Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

(Ultra)fast catalyst-free macromolecular conjugation in aqueous environment at ambient temperature

Tailor-made water-soluble macromolecules, including a glycopolymer, obtained by living/controlled RAFT-mediated polymerization are demonstrated to react in water with diene-functionalized poly(ethylene glycol)s without pre- or post-functionalization steps or the need for a catalyst at ambient temperature. As previously observed in organic solvents, hetero-Diels-Alder (HDA) conjugations reached quantitative conversion within minutes when cyclopentadienyl moieties were involved. However, while catalysts and elevated temperatures were previously necessary for open-chain diene conjugation, additive-free HDA cycloadditions occur in water within a few hours at ambient temperature. Experimental evidence for efficient conjugations is provided via unambiguous ESI-MS, UV/vis, NMR, and SEC data.

The influence of vegetation and soil type on the speciation of iron in soil water

Soluble organic matter derived from exotic Pinus species has been shown to form stronger complexes with iron (Fe) than that derived from most native Australian species. It has also been proposed that the establishment of exotic Pinus plantations in coastal southeast Queensland may have enhanced the solubility of Fe in soils by increasing the amount of organically complexed Fe, but this remains inconclusive. In this study we test whether the concentration and speciation of Fe in soil water from Pinus plantations differs significantly from soil water from native vegetation areas. Both Fe redox speciation and the interaction between Fe and dissolved organic matter (DOM) were considered; Fe - DOM interaction was assessed using the Stockholm Humic Model. Iron concentrations (mainly Fe 2+) were greatest in the soil waters with the greatest DOM content collected from sandy podosols (Podzols), where they are largely controlled by redox potential. Iron concentrations were small in soil waters from clay and iron oxide-rich soils, in spite of similar redox potentials. This condition is related to stronger sorption on to the reactive clay and iron oxide mineral surfaces in these soils, which reduces the amount of DOM available for electron shuttling and microbial metabolism, restricting reductive dissolution of Fe. Vegetation type had no significant influence on the concentration and speciation of iron in soil waters, although DOM from Pinus sites had greater acidic functional group site densities than DOM from native vegetation sites. This is because Fe is mainly in the ferrous form, even in samples from the relatively well-drained podosols. However, modelling suggests that Pinus DOM can significantly increase the amount of truly dissolved ferric iron remaining in solution in oxic conditions. Therefore, the input of ferrous iron together with Pinus DOM to surface waters may reduce precipitation of hydrous ferric oxides (ferrihydrite) and increase the flux of dissolved Fe out of the catchment. Such inputs of iron are most probably derived from podosols planted with Pinus.

Modelling the flow of cane constituents in the milling process

A model has been developed to track the flow of cane constituents through the milling process. While previous models have tracked the flow of fibre, brix and water through the process, this model tracks the soluble and insoluble solid cane components using modelling theory and experiment data, assisting in further understanding the flow of constituents into mixed juice and final bagasse. The work provided an opportunity to understand the factors which affect the distribution of the cane constituents in juice and bagasse. Application of the model should lead to improvements in the overall performance of the milling train.

Adsorption of heavy metals by road deposited solids

The research study discussed in the paper investigated the adsorption/desorption behaviour of heavy metals commonly deposited on urban road surfaces, namely, Zn, Cu, Cr and Pb for different particle size ranges of solids. The study outcomes, based on field studies and batch experiments confirmed that road deposited solids particles contain a significantly high amount of vacant charge sites with the potential to adsorb additional heavy metals. Kinetic study and adsorption experiments indicated that Cr is the most preferred metal element to associate with solids due to the relatively high electro negativity and high charge density of trivalent cation (Cr3+). However, the relatively low availability of Cr in the urban road environment could influence this behaviour. Comparing total adsorbed metals present in solids particles, it was found that Zn has the highest capacity for adsorption to solids. Desorption experiments confirmed that a low concentration of Cu, Cr and Pb in solids was present in water-soluble and exchangeable form, whilst a significant fraction of adsorbed Zn has a high likelihood of being released back into solution. Among heavy metals, Zn is considered to be the most commonly available metal among road surface pollutants.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

Natural improvements of geochemical conditions of acid sulfate soils caused by free tidal inundation and its effects on the mangrove seedlings

