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.

Playing with Grammar in the Early Years : Learning about Language in the Australian Curriculum : English

As teacher/researchers interested in the pursuit of socially-just outcomes in early childhood education, the form and function of language occupies a special position in our work. We believe that mastering a range of literacy competences includes not only the technical skills for learning, but also the resources for viewing and constructing the world (Freire and Macdeo, 1987). Rather than seeing knowledge about language as the accumulation of technical skills alone, the viewpoint to which we subscribe treats knowledge about language as a dialectic that evolves from, is situated in, and contributes to a social arena (Halliday, 1978). We do not shy away from this position just because children are in the early years of schooling. In ‘Playing with Grammar’, we focus on the Foundation to Year 2 grouping, in line with the Australian Curriculum, Assessment and Reporting Authority’s (hereafter ACARA) advice on the ‘nature of learners’ (ACARA, 2013). With our focus on the early years of schooling comes our acknowledgement of the importance and complexity of play. At a time where accountability in education has moved many teachers to a sense of urgency to prove language and literacy achievement (Genishi and Dyson, 2009), we encourage space to revisit what we know about literature choices and learning experiences and bring these together to facilitate language learning. We can neither ignore, nor overemphasise, the importance of play for the development of language through: the opportunities presented for creative use and practice; social interactions for real purposes; and, identifying and solving problems in the lives of young children (Marsh and Hallet, 2008). We argue that by engaging young children in opportunities to play with language we are ultimately empowering them to be active in their language learning and in the process fostering a love of language and the intricacies it holds. Our goal in this publication is to provide a range of highly practical strategies for scaffolding young children through some of the Content Descriptions from the Australian Curriculum English Version 5.0, hereafter AC:E V5.0 (ACARA, 2013). This recently released curriculum offers a new theoretical approach to building children’s knowledge about language. The AC:E V5.0 uses selected traditional terms through an approach developed in systemic functional linguistics (see Halliday and Matthiessen, 2004) to highlight the dynamic forms and functions of multimodal language in texts. For example, the following statement, taken from the ‘Language: Knowing about the English language’ strand states: English uses standard grammatical terminology within a contextual framework, in which language choices are seen to vary according to the topics at hand, the nature and proximity of the relationships between the language users, and the modalities or channels of communication available (ACARA, 2013). Put simply, traditional grammar terms are used within a functional framework made up of field, tenor, and mode. An understanding of genre is noted with the reference to a ‘contextual framework’. The ‘topics at hand’ concern the field or subject matter of the text. The ‘relationships between the language users’ is a description of tenor. There is reference to ‘modalities’, such as spoken, written or visual text. We posit that this innovative approach is necessary for working with contemporary multimodal and cross-cultural texts (see Exley and Mills, 2012). We believe there is enormous power in using literature to expose children to the richness of language and in turn develop language and literacy skills. Taking time to look at language patterns within actual literature is a pathway to ‘…capture interest, stir the imagination and absorb the [child]’ into the world of language and literacy (Saxby, 1993, p. 55). In the following three sections, we have tried to remain faithful to our interpretation of the AC:E V5.0 Content Descriptions without giving an exhaustive explanation of the grammatical terms. Other excellent tomes, such as Derewianka (2011), Humphrey, Droga and Feez (2012), and Rossbridge and Rushton (2011) provide these more comprehensive explanations as does the AC:E V5.0 Glossary. We’ve reproduced some of the AC:E V5.0 glossary at the end of this publication. Our focus is on the structure and unfolding of the learning experiences. We outline strategies for working with children in Foundation, Year 1 and Year 2 by providing some demonstration learning experiences based on texts we’ve selected, but maintain that the affordances of these strategies will only be realised when teaching and learning is purposively tied to authentic projects in local contexts. We strongly encourage you not to use only the resource texts we’ve selected, but to capitalise upon your skill for identifying the language features in the texts you and the children are studying and adapt some of the strategies we have outlined. Each learning experience is connected to one of the Content Descriptions from the AC:E V5.0 and contains an experience specific purpose, a suggested resource text and a sequence for the experience that always commences with an orientation to text followed by an examination of a particular grammatical resource. We expect that each of these learning experiences will take a couple if not a few teaching episodes to work through, especially if children are meeting a concept for the first time. We hope you use as much, or as little, of each experience as is needed. Our plans allow for focused discussion, shared exploration and opportunities to revisit the same text for the purpose of enhancing meaning making. We do not want the teaching of grammar to slip into a crisis of irrelevance or to be seen as a series of worksheet drills with finite answers. Strategies for effective practice, however, have much portability. We are both very keen to hear from teachers who are adopting and adapting these learning experiences in their classrooms. Please email us on b.exley@qut.edu.au or lkervin@uow.edu.au. We’d love to continue the conversation with you over time.

