Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Fractal and complex network analyses of protein molecular dynamics

Based on protein molecular dynamics, we investigate the fractal properties of energy, pressure and volume time series using the multifractal detrended fluctuation analysis (MF-DFA) and the topological and fractal properties of their converted horizontal visibility graphs (HVGs). The energy parameters of protein dynamics we considered are bonded potential, angle potential, dihedral potential, improper potential, kinetic energy, Van der Waals potential, electrostatic potential, total energy and potential energy. The shape of the h(q)h(q) curves from MF-DFA indicates that these time series are multifractal. The numerical values of the exponent h(2)h(2) of MF-DFA show that the series of total energy and potential energy are non-stationary and anti-persistent; the other time series are stationary and persistent apart from series of pressure (with H≈0.5H≈0.5 indicating the absence of long-range correlation). The degree distributions of their converted HVGs show that these networks are exponential. The results of fractal analysis show that fractality exists in these converted HVGs. For each energy, pressure or volume parameter, it is found that the values of h(2)h(2) of MF-DFA on the time series, exponent λλ of the exponential degree distribution and fractal dimension dBdB of their converted HVGs do not change much for different proteins (indicating some universality). We also found that after taking average over all proteins, there is a linear relationship between 〈h(2)〉〈h(2)〉 (from MF-DFA on time series) and 〈dB〉〈dB〉 of the converted HVGs for different energy, pressure and volume.

Molecular orientational order of nitroxide radicals in liquid crystalline media

The orientational distribution of a set of stable nitroxide radicals in aligned liquid crystals 5CB (nematic) and 8CB (smectic A) was studied in detail by numerical simulation of EPR spectra. The order parameters up to the 10th rank were measured. The directions of the principal orientation axes of the radicals were determined. It was shown that the ordering of the probe molecules is controlled by their interaction with the matrix molecules more than the inherent geometry of the probes themselves. The rigid fused phenanthrene-based (A5) and 2-azaphenalene (A4) nitroxides as well as the rigid core elongated C11 and 5α-cholestane (CLS) nitroxides were found to be most sensitive to the orientation of the liquid crystal matrixes.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

Molecular imaging with polymers

Polymers open up new possibilities in the field of molecular imaging, allowing sensitive and robust agents that can be imaged over long periods of time. This review highlights some recent advances in polymeric molecular imaging agents in both (pre)clinical and emerging applications.

Genome-wide association study of intraocular pressure identifies the GLCCI1/ICA1 region as a glaucoma susceptibility locus

To discover quantitative trait loci for intraocular pressure, amajor risk factor for glaucoma and the only modifiable one,weperformed agenome-wide association studyonadiscoverycohort of2175individualsfromSydney, Australia. We found a novel association between intraocular pressure and a common variant at 7p21 near to GLCCI1 and ICA1. The findings in this region were confirmed through two UK replication cohorts totalling 4866 individuals (rs59072263, Pcombined = 1.10 × 10-8). A copy of the G allele at this SNP is associated with an increase in mean IOP of 0.45 mmHg (95%CI = 0.30-0.61 mmHg). These results lend support to the implication of vesicle trafficking and glucocorticoid inducibility pathways in the determination of intraocular pressure and in the pathogenesis of primary open-angle glaucoma.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Identification of IL6R and chromosome 11q13.5 as risk loci for asthma

We aimed to identify novel genetic variants affecting asthma risk, since these might provide novel insights into molecular mechanisms underlying the disease. We did a genome-wide association study (GWAS) in 2669 physician-diagnosed asthmatics and 4528 controls from Australia. Seven loci were prioritised for replication after combining our results with those from the GABRIEL consortium (n=26 475), and these were tested in an additional 25 358 independent samples from four in-silico cohorts. Quantitative multi-marker scores of genetic load were constructed on the basis of results from the GABRIEL study and tested for association with asthma in our Australian GWAS dataset. Two loci were confirmed to associate with asthma risk in the replication cohorts and reached genome-wide significance in the combined analysis of all available studies (n=57 800): rs4129267 (OR 1·09, combined p= 2·4×10-8) in the interleukin-6 receptor (IL6R) gene and rs7130588 (OR 1·09, p=1·8×10-8) on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene (LRRC32, also known as GARP). The 11q13.5 locus was significantly associated with atopic status among asthmatics (OR 1·33, p=7×10-4), suggesting that it is a risk factor for allergic but not non-allergic asthma. Multi-marker association results are consistent with a highly polygenic contribution to asthma risk, including loci with weak effects that might be shared with other immune-related diseases, such as NDFIP1, HLA-B, LPP, and BACH2. The IL6R association further supports the hypothesis that cytokine signalling dysregulation affects asthma risk, and raises the possibility that an IL6R antagonist (tocilizumab) may be effective to treat the disease, perhaps in a genotype-dependent manner. Results for the 11q13.5 locus suggest that it directly increases the risk of allergic sensitisation which, in turn, increases the risk of subsequent development of asthma. Larger or more functionally focused studies are needed to characterise the many loci with modest effects that remain to be identified for asthma. National Health and Medical Research Council of Australia. A full list of funding sources is provided in the webappendix. © 2011 Elsevier Ltd.

