Pattern-oriented modelling as a novel way to verify and validate functional-structural plant models: a demonstration with the annual growth module of avocado.

Background and Aims: Functional-structural plant (FSP) models have been widely used to understand the complex interactions between plant architecture and underlying developmental mechanisms. However, to obtain evidence that a model captures these mechanisms correctly, a clear distinction must be made between model outputs used for calibration and thus verification, and outputs used for validation. In pattern-oriented modelling (POM), multiple verification patterns are used as filters for rejecting unrealistic model structures and parameter combinations, while a second, independent set of patterns is used for validation. Methods: To test the potential of POM for FSP modelling, a model of avocado (Persea americana 'Hass') was developed. The model of shoot growth is based on a conceptual model, the annual growth module (AGM), and simulates photosynthesis and adaptive carbon allocation at the organ level. The model was first calibrated using a set of observed patterns from a published article. Then, for validation, model predictions were compared with a different set of empirical patterns from various field studies that were not used for calibration. Key Results: After calibration, our model simultaneously reproduced multiple observed architectural patterns. The model then successfully predicted, without further calibration, the validation patterns. The model supports the hypothesis that carbon allocation can be modelled as being dependent on current organ biomass and sink strength of each organ type, and also predicted the observed developmental timing of the leaf sink-source transition stage. Conclusions: These findings suggest that POM can help to improve the 'structural realism' of FSP models, i.e. the likelihood that a model reproduces observed patterns for the right reasons. Structural realism increases predictive power so that the response of an AGM to changing environmental conditions can be predicted. Accordingly, our FSP model provides a better but still parsimonious understanding of the mechanisms underlying known patterns of AGM growth.

Rationalisation of a mechanism for sensing single point variants in target DNA using anthracene-tagged base discriminating probes.

The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively underpin and rationalise the BDP approach to point variant sensing, which relies on different responses to duplex formation rather than different duplex binding strengths.

De novo design of Ln(III) coiled coils for imaging applications.

A new peptide sequence (MB1) has been designed which, in the presence of a trivalent lanthanide ion, has been programmed to self-assemble to form a three stranded metallo-coiled coil, Ln(III)(MB1)3. The binding site has been incorporated into the hydrophobic core using natural amino acids, restricting water access to the lanthanide. The resulting terbium coiled coil displays luminescent properties consistent with a lack of first coordination sphere water molecules. Despite this the gadolinium coiled coil, the first to be reported, displays promising magnetic resonance contrast capabilities.

Molecular Cues for Phenological Events in the Flowering Cycle in Avocado.

Reproductively mature horticultural trees undergo an annual flowering cycle that repeats each year of their reproductive life. This annual flowering cycle is critical for horticultural tree productivity. However, the molecular events underlying the regulation of flowering in tropical tree crops such as avocado are not fully understood or documented. In this study, we investigated the potential molecular cues regulating the yearly flowering cycle in avocado for two consecutive crop cycles. Homologues of flowering-related genes were identified and assessed for their expression profiles in various tissues throughout the year. Avocado homologues of known floral genes FT, AP1, LFY, FUL, SPL9, CO and SEP2/AGL4 were upregulated at the typical time of floral induction for avocado trees growing in Queensland, Australia. We suggest these are potential candidate markers for floral initiation in these crops. In addition, DAM and DRM1, which are associated with endodormancy, were downregulated at the time of floral bud break. In this study, a positive correlation between CO activation and FT in avocado leaves to regulate flowering was not seen. Furthermore, the SOC1-SPL4 model described in annual plants appears to be conserved in avocado. Lastly, no correlation of juvenility-related miRNAs miR156, miR172 with any phenological event was observed.

The effect of leaving group on mechanistic preference in phosphate monoester hydrolysis.

We present 2-dimensional potential energy surfaces and optimised transition states (TS) for water attack on a series of substituted phosphate monoester monoanions at the DFT level of theory, comparing a standard 6-31++g(d,p) basis set with a larger triple-zeta (augmented cc-pVTZ) basis set. Small fluorinated model compounds are used to simulate increasing leaving group stability without adding further geometrical complexity to the system. We demonstrate that whilst changing the leaving group causes little qualitative change in the potential energy surfaces (with the exception of the system with the most electron withdrawing leaving group, CF(3)O(-), in which the associative pathway changes from a stepwise A(N) + D(N) pathway to a concerted A(N)D(N) pathway), there is a quantitative change in relative gas-phase and solution barriers for the two competing pathways. In line with previous studies, in the case of OCH(3), the barriers for the associative and dissociative pathways are similar in solution, and the two pathways are equally viable and indistinguishable in solution. However, significantly increasing the stability of the leaving group (decreasing proton affinity, PA) results in the progressive favouring of a stepwise dissociative, D(N) + A(N), mechanism over associative mechanisms.

