Systematics and biology of the iconic Australian scribbly gum moths Ogmograptis Meyrick (Lepidoptera: Bucculatricidae) and their unique insect-plant interaction

The larvae of particular Ogmograptis spp. produce distinctive scribbles on some smooth-barked Eucalyptus spp. which are a common feature on many ornamental and forest trees in Australia. However, although they are conspicuous in the environment the systematics and biology of the genus has been poorly studied. This has been addressed through detailed field and laboratory studies of their biology of three species (O. racemosa Horak sp. nov., O. fraxinoides Horak sp. nov., O. scribula Meyrick), in conjunction with a comprehensive taxonomic revision support by a molecular phylogeny utilising the mitochondrial Cox1 and nuclear 18S genes. In brief, eggs are laid in bark depressions and the first instar larvae bore into the bark to the level where the future cork cambium forms (the phellegen). Early instar larvae bore wide, arcing tracks in this layer before forming a tighter zig-zag shaped pattern. The second last instar turns and bores either closely parallel to the initial mine or doubles its width, along the zig-zag shaped mine. The final instar possesses legs and a spinneret (unlike the earlier instars) and feeds exclusively on callus tissue which forms within the zig-zag shaped mine formed by the previous instar, before emerging from the bark to pupate at the base of the tree. The scars of mines them become visible scribble following the shedding of bark. Sequence data confirm the placement of Ogmograptis within the Bucculatricidae, suggest that the larvae responsible for the ‘ghost scribbles’ (unpigmented, raised scars found on smooth-barked eucalypts) are members of the genus Tritymba, and support the morphology-based species groups proposed for Ogmograptis. The formerly monotypic genus Ogmograptis Meyrick is revised and divided into three species groups. Eleven new species are described: Ogmograptis fraxinoides Horak sp. nov., Ogmograptis racemosa Horak sp. nov. and Ogmograptis pilularis Horak sp. nov. forming the scribula group with Ogmograptis scribula Meyrick; Ogmograptis maxdayi Horak sp. nov., Ogmograptis barloworum Horak sp. nov., Ogmograptis paucidentatus Horak sp. nov., Ogmograptis rodens Horak sp. nov., Ogmograptis bignathifer Horak sp. nov. and Ogmograptis inornatus Horak sp. nov. as the maxdayi group; Ogmograptis bipunctatus Horak sp. nov., Ogmograptis pulcher Horak sp. nov., Ogmograptis triradiata (Turner) comb. nov. and Ogmograptis centrospila (Turner) comb. nov. as the triradiata group. Ogmograptis notosema (Meyrick) cannot be assigned to a species group as the holotype has not been located. Three unique synapomorphies, all derived from immatures, redefine the family Bucculatricidae, uniting Ogmograptis, Tritymba Meyrick (both Australian) and Leucoedemia Scoble & Scholtz (African) with Bucculatrix Zeller, which is the sister group of the southern hemisphere genera. The systematic history of Ogmograptis and the Bucculatricidae is discussed.

Continuum mechanics modelling of microfilament networks with different architectures based on molecular investigation of single F-actin

The actin microfilament plays a critical role in many cellular processes including embryonic development, wound healing, immune response, and tissue development. It is commonly organized in the form of networks whose mechanical properties change with changes in their architecture due to cell evolution processes. This paper presents a new nonlinear continuum mechanics model of single filamentous actin (F-actin) that is based on nanoscale molecular simulations. Following this continuum model of the single F-actin, mechanical properties of differently architected lamellipodia are studied. The results provide insight that can contribute to the understanding of the cell edge motions of living cells.

Exercise and Cellular Mechanisms of Muscle Injury

The general aim of this book is to provide a comprehensive summary of the characteristics of exercise-induced muscle damage and the mechanisms of tissue inflammation. The authors present a large amount of our own original data and have summarised the research of others.

