatible. This hydrogel prepared from oxidized pectin and CMC without employing any extraneous cross-linking agents is expected to have potential as wound-dressing material.

  17. Enzymatic cross-linking of soy proteins within non-fat set yogurt gel.

    Soleymanpuori, Rana; Madadlou, Ashkan; Zeynali, Fariba; Khosrowshahi, Asghar


    Soy proteins as the health-promoting ingredients and candidate fat substitutes in dairy products are good substrates for the cross-linking action of the enzyme transglutaminase. Non-fat set yogurt samples were prepared from the milks enriched with soy protein isolate (SPI) and/or treated with the enzyme transglutaminase. The highest titrable acidity was recorded for the yogurt enriched with SPI and treated with the enzyme throughout the cold storage for 21 d. SPI-enrichment of yogurt milk increased the water holding capacity. Although enrichment with SPI did not influence the count of Streptococcus themophilus, increased that of Lactobacillus bulgaricus ∼3 log cycles. The enzymatic treatment of SPI-enriched milk however, suppressed the bacteria growth-promoting influence of SPI due probably to making the soy proteins inaccessible for Lactobacillus. SPI-enrichment and enzymatic treatment of milk decreased the various organic acids content in yoghurt samples; influence of the former was more significant. The cross-linking of milk proteins to soy proteins was confirmed with the gel electrophoresis results.

  18. Evaluation of Epithelial Integrity with Various Transepithelial Corneal Cross-Linking Protocols for Treatment of Keratoconus

    Suphi Taneri


    Full Text Available Purpose. Corneal collagen cross-linking (CXL has been demonstrated to stiffen cornea and halt progression of ectasia. The original protocol requires debridement of central corneal epithelium to facilitate diffusion of a riboflavin solution to stroma. Recently, transepithelial CXL has been proposed to reduce risk of complications associated with epithelial removal. Aim of the study is to evaluate the impact of various transepithelial riboflavin delivery protocols on corneal epithelium in regard to pain and epithelial integrity in the early postoperative period. Methods. One hundred and sixty six eyes of 104 subjects affected by progressive keratoconus underwent transepithelial CXL using 6 different riboflavin application protocols. Postoperatively, epithelial integrity was evaluated at slit lamp and patients were queried regarding their ocular pain level. Results. One eye had a corneal infection associated with an epithelial defect. No other adverse event including endothelial decompensation or endothelial damage was observed, except for epithelial damages. Incidence of epithelial defects varied from 0 to 63%. Incidence of reported pain varied from 0 to 83%. Conclusion. Different transepithelial cross-linking protocols have varying impacts on epithelial integrity. At present, it seems impossible to have sufficient riboflavin penetration without any epithelial disruption. A compromise between efficacy and epithelial integrity has to be found.

  19. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin


    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

  20. Numerical Modeling of Force-Stiffness Response of Cross-Linked Actin Networks Using Tensegrity Systems

    Xian Xu


    Full Text Available A three-dimensional tensegrity structure is used as a computational model for cross-linked actin networks. The postbuckling behavior of the members under compression is considered and the constitutive relation of the postbuckling members is modeled as a second-order polynomial. A numerical scheme incorporating the equivalent constitution of the postbuckling members is used to predict the structural response of the tensegrity model under compression loads. The numerical simulation shows that the stiffness of the tensegrity structure nonlinearly increases before member buckling and abruptly decreases to a lower level as soon as members buckle. This result qualitatively mimics the experimentally observed stiffness to compression stress response of cross-linked actin networks. In order to take member length variety into account, a large number of simulations with the length of buckling members varying in the given range are also carried out. It is found that the mean response of the simulations using different buckling member length exhibits more resemblance to the experimental observation.

  1. Preparation and adsorption property of aminated cross linking microbeads of GMA/EGDMA for bilirubin

    Zhiping Chen; Baojiao Gao; Xiaofeng Yang


    Cross linking microbeads with a controllable diameter were synthesized by suspension copolymerization of the monomer glycidyl methacrylate (GMA) and the cross linking agent ethylene glycol dimethylacrylate (EGDMA). By the ring-opening reaction of the epoxy groups, the microbeads GMA/EGDMA were modified with different aminating agents and resulting in the aminated microbeads. The morphology of the microbeads was characterized by SEM. The adsorption property of aminated microbeads for bilirubin was investigated, and the effects of various factors, such as the chemical structures of the aminating agents, pH values of the medium and the presence of bovine serum albumin in the adsorption medium, on the adsorption property were examined. The experimental results show that the aminated microbeads have strong adsorption ability for bilirubin, and the isotherm adsorption behaviour is fitted to Freundlich equation satisfactorily. The adsorption ability of the aminated microbeads modified with hexanediamine is stronger than that of others, and the longer the molecule of multi-ethylene multiamine, the weaker the adsorption ability for bilirubin. The pH value of the medium affects the adsorption ability greatly, as pH = 6, the adsorption ability is strongest. In the presence of BSA, the microbeads still have a higher adsorption capacity towards bilirubin.

  2. Size-dependent release of fluorescent macromolecules and nanoparticles from radically cross-linked hydrogels.

    Henke, Matthias; Brandl, Ferdinand; Goepferich, Achim M; Tessmar, Joerg K


    Hydrogels play an important role in drug delivery and tissue engineering applications due to their excellent biocompatibility and their variable mechanical and physical properties, which allow their optimization for many different aspects of the intended use. In this study, we examined the suitability of poly(ethylene glycol) (PEG)-based hydrogels as release systems for nanometer-sized drugs or drug carriers, like nanoparticles, using the radically cross-linkable oligo(poly(ethylene glycol)fumarate) (OPF) together with two cross-linking agents. Different fluorescent nanoparticulate probes with respect to size and physical structure were incorporated in the cross-linked hydrogels, and the obtained release profiles were correlated with the physical properties and the chemical structure of the gels, indicating a strong dependence of the release on the chosen PEG prepolymers. The prepared hydrogels were characterized by oscillatory rheometry and swelling experiments. Release experiments as well as diffusion measurements using fluorescence recovery after photobleaching showed the great potential of this type of hydrogels for the preparation of adjustable release systems by altering the molecular weights of the used PEG molecules.