Acid sulfate soils (ASS) are one of the stressor factors that cause many mangrove restoration projects to fail. Achieving successful rehabilitation in an ASS affected area requires an understanding of the geochemical conditions that influence the establishment and growth of mangrove seedlings. This study evaluated the effect of tidal inundation on geochemical conditions on sub layer to better understand their impacts on the density, establishment, and growth of mangrove seedlings. This study also examined the geochemical conditions under which mangrove seedlings can establish naturally, and/or be replanted in abandoned aquaculture ponds. The study area was in an area of abandoned aquaculture ponds situated in the Mare District, adjacent to Bone Bay, South Sulawesi, Indonesia.The pH, pHfox, redox potential, organic content, water soluble sulfate, SKCl, SPOS, and grain size of the soil from the sediment core at + 10 - 15 cm depth near roots were measured using. Pyrite analysis were conducted for the top and sub sediments. The density, establishment, and the relative root growth of Rhizophoraceae were also determined. Free tidal inundation at abandoned pond sites improved the sediment quality. The high density, establishment, and growth of mangrove seedlings were characterized by freely drained areas with a higher pH (field and oxidisable), lower organic content, and high proportion of silt/clay. Higher density and growth also correlated to reduced environments. Sulfur species did not influence the density, establishment, and growth of the seedlings directly. The supply of propagules from the mangrove stands, or access from good waterways were also important for seedlings to establish naturally.

K from zeolite : glasshouse trial for testing K availability to the plants

MesoLite, a zeolite material manufactured by NanoChem Holdings Pty Ltd is made by caustic reaction of kaolin at temperatures between 80-95°C. This material has a moderate surface area (9~12 m2/g) and very high cation exchange capacity (500meq/100g). To measure the availability of K in K-MesoLite to plants, wheat was grown with K-MesoLite or a soluble fertiliser (e.g. KCl) in non-leached pots in a glasshouse. The weights and elemental compositions of the plants were compared after four weeks growth. Plants grown with K-MesoLite were slightly larger than those grown with KCl. The elemental compositions of the plants were similar except for Si, which was significantly higher in the plants grown with K-MesoLite than in those fertilised with KCl. K from K-MesoLite is readily available to plants.

Aluminosilicates of zeolite N structure

A process for making aluminosilicates of zeolite N structure comprising the steps of: (i) combining a water soluble monovalent cation, a solution of hydroxyl anions and an aluminosilicate to form a resultant mixture having a pH greater than 10 and a H.sub.2O/Al.sub.2O.sub.3 ratio in the range 30 to 220; (ii) heating the resultant mixture to a temperature of between 50.degree. C. and boiling point of the mixture for a time between 1 minute and 100 hours until a crystalline product of zeolite N structure is formed as determined by X-ray diffraction or other suitable characteristic; and (iii) separating the zeolite N product as a solid from the mixture.

Recovery and reuse of ammonium from wastewater treatment plants : an application for agriculture

In wastewater treatment plants based on anaerobic digestion, supernatant and outflows from sludge dewatering systems contain significantly high amount of ammonium. Generally, these waters are returned to the head of wastewater treatment plant (WWTP), thereby increasing the total nitrogen load of the influent flow. Ammonium from these waters can be recovered and commercially utilised using novel ion-exchange materials. Mackinnon et al. have described an approach for removal and recovery of ammonium from side stream centrate returns obtained from anaerobic digester of a typical WWTP. Most of the ammonium from side streams can potentially be removed, which significantly reduces overall inlet demand at a WWTP. However, the extent of reduction achieved depends on the level of ammonium and flow-rate in the side stream. The exchange efficiency of the ion-exchange material, MesoLite, used in the ammonium recovery process deteriorates with long-term use due to mechanical degradation and use of regenerant. To ensure that a sustainable process is utilised a range of potential applications for this “spent” MesoLite have been evaluated. The primary focus of evaluations has been use of ammonium-loaded MesoLite as a source of nitrogen and growth medium for plants. A MesoLite fertiliser has advantage over soluble fertilisers in that N is held on an insoluble matrix and is gradually released according to exchange equilibria. Many conventional N fertilisers are water-soluble and thus, instantly release all applied N into the soil solution. Loss of nutrient commonly occurs through volatilisation and/or leaching. On average, up to half of the N delivered by a typical soluble fertiliser can be lost through these processes. In this context, use of ammonium-loaded MesoLite as a fertiliser has been evaluated using standard greenhouse and field-based experiments for low fertility soils. Rye grass, a suitable test species for greenhouse trials, was grown in 1kg pots over a period of several weeks with regular irrigation. Nitrogen was applied at a range of rates using a chemical fertiliser as a control and using two MesoLite fertilisers. All other nutrients were applied in adequate amounts. All treatments were replicated three times. Plants were harvested after four weeks, and dry plant mass and N concentrations were determined. At all nitrogen application rates, ammonium-loaded MesoLite produced higher plant mass than plants fertilised by the chemical fertiliser. The lower fertiliser effectiveness of the chemical fertliser is attributed to possible loss of some N through volatilisation. The MesoLite fertilisers did not show any adverse effect on availability of macro and trace nutrients, as shown by lack of deficiency symptoms, dry matter yield and plant analyses. Nitrogen loaded on to MesoLite in the form of exchanged ammonium is readily available to plants while remaining protected from losses via leaching and volatilisation. Spent MesoLite appears to be a suitable and effective fertiliser for a wide range of soils, particularly sandy soils with poor nutrient holding capacity.