SECIS-binding protein 2 promotes cell survival by protecting against oxidative stress

Reactive oxygen species (ROS) are a primary cause of cellular damage that leads to cell death. In cells, protection from ROS-induced damage and maintenance of the redox balance is mediated to a large extent by selenoproteins, a distinct family of proteins that contain selenium in form of selenocysteine (Sec) within their active site. Incorporation of Sec requires the Sec-insertion sequence element (SECIS) in the 3'-untranslated region of selenoproteins mRNAs and the SECIS-binding protein 2 (SBP2). Previous studies have shown that SBP2 is required for the Sec-incorporation mechanism; however, additional roles of SBP2 in the cell have remained undefined. We herein show that depletion of SBP2 by using antisense oligonucleotides (ASOs) causes oxidative stress and induction of caspase- and cytochrome c-dependent apoptosis. Cells depleted of SBP2 have increased levels of ROS, which lead to cellular stress manifested as 8-oxo-7,8-dihydroguanine (8-oxo-dG) DNA lesions, stress granules, and lipid peroxidation. Small-molecule antioxidants N-acetylcysteine, glutathione, and α-tocopherol only marginally reduced ROS and were unable to rescue cells fully from apoptosis, indicating that apoptosis might be directly mediated by selenoproteins. Our results demonstrate that SBP2 is required for protection against ROS-induced cellular damage and cell survival. Antioxid. Redox Signal. 12, 797–808.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

Cell migration and proliferation on homogeneous and non-homogeneous domains : modelling on the scale of individuals and populations

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

Metabolomics coupled with proteomics advancing drug discovery toward more agile development of targeted combination therapies

To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations of plant species and/or minerals, called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics, and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of hepatic injury. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect, and activates both intrinsic and extrinsic pathways.

Induction of antioxidative Nrf2 gene transcription by coffee in humans : depending on genotype?

The Nrf2/ARE pathway is a major cellular defense mechanism that prevents damage by reactive oxygen species through induction of antioxidative phase II enzymes. However, the activity of the Nrf2/ARE system is not uniform with variability in response presumed to be dependent on the Nrf2 genotype. We recently completed a pilot human coffee intervention trial with healthy humans, where large interindividual differences in the antioxidative response to the study coffee were examined. Here, we address the question whether differences in the modulation of Nrf2 gene transcription, assessed as an induction of Nrf2 gene transcription by Q-PCR, might be correlated with specific Nrf2 genotypes. To date, nine single nucleotide polymorphisms (SNPs) have been identified in the Nrf2 (NFE2L2) gene. Two of these, the -617C/A and -651G/A SNPs are located within the promoter region and have previously been reported to influence the activity of the Nrf2/ARE pathway by reducing Nrf2 transcriptional activity. Sequencing of the critical Nrf2 gene promoter region not only confirmed the existence of these SNPs within the participants of the trial at the expected frequency (33% carrying the -617C/A, 17% the -651G/A and 56% the -653A/G SNP) but also indicated reduced Nrf2 gene transcription associated with a normal diet if the SNPs at position -617, -651 or -653 were present. Of note, the data also indicated the study coffee increased Nrf2 gene transcription even in SNP carriers. This further highlights the relevance of genotype-dependent induction of Nrf2 gene transcription that appears to be largely influenced by dietary factors.

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

The biology of the glutamatergic system and potential role in migraine

Migraine is a common genetically linked neurovascular disorder. Approximately ~12% of the Caucasian population are affected including 18% of adult women and 6% of adult men (1, 2). A notable female bias is observed in migraine prevalence studies with females affected ~3 times more than males and is credited to differences in hormone levels arising from reproductive achievements. Migraine is extremely debilitating with wide-ranging socioeconomic impact significantly affecting people's health and quality of life. A number of neurotransmitter systems have been implicated in migraine, the most studied include the serotonergic and dopaminergic systems. Extensive genetic research has been carried out to identify genetic variants that may alter the activity of a number of genes involved in synthesis and transport of neurotransmitters of these systems. The biology of the Glutamatergic system in migraine is the least studied however there is mounting evidence that its constituents could contribute to migraine. The discovery of antagonists that selectively block glutamate receptors has enabled studies on the physiologic role of glutamate, on one hand, and opened new perspectives pertaining to the potential therapeutic applications of glutamate receptor antagonists in diverse neurologic diseases. In this brief review, we discuss the biology of the Glutamatergic system in migraine outlining recent findings that support a role for altered Glutamatergic neurotransmission from biochemical and genetic studies in the manifestation of migraine and the implications of this on migraine treatment.