  3. Controlled Release of Salicylic Acid from Biodegradable Cross-Linked Polyesters.

    Dasgupta, Queeny; Chatterjee, Kaushik; Madras, Giridhar


    The purpose of this work was to develop a family of cross-linked poly(xylitol adipate salicylate)s with a wide range of tunable release properties for delivering pharmacologically active salicylic acid. The synthesis parameters and release conditions were varied to modulate polyester properties and to understand the mechanism of release. Varying release rates were obtained upon longer curing (35% in the noncured polymer to 10% in the cured polymer in 7 days). Differential salicylic acid loading led to the synthesis of polymers with variable cross-linking and the release could be tuned (100% release for the lowest loading to 30% in the highest loading). Controlled release was monitored by changing various factors, and the release profiles were dependent on the stoichiometric composition, pH, curing time, and presence of enzyme. The polymer released a combination of salicylic acid and disalicylic acid, and the released products were found to be nontoxic. Minimal hemolysis and platelet activation indicated good blood compatibility. These polymers qualify as "bioactive" and "resorbable" and can, therefore, find applications as immunomodulatory resorbable biomaterials with tunable release properties.

  4. Neutron scattering and ab initio molecular dynamics study of cross-linking in biomedical phosphate glasses

    Parsons, A J; Ahmed, I; Rudd, C D [Division of Materials, Mechanics and Structures, Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Cuello, G J; Pellegrini, E; Richard, D; Johnson, M R, E-mail: [Institut Laue-Langevin, BP 156, 38042 Grenoble (France)


    Details of the microscopic structure of phosphate glasses destined for biomedical applications, which include sodium, magnesium and calcium cations, have been obtained from the static structure factor measured by means of neutron scattering. A complementary, molecular dynamics study has been performed on a range of phosphate glasses using density functional theory methods, which allow structural fluctuations, including bond breaking, in the liquid phase before quenching to the glass phase. Good agreement between experiment and simulation allows the molecular dynamics trajectories to be analysed in detail. In particular, attention is focused on the cross-linking of divalent cations in contrast with the structural aspects associated with monovalent cations. Magnesium cations are found equidistant and bridging between the phosphorus atoms of different phosphate chains, leading to a shorter phosphorus-phosphorus second neighbour distance (that is, a more compact packing of neighbouring phosphate chains) compared to the effect of sodium cations. Calcium cations show behaviour intermediate between those of magnesium and sodium. Molecular dynamics simulations give access to the cation mobility, which is lowest for magnesium, reflecting its structural, cross-linking role.

  5. Swelling Studies of Chitosan-Gelatin Films Cross-Linked by Sulfate

    Xiao Ling; Yu Zu-yu; Yang Chao; Zhu Hua-yue; Du Yu-min


    Swelling properties of chitosan-gelatin films cross-linked by sulfate were investigated. Sulfate cross-linked chitosan-gelatin films (SCG) were prepared simply by dipping chitosan-gelatin films into sodium sulfate solution. The swelling behavior of SCG was investigated as a function of pH and ionic strength. Under acidic conditions pH less than 4, SCG swelled less than 120%, while under the conditions pH larger than 7.4, SCG swelled very significantly, the swelling ratio was over 350%. Sodium chloride weakened the electrostatic interaction between sulfate and amine ions of chitosan and gelatin, therefore facilitated the film swelling. The swelling ratio increased with increasing sodium chloride concentration, the SCG dissociated in the sodium chloride concentration of 0.20 mol·L-1. The parameters of film preparation such as sulfate concentration, dipping time, sulfate solution pH, influenced the film swelling behavior. The lower concentration and the higher pH of sulfate solution resulted in a larger swelling ratio.

  6. Laser cross-linking protein captures for living cells on a biochip

    Lin, Chih-Lang; Pan, Ming-Jeng; Chen, Hai-Wen; Lin, Che-Kuan; Lin, Chuen-Fu; Baldeck, Patrice L.


    In this study, bio-sensing pads are proposed to capture living cells, which are fabricated on cover glasses by cross-linking proteins/antibodies using laser induced photochemistry. The biological functions of the cross-linked protein/antibody were verified by capturing Staphylococcus aureus (S. aureus), Leptospira, and red blood cells (RBCs), separately, with associated protein/antibody sensing pads. The experimental results show that S. aureus were bound on GFP-AcmA' pad after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed with reference pad made of neutral bovine serum albumin (BSA). Second, A-type RBCs were chosen as the model cell to demonstrate the blood typing feasibility of the anti-A pad in microchannel. The A-type RBCs were captured only by the anti-A pad, but not the reference pad made of BSA. The same experimental model was carried out on the Leptospira, which stuck on the blood serum pad after PBS rinsing, but not BSA pad. This study provides a potential platform for simple and direct detection of living full cells without culture that could be used in point-of-care settings.

  7. Study the adsorption of sulfates by high cross-linked polystyrene divinylbenzene anion-exchange resin

    Fathy, Mahmoud; Moghny, Th. Abdel; Awadallah, Ahmed E.; El-Bellihi, Abdel-Hameed A.-A.


    In response to rising concerns about the effect of sulfate on water quality, human health, and agriculture, many jurisdictions around the world are imposing tighter regulations for sulfate discharge. This is driving the need for environmental compliance in industries like mining, metal processing, pulp and paper, sewage treatment, and chemical manufacturing. The sulfate removal from synthetic water by high cross-linked polystyrene divinylbenzene resin was studied at batch experiments in this study. The effect of pH, contact time, sulfates concentration, and adsorbent dose on the sulfate sequestration was investigated. The optimum conditions were studied on Saline water as a case study. The results showed that with increasing of the absorbent amount; contact time, and pH improve the efficiency of sulfate removal. The maximum sulfates uptake was obtained in pH and contact time 3.0 and 120 min, respectively. Also, with increasing initial concentration of sulfates in water, the efficiency of sulfate removal decreased. The obtained results in this study were matched with Freundlich isotherm and pseudo-second-order kinetic. The maximum adsorption capacity (Qm) and constant rate were found 0.318 (mg/g) and 0.21 (mg/g.min), respectively. This study also showed that in the optimum conditions, the sulfate removal efficiency from Saline water by 0.1 mg/L sulfates was 65.64 %. Eventually, high cross-linked polystyrene divinylbenzene resin is recommended as a suitable and low cost absorbent to sulfate removal from aqueous solutions.

