Spatially orthogonal chemical functionalization of a hierarchical pore network for catalytic cascade reactions

The chemical functionality within porous architectures dictates their performance as heterogeneous catalysts; however, synthetic routes to control the spatial distribution of individual functions within porous solids are limited. Here we report the fabrication of spatially orthogonal bifunctional porous catalysts, through the stepwise template removal and chemical functionalization of an interconnected silica framework. Selective removal of polystyrene nanosphere templates from a lyotropic liquid crystal-templated silica sol–gel matrix, followed by extraction of the liquid crystal template, affords a hierarchical macroporous–mesoporous architecture. Decoupling of the individual template extractions allows independent functionalization of macropore and mesopore networks on the basis of chemical and/or size specificity. Spatial compartmentalization of, and directed molecular transport between, chemical functionalities affords control over the reaction sequence in catalytic cascades; herein illustrated by the Pd/Pt-catalysed oxidation of cinnamyl alcohol to cinnamic acid. We anticipate that our methodology will prompt further design of multifunctional materials comprising spatially compartmentalized functions.

Functionalisation of polymers: reactive processing, structure and performance characteristics

The main aim of this work was to study the effect of two comonomers, trimethylolpropane trimethacrylate (TRIS) and divinylbenzene (DVB) on the nature and efficiency of grafting of two different monomers, glycidyl methacrylate (GMA) and maleic anhydride (MA) on polypropylene (P) and on natural rubber (NR) using reactive processing methods. Four different peroxides, benzoyl peroxide (BPO), dicumyl peroxide (DCP), 2,5-dimethyl-2,5-bis-(tert-butyl peroxy) hexane (t-101), and 1,1-di(tert-butylperoxy)-3,3,5-trimethyl cyclohexene (T-29B90) were examined as free radical initiators. An appropriate methodology was established and chemical composition and reactive processing parameters were examined and optimised. It was found that in the absence of the coagents DVB and TRIS, the grafting degree of GMA and MA increased with increasing peroxide concentration, but the level of grafting was low and the homopolymerisaton of GMA and the crosslinking of NR or chain scission of PP were identified as the main side reactions that competed with the desired grafting reaction in the polymers. At high concentrations of the peroxide T-101 (>0.02 mr) cross linking of NR and chain scission of PP became dominant and unacceptable. An attempt to add a reactive coagent, e.g. TRIS during grafting of GMA on natural rubber resulted in excessive crosslinking because of the very high reactivity of this comonomer with the C=C of the rubber. Therefore, the use of any multifunctional and highly reactive coagent such as TRIS, could not be applied in the grafting of GAM onto natural rubber. In the case of PP, however, the use of TRIS and DVB was shown to greatly enhance the grafting degree and reduce the chain scission with very little extent of monomer homopolymerisation taking place. The results showed that the grafting degree was increased with increasing GMA and MA concentrations. It was also found that T-101 was a suitable peroxide to initiate the grafting reaction of these monomers on NR and PP and the optimum temperature for this peroxide was =160°C. A very preliminary work was also conducted on the use of the functionalised-PP (f-PP) in the absence and presence of the two comonomers (f-PP-DVB or f-PP-TRIS) for the purpose of compatibilising PP-PBT blends through reactive blending. Examination of the morphology of the blends suggested that an effective compatibilisation has been achieved when using f-PP-DVB and f-PP-TRIS, however more work is required in this area.

Cationic ring opening polymerisation (CROP) of oxetane and its derivatives using oxonium derived initiators