Mechanism of CB1954 reduction by Escherichia coli nitroreductase.

NTR (nitroreductase NfsB from Escherichia coli) is a flavoprotein with broad substrate specificity, reducing nitroaromatics and quinones using either NADPH or NADH. One of its substrates is the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which is converted into a cytotoxic agent; so NTR/CB1954 has potential for use in cancer gene therapy. However, wild-type NTR has poor kinetics and binding with CB1954, and the mechanism for the reduction of CB1954 by NTR is poorly understood. Computational methods have been utilized to study potential underlying reaction mechanisms so as to identify the order of electron and proton transfers that make up the initial reduction step and the sources of the protons. We have used Molecular Dynamics to examine the nature of the active site of the wild-type enzyme and the preferred binding mode of the substrate. A combination of these results has allowed us to unequivocally identify the reaction mechanism for the reduction of CB1954 by NTR.

Regulation of floral initiation in horticultural trees.

The intention of this review is to discuss floral initiation of horticultural trees. Floral initiation is best understood for herbaceous species, especially at the molecular level, so a brief overview of the control of floral initiation of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) precedes the discussion of trees. Four major pathways to flowering have been characterized in Arabidopsis, including environmental induction through photoperiod and temperature, autonomous floral initiation, and regulation by gibberellins. Tropical trees are generally induced to flower through environmental cues, whereas floral initiation of temperate deciduous trees is often autonomous. In the tropical evergreen tree mango, Mangifera indica L., cool temperature is the only factor known to induce flowering, but does not ensure floral initiation will occur because there are important interactions with vegetative growth. The temperate deciduous tree apple, Malus domestica Borkh., flowers autonomously, with floral initiation dependent on aspects of vegetative development in the growing season before anthesis, although with respect to the floral initiation of trees in general: the effect of the environment, interactions with vegetative growth, the roles of plant growth regulators and carbohydrates, and recent advances in molecular biology, are discussed.

Anion complexation via C-H...X interactions using a palladacyclic receptor.

A novel organometallic receptor binds anions in solution and in the solid state, with complexes stabilised through a series of C-HX interactions, as evidenced by 1H NMR spectroscopy, X-ray crystallography and computational models.

BTN3A1 Discriminates γδ T Cell Phosphoantigens from Nonantigenic Small Molecules via a Conformational Sensor in Its B30.2 Domain.

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag's are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.

Two-year preclinical testing of perfluoropolyether polymer as a corneal inlay.

PURPOSE: To assess the long-term biocompatibility and optical clarity of a perfluoropolyether (PFPE) polymer as a corneal inlay. METHODS: A 4-mm-diameter PFPE inlay was implanted under a microkeratome flap in the corneas of rabbits (n = 16) and maintained for predetermined time points of 6, 12, or 24 months. These were compared with normal (n = 3) and time-matched sham-wounded rabbit corneas (n = 8). All corneas were monitored clinically with a slit lamp. Histology was performed on all eyes on termination to assess the tissue response. RESULTS: Some sham and implanted animals were discontinued from study 1 to 2 days after surgery because of flap dislodgement. Ten animals with PFPE inlays remained in the study, and 7 of these were maintained to their predetermined time point for up to 2 years (3 were discontinued because of peripheral corneal defects). The corneas of these 7 animals remained clear and healthy, tear film remained normal, and there were no signs of inflammation, neovascularization, or increased conjunctival redness. All inlays remained centered and optically clear (clarity 85% or greater). Histology showed PFPE was biostable. The epithelia of operated corneas were stratified but slightly thinned compared with those of normal corneas. Stromal tissue anterior and posterior to each inlay appeared normal. Keratocytes in the vicinity of the inlay were normal in distribution but showed increased vacuolation, indicating tissue repair after the surgery. CONCLUSIONS: The PFPE polymer maintained a high level of optical clarity and showed long-term biocompatibility for up to 2 years when implanted as an inlay in the rabbit cornea.

Approaches to improving the biocompatibility of porous perfluoropolyethers for ophthalmic applications.

Porous perfluoropolyether (PFPE) membranes for ophthalmic applications were prepared with a zwitterion monomer, 3-[[2-(methacryloxy) ethyl](N,N-dimethyl)ammonio]-propane-1-sulphonate, copolymerized in weight ratios of 0-10%. The polymer samples were assessed for a range of physical properties, including equilibrium water content, bovine serum albumin permeability, transparency, refractive index and the ability to support corneal epithelial cell and tissue attachment, growth and migration. In vitro assessment of the polymers using bovine corneal epithelial cells and tissue showed that a zwitterion incorporation level of between 0% and 6% in the PFPE membranes supported the migration of an intact sheet of epithelial tissue without compromising epithelial cell attachment and growth, with 4-6% being the optimal level for these properties. Binding patterns of the cell adhesion glycoprotein fibronectin were also found to reflect the cell and tissue response. Effective nutrient permeability, refractive index and optical transparency were also maintained by the porous PFPE polymers containing this concentration of zwitterionic monomer. The presence of amounts of zwitterion greater than 6% was inhibitory to both tissue migration and cell growth and was associated with increased optical haze. These results demonstrated that it is possible to achieve the potential for increased biocompatibility in zwitterion-containing PFPE polymers without compromising existing beneficial characteristics.