  8. Genipin-cross-linked fucose-chitosan/heparin nanoparticles for the eradication of Helicobacter pylori.

    Lin, Yu-Hsin; Tsai, Shih-Chang; Lai, Chih-Ho; Lee, Che-Hsin; He, Zih Sian; Tseng, Guan-Chin


    Helicobacter pylori is a significant human pathogen that recognizes specific carbohydrate receptors, such as the fucose receptor, and produces the vacuolating cytotoxin, which induces inflammatory responses and modulates the cell-cell junction integrity of the gastric epithelium. The clinical applicability of topical antimicrobial agents was needed to complete the eradication of H. pylori in the infected fundal area. In the present study, we combined fucose-conjugated chitosan and genipin-cross-linking technologies in preparing multifunctional genipin-cross-linked fucose-chitosan/heparin nanoparticles to encapsulate amoxicillin of targeting and directly make contact with the region of microorganism on the gastric epithelium. The results show that the nanoparticles effectively reduced drug release at gastric acids and then released amoxicillin in an H. pylori survival situation to inhibit H. pylori growth and reduce disruption of the cell-cell junction protein in areas of H. pylori infection. Furthermore, with amoxicillin-loaded nanoparticles, a more complete H. pylori clearance effect was observed, and H. pylori-associated gastric inflammation in an infected animal model was effectively reduced.

  9. Mono-dispersed cross-linked polystyrene micro-spheres prepared by seed swelling polymerization method

    Dongsha WANG; Yanjun LIU


    A two-step swelling procedure was adopted to synthesize mono-dispersed and highly cross-linked poly (St-divinylbenzene) particles with PSt micro-spheres (1.80 μmin diameter). The PSt micro-spheres were prepared by a dispersion polymerization method and used as seeds. The effects of monomer concentration, ratio of ethanol to water, swelling reagents, crosslinking reagents, swelling temper-ature and agitation speed on particle size were investigated in detail. The morphologies and size distributions of these micro-spheres were examined by SEM and particle size analysis (PSA). The Tg of the micro-spheres was measured by DSC. The results indicate that the particles (6.20 μm in diameter) exhibit excellent mono dispersed property and high crosslinking degree when the concentration of the swelling reagent was 25%, the concentration of the cross-linking reagents was 23%, the swelling temperature was 30℃ and the stirring speed was 150 r/min.

  10. Continuous Sensing Photonic Lab-on-a-Chip Platform Based on Cross-Linked Enzyme Crystals.

    Conejero-Muriel, Mayte; Rodríguez-Ruiz, Isaac; Verdugo-Escamilla, Cristóbal; Llobera, Andreu; Gavira, José A


    Microfluidics or lab-on-a-chip technology offer clear advantages over conventional systems such as a dramatic reduction of reagent consumption or a shorter analysis time, which are translated into cost-effective systems. In this work, we present a photonic enzymatic lab-on-a-chip reactor based on cross-linked enzyme crystals (CLECs), able to work in continuous flow, as a highly sensitive, robust, reusable, and stable platform for continuous sensing with superior performance as compared to the state of the art. The microreactor is designed to facilitate the in situ crystallization and crystal cross-linking generating enzymatically active material that can be stored for months/years. Thus, and by means of monolithically integrated micro-optics elements, continuous enzymatic reactions can be spectrophotometrically monitored. Lipase, an enzyme with industrial significance for catalyzed transesterification, hydrolysis, and esterification reactions, is used to demonstrate the potential of the microplatforms as both a continuous biosensor and a microreactor for the synthesis of high value compounds.

  11. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    Xuefei Song


    Full Text Available Purpose. The purpose of this study was to determine the impact of cross-linking (CXL on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham’s F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2 during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA. Results. Following CXL, cell viability and proliferation decreased (P0.06. Five hours after CXL, FGFb secretion increased significantly (P=0.037; however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P>0.12. Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours, normalizing after 24 hours.

  12. A covalently cross-linked gel derived from the epidermis of the pilot whale Globicephala melas.

    Baum, C; Fleischer, L-G; Roessner, D; Meyer, W; Siebers, D


    The rheological properties of the stratum corneum of the pilot whale (Globicephala melas) were investigated with emphasis on their significance to the self-cleaning abilities of the skin surface smoothed by a jelly material enriched with various hydrolytic enzymes. The gel formation of the collected fluid was monitored by applying periodic-harmonic oscillating loads using a stress-controlled rheometer. In the mechanical spectrum of the gel, the plateau region of the storage modulus G' (120 Pa) were independent of frequency (omega = 43.98 to 0.13 rad x s(-1), tau = 15 Pa, T = 20 degrees C), indicating high elastic performance of a covalently cross-linked viscoelastic solid. In addition, multi-angle laser light scattering experiments (MALLS) were performed to analyse the potential time-dependent changes in the weight-average molar mass of the samples. The observed increase showed that the gel formation is based on the assembly of covalently cross-linked aggregates. The viscoelastic properties and the shear resistance of the gel assure that the enzyme-containing jelly material smoothing the skin surface is not removed from the stratum corneum by shear regimes during dolphin jumping. The even skin surface is considered to be most important for the self-cleaning abilities of the dolphin skin against biofouling.

  13. Internal acetylene unit as a cross-link site for polyimides

    Takeichi, T.; Tanikawa, M. [Toyohashi Univ. of Technology (Japan)


    We have been studying on the cross-linking behavior of internal acetylenes linked meta-meta to aromatic connecting units which were introduced into the polyimide backbone utilizing 3,3`-diaminodiphenylacetylene (m-intA). In this study, we studied on the cross-linking behavior of internal acetylenes linked para-para to aromatic connecting units. The internal acetylene units were introduced into the polyimide backbone by the reaction of 4,4`-diaminodiphenylacetylene (p-intA) with such acid anhydrides as biphenyltetracarboxylic dianhydride (BPDA), pyromellitic dianhydride (PMDA), and 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6FDA). The polyimides showed exotherm on DSC: The onset of the exotherm of p-intA appeared at around 330-390{degrees}C, which is 10-50{degrees}C higher than that of m-intA. The exotherm disappeared after thermal treatment at 400{degrees}C, suggesting the progress of crosslinking between acetylene units. The polyimides cured at 350{degrees}C or 400{degrees}C showed increased Tg and improved physical properties at high temperatures as confirmed by viscoelastic analyses. It was also made clear that polyimides containing p-intA showed higher modulus compared with polyimides containing m-intA, especially when coupled with BPDA and PMDA.