Several cationic initiator systems were developed and used to polymerise oxetane with two oxonium ion initiator systems being investigated in depth. The first initiator system was generated by the elimination of a chloride group from a chloro methyl ethyl ether. Adding a carbonyl co-catalyst to a carbocationic centre generated the second initiator system. It was found that the anion used to stabilise the initiator was critical to the initial rate of polymerisation of oxetane with hexafluoroantimonate resulting in the fastest polymerisations. Both initiator systems could be used at varying monomer to initiator concentrations to control the molecular number average, Mn, of the resultant polymer. Both initiator systems showed living characteristics and were used to polymerise further monomers and generate higher molecular weight material and block copolymers. Oxetane and 3,3-dimethyl oxetane can both be polymerised using either oxonium ion initiator system in a variety of DCM or DCM/1,4-dioxane solvent mixtures. The level of 1,4-dioxane does have an impact on the initial rate of polymerisation with higher levels resulting in lower initial rates of polymerisation but do tend to result in higher polydispersities. The level of oligomer formation is also reduced as the level of 1,4-dioxane is increased. 3,3-bis-bromomethyl oxetane was also polymerised but a large amount of hyperbranching was seen at the bromide site resulting in a difficult to solvate polymer system. Multifunctional initiator systems were also generated using the halide elimination reactions with some success being achieved with 1,3,5-tris-bromomethyl-2,4,6-tris-methyl-benzene derived initiator system. This offered some control over the molecular number average of the resultant polymer system.

A novel role for the adipokine visfatin/pre-B cell colony-enhancing factor 1 in prostate carcinogenesis

Adipose tissue is now well established as an endocrine organ and multiple hormones termed ‘adipokines’ are released from it. With the rapidly increasing obese population and the increased risk mortality from prostate cancer within the obese population we looked to investigate the role of the adipokine visfatin in LNCaP and PC3 prostate cancer cell lines. Using immunohistochemistry and immunocytochemistry we demonstrate visfatin expression in LNCaP (androgen-sensitive) and PC3 (androgen-insensitive) human prostate cancer cell lines as well as human prostate cancer tissue. Additionally, we show that visfatin increases PC3 cell proliferation and demonstrate the activation of the MAPKs ERK-1/2 and p38. Moreover we also demonstrate that visfatin promotes the expression and activity of MMP-2/9 which are important proteases involved in the breakdown of the extracellular matrix, suggesting a possible role for visfatin in prostate cancer metastases. These data suggest a contributory and multifunctional role for visfatin in prostate cancer progression, with particular relevance and emphasis in an obese population.

A sulphonate based hydrogel for nucleus pulposus repair

Introduction: Lower back pain treatment and compensation costs >$80 billion overall in the US. 75% of back pain is due to disc degeneration in the lumbar region of the spine. Current treatment comprises of painkillers and bed rest or as a more radical solution – interbody cage fusion. In the early stages of disc degeneration the patient would benefit from addition of an injectable gel which polymerises in situ to support the degenerated nucleus pulposus. This involves a material which is an analogue of the natural tissue capable of restoring the biomechanical properties of the natural disc. The nucleus pulposus of the intervertebral disc is an example of a natural proteoglycan consisting of a protein core with negatively charged keratin and chondroitin sulphate attached. As a result of the high fixed charge density of the proteoglycan, the matrix exerts an osmotic swelling pressure drawing sufficient water into support the spinal system. Materials and Methods: NaAMPs (sodium 2- acrylamido 2-methyl propane sulphonic acid) and KSPA (potassium 3- sulphopropyl acrylate) were selected as monomers, the sulphonate group being used to mimic the natural sulphate group. These are used in dermal applications involving chronic wounds and have acceptably low cytotoxicity. Other hydrophilic carboxyl, amide and hydroxyl monomers such as 2-hydroxyethyl acrylamide, ß-carboxyethyl acrylate, acryloyl morpholine, and polyethylene glycol (meth)acrylate were used as diluents together with polyethyleneglycol di(meth)acrylate and hydrophilic multifunctional macromers as cross-linker. Redox was the chosen method of polymerisation and a range of initiators were investigated. Components were packaged in two solutions each containing a redox pair. A dual syringe method of injection into the cavity was used, the required time for polymerisation is circa 3-7 minutes. The final materials were tested using a Bohlin CVO Rheometer cycling from 0.5-25Hz at 37oC to measure the modulus. An in-house compression testing method was developed, using dialysis tubing to mimic the cavity, the gels were swelled in solutions of various osmolarity and compressed to ~ 20%. The pre-gel has also been injected into sheep spinal segments for mechanical compression testing to demonstrate the restoration of properties upon use of the gel. Results and Discussion: Two systems resulted using similar monomer compositions but different initiation and crosslinking agents. NaAMPs and KSPA were used together at a ratio of ~1:1 in both systems with 0.25-2% crosslinking agent, diacrylate or methacrylate. The two initiation systems were ascorbic acid/oxone, and N,N,N,N - tetramethylethylenediamine (TEMED)/ potassium persulphate. These systems produced gelation within 3-7 and 3-5 minutes respectively. Storage of the two component systems was shown to be stable for approximately one month after mixing, in the dark, refrigerated at 1-4oC. The gelation was carried out at 37oC. Literature values for the natural disc give elastic constants ranging from 3-8kPa. The properties of the polymer can be tailored by altering crosslink density and monomer composition and are able to match those of the natural disc. It is possible to incorporate a radio-opaque (histodenz) to enable x-ray luminescence during and after injection. At an inclusion level of 5% the gel is clearly visible and polymerisation and mechanical properties are not altered. Conclusion: A two-pac injection system which will polymerise in situ, that can incorporate a radio-opaque, has been developed. This will reinforce the damaged nucleus pulposus in degenerative disc disease restoring adequate hydration and thus biomechanical properties. Tests on sheep spine segments are currently being carried out to demonstrate that a disc containing the gel has similar properties to an intact disc in comparison to one with a damaged nucleus.

