A priori calculations of the free energy of formation from solution of polymorphic self-assembled monolayers.

Modern quantum chemical electronic structure methods typically applied to localized chemical bonding are developed to predict atomic structures and free energies for meso-tetraalkylporphyrin self-assembled monolayer (SAM) polymorph formation from organic solution on highly ordered pyrolytic graphite surfaces. Large polymorph-dependent dispersion-induced substrate-molecule interactions (e.g., -100 kcal mol(-1) to -150 kcal mol(-1) for tetratrisdecylporphyrin) are found to drive SAM formation, opposed nearly completely by large polymorph-dependent dispersion-induced solvent interactions (70-110 kcal mol(-1)) and entropy effects (25-40 kcal mol(-1) at 298 K) favoring dissolution. Dielectric continuum models of the solvent are used, facilitating consideration of many possible SAM polymorphs, along with quantum mechanical/molecular mechanical and dispersion-corrected density functional theory calculations. These predict and interpret newly measured and existing high-resolution scanning tunnelling microscopy images of SAM structure, rationalizing polymorph formation conditions. A wide range of molecular condensed matter properties at room temperature now appear suitable for prediction and analysis using electronic structure calculations.

Third-Generation Light-Driven Symmetric Molecular Motors.

Symmetric molecular motors based on two overcrowded alkenes with a notable absence of a stereogenic center show potential to function as novel mechanical systems in the development of more advanced nanomachines offering controlled motion over surfaces. Elucidation of the key parameters and limitations of these third-generation motors is essential for the design of optimized molecular machines based on light-driven rotary motion. Herein we demonstrate the thermal and photochemical rotational behavior of a series of third-generation light-driven molecular motors. The steric hindrance of the core unit exerted upon the rotors proved pivotal in controlling the speed of rotation, where a smaller size results in lower barriers. The presence of a pseudo-asymmetric carbon center provides the motor with unidirectionality. Tuning of the steric effects of the substituents at the bridgehead allows for the precise control of the direction of disrotary motion, illustrated by the design of two motors which show opposite rotation with respect to a methyl substituent. A third-generation molecular motor with the potential to be the fastest based on overcrowded alkenes to date was used to visualize the equal rate of rotation of both its rotor units. The autonomous rotational behavior perfectly followed the predicted model, setting the stage for more advanced motors for functional dynamic systems.

Enantioselective synthesis of the strigolactone mimic (+)-GR24.

A short and cost-effective synthesis of the important strigolactone analogue (+)-GR24 is described. Central to this new approach is the concise, enantioselective synthesis of the A-C ring system.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

Balancing between competition and regulation in healthcare markets.

Systems of managed competition naturally seek the middle ground between competition and regulation. This debate essay makes the case for adjusting the level of regulation according to the characteristics of the submarket in question. We first develop a theoretical framework that can be used to identify the services in which relatively free competition will be beneficial. The framework is grounded in the economic literature and consists of eight criteria. Targeted regulatory tools are then discussed that can be used to structure submarkets in which these criteria are not (fully) met. Applying this framework and targeted interventions, regulators gain the flexibility to react to potential market failures, without foregoing the benefits of managed competition where it works well. This analysis is highly relevant for countries in transition to managed competition. Regulators can identify potential failure in submarkets for medical services, and apply the necessary regulatory tools to prepare for a smooth transition.

An assessment of molecular variability and recombination patterns in South African isolates of Potato virus Y.

The coat protein (CP) gene of 75 South African Potato virus Y (PVY) isolates was amplified using reverse-transcriptase polymerase chain reaction (RT-PCR). The resulting cDNA products were cloned and sequenced. These sequences were used to identify the strains to which the isolates belonged. Some, when compared to reference sequences, belonged to the PVY(N) and PVY(O) strains. A number of isolates were found to demonstrate significant homology to PVY(N) strains from China. A large number of South African isolates possessed CP sequences showing evidence of recombination between PVY(N) and PVY(O) strains, similar to those of PVY(NTN) isolates. Multiplex RT-PCR analysis allowed further differentiation of PVY(O) isolates and revealed that the majority were of the PVY(N)-Wilga strain. It was deduced that the most likely way in which these isolates reached South Africa was via the importation of infected material.

Harnessing Macrophages for Controlled-Release Drug Delivery: Lessons From Microbes.

With the effectiveness of therapeutic agents ever decreasing and the increased incidence of multi-drug resistant pathogens, there is a clear need for administration of more potent, potentially more toxic, drugs. Alternatively, biopharmaceuticals may hold potential but require specialized protection from premature in vivo degradation. Thus, a paralleled need for specialized drug delivery systems has arisen. Although cell-mediated drug delivery is not a completely novel concept, the few applications described to date are not yet ready for in vivo application, for various reasons such as drug-induced carrier cell death, limited control over the site and timing of drug release and/or drug degradation by the host immune system. Here, we present our hypothesis for a new drug delivery system, which aims to negate these limitations. We propose transport of nanoparticle-encapsulated drugs inside autologous macrophages polarized to M1 phenotype for high mobility and treated to induce transient phagosome maturation arrest. In addition, we propose a significant shift of existing paradigms in the study of host-microbe interactions, in order to study microbial host immune evasion and dissemination patterns for their therapeutic utilization in the context of drug delivery. We describe a system in which microbial strategies may be adopted to facilitate absolute control over drug delivery, and without sacrificing the host carrier cells. We provide a comprehensive summary of the lessons we can learn from microbes in the context of drug delivery and discuss their feasibility for in vivo therapeutic application. We then describe our proposed "synthetic microbe drug delivery system" in detail. In our opinion, this multidisciplinary approach may hold the solution to effective, controlled drug delivery.

