Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

Atomistic simulations of 2D bicomponent self-assembly: from molecular recognition to self-healing

Supramolecular two-dimensional engineering epitomizes the design of complex molecular architectures through recognition events in multicomponent self-assembly. Despite being the subject of in-depth experimental studies, such articulated phenomena have not been yet elucidated in time and space with atomic precision. Here we use atomistic molecular dynamics to simulate the recognition of complementary hydrogen-bonding modules forming 2D porous networks on graphite. We describe the transition path from the melt to the crystalline hexagonal phase and show that self-assembly proceeds through a series of intermediate states featuring a plethora of polygonal types. Finally, we design a novel bicomponent system possessing kinetically improved self-healing ability in silico, thus demonstrating that a priori engineering of 2D self-assembly is possible.

Simulating Cortical Development as a Self Constructing Process: A Novel Multi-Scale Approach Combining Molecular and Physical Aspects

Current models of embryological development focus on intracellular processes such as gene expression and protein networks, rather than on the complex relationship between subcellular processes and the collective cellular organization these processes support. We have explored this collective behavior in the context of neocortical development, by modeling the expansion of a small number of progenitor cells into a laminated cortex with layer and cell type specific projections. The developmental process is steered by a formal language analogous to genomic instructions, and takes place in a physically realistic three-dimensional environment. A common genome inserted into individual cells control their individual behaviors, and thereby gives rise to collective developmental sequences in a biologically plausible manner. The simulation begins with a single progenitor cell containing the artificial genome. This progenitor then gives rise through a lineage of offspring to distinct populations of neuronal precursors that migrate to form the cortical laminae. The precursors differentiate by extending dendrites and axons, which reproduce the experimentally determined branching patterns of a number of different neuronal cell types observed in the cat visual cortex. This result is the first comprehensive demonstration of the principles of self-construction whereby the cortical architecture develops. In addition, our model makes several testable predictions concerning cell migration and branching mechanisms.

The Exploitation of Versatile Building Blocks for the Self-Assembly of Novel Molecular Magnets

Using molecular building blocks to self-assemble lattices supporting long-range magnetic order is currently an active area of solid-state chemistry. Consequently, it is the realm of supramolecular chemistry that synthetic chemists are turning to in order to develop techniques for the synthesis of structurally well-defined supramolecular materials. In recent years we have investigated the versatility and usefulness of two classes of molecular building blocks, namely, tris-oxalato transition-metal (M. Pilkington and S. Decurtins, in “Magnetoscience—From Molecules to Materials,” Wiley–VCH, 2000), and octacyanometalate complexes (Pilkington and Decurtins, Chimia 54, 593 (2001)), for applications in the field of molecule-based magnets. Anionic, tris-chelated oxalato building blocks are able to build up two-dimensional honeycomb-layered structural motifs as well as three-dimensional decagon frameworks. The discrimination between the crystallization of the two- or three-dimensional structures relies on the choice of the templating counterions (Decurtins, Chimia 52, 539 (1998); Decurtins et al. Mol. Cryst. Liq. Cryst. 273, 167 (1995); New J. Chem. 117 (1998)). These structural types display a range of ferro, ferri, and antiferromagnetic properties (Pilkington and Decurtins, in “Magnetoscience—From Molecules to Materials”). Octacyanometalate building blocks self-assemble to afford two new classes of cyano-bridged compounds namely, molecular clusters and extended three dimensional networks (J. Larionova et al., Angew. Chem. Int. Ed. 39, 1605 (2000); Pilkington et al., in preparation). The molecular cluster with a MnII9MoV6 core has the highest ground state spin value, S=51/2, reported to-date (Larionova et al., Angew. Chem. Int. Ed. 39, 1605 (2000)). In the high-temperature regime, the magnetic properties are characterized by ferromagnetic intracluster coupling. In the magnetic range below 44 K, the magnetic cluster signature is lost as possibly a bulk behavior starts to emerge. The three-dimensional networks exhibit both paramagnetic and ferromagnetic behavior, since the magnetic properties of these materials directly reflect the electronic configuration of the metal ion incorporated into the octacyanometalate building blocks (Pilkington et al., in preparation). For both the oxalate- and cyanide-bridged materials, we are able to manipulate the magnetic properties of the supramolecular assemblies by tuning the electronic configurations of the metal ions incorporated into the appropriate molecular building blocks (Pilkington and Decurtins, in “Magnetoscience—From Molecules to Materials,” Chimia 54, 593 (2000)).

The Design and Synthesis of Versatile Molecular Building Blocks: Working Towards the Controlled Self-Assembly of Novel Functional Materials

Our research goals are focused on the preparation of novel molecule-based materials that possess specifically designed properties in solution or in the solid state e.g. self-assembly, magnetism, conductivity and spin crossover phenomena. Most of our systems incorporate paramagnetic transition metal ions and the search for new molecule-based magnetic materials is a prominent theme. Specific areas of research include the preparation and study of oxalate based 2D and 3D magnets, probing the versatility of octacyanometalate building blocks as precursors for new molecular magnets, and the preparation of new tetrathiafulvalene (TIF) derivatives for applications in molecular and supramolecular chemistry.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

BACKGROUND Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. RESULTS Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. CONCLUSION Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Orientation dependent molecular friction on organic layer compound crystals

High resolution friction force maps of the benzylammonium terminated crystalline surface of a layer compound are presented. The lateral force map acquired with an atomic force microscope, reveals a significant contrast between different molecular orientations yielding molecular rows which differ from their neighboring ones. The single crystals are formed by stacks of copper oxalate sheets sandwiched between stereoregular organic cations, resulting in highly organized surface structures. Single molecular defects are observed at small loads. The experimental results are compared with numerical calculations which indicate a transition from an unperturbed state at small loads to a distorted state at higher loads. (C) 2011 American Institute of Physics.

Molecular cloning and characterization of equine thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that plays a central role in T helper 2 (Th2) cell differentiation and allergic inflammation. It is predominantly expressed by epithelial cells, and its expression is increased in patients with atopic dermatitis and asthma. Mice overexpressing TSLP in the skin develop allergic dermatitis and mice overexpressing TSLP in lungs develop asthma-like disease. However, it is not known whether TSLP plays an important role in equine allergies. Therefore, we cloned and sequenced the complete translated region of equine TSLP gene and measured its expression in various tissues. The equine TSLP gene is organized in 4 exons and encodes a protein of 143 amino acids, which has 62% amino acid identity with human TSLP.