A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

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.

Stereoselective synthesis of vinyl germanes and silanes. Applications of tris(trimethyl)germanes and silanes in Pd-catalyzed cross -coupling reactions

Although group 14 organometallic compounds (Si, Sn) have been well developed as transmetallation reagents in cross-coupling reactions, the application of organogermanium compounds as cross-coupling reagents is still a relatively new area with few papers published. This study aimed to develop methods for the synthesis of new classes of vinyl germane and vinyl silane compounds, mainly Z and E tris(trimethylsilyl)germanes and silanes, which were then applied to Pd-catalyzed cross-couplings with aryl and alkenyl halides. The stereoselective radical-mediated desulfonylation of vinyl sulfones with tris(trimethyl)germanium or silane hydrides provided access to the synthesis of trans vinyl germanes or silanes. Alternatively hydrogermylation or hydrosilylation of terminal alkynes gave cis vinyl germanes or silanes. The application of these new classes of organometallic compounds in cross-coupling reactions with various aryl and alkenyl halides under aqueous [NaOH/H2O2/Pd(PPh 3)4] and anhydrous [KH/t-BuOOH/Pd(PPh 3)4] oxidative conditions were investigated. ^ It was found that the vinyl tris(trimethylsilyl)germanes successfully underwent Pd-catalyzed cross-couplings with aryl and alkenyl halides and aryl triflates under aqueous and anhydrous oxidative conditions. These procedures provided examples of "ligand-free" Pd-catalyzed coupling of organogermanes with aryl and alkenyl halides. Interestingly, couplings with fluorinated vinyl germanes appeared to occur more easily than with the corresponding (α-fluoro)vinyl stannanes and silanes since neither addition of an extra ligand nor activation with fluoride was necessary. The vinyl tris(trimethyl)silanes were found to be alternative substrates for the Hiyama reaction. The coupling of TTMS-silanes with various aryl, heteroaryl as well as alkenyl halides proceeded smoothly upon treatment with hydrogen peroxide in the presence of sodium hydroxide and fluoride ion. ^

Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritionaland physiological properties of food proteins. In this study, cross-linked enzyme aggre-gates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillusdelbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using severalfood proteins. We have optimized cross-linking conditions including ammonium sulphateconcentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker con-centration, reaction time and the addition of proteic feeders. Particularly, the presence ofBSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%.Blocking unreacted cross-linking groups on aggregates is important to enhance recyclabil-ity of CLCEPAs. CLCEPAs had attractive thermal stability at 50◦C and it showed enhancedcatalytic activity over long-term storage after lyophilization. We have demonstrated thatCLCEPAs has proteolytic properties on different food proteins including complex (chickenegg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), andpurified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilliCEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation ofvarious food proteins.

External pressure in the hardening of phosphate in tribofilm on iron surfaces

Purpose: In the present work we consider our (in progress) spectroscopy study of zinc and iron phosphates under the influence external high pressure to determine zinc ion change coordination from tetrahedral to octahedral (or hexahedral) structure.----- Design/methodology/approach: The standard equipment is the optical high pressure cell with diamond (DAC). The DAC is assembled and then vibrational or electronic spectra are collected by mounting the cell in an infrared, Raman, EXAFS or UV-visible spectrometer.----- Findings: Mechanism by which zinc and iron methaphosphate material is transformed to glassy meta-phosphate is enhancing mechanical properties of tribofilm. The two decades of intensive study demonstrates that Zn (II) and Fe (III) ions participate to cross-link network under friction, hardening the phosphate.----- Research limitations/implications: Transition metal atoms with d orbital have flexible coordination numbers, for example zinc acts as a cross-linking agent increasing hardness, by changing coordination from tetrahedral to octahedral. Perhaps the external pressure effect on the [Zn–(O-P-)4 ] complex causes a transformation to an [Zn –(O-P-)6] grouping.----- Originality/value: This paper analyses high-pressure spectroscopy which has been applied for the investigation of 3D transition metal ions in solids. When studying pressure effects on coordination compounds structure, we can expect changes in ground electronic state (spin-crossovers), electronic spectra due to structural distortions (piezochromism), and changes in the ligand field causing shifts in the electronic transitions.