A point prevalence study of cancer nursing practices for managing intravascular devices in an Australian tertiary cancer center

Purpose The use of intravascular devices is associated with a number of potential complications. Despite a number of evidence-based clinical guidelines in this area, there continues to be nursing practice discrepancies. This study aims to examine nursing practice in a cancer care setting to identify nursing practice and areas for improvement respective to best available evidence. Methods A point prevalence survey was undertaken in a tertiary cancer care centre in Queensland, Australia. On a randomly selected day, four nurses assessed intravascular device related nursing practices and collected data using a standardized survey tool. Results 58 inpatients (100%) were assessed. Forty-eight (83%) had a device in situ, comprising 14 Peripheral Intravenous Catheters (29.2%), 14 Peripherally Inserted Central Catheters (29.2%), 14 Hickman catheters (29.2%) and six Port-a-Caths (12.4%). Suboptimal outcomes such as incidences of local site complications, incorrect/inadequate documentation, lack of flushing orders, and unclean/non intact dressings were observed. Conclusions This study has highlighted a number of intravascular device related nursing practice discrepancies compared with current hospital policy. Education and other implementation strategies can be applied to improve nursing practice. Following education strategies, it will be valuable to repeat this survey on a regular basis to provide feedback to nursing staff and implement strategies to improve practice. More research is required to provide evidence to clinical practice with regards to intravascular device related consumables, flushing technique and protocols.

Author response: "NPO practices and preoperative oral carbohydrate intake"

"We thank Dr Marsh for his thoughtful comments and acknowledge the limitations of our study, which we clearly outlined in the article. We also thank Dr Marsh for supporting our call for larger, independent trials to test the effectiveness of preoperative consumption of highcarbohydrate fluids to improve patient outcomes..."

The protease inhibitor JO146 demonstrates a critical role for CtHtrA for Chlamydia trachomatis reversion from penicillin persistence

The Chlamydia trachomatis serine protease HtrA (CtHtrA) has recently been demonstrated to be essential during the replicative phase of the chlamydial developmental cycle. A chemical inhibition strategy (serine protease inhibitor JO146) was used to demonstrate this essential role and it was found that the chlamydial inclusions diminish in size and are lost from the cell after CtHtrA inhibition without formation of viable elementary bodies. The inhibitor (JO146) was used in this study to investigate the role of CtHtrA for penicillin persistence and heat stress model conditionscultures for Chlamydia trachomatis. JO146 addition during penicillin persistence resulted in only minor reductions (~1 log) in the final viable infectious yield after persistent Chlamydia were reverted from persistence. However, JO146 treatment during the reversion and recovery from penicillin persistence was completely lethal for Chlamydia trachomatis. JO146 was completely lethal when added either during heat stress conditions, or during the recovery from heat stress conditions. These data together indicate that CtHtrA has essential roles during some stress environments (heat shock), recovery from stress environments (heat shock and penicillin persistence), as well as the previously characterised essential role during the replicative phase of the chlamydial developmental cycle. Thus, CtHtrA is an essential protease with both replicative phase and stress condition functions for Chlamydia trachomatis.