Single Site Discrimination of Cytosine, 5-Methylcytosine, and 5-Hydroxymethylcytosine in Target DNA Using Anthracene-Tagged Fluorescent Probes.

The ability to discriminate between epigenetic variants in DNA is a necessary tool if we are to increase our understanding of the roles that they play in various biological processes and medical conditions. Herein, it is demonstrated how a simple two-step fluorescent probe assay can be used to differentiate all three major epigenetic variants of cytosine at a single locus site in a target strand of DNA.

Progress in the development of a synthetic corneal onlay.

PURPOSE: This study evaluated an improved perfluoropolyether polymer formulation designed for use as a corneal onlay to correct refractive error. METHODS: Collagen I coated perfluoropolyether lenticules were implanted in feline corneas exposing a 6-mm diameter area of lenticule surface for epithelial growth. A parallel series of sham-wounded corneas were also studied. All corneas were monitored clinically for 4 or 8 weeks after surgery when animals were terminated and corneas used for histology with light and electron microscopy. RESULTS: Postoperative epithelial growth began on days 1 and 2. Lenticule surfaces were fully epithelialized by days 5 to 11. Corneas remained clear, and the lenticules maintained epithelial cover until the designated time points. Histology of the implanted corneas showed that the lenticules were well tolerated by the cornea. Each lenticule was fully covered by a multilayered epithelium with microvilli, desmosomes, and a differentiated basal cell layer. Epithelial adhesive structures (basal lamina, hemidesmosomes, and anchoring fibrils) had assembled at the tissue-lenticule interface. CONCLUSIONS: Collagen coated perfluoropolyether lenticules implanted in the feline cornea supported the growth of a stable stratified squamous epithelium. These encouraging results are a step further in the development of a corneal onlay for correction of refractive error.

The use of corneal organ culture in biocompatibility studies.

This study investigated the potential of a corneal organ culture system in the evaluation of polymers for ophthalmic devices that require epithelialisation. Two different polymers were tested in lenticule form to explore the sensitivity of this in vitro assay. Polycarbonate and perfluoropolyether-based lenticules were surgically implanted into bovine corneas and compared with a parallel series of sham-wounded corneas. Following surgery, all corneas were maintained in an air/liquid organ culture system for up to 8 days during which time they were evaluated clinically to monitor the rate of epithelial growth across the lenticule surface (implanted) or wound bed (sham). Data showed differences in the kinetics of epithelial migration according to the underlying surface with full epithelialisation of the sham series occurring on day 5+/-0.5, the perfluoropolyether lenticules on day 6+0.5 and polycarbonate lenticules on day 8+/-0.5. Histology revealed differences in the structure and morphology of the migrating and stable epithelium in each series of corneas. The differential response of the corneal epithelium was related to the physiochemical characteristics of the natural (sham) or synthetic (perfluoropolyether or polycarbonate) substrata which the epithelium could detect when maintained in organ culture. This assay system has utility for screening candidate polymers for certain ophthalmic applications.

NMR and molecular dynamics study of the size, shape, and composition of reverse micelles in a cetyltrimethylammonium bromide (CTAB)/n-hexane/pentanol/water microemulsion.

The size, shape, and composition of reverse micelles (RMs) in a cetyltrimethylammonium bromide (CTAB)/pentanol/n-hexane/water microemulsion were investigated using pulsed gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) measurements and molecular modeling. PGSTE data were collected at observation times (Δ) of 10, 40, and 450 ms. At long observation times, CTAB and pentanol exhibited single diffusion coefficients. However, at short (Δ ≤ 40 ms) observation times both CTAB and pentanol exhibited slow and fast diffusion coefficients. These NMR data indicate that both CTAB and pentanol molecules reside in different environments within the microemulsion and that there is exchange between regions on the millisecond time scale. Molecular dynamic simulations of the CTAB RM, in a solvent box containing n-hexane and pentanol, produced an ellipsoid shaped RM. Using structural parameters from these simulations and the Stokes-Einstein relation, the structure factor and dimensions of the reverse micelle were determined. Analysis of the composition of the interphase also showed that there was a variation in the ratio of surfactant to cosurfactant molecules depending on the curvature of the interphase.

A perfluoropolyether corneal inlay for the correction of refractive error.

