Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8x10(-4) nucleotide substitutions per site year(-1)), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Molecular structure of Mg-Al, Mn-Al and Zn-Al halotrichites : an infrared spectroscopic study

Near infrared (NIR), X-ray diffraction (XRD) and infrared (IR) spectroscopy have been applied to halotrichites of the formula MgAl2(SO4)4∙22H2O, MnAl2(SO4)4∙22H2O and ZnAl2(SO4)4∙22H2O. Comparison of the halotrichites in different spectral regions has shown that the incorporation of a divalent transition metal into the halotrichite structure causes a shift in OH stretching band positions to lower wavenumbers. Therefore, an increase in hydrogen bonded water is observed for divalent cations with a larger molecular mass. XRD has confirmed the formation of halotrichite for all three samples and characteristic peaks of halotrichite have been identified at 18.5 and 24.5° 2θ, along with a group of six peaks between 5 and 15° 2θ. It has been observed that Mg-Al and Mn-Al halotrichite are very similar in structure, while Zn-Al showed several differences particularly in the NIR spectra. This work has shown that halotrichite structures can be synthesised and characterised by infrared and NIR spectroscopy.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Genome-wide identification and expression analysis of the NF-Y family of transcription factors in Triticum aestivum

Nuclear Factor Y (NF-Y) is a trimeric complex that binds to the CCAAT box, a ubiquitous eukaryotic promoter element. The three subunits NF-YA, NF-YB and NF-YC are represented by single genes in yeast and mammals. However, in model plant species (Arabidopsis and rice) multiple genes encode each subunit providing the impetus for the investigation of the NF-Y transcription factor family in wheat. A total of 37 NF-Y and Dr1 genes (10 NF-YA, 11 NF-YB, 14 NF-YC and 2 Dr1) in Triticum aestivum were identified in the global DNA databases by computational analysis in this study. Each of the wheat NF-Y subunit families could be further divided into 4-5 clades based on their conserved core region sequences. Several conserved motifs outside of the NF-Y core regions were also identified by comparison of NF-Y members from wheat, rice and Arabidopsis. Quantitative RT-PCR analysis revealed that some of the wheat NF-Y genes were expressed ubiquitously, while others were expressed in an organ-specific manner. In particular, each TaNF-Y subunit family had members that were expressed predominantly in the endosperm. The expression of nine NF-Y and two Dr1 genes in wheat leaves appeared to be responsive to drought stress. Three of these genes were up-regulated under drought conditions, indicating that these members of the NF-Y and Dr1 families are potentially involved in plant drought adaptation. The combined expression and phylogenetic analyses revealed that members within the same phylogenetic clade generally shared a similar expression profile. Organ-specific expression and differential response to drought indicate a plant-specific biological role for various members of this transcription factor family.

Ecological aspects of biosecurity surveillance design for the detection of multiple invasive animal species

Complex surveillance problems are common in biosecurity, such as prioritizing detection among multiple invasive species, specifying risk over a heterogeneous landscape, combining multiple sources of surveillance data, designing for specified power to detect, resource management, and collateral effects on the environment. Moreover, when designing for multiple target species, inherent biological differences among species result in different ecological models underpinning the individual surveillance systems for each. Species are likely to have different habitat requirements, different introduction mechanisms and locations, require different methods of detection, have different levels of detectability, and vary in rates of movement and spread. Often there is a further challenge of a lack of knowledge, literature, or data, for any number of the above problems. Even so, governments and industry need to proceed with surveillance programs which aim to detect incursions in order to meet environmental, social and political requirements. We present an approach taken to meet these challenges in one comprehensive and statistically powerful surveillance design for non-indigenous terrestrial vertebrates on Barrow Island, a high conservation nature reserve off the Western Australian coast. Here, the possibility of incursions is increased due to construction and expanding industry on the island. The design, which includes mammals, amphibians and reptiles, provides a complete surveillance program for most potential terrestrial vertebrate invaders. Individual surveillance systems were developed for various potential invaders, and then integrated into an overall surveillance system which meets the above challenges using a statistical model and expert elicitation. We discuss the ecological basis for the design, the flexibility of the surveillance scheme, how it meets the above challenges, design limitations, and how it can be updated as data are collected as a basis for adaptive management.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

What aspects of the scoliosis correction are most important to the teenager who has keyhole scoliosis correction surgery? A prospective series of 100 patients

Introduction. Surgical treatment of scoliosis is assessed in the spine clinic by the surgeon making numerous measurements on X-Rays as well as the rib hump. But it is important to understand which of these measures correlate with self-reported improvements in patients’ quality of life following surgery. The objective of this study was to examine the relationship between patient satisfaction after thoracoscopic (keyhole) anterior scoliosis surgery and standard deformity correction measures using the Scoliosis Research Society (SRS) adolescent questionnaire. Methods. A series of 100 consecutive adolescent idiopathic scoliosis patients received a single anterior rod via a keyhole approach at the Mater Children’s Hospital, Brisbane. Patients completed SRS outcomes questionnaires before surgery and again at 24 months after surgery. Multiple regression and t-tests were used to investigate the relationship between SRS scores and deformity correction achieved after surgery. Results. There were 94 females and 6 males with a mean age of 16.1 years. The mean Cobb angle improved from 52º pre-operatively to 21º for the instrumented levels post-operatively (59% correction) and the mean rib hump improved from 16º to 8º (51% correction). The mean total SRS score for the cohort was 99.4/120 which indicated a high level of satisfaction with the results of their scoliosis surgery. None of the deformity related parameters in the multiple regressions were significant. However, the twenty patients with the smallest Cobb angles after surgery reported significantly higher SRS scores than the twenty patients with the largest Cobb angles after surgery, but there was no difference on the basis of rib hump correction. Discussion. Patients undergoing thoracoscopic (keyhole) anterior scoliosis correction report good SRS scores which are comparable to those in previous studies. We suggest that the absence of any statistically significant difference in SRS scores between patients with and without rod or screw complications is because these complications are not associated with any clinically significant loss of correction in our patient group. The Cobb angle after surgery was the only significant predictor of patient satisfaction when comparing subgroups of patients with the largest and smallest Cobb angles after surgery.

Molecular dynamics study of dynamic buckling properties of nanowires with defects

Based on the embedded atom method (EAM) and molecular dynamics (MD) method, the deformation properties of Cu nanowires with different single defects under dynamic compression have been studied. The mechanical behaviours of the perfect nanowire are first studied, and the critical stress decreases with the increase of the nanowire’s length, which is well agreed with the modified Euler theory. We then consider the effects to the buckling phenomenon resulted from different defects. It is found that obvious decrease of the critical stress is resulted from different defects, and the largest decrease is found in nanowire with the surface vertical defect. Surface defects are found exerting larger influence than internal defects. The buckling duration is found shortened due to different defects except the nanowire with surface horizon defect, which is also found possessing the largest deflection. Different deflections are also observed for different defected nanowires. It is find that due to surface defects, only deflection in one direction is happened, but for internal defects, more complex deflection circumstances are observed.