  14. Polymeric redox-responsive delivery systems bearing ammonium salts cross-linked via disulfides

    Christian Dollendorf


    Full Text Available A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm and 2-(dimethylaminoethyl methacrylate (DMAEMA. N,N’-bis(4-chlorobutanoylcystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT, tris(2-carboxyethylphosphine (TCEP or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg, swelling behavior and cloud points (Tc were investigated. Redox-responsive behavior was further analyzed by rheological measurements.

  15. Biomolecule-recognition gating membrane using biomolecular cross-linking and polymer phase transition.

    Kuroki, Hidenori; Ito, Taichi; Ohashi, Hidenori; Tamaki, Takanori; Yamaguchi, Takeo


    We present for the first time a biomolecule-recognition gating system that responds to small signals of biomolecules by the cooperation of biorecognition cross-linking and polymer phase transition in nanosized pores. The biomolecule-recognition gating membrane immobilizes the stimuli-responsive polymer, including the biomolecule-recognition receptor, onto the pore surface of a porous membrane. The pore state (open/closed) of this gating membrane depends on the formation of specific biorecognition cross-linking in the pores: a specific biomolecule having multibinding sites can be recognized by several receptors and acts as the cross-linker of the grafted polymer, whereas a nonspecific molecule cannot. The pore state can be distinguished by a volume phase transition of the grafted polymer. In the present study, the principle of the proposed system is demonstrated using poly(N-isopropylacrylamide) as the stimuli-responsive polymer and avidin-biotin as a multibindable biomolecule-specific receptor. As a result of the selective response to the specific biomolecule, a clear permeability change of an order of magnitude was achieved. The principle is versatile and can be applied to many combinations of multibindable analyte-specific receptors, including antibody-antigen and lectin-sugar analogues. The new gating system can find wide application in the bioanalytical field and aid the design of novel biodevices.

  16. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin


    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Dual Cross-Linked Carboxymethyl Sago Pulp-Gelatine Complex Coacervates for Sustained Drug Delivery

    Saravanan Muniyandy


    Full Text Available In the present work, we report for the first time the complex coacervation of carboxymethyl sago pulp (CMSP with gelatine for sustained drug delivery. Toluene saturated with glutaraldehyde and aqueous aluminum chloride was employed as cross-linkers. Measurements of zeta potential confirm neutralization of two oppositely charged colloids due to complexation, which was further supported by infrared spectroscopy. The coacervates encapsulated a model drug ibuprofen and formed microcapsules with a loading of 29%–56% w/w and an entrapment efficiency of 85%–93% w/w. Fresh coacervates loaded with drug had an average diameter of 10.8 ± 1.93 µm (n = 3 ± s.d.. The coacervates could encapsulate only the micronized form of ibuprofen in the absence of surfactant. Analysis through an optical microscope evidenced the encapsulation of the drug in wet spherical coacervates. Scanning electron microscopy revealed the non-spherical geometry and surface roughness of dried drug-loaded microcapsules. X-ray diffraction, differential scanning calorimetry and thermal analysis confirmed intact and crystalline ibuprofen in the coacervates. Gas chromatography indicated the absence of residual glutaraldehyde in the microcapsules. Dual cross-linked microcapsules exhibited a slower release than mono-cross-linked microcapsules and could sustain the drug release over the period of 6 h following Fickian diffusion.

  18. Removal of anionic azo dyes from aqueous solution by functional ionic liquid cross-linked polymer

    Gao, Hejun [Key Laboratory of Colloid and Interface Chemistry, Shandong University, Ministry of Education, Jinan 250100 (China); Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, China West Normal University, Nanchong 637000 (China); Kan, Taotao [CNOOC Energy Technology and Services-oilfield Technology Services Co., Tanggu, Tianjin 300452 (China); Zhao, Siyuan; Qian, Yixia; Cheng, Xiyuan; Wu, Wenli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xiaodong [Shandong Provincial Analysis and Test Center, Jinan 250100 (China); Zheng, Liqiang, E-mail: [Key Laboratory of Colloid and Interface Chemistry, Shandong University, Ministry of Education, Jinan 250100 (China)


    Highlights: • Equilibrium, kinetic and thermodynamic of adsorption of dyes onto PDVB-IL was investigated. • PDVB-IL has a high adsorption capacity to treat dyes solution. • Higher adsorption capacity is due to the functional groups of PDVB-IL. • Molecular structure of dyes influences the adsorption capacity. -- Abstract: A novel functional ionic liquid based cross-linked polymer (PDVB-IL) was synthesized from 1-aminoethyl-3-vinylimidazolium chloride and divinylbenzene for use as an adsorbent. The physicochemical properties of PDVB-IL were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. The adsorptive capacity was investigated using anionic azo dyes of orange II, sunset yellow FCF, and amaranth as adsorbates. The maximum adsorption capacity could reach 925.09, 734.62, and 547.17 mg/g for orange II, sunset yellow FCF and amaranth at 25 °C, respectively, which are much better than most of the other adsorbents reported earlier. The effect of pH value was investigated in the range of 1–8. The result shows that a low pH value is found to favor the adsorption of those anionic azo dyes. The adsorption kinetics and isotherms are well fitted by a pseudo second-order model and Langmuir model, respectively. The adsorption process is found to be dominated by physisorption. The introduction of functional ionic liquid moieties into cross-linked poly(divinylbenzene) polymer constitutes a new and efficient kind of adsorbent.

  19. Multivalent anchoring and cross-linking of mussel-inspired antifouling surface coatings.

    Wei, Qiang; Becherer, Tobias; Mutihac, Radu-Cristian; Noeske, Paul-Ludwig Michael; Paulus, Florian; Haag, Rainer; Grunwald, Ingo


    In this work, we combine nature's amazing bioadhesive catechol with the excellent bioinert synthetic macromolecule hyperbranched polyglycerol (hPG) to prepare antifouling surfaces. hPG can be functionalized by different amounts of catechol groups for multivalent anchoring and cross-linking because of its highly branched architecture. The catecholic hPGs can be immobilized on various surfaces including metal oxides, noble metals, ceramics, and polymers via simple incubation procedures. The effect of the catechol amount on the immobilization, surface morphology, stability, and antifouling performance of the coatings was studied. Both anchoring and cross-linking interactions provided by catechols can enhance the stability of the coatings. When the catechol groups on the hPG are underrepresented, the tethering of the coating is not effective; while an overrepresentation of catechol groups leads to protein adsorption and cell adhesion. Thus, only a well-balanced amount of catechols as optimized and described in this work can supply the coatings with both good stability and antifouling ability.