Crystallographic characterization of the first reported crystalline form of the potent hallucinogen (R)-2-amino-1-(8-bromobenzo[1,2-b;5,4-b']difuran-4-yl)propane or `bromodragonfly': the 1:1 anhydrous proton-transfer compound with 3,5-dinitrosalicylic acid

The 1:1 proton-transfer compound of the potent substituted amphetamine hallucinogen (R)-1-(8-bromobenzo[1,2-b; 4,5-b']difuran-4-yl)-2-aminopropane (common trivial name 'bromodragonfly') with 3,5-dinitrosalicylic acid, 1-(8-bromobenzo[1,2-b;4,5-b']difuran-4-yl)-2-mmoniopropane 2-carboxy-4,6-dinitrophenolate, C13H13BrNO2+ C7H3N2O7- forms hydrogen-bonded cation-anion chain substructures comprising undulating head-to-tail anion chains formed through C(8) carboxyl O-H...O(nitro) associations and incorporating the aminium groups of the cations. The intra-chain cation-anion hydrogen-bonding associations feature proximal cyclic R33(8) interactions involving both a N+-H...O(phenolate) and the carboxyl O--H...O(nitro)associations. Also present are aromatic pi-pi ring interactions [minimum ring centroid separation, 3.566(2)A; inter-plane dihedral angle, 5.13(1)deg]. A lateral hydrogen-bonding interaction between the third aminium proton and a carboxyl O acceptor link the chain substructures giving a two-dimensional sheet structure. This determination represents the first of any form of this compound and confirms that it has the (R) absolute configuration. The atypical crystal stability is attributed both to the hydrogen-bonded chain substructures provided by the anions, which accommodate the aminium proton-donor groups of the cations and give cross-linking, and to the presence of cation--anion aromatic ring pi-pi interactions.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

An audio wiki supporting mobile collaboration

Wikis have proved to be very effective collaboration and knowledge management tools in large variety of fields thanks to their simplicity and flexible nature. Another important development for the internet is the emergence of powerful mobile devices supported by fast and reliable wireless networks. The combination of these developments begs the question of how to extend wikis on mobile devices and how to leverage mobile devices' rich modalities to supplement current wikis. Realizing that composing and consuming through auditory channel is the most natural and efficient way for mobile device user, this paper explores the use of audio as the medium of wiki. Our work, as the first step towards this direction, creates a framework called Mobile Audio Wiki which facilitates asynchronous audio-mediated collaboration on the move. In this paper, we present the design of Mobile Audio Wiki. As a part of such design, we propose an innovative approach for a light-weight audio content annotation system for enabling group editing, versioning and cross-linking among audio clips. To elucidate the novel collaboration model introduced by Mobile Audio Wiki, its four usage modes are identified and presented in storyboard format. Finally, we describe the initial design for presentation and navigation of Mobile Audio Wiki.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Synthesis of an erodible biomimetic hydrogel for drug delivery using native chemical ligation

Hydrogels are hydrophilic, three dimensional polymers that imbibe large quantities of water while remaining insoluble in aqueous solutions due to chemical or physical cross-linking. The polymers swell in water or biological fluids, immobilizing the bioactive agent, leading to drug release in a well-defined specific manner. Thus the hydrogels’ elastic properties, swellability and biocompatibility make them excellent formulations for drug delivery. Currently, many drug potencies and therapeutic effects are limited or otherwise reduced because of the partial degradation that occurs before the administered drug reaches the desired site of action. On the other hand, sustained release medications release drugs continually, rather than providing relief of symptoms and protection solely when necessary. In fact, it would be much better if drugs could be administered in a manner that precisely matches physiological needs at desired times and at the desired site (site specific targeting). There is therefore an unmet need to develop controlled drug delivery systems especially for delivery of peptide and protein bound drugs. The purpose of this project is to produce hydrogels for structural drug delivery and time-dependent sustained release of drugs (bioactive agents). We use an innovative polymerisation strategy based on native chemical ligation (NCL) to covalently cross-link polymers to form hydrogels. When mixed in aqueous solution, four armed (polyethylene glycol) amine (PEG-4A) end functionalised with thioester and four branched Nterminal cysteine peptide dendrimers spontaneously conjugated to produce biomimetic hydrogels. These hydrogels showed superior resistance to shear stress compared to an equivalent PEG macromonomer system and were shown to be proteolytically degradable with concomitant release of a model payload molecule. This is the first report of a peptide dendrimers/PEG macromonomer approach to hydrogel production and opens up the prospect of facile hydrogel synthesis together with tailored payload release.

Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering

In this study, a hierarchical nano/microfibrous chitosan/collagen scaffold that approximates structural and functional attributes of native extracellular matrix (ECM), has been developed for applicability in skin tissue engineering. Scaffolds were produced by electrospinning of chitosan followed by imbibing of collagen solution, freeze-drying and subsequent cross-linking of two polymers. Scanning electron microscopy showed formation of layered scaffolds with nano/microfibrous architechture. Physico-chemical properties of scaffolds including tensile strength, swelling behavior and biodegradability were found satisfactory for intended application. 3T3 fibroblasts and HaCaT keratinocytes showed good in vitro cellular response on scaffolds thereby indicating the matrices′ cytocompatible nature. Scaffolds tested in an ex vivo human skin equivalent (HSE) wound model, as a preliminary alternative to animal testing, showed keratinocyte migration and wound re-epithelization — a pre-requisite for healing and regeneration. Taken together, the herein proposed chitosan/collagen scaffold, shows good potential for skin tissue engineering.

trans-Polyacetylene on sodium and cesium mordenites: a resonance Raman spectroscopic study

Sodium and cesium mordenite (denoted NaM and CsM, respectively) were investigated as potential catalysts for the synthesis of polyacetylene ((CH) x). Both were successful in initiating polymerization of purified gaseous acetylene at room temperature as evidenced by Raman spectroscopic studies. The polyacetylene synthesised in this way exhibited resonance enhancement of the polyene skeletal vibrations. trans-Polyacetylene, but no cis-(CH) x, was detected. As no apparent coloration of the NaM and CsM substrates accompanied the formation of trans-(CH) x it was concluded that only small quantities of the polymer were present. The number of conjugated double bonds was estimated from the frequencies of the Raman active C-C and C=C stretching vibrations, and it was shown that the trans-(CH) x formed on CsM has a distribution of conjugation lengths ranging from less than 6 to at least 30 double bonds. The polyacetylene formed on NaM was significantly shorter and was produced in lower yields than that synthesized on CsM. "Sliced" resonance excitation profiles of polyacetylene formed on CsM were obtained using nearly 40 different excitation wavelengths and these confirmed that the adsorbed trans-(CH) x was composed of segments having a distribution of conjugated lengths. The architecture of the mordenite pore system permitted only a single polymer molecule per channel, thereby preventing cross-linking. Raman spectroscopic studies of the effects of exposure to air revealed that progressive oxidative degradation occurred with a reduction in the number of conjugated double bond

Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs

Gelatin-methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross-linking parameters. Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.

Synthesis and modification of Alumina nanofibres and its applications

This thesis is a forward study of alumina nanofiber material in developing its applications biology field. It demonstrates that by applying proper modification strategy, alumina nanofiber is a promising material in protein purification and enzyme immobilization. The hydrophobic modification has dramatically improved the rejecting of protein molecular in purification system. On the other hand, utilisation of cross-linking agent firmly combined alumina nanofiber and target enzyme for immobilisation purpose. This step of progress could lead to inspiration of alumina nanofiber’s application in various area.

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

Non-invasive assessment of corneal crosslinking changes using polarization sensitive optical coherence tomography

Collagen crosslinking (CXL) has shown promising results in the prevention of the progression of keratoconus and corneal ectasia. However, techniques for in vivo and in situ assessment of the treatment are limited. In this study, ex vivo porcine eyes were treated with a chemical CXL agent (glutaraldehyde), during which polarization sensitive optical coherence tomography (PS-OCT) recordings were acquired simultaneously to assess the sensitivity of the technique to assess changes in the cornea. The results obtained in this study suggest that PS-OCT may be a suitable technique to measure CXL changes in situ and to assess the local changes in the treated region of the cornea.