Nucleoside diphosphate kinase--a component of the [Na+1]- and [Cl-]-sensitive phosphorylation cascade in human and murine airway epithelium

We have shown that proteins within apically enriched fractions of human nasal respiratory epithelium vary their phosphohistidine content with ambient [Cl-] and other anion concentrations. This membrane-delimited phosphorylation cascade includes a multifunctional protein histidine kinase - nucleoside diphosphate kinase (NDPK). NDPK is itself a cascade component in both human and ovine airway, the self-phosphorylation of which is inhibited selectively by [Na+] in the presence of ATP (but not GTP). These findings led us to propose the existence of a dual anion-/cation-controlled phosphorylation-based "sensor" bound to the apical membrane. The present study showed that this cascade uses ATP to phosphorylate a group of proteins above 45 kDa (p45-group, identities unknown). Additionally, the Cl- dependence of ATP (but not GTP) phosphorylation is conditional on phosphatase activity and that interactions exist between the ATP- and GTP-phosphorylated components of the cascade under Cl--free conditions. As a prelude to studies in cystic fibrosis (CF) mice, we showed in the present study that NDPK is present and functionally active in normal murine airway. Since NDPK is essential for UTP synthesis and regulates fetal gut development, G proteins, K+channels, neutrophil-mediated inflammation and pancreatic secretion, the presence of ion-regulated NDPK protein in mouse airway epithelium might aid understanding of the pathogenesis of CF.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Understanding the link between transglutaminase and the induction of fibrosis in cystic fibrosis (CF)