  20. Cross-linked self-assembled micelle based nanosensor for intracellular pH measurements

    Ek, Pramod Kumar; Søndergaard, Rikke Vicki; Windschiegl, Barbara


    A micelle based nanosensor was synthesized and investigated as a ratiometric pH sensor for use in measurements in living cells by fluorescent microscopy. The nanosensor synthesis was based on self-assembly of an amphiphilic triblock copolymer, which was chemically cross-linked after micelle forma...... provided a sensor with a very broad measurement range that seems to be influenced by the chemical design of the sensor. Cell experiments show that the sensor is capable of monitoring the pH distributions in HeLa cells.......A micelle based nanosensor was synthesized and investigated as a ratiometric pH sensor for use in measurements in living cells by fluorescent microscopy. The nanosensor synthesis was based on self-assembly of an amphiphilic triblock copolymer, which was chemically cross-linked after micelle...... formation. The copolymer, poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate)-b-poly(styrene) (PEG-b-PAEMA-b-PS), was synthesized by isolated macroinitiator atom transfer radical polymerization that forms micelles spontaneously in water. The PAEMA shell of the micelle was hereafter cross...

  1. Synthesis of Covalently Cross-Linked Colloidosomes from Peroxidized Pickering Emulsions

    Nadiya Popadyuk


    Full Text Available A new approach to the formation of cross-linked colloidosomes was developed on the basis of Pickering emulsions that were stabilized exclusively by peroxidized colloidal particles. Free radical polymerization and a soft template technique were used to convert droplets of a Pickering emulsion into colloidosomes. The peroxidized latex particles were synthesized in the emulsion polymerization process using amphiphilic polyperoxide copolymers poly(2-tert-butylperoxy-2-methyl-5-hexen-3-ine-co-maleic acid (PM-1-MAc or poly[N-(tert-butylperoxymethylacrylamide]-co-maleic acid (PM-2-MAc, which were applied as both initiators and surfactants (inisurfs. The polymerization in the presence of the inisurfs results in latexes with a controllable amount of peroxide and carboxyl groups at the particle surface. Peroxidized polystyrene latex particles with a covalently grafted layer of inisurf PM-1-MAc or PM-2-MAc were used as Pickering stabilizers to form Pickering emulsions. A mixture of styrene and/or butyl acrylate with divinylbenzene and hexadecane was applied as a template for the synthesis of colloidosomes. Peroxidized latex particles located at the interface are involved in the radical reactions of colloidosomes formation. As a result, covalently cross-linked colloidosomes were obtained. It was demonstrated that the structure of the synthesized (using peroxidized latex particles colloidosomes depends on the amount of functional groups and pH during the synthesis. Therefore, the size and morphology of colloidosomes can be controlled by latex particle surface properties.

  2. Development and characterization of films based on chemically cross-linked gliadins.

    Hernández-Muñoz, Pilar; Kanavouras, Antonis; Lagaron, José M; Gavara, Rafael


    The aim of the present work has been to study the possibility of obtaining modified gliadin films with improved water resistance and mechanical properties by means of promoting intermolecular covalent bonds between polypeptide chains. Prior to casting films, formaldehyde, glutaraldehyde, and glyoxal were used to cross-link proteins at concentrations ranging from 1% to 4% (grams per 100 g of protein). Mechanical properties (tensile strength and elongation at break), water vapor permeability, moisture sorption isotherms, and optical properties of the films produced were evaluated as a function of the cross-linker used. Experimental results showed that some properties of gliadin films were considerably modified. Cross-linking improved the water resistance of films, avoiding their disintegration. Their water barrier properties were also enhanced, but their moisture sorption properties remained unchanged. Formaldehyde imparted greater mechanical strength to films than glutaraldehyde or glyoxal, increasing tensile strength values 10-fold. Addition of the cross-linkers at concentrations in excess of 2.5% did not further improve the mechanical or barrier properties. However, modification with glutaraldehyde or glyoxal imparted an increasingly yellowish tint to the films.

  3. Angiotensin I-converting enzyme inhibitor derived from cross-linked oyster protein.

    Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young; Choi, Yeung-Joon


    Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

  4. Effects of Composition of Iron-Cross-Linked Alginate Hydrogels for Cultivation of Human Dermal Fibroblasts

    Ikuko Machida-Sano


    Full Text Available We investigated the suitability of ferric-ion-cross-linked alginates (Fe-alginate with various proportions of L-guluronic acid (G and D-mannuronic acid (M residues as a culture substrate for human dermal fibroblasts. High-G and high-M Fe-alginate gels showed comparable efficacy in promoting initial cell adhesion and similar protein adsorption capacities, but superior cell proliferation was observed on high-G than on high-M Fe-alginate as culture time progressed. During immersion in culture medium, high-G Fe-alginate showed little change in gel properties in terms of swelling and polymer content, but the properties of high-M Fe-alginate gel were altered due to loss of ion cross-linking. However, the degree of cell proliferation on high-M Fe-alginate gel was improved after it had been stabilized by immersion in culture medium until no further changes occurred. These results suggest that the mode of cross-linkage between ferric ions and alginate differs depending on alginate composition and that the major factor giving rise to differences in cell growth on the two types of Fe-alginate films is gel stability during culture, rather than swelling of the original gel, polymer content, or protein adsorption ability. Our findings may be useful for extending the application of Fe-alginate to diverse biomedical fields.

  5. Low-Temperature Cross-Linking of PEDOT:PSS Films Using Divinylsulfone.

    Mantione, Daniele; Del Agua, Isabel; Schaafsma, Wandert; ElMahmoudy, Mohammed; Uguz, Ilke; Sanchez-Sanchez, Ana; Sardon, Haritz; Castro, Begoña; Malliaras, George G; Mecerreyes, David


    Recent interest in bioelectronics has prompted the exploration of properties of conducting polymer films at the interface with biological milieus. Poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) from a commercially available source has been used as a model system for these studies. Different cross-linking schemes have been used to stabilize films of this material against delamination and redispersion, but the cost is a decrease in the electrical conductivity and/or additional heat treatment. Here we introduce divinylsulfone (DVS) as a new cross-linker for PEDOT:PSS. Thanks to the higher reactiveness of the vinyl groups of DVS, the cross-linking can be performed at room temperature. In addition, DVS does not reduce electronic conductivity of PEDOT:PSS but rather increases it by acting as a secondary dopant. Cell culture studies show that PEDOT:PSS:DVS films are cytocompatible and support neuroregeneration. As an example, we showed that this material improved the transconductance value and stability of an organic electrochemical transistor (OECT) device. These results open the way for the utilization of DVS as an effective cross-linker for PEDOT:PSS in bioelectronics applications.