This study assessed the long-term biological response of a perfluoropolyether-based polymer developed as a corneal inlay to correct refractive error. The polymer formulation met chemical and physical specifications and was non-cytotoxic when tested using standard in vitro techniques. It was cast into small microporous membranes that were implanted as inlays into corneas of rabbits (n = 5) and unsighted humans (n = 5 + 1 surgical control) which were monitored for up to 23 and 48 months respectively. Overall, the inlays were well tolerated during study period with the corneas remaining clear and holding a normal tear film and with no increased vascularisation or redness recorded. Inlays in three human corneas continued past 48 months without sequelae. Inlays in two human corneas were removed early due to small, focal erosions developing 5 and 24 months post-implantation. Polymer inlays maintained their integrity and corneal position for the study duration although the optical clarity of the inlays reduced slowly with time. Inlays induced corneal curvature changes in human subjects that showed stability with time and the refractive effect was reversed when the inlay was removed. Outcomes showed the potential of a perfluoropolyether inlay as a biologically acceptable corneal implant with which to provide stable correction of refractive error.

The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion.

Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.

Magnetic resonance studies of a redox probe in a reverse sodium bis(2-ethylhexyl)sulfosuccinate/octane/water microemulsion.

The location and dynamics of the [Ru(bpy)(3)](2+) complex inside sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/octane/water microemulsions were studied, over a range of droplet sizes, using magnetic resonance spectroscopy, dynamic light scattering, and molecular modeling. The T(1) magnetic resonance relaxation times of water inside the AOT reverse micelles (RMs) were measured in both the presence and the absence of the [Ru(bpy)(3)](2+) complex. Large size droplet RMs (ω(0) > 20) were found to be sensitive to the presence of the [Ru(bpy)(3)](2+) complex, which was detected through a decrease in the T(1) relaxation time of the water inside the RM core, as compared to RMs containing no [Ru(bpy)(3)](2+). However, no difference in T(1) relaxation time was observed for water in small RMs (ω(0) < 20). Two-dimensional (1)H-(1)H NOESY spectroscopy was performed to probe the location of the [Ru(bpy)(3)](2+) complex in both small (ω(0) = 9.2) and large droplets (ω(0) = 34.9). Cross-peaks between protons in the AOT tail groups and bipyridyl ligands were observed, showing that the [Ru(bpy)(3)](2+) complex resided in the RM interface. Finally, molecular modeling simulations were performed to probe the location of the [Ru(bpy)(3)](2+) complex and the structure of the RM. Molecular dynamics simulations confirmed the location of the [Ru(bpy)(3)](2+) complex in the RM interface and detected differences in the surfactant layer and the amount of water penetration into this layer with changing droplet size.

Functionalised polysiloxanes as injectable, in situ curable accommodating intraocular lenses.

The aged eye's ability to change focus (accommodation) may be restored by replacing the hardened natural lens with a soft gel. Functionalised polysiloxane macromonomers, designed for application as an injectable, in situ curable accommodating intraocular lens (A-IOL), were prepared via a two-step synthesis. Prepolymers were synthesised via ring opening polymerisation (ROP) of octamethylcyclotetrasiloxane (D(4)) and 2,4,6,8-tetramethylcyclotetrasiloxane (D(4)(H)) in toluene using trifluoromethanesulfonic acid (TfOH) as catalyst. Hexaethyldisiloxane (HEDS) was used as the end group to control the molecular weight of the prepolymers, which were then converted to macromonomers by hydrosilylation of the SiH groups with allyl methacrylate (AM) to introduce polymerisable groups. The resulting macromonomers had an injectable consistency and thus, were able to be injected into and refill the empty lens capsular bag. The macromonomers also contained a low ratio of polymerisable groups so that they may be cured on demand, in situ, under irradiation of blue light, in the presence of a photo-initiator, to form a soft polysiloxane gel (an intraocular lens) in the eye. The pre-cure viscosity and post-cure modulus of the polysiloxanes, which are crucial factors for an injectable, in situ curable A-IOL application, were controlled by adjusting the end group and D(4)(H) concentrations, respectively, in the ROP. The macromonomers were fully cured within 5 min under light irradiation, as shown by the rapid change in modulus monitored by photo-rheology. Ex vivo primate lens stretching experiments on an Ex Vivo Accommodation Simulator (EVAS) showed that the polysiloxane gel refilled lenses achieved over 60% of the accommodation amplitude of the natural lens. An in vivo biocompatibility study in rabbits using the lens refilling (Phaco-Ersatz) procedure demonstrated that the soft gels had good biocompatibility with the ocular tissue. The polysiloxane macromonomers meet the targeted optical and mechanical properties of a young natural crystalline lens and show promise as candidate materials for use as injectable, in situ curable A-IOLs for lens refilling procedures.

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.