Increased generation of platelet-derived microparticles following percutaneous transluminal coronary angioplasty

Platelet-derived microparticles (PMPs) which are produced during platelet activation contribute to coagulation1 and bind to traumatized endothelium in an animal model2. Such endothelial injury occurs during percutaneous transluminal coronary angioplasty (PTCA), a procedure which restores the diameter of occluded coronary arteries using balloon inflations. However, re-occlusions subsequently develop in 20-25% of patients3, although this is limited by treatment with anti-platelet glycoprotein IIb/IIIa receptor drugs such as abciximab4. However, abciximab only partially decreases the need for revascularisation5, and therefore other mechanisms appear to be involved. As platelet activation occurs during PTCA, it is likely that PMPs may be produced and contribute to restenosis. This study population consisted of 113 PTCA patients, of whom 38 received abciximab. Paired peripheral arterial blood samples were obtained from the PTCA sheath: 1) following heparinisation (baseline); and 2) subsequent to all vessel manipulation (post-PTCA). Blood was prepared with an anti-CD61 (glycoprotein IIIa) fluorescence conjugated antibody to identify PMPs using flow cytometry, and PMP results expressed as a percentage of all CD61 events. The level of PMPs increased significantly from baseline following PTCA in the without abciximab group (paired t test, P=0.019). However, there was no significant change in the level of PMPs following PTCA in patients who received abciximab. Baseline clinical characteristics between patient groups were similar, although patients administered abciximab had more complex PTCA procedures, such as increased balloon inflation pressures (ANOVA, P=0.0219). In this study, we have clearly demonstrated that the level of CD61-positive PMPs increased during PTCA. This trend has been demonstrated previously, although a low sample size prevented statistical significance being attained6. The results of our work also demonstrate that there was no increase in PMPs after PTCA with abiciximab treatment. The increased PMPs may adhere to traumatized endothelium, contributing to re-occlusion of the arteries, but this remains to be determined. References: (1) Holme PA, Brosstad F, Solum NO. Blood Coagulation and Fibrinolysis. 1995;6:302-310. (2) Merten M, Pakala R, Thiagarajan P, Benedict CR. Circulation. 1999;99:2577-2582. (3) Califf RM. American Heart Journal.1995;130:680-684. (4) Coller BS, Scudder LE. Blood. 1985;66:1456-1459. (5) Topol EJ, Califf RM, Weisman HF, Ellis SG, Tcheng JE, Worley S, Ivanhoe R, George BS, Fintel D, Weston M, Sigmon K, Anderson KM, Lee KL, Willerson JT on behalf of the EPIC investigators. Lancet. 1994;343:881-886. (6) Scharf RE, Tomer A, Marzec UM, Teirstein PS, Ruggeri ZM, Harker LA. Arteriosclerosis and Thrombosis. 1992;12:1475-87.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population

Background Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. Results Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals. A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia. The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. Conclusions The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Increased platelet-derived microparticles in the coronary circulation of percutaneous transluminal coronary angioplasty patients

Platelet-derived microparticles that are produced during platelet activation are capable of adhesion and aggregation. Endothelial trauma that occurs during percutaneous transluminal coronary angioplasty (PTCA) may support platelet-derived microparticle adhesion and contribute to development of restenosis. We have previously reported an increase in platelet-derived microparticles in peripheral arterial blood with angioplasty. This finding raised concerns regarding the role of plateletderived microparticles in restenosis, and therefore the aim of this study was to monitor levels in the coronary circulation. The study population consisted of 19 angioplasty patients. Paired coronary artery and sinus samples were obtained following heparinization, following contrast administration, and subsequent to all vessel manipulation. Platelet-derived microparticles were identified with an anti-CD61 (glycoprotein IIIa) fluorescence-conjugated antibody using flow cytometry. There was a significant decrease in arterial platelet-derived microparticles from heparinization to contrast administration (P 0.001), followed by a significant increase to the end of angioplasty (P 0.004). However, there was no significant change throughout the venous samples. These results indicate that the higher level of platelet-derived microparticles after angioplasty in arterial blood remained in the coronary circulation. Interestingly, levels of thrombin–antithrombin complexes did not rise during PTCA. This may have implications for the development of coronary restenosis post-PTCA, although this remains to be determined.