  6. Nanomechanical characterization and molecular mechanism study of nanoparticle reinforced and cross-linked chitosan biopolymer.

    Rath, Amrita; Mathesan, Santhosh; Ghosh, Pijush


    Chitosan (CS) is a biomaterial that offers many sophisticated and innovative applications in the biomedical field owing to its excellent characteristics of biodegradability, biocompatibility and non-toxicity. However, very low mechanical properties of chitosan polymer impose restriction on its further development. Cross-linking and nanoparticle reinforcement are the two possible methods to improve the mechanical properties of chitosan films. In this research, these two methods are adopted individually by using tripolyphosphate as cross-linker and nano-hydroxyapatite as particle reinforcement. The nanomechanical characterizations under static loading conditions are performed on these modified chitosan films. It is observed that nanoparticle reinforcement provided necessary mechanical properties such as ductility and modulus. The mechanisms involved in improvement of mechanical properties due to particle reinforcement are studied by molecular dynamics (MD). Further, improvement in mechanical properties due to combination of particle reinforcement and cross-linking agent with chitosan is investigated. The stress relaxation behavior for all these types of films is characterized under dynamic loading conditions using dynamic mechanical analysis (nanoDMA) experiment. A viscoelastic solid like response is observed for all types of film with modulus relaxing by 3-6% of its initial value. A suitable generalized Maxwell model is fitted with the obtained viscoelastic response of these films. The response to nano-scratch behavior is also studied for particle reinforced composite films.

  7. In situ cross-linking of stimuli-responsive hemicellulose microgels during spray drying.

    Zhao, Weifeng; Nugroho, Robertus Wahyu N; Odelius, Karin; Edlund, Ulrica; Zhao, Changsheng; Albertsson, Ann-Christine


    Chemical cross-linking during spray drying offers the potential for green fabrication of microgels with a rapid stimuli response and good blood compatibility and provides a platform for stimuli-responsive hemicellulose microgels (SRHMGs). The cross-linking reaction occurs rapidly in situ at elevated temperature during spray drying, enabling the production of microgels in a large scale within a few minutes. The SRHMGs with an average size range of ∼ 1-4 μm contain O-acetyl-galactoglucomannan as a matrix and poly(acrylic acid), aniline pentamer (AP), and iron as functional additives, which are responsive to external changes in pH, electrochemical stimuli, magnetic field, or dual-stimuli. The surface morphologies, chemical compositions, charge, pH, and mechanical properties of these smart microgels were evaluated using scanning electron microscopy, IR, zeta potential measurements, pH evaluation, and quantitative nanomechanical mapping, respectively. Different oxidation states were observed when AP was introduced, as confirmed by UV spectroscopy and cyclic voltammetry. Systematic blood compatibility evaluations revealed that the SRHMGs have good blood compatibility. This bottom-up strategy to synthesize SRHMGs enables a new route to the production of smart microgels for biomedical applications.

  8. Synthesis and antimicrobial activity of some novel cross-linked chitosan hydrogels.

    Mohamed, Nadia Ahmed; Fahmy, Mona Mohamed


    Four novel hydrogels based on chitosan were synthesized via a cross-linking reaction of chitosan with different concentrations of oxalyl bis 4-(2,5-dioxo-2H-pyrrol- 1(5H)-yl)benzamide. Their structures were confirmed by fourier transform infrared X-ray (FTIR), scanning electron microscopy (SEM) and X-ray diffraction. The antimicrobial activities of the hydrogels against two crop-threatening pathogenic fungi namely: Aspergillus fumigatus (A. fumigatus, RCMBA 06002), and Aspergillus niger (A. niger, RCMBA 06106), and five bacterial species namely: Bacillis subtilis (B. subtilis, RCMBA 6005), Staphylococcus aureus (S. aureus, RCMBA 2004), Streptococcus pneumoniae (S. pneumonia, RCMB 000101) as Gram positive bacteria, and Salmonella typhimurium (S. typhimurium, RCMB 000104), and Escherichia coli (E. coli, RCMBA 5003) as Gram negative bacteria have been investigated. The prepared hydrogels showed much higher antimicrobial activities than that of the parent chitosan. The hydrogels were more potent in case of Gram-positive bacteria than Gram-negative bacteria. Increasing the degree of cross-linking in the hydrogels resulted in a weaker antimicrobial activity.

  9. Synthesis and Antimicrobial Activity of Some Novel Cross-Linked Chitosan Hydrogels

    Mona Mohamed Fahmy


    Full Text Available Four novel hydrogels based on chitosan were synthesized via a cross-linking reaction of chitosan with different concentrations of oxalyl bis 4-(2,5-dioxo-2H-pyrrol-1(5H-ylbenzamide. Their structures were confirmed by fourier transform infrared X-ray (FTIR, scanning electron microscopy (SEM and X-ray diffraction. The antimicrobial activities of the hydrogels against two crop-threatening pathogenic fungi namely: Aspergillus fumigatus (A. fumigatus, RCMBA 06002, and Aspergillus niger (A. niger, RCMBA 06106, and five bacterial species namely: Bacillis subtilis (B. subtilis, RCMBA 6005, Staphylococcus aureus (S. aureus, RCMBA 2004, Streptococcus pneumoniae (S. pneumonia, RCMB 000101 as Gram positive bacteria, and Salmonella typhimurium (S. typhimurium, RCMB 000104, and Escherichia coli (E. coli, RCMBA 5003 as Gram negative bacteria have been investigated. The prepared hydrogels showed much higher antimicrobial activities than that of the parent chitosan. The hydrogels were more potent in case of Gram-positive bacteria than Gram-negative bacteria. Increasing the degree of cross-linking in the hydrogels resulted in a weaker antimicrobial activity.