Development of a transendothelial shuttle by macrophage modification.

One of the limiting factors in tissue regeneration, particularly in the context of chronic disease such as myodystrophy, motor neuron disease, sarcopenia, and cardiovascular disease, is limited availability of stem cells. We propose employing autologous macrophages to deliver stem cells, thereby facilitating tissue regeneration, by a novel and relatively non-invasive therapeutic intervention. Circulatory monocytic cells of M1 phenotype have capacity for transendothelial migration to infiltrate damaged tissue, making them ideal delivery vehicles. However, in order to deliver viable stem cells, these macrophages must undergo phagosome maturation arrest. Our aim was to induce phagosome maturation arrest in prepolarised M1 macrophages, whilst maintaining capacity for phagocytic engulfment (including phagosome formation) and transendothelial migration. Primary human M1 macrophages were treated with a wortmannin-concanamycin A-chloroquine cocktail to induce arrest. Modified cells were allowed to ingest 4.5 µm protein-coated fluorescent latex beads (simulated stem cells), before migratory capacity in response to MCP-1 was assessed over a 2-hr period in a Transwell co-culture system. Data indicate that phagosome acidification (as indicated by pHrodo®) was prevented in treated cells, effectively limiting digestion of ingested "cargo" (1.23 ± 0.26% vs. 7.52 ± 0.98% in controls; p < .0001). Neither phagocytic engulfment capacity (68.67 ± 3.51% vs. 61.19 ± 4.68%) nor migratory capacity (70.14 ± 12.6 vs. 72.86 ± 16.0 migrated cells per well) was compromised. We conclude that macrophages were successfully modified into transendothelial delivery vehicles, without compromising required functionality. This delivery system can be exploited to develop a novel method for focussed stem cell and/or drug delivery.

Unidirectional rotary motion in achiral molecular motors.

Control of the direction of motion is an essential feature of biological rotary motors and results from the intrinsic chirality of the amino acids from which the motors are made. In synthetic autonomous light-driven rotary motors, point chirality is transferred to helical chirality, and this governs their unidirectional rotation. However, achieving directional rotary motion in an achiral molecular system in an autonomous fashion remains a fundamental challenge. Here, we report an achiral molecular motor in which the presence of a pseudo-asymmetric carbon atom proved to be sufficient for exclusive autonomous disrotary motion of two appended rotor moieties. Isomerization around the two double bonds enables both rotors to move in the same direction with respect to their surroundings--like wheels on an axle--demonstrating that autonomous unidirectional rotary motion can be achieved in a symmetric system.

The recent recombinant evolution of a major crop pathogen, potato virus Y.

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVY(NTN) occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences.

Phylogeography and molecular evolution of potato virus Y.

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.

Two-dimensional molecular patterning by surface-enhanced Zn-porphyrin coordination.

In this contribution, we show how zinc-5,10,15,20-meso-tetradodecylporphyrins (Zn-TDPs) self-assemble into stable organized arrays on the surface of graphite, thus positioning their metal center at regular distances from each other, creating a molecular pattern, while retaining the possibility to coordinate additional ligands. We also demonstrate that Zn-TDPs coordinated to 3-nitropyridine display a higher tendency to be adsorbed at the surface of highly oriented pyrolytic graphite (HOPG) than noncoordinated ones. In order to investigate the two-dimensional (2D) self-assembly of coordinated Zn-TDPs, solutions with different relative concentrations of 3-nitropyridine and Zn-TDP were prepared and deposited on the surface of HOPG. STM measurements at the liquid-solid interface reveal that the ratio of coordinated Zn-TDPs over noncoordinated Zn-TDPs is higher at the n-tetradecane/HOPG interface than in n-tetradecane solution. This enhanced binding of the axial ligand at the liquid/solid interface is likely related to the fact that physisorbed Zn-TDPs are better binding sites for nitropyridines.

Self-organized monolayer of meso-tetradodecylporphyrin coordinated to Au(111).

The structure of molecular monolayers formed at the interface between atomically flat surfaces and a solution of free-base meso-tetradodecylporphyrins (H2Ps) was examined by scanning tunneling microscopy (STM) at the liquid/solid interface. On the surface of graphite (HOPG), H2Ps form a well-ordered monolayer characterized by an oblique unit cell. On Au(111), H2Ps form a self-organized monolayer comprised of two distinct domain types. In both types of domains, the density of the porphyrin cores is increased in comparison to the arrangement observed on HOPG. Also, high-resolution STM images reveal that, in contrast to what is observed on HOPG, physisorption on Au(111) induces a distortion of the porphyrin macrocycle out of planarity. By using X-ray photoelectron spectroscopy, we demonstrate that this is likely to be due to the coordination of the lone pairs of the iminic (-C=N-) nitrogen atoms of the porphyrin macrocycle to Au(111).