Increased generation of platelet-derived microparticles following percutaneous transluminal coronary angioplasty

Platelet-derived microparticles that are produced during platelet activation bind to traumatized endothelium. Such endothelial injury occurs during percutaneous transluminal coronary angioplasty. Approximately 20% of these patients subsequently develop restenosis, although this is improved by treatment with the anti-platelet glycoprotein IIb/IIIa receptor drug abciximab. As platelet activation occurs during angioplasty, it is likely that platelet-derived microparticles may be produced and hence contribute to restenosis. This study population consisted of 113 angioplasty patients, of whom 38 received abciximab. Paired peripheral arterial blood samples were obtained following heparinization and subsequent to all vessel manipulation. Platelet-derived microparticles were identified using an anti-CD61 (glycoprotein IIIa) fluorescence-conjugated antibody and flow cytometry. Baseline clinical characteristics between patient groups were similar. The level of platelet-derived microparticles increased significantly following angioplasty in the group without abciximab (paired t test, P 0.019). However, there was no significant change in the level of platelet-derived microparticles following angioplasty in patients who received abciximab, despite requiring more complex angioplasty procedures. In this study, we have demonstrated that the level of platelet-derived microparticles increased during percutaneous transluminal coronary angioplasty, with no such increase with abciximab treatment. The increased platelet-derived microparticles may adhere to traumatized endothelium, contributing to re-occlusion of the arteries, but this remains to be determined.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between mitochondrial biogenesis, cell signalling and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats treated with DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise, and; (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P<0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P<0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P<0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) mRNA compared to the control animals that were exercised (P<0.05). This study provides novel evidence that by reducing the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Toward making inroads in reducing the disparity of lung health in Australian Indigenous and New Zealand Maori children

Globally, Indigenous populations, which include Aboriginal and Torres Strait islanders in Australia and Māori people in New Zealand (NZ), have poorer health than their non-Indigenous counterparts. Indigenous peoples worldwide face substantial challenges in poverty, education, employment, housing and disconnection from ancestral lands. While addressing social determinants of health is a priority, solving clinical issues is equally important. Indeed, ignoring the latter until social issues improve risks further disparity as this may take generations. A systematic overview of interventions addressing social determinants of health found a striking lack of reliable evaluations.Where evidence was available, health improvement associated with interventions was modest or uncertain. 10 Thus advances in healthcare remain essential and these require the best evidence available in 11 preventing and managing common illnesses, including respiratory illnesses.

Managing moose harvests by the seat of your pants

Moose populations are managed for sustainable yield balanced against costs caused by damage to forestry or agriculture and collisions with vehicles. Optimal harvests can be calculated based on a structured population model driven by data on abundance and the composition of bulls, cows, and calves obtained by aerial-survey monitoring during winter. Quotas are established by the respective government agency and licenses are issued to hunters to harvest an animal of specified age or sex during the following autumn. Because the cost of aerial monitoring is high, we use a Management Strategy Evaluation to evaluate the costs and benefits of periodic aerial surveys in the context of moose management. Our on-the-fly "seat of your pants" alternative to independent monitoring is management based solely on the kill of moose by hunters, which is usually sufficient to alert the manager to declines in moose abundance that warrant adjustments to harvest strategies. Harvests are relatively cheap to monitor; therefore, data can be obtained each year facilitating annual adjustments to quotas. Other sources of "cheap" monitoring data such as records of the number of moose seen by hunters while hunting also might be obtained, and may provide further useful insight into population abundance, structure and health. Because conservation dollars are usually limited, the high cost of aerial surveys is difficult to justify when alternative methods exist. © 2012 Elsevier Inc.

Critical literacy

In this chapter, we provide an overview of the concept of critical literacy and outline how it can inform classroom practice. This is no simple task, because there is no singular, normative version of critical literacy. Rather, it is a concept that has been shaped by various theoretical paradigms over the years and formulated in practice in ways that are deeply contextualized and specifically situated. This means that providing a historical overview of its development is well nigh impossible. Nevertheless, we will, in this introductory session, trace critical literacy's origins in critical theory and pedagogy, and offer a brief review of other theoretical influences on its development