Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer

Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2’–deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

A genome-wide linkage scan provides evidence for both new and previously reported loci influencing common migraine

Latent class analysis was performed on migraine symptom data collected in a Dutch population sample (N = 12,210, 59% female) in order to obtain empirical groupings of individuals suffering from symptoms of migraine headache. Based on these heritable groupings (h(2) = 0.49, 95% CI: 0.41-0.57) individuals were classified as affected (migrainous headache) or unaffected. Genome-wide linkage analysis was performed using genotype data from 105 families with at least 2 affected siblings. In addition to this primary phenotype, linkage analyses were performed for the individual migraine symptoms. Significance levels, corrected for the analysis of multiple traits, were determined empirically via a novel simulation approach. Suggestive linkage for migrainous headache was found on chromosomes 1 (LOD = 1.63; pointwise P = 0.0031), 13 (LOD = 1.63; P = 0.0031), and 20 (LOD = 1.85; P = 0.0018). Interestingly, the chromosome 1 peak was located close to the ATP1A2 gene, associated with familial hemiplegic migraine type 2 (FHM2). Individual symptom analysis produced a LOD score of 1.97 (P = 0.0013) on chromosome 5 (photo/phonophobia), a LOD score of 2.13 (P = 0.0009) on chromosome 10 (moderate/severe pain intensity) and a near significant LOD score of 3.31 (P = 0.00005) on chromosome 13 (pulsating headache). These peaks were all located near regions previously reported in migraine linkage studies. Our results provide important replication and support for the presence of migraine susceptibility genes within these regions, and further support the utility of an LCA-based phenotyping approach and analysis of individual symptoms in migraine genetic research. Additionally, our novel "2-step" analysis and simulation approach provides a powerful means to investigate linkage to individual trait components.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.

Juggling priorities: Balancing economic and social drivers to address the LLN needs of students in the VET sector

Economic success, and a commitment to the social benefits of inclusive training opportunities are important goals for public vocational education and training (VET). Currently, in Australia, VET policy is a shared responsibility between the Commonwealth and the States and Territories. Priorities for investment are juggled between: a) improving efficiency and responsiveness; and b) providing societal prosperity. Amid recent VET educational reforms and policy directives the authors of this paper undertook a pilot study examining language, literacy and numeracy support and inclusive teaching and learning practices in a Diploma of Nursing course. The data highlighted implications arising from new market driven education reforms. This article reports on identified factors that influenced inclusive learning opportunities, noticeably associated with two recent policy developments: the release of the FSK Foundation Skills Training Package (IBSA 2014); and the Queensland's Higher Skills Program Policy 2014-15.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Triosephosphate isomerase from Plasmodium falciparum:. the crystal structure provides insights into antimalarial drug design

Background: Malaria caused by the parasite Plasmodium falciparum is a major public health concern. The parasite lacks a functional tricarboxylic acid cycle, making glycolysis its sole energy source. Although parasite enzymes have been considered as potential antimalarial drug targets, little is known about their structural biology. Here we report the crystal structure of triosephosphate isomerase (TIM) from P. falciparum at 2.2 Angstrom resolution. Results: The crystal structure of P. falciparum TIM (PfTIM), expressed in Escherichia coli, was determined by the molecular replacement method using the structure of trypanosomal TIM as the starting model. Comparison of the PfTIM structure with other TIM structures, particularly human TIM, revealed several differences, In most TIMs the residue at position 183 is a glutamate but in PtTIM it is a leucine, This leucine residue is completely exposed and together with the surrounding positively charged patch, may be responsible for binding TIM to the erythrocyte membrane. Another interesting feature is the occurrence of a cysteine residue at the dimer interface of PfTIM (Cys13), in contrast to human TIM where this residue is a methionine. Finally, residue 96 of human TIM (Ser96), which occurs near the active site, has been replaced by phenylalanine in PfTIM.

Solution structures of conformationally equilibrium forms of holo-acyl carrier protein (PfACP) from Plasmodium falciparum provides insight into the mechanism of activation of ACPs

Acyl Carrier Protein (ACP) from the malaria parasite, Plasmodium falciparum (PfACP) in its holo form is found to exist in two conformational states in solution. Unique 3D solution structures of holo-PfACP have been determined for both equilibrium conformations, using high-resolution NMR methods. Twenty high-resolution solution structures for each of the two forms of holo-PfACP have been determined on the basis of 1226 and 1218 unambiguously assigned NOEs (including NOEs between 4 '-phosphopantetheine prosthetic group (4 '-PP) and protein), 55 backbone dihedral angles and 26 hydrogen bonds. The atomic rmsd values of the determined structures of two equilibrium forms, about the mean coordinates of the backbone and heavy atoms, are 0.48 +/- 0.09 and 0.92 +/- 0.10 and 0.49 +/- 0.08 and 0.97 +/- 0.11 angstrom, respectively. The interaction of 4 '-PP with the polypeptide backbone is reported here for the first time for any of the ACPs. The structures of holo-PfACP consist of three well-defined helices that are tightly packed. The structured regions of the molecule are stabilized by extensive hydrophobic interactions. The difference between the two forms arises from a reorientation of the 4 '-PP group. The enthalpy difference between the two forms, although small, implies that a conformational switch is essential for the activation of holo-ACP. Sequence and structures of holo-PfACP have been compared with those of the ACPs from type I and type II fatty acid biosynthesis pathways (FAS), in particular with the ACP from rat and the butyryl-ACP from E. coli. The PfACP structure, thus determined has several novel features hitherto not seen in other ACPs.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.