The emerging role of the multifunctional enzyme, Transglutaminase 2 (TG2) in Cystic Fibrosis (CF) has been linked to its increased expression and intracellular transamidating activity. However, a full understanding of the molecular mechanisms involved still remains unclear despite numerous studies that have attempted to delineate this process. These mechanisms include the NFκB and TGFβ1 pathway amongst others. This study reveals for the first time that the development of fibrosis in CF is due to a TG2-driven epithelial to mesenchymal transition (EMT) via a mechanism involving the activation of the pro-fibrotic cytokine TGFβ1. Using a human ΔF508/W1282X CFTR CF mutant bronchial cell (IB3-1), its CFTR corrected “add-back” cell (C38) as well as a primary human bronchial epithelial cell (HBEC), elevated TG2 levels in the CFTR mutant IB3 cell were shown to activate latent TGFβ1 leading to increased levels found in the culture medium. This activation process was blocked by the presence of cell-permeable and impermeable TG2 inhibitors while inhibition of TGFβ1 receptors blocked TG2 expression. This demonstrates the direct link between TG2 and TGFβ1 in CF. The presence of active cell surface TG2 correlated with an increase in the expression of EMT markers, associated with the CF mutant cells, which could be blocked by the presence of TG2 inhibitors. This was mimicked using the “addback” C38 cell and the primary human bronchial epithelial cell, HBEC, where an increase in TG2 expression and activity in the presence of TGFβ1 concurred with a change in cell morphology and an elevation in EMT marker expression. Conversely, a knockdown of TG2 in the CF mutant IB3 cells illustrated that an inhibition of TG2 blocks the increase in EMT marker expression as well as causing an increase in TEER measurement. This together with an increase in the migration profile of the CF mutant IB3 cell against the “add-back” C38 cell suggests that TG2 drives a mesenchymal phenotype in CF. The involvement of TG2 activated TGFβ1 in CF was further demonstrated with an elevation/inhibition of p- SMAD 2 and 3 activation in the presence of TGFβ1/TG2 cell-permeable/impermeable inhibitors respectively. The use of a comparative airway cell model where bronchial epithelial cells were cultured at the air liquid interface (ALI) confirmed the observations in submerged culture depicting the robustness of the model and reiterated the importance of TG2 in CF. Using a CFTR corrector combined with TG2 inhibitors, this study showed that the correction and stabilisation of the ΔF508 CFTR mutation in the mutant cell forged an increase in matured CFTR copies trafficking to the apical surface by circumventing proteosomal degradation. Thus the results presented here suggests that TG2 expression is elevated in the CFTR mutant bronchial cell via a TGFβ1 driven positive feedback cycle whereby activation of latent TGFβ1 by TG2 leads in turn to an elevation in its own expression by TGFβ1. This vicious cycle then drives EMT in CF ultimately leading to lung remodelling and fibrosis. Importantly, TG2 inhibition blocks TGFβ1 activation leading to an inhibition of EMT and further blocks the emerging fibrosis, thus stabilizing and supporting the maturation, trafficking and conductance of CFTR channels at the apical surface.

Isopeptidase activity of human transglutaminase 2:disconnection from transamidation and characterization by kinetic parameters

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 -driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

A sensing mechanism for the detection of carbon nanotubes using selective photoluminescent probes based on ionic complexes with organic dyes

The multifunctional properties of carbon nanotubes (CNTs) make them a powerful platform for unprecedented innovations in a variety of practical applications. As a result of the surging growth of nanotechnology, nanotubes present a potential problem as an environmental pollutant, and as such, an efficient method for their rapid detection must be established. Here, we propose a novel type of ionic sensor complex for detecting CNTs – an organic dye that responds sensitively and selectively to CNTs with a photoluminescent signal. The complexes are formed through Coulomb attractions between dye molecules with uncompensated charges and CNTs covered with an ionic surfactant in water. We demonstrate that the photoluminescent excitation of the dye can be transferred to the nanotubes, resulting in selective and strong amplification (up to a factor of 6) of the light emission from the excitonic levels of CNTs in the near-infrared spectral range, as experimentally observed via excitation-emission photoluminescence (PL) mapping. The chirality of the nanotubes and the type of ionic surfactant used to disperse the nanotubes both strongly affect the amplification; thus, the complexation provides sensing selectivity towards specific CNTs. Additionally, neither similar uncharged dyes nor CNTs covered with neutral surfactant form such complexes. As model organic molecules, we use a family of polymethine dyes with an easily tailorable molecular structure and, consequently, tunable absorbance and PL characteristics. This provides us with a versatile tool for the controllable photonic and electronic engineering of an efficient probe for CNT detection.

Spray drying lactoferrin produces inhalable antimicrobial particles

The use of antimicrobial peptides and proteins as potential therapeutic agents in the management of multi-drug resistant infections is considered an attractive concept especially since such compounds should theoretically have low immunogenicity, high bioavailability with negligible toxicity. In this study we investigated the potential of developing a dry powder inhaler formulation of lactoferrin (a multifunctional iron binding protein). To achieve this, the protein was spray dried from a water only feedstock with suitably adjusted spray drying parameters. The particle size, degree of crystallinity, moisture content and yield of the spray dried powders along with the minimum bactericidal concentration (MBC) against Pseudomonas aeruginosa strain PAO1, were assessed. Dry powder inhaler formulations were prepared, and in vitro assessment studies using the multistage impinger were carried out to assess the aerosolisation performance of the formulations. Data obtained indicate that spray dried lactoferrin retains activity against biofilms and may be successfully employed in the treatment of chronic airway infections.

Computational model of bladder tissue based on its measured optical properties

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a "generic" computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system "LAKK-M" was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.