Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

Chemistry and biology of maculalactone A from the marine cyanobacterium Kyrtuthrix maculans

Maculalactone A is the most abundant secondary metabolite in Kyrtuthrix maculans, a marine cyanobacterium found in the mid-high shore of moderately exposed to sheltered rocky shores in Hong Kong and South East Asia. This species appears to survive as pure colonies forming distinct black zones on the rock. Maculalactone A may provide K. maculans with a chemical defense against several marine organisms, including the common grazer, Chlorostoma argyrostoma and settlement by larvae of the barnacles, Tetraclita japonica, Balanus amphitrite and Ibla cumingii. The natural concentration of maculalactone A varied with season and also with tidal height on the shore and although a strong positive linear correlation was observed between maculalactone A concentration and herbivore grazing pressure, manipulative experiments demonstrated that grazing pressure was not directly responsible for inducing the biosynthesis of this metabolite. The potential of maculalactone A as a natural marine anti-fouling agent (i.e. as an alternative to environmentally-damaging copper- and tin-based anti-fouling paints) was investigated after achieving a gram-scale synthesis of this compound. Preliminary field trials with anti-fouling paints which contained synthetic maculalactone A as the active principle have confirmed that this compound seems to have a specific activity against molluscan settlers.

Self-assembly of a designed amyloid peptide containing the functional thienylalanine unit

The self-assembly of a peptide based on a sequence from the amyloid beta peptide but incorporating the non-natural amino acid beta-2-thienylalanine (2-Thi) has been investigated in aqueous and methanol solutions. The peptide AAKLVFF was used as a design motif, replacing the phenylalanine residues (F) with 2-Thi units to yield (2-Thi)(2-Thi)VLKAA. The 2-Thi residues are expected to confer interesting electronic properties due to charge delocalization and pi-stacking. The peptide is shown to form beta-sheet-rich amyloid fibrils with a twisted morphology, in both water and methanol solutions at sufficiently high concentration. The formation of a self-assembling hydrogel is observed at high concentration. Detailed molecular modeling using molecular dynamics methods was performed using NOE constraints provided by 2D-NMR experiments. The conformational and charge properties of 2-Thi were modeled using quantum mechanical methods, and found to be similar to those previously reported for the beta-3-thienylalanine analogue. The molecular dynamics simulations reveal well-defined folded structures (turn-like) in dilute aqueous solution, driven by self-assembly of the hydrophobic aromatic units, with charged lysine groups exposed to water.

Influence of the solvent on the self-assembly of a modified amyloid beta peptide fragment. II. NMR and computer simulation investigation

The conformation of a model peptide AAKLVFF based on a fragment of the amyloid beta peptide A beta 16-20, KLVFF, is investigated in methanol and water via solution NMR experiments and Molecular dynamics computer simulations. In previous work, we have shown that AAKLVFF forms peptide nanotubes in methanol and twisted fibrils in water. Chemical shift measurements were used to investigate the solubility of the peptide as a function of concentration in methanol and water. This enabled the determination of critical aggregation concentrations, The Solubility was lower in water. In dilute solution, diffusion coefficients revealed the presence of intermediate aggregates in concentrated solution, coexisting with NMR-silent larger aggregates, presumed to be beta-sheets. In water, diffusion coefficients did not change appreciably with concentration, indicating the presence mainly of monomers, coexisting with larger aggregates in more concentrated solution. Concentration-dependent chemical shift measurements indicated a folded conformation for the monomers/intermediate aggregates in dilute methanol, with unfolding at higher concentration. In water, an antiparallel arrangement of strands was indicated by certain ROESY peak correlations. The temperature-dependent solubility of AAKLVFF in methanol was well described by a van't Hoff analysis, providing a solubilization enthalpy and entropy. This pointed to the importance of solvophobic interactions in the self-assembly process. Molecular dynamics Simulations constrained by NOE values from NMR suggested disordered reverse turn structures for the monomer, with an antiparallel twisted conformation for dimers. To model the beta-sheet structures formed at higher concentration, possible model arrangements of strands into beta-sheets with parallel and antiparallel configurations and different stacking sequences were used as the basis for MD simulations; two particular arrangements of antiparallel beta-sheets were found to be stable, one being linear and twisted and the other twisted in two directions. These structures Were used to simulate Circular dichroism spectra. The roles of aromatic stacking interactions and charge transfer effects were also examined. Simulated spectra were found to be similar to those observed experimentally.(in water or methanol) which show a maximum at 215 or 218 nm due to pi-pi* interactions, when allowance is made for a 15-18 nm red-shift that may be due to light scattering effects.

Goal sketching: an agile approach to clarifying requirements

This paper describes a technique that can be used as part of a simple and practical agile method for requirements engineering. It is based on disciplined goal-responsibility modelling but eschews formality in favour of a set of practicality objectives. The technique can be used together with Agile Programming to develop software in internet time. We illustrate the technique and introduce lazy refinement, responsibility composition and context sketching. Goal sketching has been used in a number of real-world development.