  10. Covalent Immobilization of Catalase onto Regenerated Silk Fibroins via Tyrosinase-Catalyzed Cross-Linking.

    Wang, Ping; Qi, Chenglong; Yu, Yuanyuan; Yuan, Jiugang; Cui, Li; Tang, Gengtie; Wang, Qiang; Fan, Xuerong


    Regenerated silk fibroins could be used as medical scaffolds and carrier materials for enzyme immobilization. In the present work, tyrosinase enzyme was used for enzymatic oxidation of silk fibroins, followed by immobilization of catalase onto the fibroin surfaces through physical adsorption and covalent cross-linking as well. Spectrophotometry, SDS-PAGE, and Fourier transform infrared spectroscopy (FTIR) were used to examine the efficiency of enzymatic oxidation and catalase immobilization, respectively. The results indicate that tyrosine residues in silk fibroins could be oxidized and converted to the active o-quinones. Incubating silk fibroins with catalase and tyrosinase led to a noticeable change of molecular weight distribution, indicating the occurrence of the cross-links between silk fibroins and catalase molecules. Two different pathways were proposed for the catalase immobilizations, and the method based on grafting of catalase onto the freeze-dried fibroin membrane is more acceptable. The residual enzyme activity for the immobilized catalase exhibited higher than that of the control after repeated washing cycles. Meanwhile, the thermal stability and alkali resistance were also slightly improved as compared to free catalase. The mechanisms of enzymatic immobilization are also concerned.

  11. Cross-linked chitosan/liposome hybrid system for the intestinal delivery of quercetin.

    Caddeo, Carla; Díez-Sales, Octavio; Pons, Ramon; Carbone, Claudia; Ennas, Guido; Puglisi, Giovanni; Fadda, Anna Maria; Manconi, Maria


    Quercetin is a flavonoid with antioxidant/anti-inflammatory properties, poorly absorbed when administered orally. To increase its bioavailability and optimize its release in the intestine, a hybrid system made of liposomes coated with cross-linked chitosan, named TPP-chitosomes, was developed and characterized by light scattering, transmission electron microscopy, differential scanning calorimetry, X-ray powder diffraction and Turbiscan® technology. The TPP-chitosomes were nanosized (∼180 nm), fairly spherical in shape and unilamellar. The actual coating of the surface of liposomes with the cross-linked chitosan was demonstrated by Small-Angle X-ray Scattering. The release of quercetin in simulated gastric and intestinal pH was investigated, the results showing that the system provided resistance to acidic conditions, and promoted the release in alkaline pH, mimicking the intestinal environment. The proposed hybrid system represents a promising combination of nanovesicles and chitosan for the delivery of quercetin to the intestine in the therapy of oxidative stress/inflammation related disorders.

  12. Comparative Study of One-Step Cross-Linked Electrospun Chitosan-Based Membranes

    Yanet E. Aguirre-Chagala


    Full Text Available Chitosan membranes are widely applied for tissue engineering; however, a major drawback is their low resistance in aqueous phases and therefore the structure collapses impeding their long-term use. Although there is extensive research, because of chitosan’s importance as a biomaterial, studies involving chitosan-based membranes are still needed. Herein, a detailed investigation of diverse chemical routes to cross-link fibers in situ by electrospinning process is described. In case of using genipin as cross-linker, a close relationship with the content and the mean diameter values is reported, suggesting a crucial effect over the design of nanostructures. Also, the physical resistance is enhanced for the combination of two types of methods, such as chemical and physical methods. Cross-linked fibers upon exposure to long wave ultraviolet A (UVA light change their morphology, but not their chemical composition. When they are incubated in aqueous phase for 70 days, they show an extensive improvement of their macrostructural integrity which makes them attractive candidates for tissue engineering application. As a result, the thermal properties of these materials reveal less crystallinity and higher temperature of degradation.

  13. Alkaline membrane fuel cells with in-situ cross-linked ionomers

    Leng, YJ; Wang, LZ; Hickner, MA; Wang, CY


    Improving cell performance and durability through both new materials and membrane electrode processing optimization is needed for the commercialization of alkaline membrane fuel cell (AMFC) technologies. In this work, we adopted an in-situ cross-linking strategy of an anion-conducting block copolymer to prepare durable ionomers for use in alkaline membrane fuel cells (AMFCs). Our goal was to use new ionomers and binders with an aim at improving long-term stability of AMFCs, especially at high operation temperatures. At 80 degrees C, AMFCs with in-situ cross-linked ionomers showed promising stability with an operating life time of more than 350 hours at 100 mA/cm(2). We found that the optimized electrode fabrication process and operating conditions can significantly improve the durability performance of AMFCs. For example, a suitable electrode binder in addition to the ion-conducting ionomer can greatly enhance the durability performance of AMFCs. Operating fuel cells under a cathode over-humification condition can also enhance the long-term stability of AMFCs. (C) 2014 Elsevier Ltd. All rights reserved.

  14. Chemical, Physical, and Mechanical Characterization of Isocyanate Cross-linked Amine-Modified Silica Aerogels

    Katti, Atul; Shimpi, Nilesh; Roy, Samit; Lu, Hongbing; Fabrizio, Eve F.; Dass, Amala; Capadona, Lynn A.; Leventis, Nicholas


    We describe a new mechanically strong lightweight porous composite material obtained by encapsulating the skeletal framework of amine-modified silica aerogels with polyurea. The conformal polymer coating preserves the mesoporous structure of the underlying silica framework and the thermal conductivity remains low at 0.041 plus or minus 0.001 W m(sup -1 K(sup -1). The potential of the new cross-linked silica aerogels for load-carrying applications was determined through characterization of their mechanical behavior under compression, three-point bending, and dynamic mechanical analysis (DMA). A primary glass transition temperature of 130 C was identified through DMA. At room temperature, results indicate a hyperfoam behavior where in compression cross-linked aerogels are linearly elastic under small strains (less than 4%) and then exhibit yield behavior (until 40% strain), followed by densification and inelastic hardening. At room temperature the compressive Young's modulus and the Poisson's ratio were determined to be 129 plus or minus 8 MPa and 0.18, respectively, while the strain at ultimate failure is 77% and the average specific compressive stress at ultimate failure is 3.89 x 10(exp 5) N m kg(sup -1). The specific flexural strength is 2.16 x 10(exp 4) N m kg(sup -1). Effects on the compressive behavior of strain rate and low temperature were also evaluated.