Nutritional analysis of gastric contents and body condition score at a single time point in feral horses in Australia

Objective: To determine the impact of a free-choice diet on nutritional intake and body condition of feral horses. Animals: Cadavers of 41 feral horses from 5 Australian locations. Procedures: Body condition score (BCS) was determined (scale of 1 to 9), and the stomach was removed from horses during postmortem examination. Stomach contents were analyzed for nutritional variables and macroelement and microelement concentrations. Data were compared among the locations and also compared with recommended daily intakes for horses. Results: Mean BCS varied by location; all horses were judged to be moderately thin. The BCS for males was 1 to 3 points higher than that of females. Amount of protein in the stomach contents varied from 4.3% to 14.9% and was significantly associated with BCS. Amounts of water-soluble carbohydrate and ethanol-soluble carbohydrate in stomach contents of feral horses from all 5 locations were higher than those expected for horses eating high-quality forage. Some macroelement and microelement concentrations were grossly excessive, whereas others were grossly deficient. There was no evidence of ill health among the horses. Conclusions and Clinical Relevance: Results suggested that the diet for several populations of feral horses in Australia appeared less than optimal. However, neither low BCS nor trace mineral deficiency appeared to affect survival of the horses. Additional studies on food sources in these regions, including analysis of water-soluble carbohydrate, ethanol-soluble carbohydrate, and mineral concentrations, are warranted to determine the provenance of such rich sources of nutrients. Determination of the optimal diet for horses may need revision.

Effects of bovine colostrum supplementation on immune variables of highly-trained cyclists

The aim of this study was to investigate the influence of low-dose bovine colostrum protein concentrate (CPC) supplementation on selected immune variables in cyclists. Twenty-nine highly trained male road cyclists completed an initial 40-km time trial (TT(40)) and were then randomly assigned to either a supplement (n = 14, 10 g bovine CPC/day) or placebo group (n = 15, 10 g whey protein concentrate/day). After 5 wk of supplementation, the cyclists completed a second TT(40). They then completed 5 consecutive days of high-intensity training (HIT) that included a TT(40), followed by a final TT(40) in the following week. Venous blood and saliva samples were collected immediately before and after each TT(40), and upper respiratory illness symptoms were recorded over the experimental period. Compared with the placebo group, bovine CPC supplementation significantly increased preexercise serum soluble TNF receptor 1 during the HIT period (bovine CPC = 882 +/- 233 pg/ml, placebo = 468 +/- 139 pg/ml; P = 0.039). Supplementation also suppressed the postexercise decrease in cytotoxic/suppressor T cells during the HIT period (bovine CPC = -1.0 +/- 2.7%, placebo = -9.2 +/- 2.8%; P = 0.017) and during the following week (bovine CPC = 1.4 +/- 2.9%, placebo = -8.2 +/- 2.8%; P = 0.004). Bovine CPC supplementation prevented a postexercise decrease in serum IgG(2) concentration at the end of the HIT period (bovine CPC = 4.8 +/- 6.8%, P = 0.88; placebo = -9.7 +/- 6.9%, P = 0.013). There was a trend toward reduced incidence of upper respiratory illness symptoms in the bovine CPC group (P = 0.055). In summary, low-dose bovine CPC supplementation modulates immune parameters during normal training and after an acute period of intense exercise, which may have contributed to the trend toward reduced upper respiratory illness in the bovine CPC group.

Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.