The role of management and landscape context in the restoration of grassland phytophagous beetles

1. Declines in area and quality of species-rich mesotrophic and calcareous grasslands have occurred all across Europe.While the European Union has promoted schemes to restore these grasslands, the emphasis for management has remained largely focused on plants. Here we focus on restoration of the phytophagous beetles of these grasslands. Although local management, particularly that which promotes the establishment of host plants, is key to restoration success, dispersal limitation is also likely to be an important limiting factor during the restoration of phytophagous beetle assemblages. 2. Using a 3-year multi-site experiment, we investigated how restoration success of phytophagous beetles was affected by hay-spreading management (intended to introduce target plant species), success in restoration of the plant communities and the landscape context within which restoration was attempted. 3. Restoration success of the plants was greatest where green hay spreading had been used to introduce seeds into restoration sites. Beetle restoration success increased over time, although hayspreading had no direct effect. However, restoration success of the beetles was positively correlated with restoration success of the plants. 4. Overall restoration success of the phytophagous beetles was positively correlated with the proportion of species-rich grassland in the landscape, as was the restoration success of the polyphagous beetles. Restoration success for beetles capable of flight and those showing oligophagous host plant specialism were also positively correlated with connectivity to species-rich grasslands. There was no indication that beetles not capable of flight showed greater dependence on landscape scale factors than flying species. 5. Synthesis and applications. Increasing the similarity of the plant community at restoration sites to target species-rich grasslands will promote restoration success for the phytophagous beetles. However, landscape context is also important, with restoration being approximately twice as successful in those landscapes containing high as opposed to low proportions of species-rich grassland. By targeting grassland restoration within landscapes containing high proportions of species-rich grassland, dispersal limitation problems associated with restoration for invertebrate assemblages are more likely to be overcome.

The restoration of phytophagous beetles in species-rich chalk grasslands

This study focuses on the restoration of chalk grasslands over a 6-year period and tests the efficacy of two management practices, hay spreading and soil disturbance, in promoting this process for phytophagous beetles. Restoration success for the beetles, measured as similarity to target species-rich chalk grassland, was not found to be influenced by either management practice. In contrast, restoration success for the plants did increase in response to hay spreading management. Although the presence of suitable host plants was considered to dictate the earliest point at which phytophagous beetles could successfully colonized, few beetle species colonized as soon as their host plants became established. Morphological characteristics and feeding habits of 27 phytophagous beetle species were therefore tested to identify factors that limited their colonization and persistence. The lag time between host plant establishment and colonization was greatest for flightless beetles. Beetles with foliage-feeding larvae both colonized at slower rates than seed-, stem-, or root-feeding species and persisted within the swards for shorter periods. Although the use of hay spreading may benefit plant communities during chalk grassland restoration, it did not directly benefit phytophagous beetles. Without techniques for overcoming colonization limitation for invertebrate taxa, short-term success of restoration may be limited to the plants only.

c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.

Vermillion Vortex

An animated film commissioned and screened by Art Review Magazine on their website (Oct-Dec 2010), and a double page comic strip (Art Review, Oct 2010. The project addresses a key problem with contemporary debates regarding ideas of ‘performativity’ and ‘fictioning’ (Foucault/Deleuze/Butler) whereby the structural requirement for an ‘End’ pre-determines or back-codes the ‘story’ or progression of events leading up to this ‘End’ and therefore cuts against the potentials claimed for ‘performance’ and ‘performativity’. Film credits Primary soundtrack: Music: Rose Kallal. Spoken word: Mark Beasley Voices: Katie Barrington, Marnie Watts, Maria Deegan & John Russell Sound engineer: Bob Geal PLUS Special bonus track: (after 'The End'): 'Strychnine Motive' (2011) by Gum Takes Tooth

Learning enhanced 3D models for vehicle tracking

This paper presents an enhanced hypothesis verification strategy for 3D object recognition. A new learning methodology is presented which integrates the traditional dichotomic object-centred and appearance-based representations in computer vision giving improved hypothesis verification under iconic matching. The "appearance" of a 3D object is learnt using an eigenspace representation obtained as it is tracked through a scene. The feature representation implicitly models the background and the objects observed enabling the segmentation of the objects from the background. The method is shown to enhance model-based tracking, particularly in the presence of clutter and occlusion, and to provide a basis for identification. The unified approach is discussed in the context of the traffic surveillance domain. The approach is demonstrated on real-world image sequences and compared to previous (edge-based) iconic evaluation techniques.

Visual surveillance for moving vehicles

An overview is given of a vision system for locating, recognising and tracking multiple vehicles, using an image sequence taken by a single camera mounted on a moving vehicle. The camera motion is estimated by matching features on the ground plane from one image to the next. Vehicle detection and hypothesis generation are performed using template correlation and a 3D wire frame model of the vehicle is fitted to the image. Once detected and identified, vehicles are tracked using dynamic filtering. A separate batch mode filter obtains the 3D trajectories of nearby vehicles over an extended time. Results are shown for a motorway image sequence.

Visual surveillance using deformable models of vehicles

This paper presents recent developments to a vision-based traffic surveillance system which relies extensively on the use of geometrical and scene context. Firstly, a highly parametrised 3-D model is reported, able to adopt the shape of a wide variety of different classes of vehicle (e.g. cars, vans, buses etc.), and its subsequent specialisation to a generic car class which accounts for commonly encountered types of car (including saloon, batchback and estate cars). Sample data collected from video images, by means of an interactive tool, have been subjected to principal component analysis (PCA) to define a deformable model having 6 degrees of freedom. Secondly, a new pose refinement technique using “active” models is described, able to recover both the pose of a rigid object, and the structure of a deformable model; an assessment of its performance is examined in comparison with previously reported “passive” model-based techniques in the context of traffic surveillance. The new method is more stable, and requires fewer iterations, especially when the number of free parameters increases, but shows somewhat poorer convergence. Typical applications for this work include robot surveillance and navigation tasks.