A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G→A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Crystal engineering in two dimensions: An approach to molecular nanopatterning

We describe a surprising cooperative adsorption process observed by scanning tunneling microscopy (STM) at the liquid−solid interface. The process involves the association of a threefold hydrogen-bonding unit, trimesic acid (TMA), with straight-chain aliphatic alcohols of varying length (from C7 to C30), which coadsorb on highly oriented pyrolytic graphite (HOPG) to form linear patterns. In certain cases, the known TMA “flower pattern” can coexist temporarily with the linear TMA−alcohol patterns, but it eventually disappears. Time-lapsed STM imaging shows that the evolution of the flower pattern is a classical ripening phenomenon. The periodicity of the linear TMA−alcohol patterns can be modulated by choosing alcohols with appropriate chain lengths, and the precise structure of the patterns depends on the parity of the carbon count in the alkyl chain. Interactions that lead to this odd−even effect are analyzed in detail. The molecular components of the patterns are achiral, yet their association by hydrogen bonding leads to the formation of enantiomeric domains on the surface. The interrelation of these domains and the observation of superperiodic structures (moiré patterns) are rationalized by considering interactions with the underlying graphite surface and within the two-dimensional crystal of the adsorbed molecules. Comparison of the observed two-dimensional structures with the three-dimensional crystal structures of TMA−alcohol complexes determined by X-ray crystallography helps reveal the mechanism of molecular association in these two-component systems.

Stabilization of exotic minority phases in a multicomponent self-assembled molecular network

Trimesic acid (TMA) and alcohols were recently shown to self-assemble into a stable, two-component linear pattern at the solution/highly oriented pyrolytic graphite (HOPG) interface. Away from equilibrium, the TMA/alcohol self-assembled molecular network (SAMN) can coexist with pure-TMA networks. Here, we report on some novel characteristics of these non-equilibrium TMA structures, investigated by scanning tunneling microscopy (STM). We observe that both the chicken-wire and flower-structure TMA phases can host 'guest' C60 molecules within their pores, whereas the TMA/alcohol SAMN does not offer any stable adsorption sites for the C60 molecules. The presence of the C60 molecules at the solution/solid interface was found to improve the STM image quality. We have taken advantage of the high-quality imaging conditions to observe unusual TMA bonding geometries at domain boundaries in the TMA/alcohol SAMN. Boundaries between aligned TMA/alcohol domains can give rise to doubled TMA dimer rows in two different configurations, as well as a tripled-TMA row. The boundaries created between non-aligned domains can create geometries that stabilize TMA bonding configurations not observed on surfaces without TMA/alcohol SAMNs, including small regions of the previously predicted 'super flower' TMA bonding geometry and a tertiary structure related to the known TMA phases. These structures are identified as part of a homologic class of TMA bonding motifs, and we explore some of the reasons for the stabilization of these phases in our multicomponent system.

Molecular self-assembly on graphene

The formation of ordered arrays of molecules via self-assembly is a rapid, scalable route towards the realization of nanoscale architectures with tailored properties. In recent years, graphene has emerged as an appealing substrate for molecular self-assembly in two dimensions. Here, the first five years of progress in supramolecular organization on graphene are reviewed. The self-assembly process can vary depending on the type of graphene employed: epitaxial graphene, grown in situ on a metal surface, and non-epitaxial graphene, transferred onto an arbitrary substrate, can have different effects on the final structure. On epitaxial graphene, the process is sensitive to the interaction between the graphene and the substrate on which it is grown. In the case of graphene that strongly interacts with its substrate, such as graphene/Ru(0001), the inhomogeneous adsorption landscape of the graphene moiré superlattice provides a unique opportunity for guiding molecular organization, since molecules experience spatially constrained diffusion and adsorption. On weaker-interacting epitaxial graphene films, and on non-epitaxial graphene transferred onto a host substrate, self-assembly leads to films similar to those obtained on graphite surfaces. The efficacy of a graphene layer for facilitating planar adsorption of aromatic molecules has been repeatedly demonstrated, indicating that it can be used to direct molecular adsorption, and therefore carrier transport, in a certain orientation, and suggesting that the use of transferred graphene may allow for predictible molecular self-assembly on a wide range of surfaces.

Substrate, molecular structure, and solvent effects in 2D self-assembly via hydrogen and halogen bonding

Recently, halogen···halogen interactions have been demonstrated to stabilize two-dimensional supramolecular assemblies at the liquid–solid interface. Here we study the effect of changing the halogen, and report on the 2D supramolecular structures obtained by the adsorption of 2,4,6-tris(4-bromophenyl)-1,3,5-triazine (TBPT) and 2,4,6-tris(4-iodophenyl)-1,3,5-triazine (TIPT) on both highly oriented pyrolytic graphite and the (111) facet of a gold single crystal. These molecular systems were investigated by combining room-temperature scanning tunneling microscopy in ambient conditions with density functional theory, and are compared to results reported in the literature for the similar molecules 1,3,5-tri(4-bromophenyl)benzene (TBPB) and 1,3,5-tri(4-iodophenyl)benzene (TIPB). We find that the substrate exerts a much stronger effect than the nature of the halogen atoms in the molecular building blocks. Our results indicate that the triazine core, which renders TBPT and TIPT stiff and planar, leads to stronger adsorption energies and hence structures that are different from those found for TBPB and TIPB. On the reconstructed Au(111) surface we find that the TBPT network is sensitive to the fcc- and hcp-stacked regions, indicating a significant substrate effect. This makes TBPT the first molecule reported to form a continuous monolayer at room temperature in which molecular packing is altered on the differently reconstructed regions of the Au(111) surface. Solvent-dependent polymorphs with solvent coadsorption were observed for TBPT on HOPG. This is the first example of a multicomponent self-assembled molecular networks involving the rare cyclic, hydrogen-bonded hexamer of carboxylic groups, R66(24) synthon.