  15. Adsorption of Pb{sup 2+} on chitosan cross-linked with triethylene-tetramine

    Tang, X.H. [College of Chemistry and Chemical Engineering, Hunan University, Changsha (China); College of Environmental and Chemical Engineering, Nanchang Institute of Aeronautical Technology (China); Zhang, X.M.; Zhou, A.L. [College of Environmental and Chemical Engineering, Nanchang Institute of Aeronautical Technology (China); Guo, C.C. [College of Chemistry and Chemical Engineering, Hunan University, Changsha (China)


    A novel triethylene-tetramine cross-linked chitosan (CCTS) was synthesized via the cross-linking of triethylene-tetramine and epichlorohydrin activated chitosan. Its structure was characterized by elemental analysis, infrared spectroscopy and X-ray diffraction analysis, and the surface topography was determined with ESEM. The results were in agreement with expectations. The capacity of CCTS to adsorb Pb{sup 2+} ions from aqueous solutions was examined, and equilibrium and kinetic investigations were undertaken. The adsorption isotherms were fitted well by the Langmuir equation (R > 0.999). The maximum adsorbed amount, at pH 5.5, with an initial concentration of 3 mmol/L (621 ppm), was 378.8 mg/g. The adsorption process could be best described by a second-order equation (R = 1). This suggests that the rate-limiting step may be the chemical adsorption (chemisorption) step and not the mass transport. The separation factor used was 0 < R{sub L} < 1. Therefore, it can be concluded that CCTS is an effective adsorbent for the collection of Pb{sup 2+}. (Abstract Copyright [2007], Wiley Periodicals, Inc.)

  16. Kinetics of copper ion absorption by cross-linked calcium polyacrylate membranes

    Philipp, W. H.; May, C. E.


    The absorption of copper ions from aqueous copper acetate solutions by cross-linked calcium acrylate membranes was found to obey parabolic kinetics similar to that found for oxidation of metals that form protective oxide layers. For pure calcium polyacrylate membranes the rate constant was essentially independent of copper acetate concentration and film thickness. For a cross-linked copolymer film of polyvinyl alcohol and calcium polyacrylate, the rate constant was much greater and dependent on the concentration of copper acetate. The proposed mechanism in each case involves the formation of a copper polyacrylate phase on the surface of the membrane. The diffusion of the copper ion through this phase appears to be the rate controlling step for the copolymer film. The diffusion of the calcium ion is apparently the rate controlling step for the calcium polyacrylate. At low pH, the copper polyacrylate phase consists of the normal copper salt; at higher pH, the phase appears to be the basic copper salt.

  17. Kinetics of chromium ion absorption by cross-linked polyacrylate films

    May, C. E.


    Three cross-linked ion exchange membranes were studied as to their ability to absorb chromium ion from aqueous chromium III nitrate solutions. Attention was given to the mechanism of absorption, composition of the absorbed product, and the chemical bonding. The membranes were: calcium polyacrylate, polyacrylic acid, and a copolymer of acrylic acid and vinyl alcohol. For the calcium polyacrylate and the copolymer, parabolic kinetics were observed, indicating the formation of a chromium polyacrylate phase as a coating on the membrane. The rate of absorption is controlled by the diffusion of the chromium ion through this coating. The product formed in the copolymer involves the formation of a coordination complex of a chromium ion with 6 carboxylic acid groups from the same molecule. The absorption of the chromium ion by the polyacrylic acid membranes appears to be more complicated, involving cross-linking. This is due to the coordination of the chromium ion with carboxylic acid groups from more than one polymer molecule. The absorption rate of the chromium ion by the calcium salt membrane was found to be more rapid than that by the free polyacrylic acid membrane.

  18. [Healing of osseous defects by guided bone regeneration using ribose cross linked collagen membranes].

    Tal, H


    The ultimate goal of periodontal therapy has long been the complete regeneration of the periodontal attachment apparatus. Guided Tissue Regeneration (GTR) and Guided Bone Regeneration (GBR) are two regenerative procedures which converted this goal from a dream to reality. In search of a biocompatible resorbable tissue barrier, collagen, being a natural protein and a weak antigen, has attracted much interest and became the focus of much intention during the 80's and the 90's. The understanding that cross linking of collagen with aldehyde sugars, especially ribose, produces collagen which is highly resistant to resorption in vivo led to the development of a "natural" Crossed-Linked Collagen Barrier (CB-SX). Animal and Human studies have shown that the newly developed membrane is biocompatible, remains intact in the tissues 6 months and more, and results in impressive guided tissue/bone regeneration. Spontaneous early exposure of the membrane is common but the healing potential of the resulted tissue dehiscence is favorable with no tendency for bacterial infection. The commercial version of the CB-SX is especially suitable for GBR procedures; it is highly recommended that the gingival flaps involved will properly be released, will lack tension, and be thoroughly sutured.

  19. Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

    Cheng-Liang Xie


    Full Text Available Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50 of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR. The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

  20. Characterization of Chemical and Physical Properties of Hydroxypropylated and Cross-linked Arrowroot (Marantha arundinacea Starch

    Rijanti Rahaju Maulani


    Full Text Available The modern food industry and a variety of food products require tolerant starch as raw material for processing in a broad range of techniques, from preparation to storage and distribution. Dual modification of arrowroot starch using hydroxypropylation and cross-linking was carried out to overcome the lack of native arrowroot starch in food processing application. The modifications applied were: combined propylene oxide (8%, 10%, and 12%; sodium tri meta phosphate/STMP (1%, 2%, and 3%; and sodium tri poly phosphate/STPP (4%, 5%, and 6%. These modifications significantly affected the composition of the amylose and amylopectin and the amount of phosphorus in the granules. Higher amounts of phosphate salt gave a higher phosphorus content, which increased the degree of substitution (DS and the degree of cross-link. Arrowroot starch that was modified using a concentration of 8-10% propylene oxide and 1-2% STMP : 3-5% STPP produced a starch with < 0.4% phosphorus content. A higher concentration of propylene oxide provided a higher degree of hydroxypropyl. The changed physical properties of the modified granular arrowroot starch were examined through SEM testing, and its changed crystalline patterns through X-ray diffraction measurements. Especially, provision of a high concentration of propylene oxide (12% combined with 3% STMP : 6% STPP affected the granular morphology and the crystallinity.