A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility

The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5′ and 3′ regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5′ region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5′ repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5′ and 3′ genetic variants were investigated.

Analysis of Sdhd and Mmp12 in an affected solar keratosis and control cohort

The incidence of Squamous Cell Carcinoma (SCG) is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 40% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis (SK) lesion and with a small, but significant, chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. More specifically the first aim of this project was to analyse the SDHD and MMP12 genes via Dual-Labelled Probe Real-Time PCR for copy number aberrations in an affected Solar Keratosis and control cohort. It was found that 12 samples had identifiable copy-number aberrations in either the SDHD or MMP12 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The significance of this study is the contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. © 2008 Springer Science+Business Media, LLC.

The influence of cellular source on periodontal regeneration using calcium phosphate coated polycaprolactone scaffold supported cell sheets

Cell-based therapy is considered a promising approach to achieving predictable periodontal regeneration. In this study, the regenerative potential of cell sheets derived from different parts of the periodontium (gingival connective tissue, alveolar bone and periodontal ligament) were investigated in an athymic rat periodontal defect model. Periodontal ligament (PDLC), alveolar bone (ABC) and gingival margin-derived cells (GMC) were obtained from human donors. The osteogenic potential of the primary cultures was demonstrated in vitro. Cell sheets supported by a calcium phosphate coated melt electrospun polycaprolactone (CaP-PCL) scaffold were transplanted to denuded root surfaces in surgically created periodontal defects, and allowed to heal for 1 and 4 weeks. The CaP-PCL scaffold alone was able to promote alveolar bone formation within the defect after 4 weeks. The addition of ABC and PDLC sheets resulted in significant periodontal attachment formation. The GMC sheets did not promote periodontal regeneration on the root surface and inhibited bone formation within the CaP-PCL scaffold. In conclusion, the combination of either PDLC or ABC sheets with a CaP-PCL scaffold could promote periodontal regeneration, but ABC sheets were not as effective as PDLC sheets in promoting new attachment formation.

Bridging the biology of the ovine TRALI model – human inflammatory cell responses to TRALI inducing supernatants

Background Transfusion-related acute lung injury (TRALI) is a serious and potentially fatal consequence of transfusion. A two-event TRALI model demonstrated date-of-expiry - day (D) 5 platelet (PLT) and D42 packed red blood cell (PRBC) supernatants (SN) induced TRALI in LPS-treated sheep. We have adapted a whole blood transfusion culture model as an investigative bridge between the ovine TRALI model human responses to transfusion. Methods A whole blood transfusion model was adapted to replicate the ovine model - specifically +/- 0.23μg/mL LPS as the first event and 10% SN volume (transfusion) as the second event. Four pooled SN from blood products, previously used in the TRALI ovine model, were investigated: D1-PLT, D5-PLT, D1-PRBC, and D42-PRBC. Fresh human whole blood (recipient) was mixed with combinations of LPS and BP-SN stimuli and incubated in vitro for 6 hrs. Addition of golgi plug enabled measurement of monocyte cytokine production (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1α, CXCL-5, IP-10, MIP-1α, MCP-1) using multi-colour flow cytometry. Responses for 6 recipients were assessed. Results In the presence of LPS, D42-PRBC-SN significantly increased monocyte IL-6 (P=0.031), IL-8 (P=0.016) and IL-1α (P=0.008) production compared to D1-PRBC-SN. This response to D42-PRBC-SN was LPS-dependent, and was not evident in non-LPSstimulated controls. This response was also specific to D42-PRBC-SN, as similar changes were not evident for the D5-PLT-SN, compared to the D1-PLT-SN, regardless of the presence of LPS. D5-PLT-SN significantly increased IL-12 production (P=0.024) compared to D1-PLT-SN. This response was again LPS-dependent. Conclusions These data demonstrate a novel two-event mechanism of monocyte inflammatory response that was dependent upon both the presence of date-of-expiry blood product SN and LPS. Further, these results demonstrate different cytokines responses induced by date-of-expiry PLT-SN and PRBC-SN. These data are consistent with the evidence from the ovine TRALI model, and enhancing its relevance to transfusion related changes in humans.