Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on in vitro growth. To address the in vivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbΔMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbΔMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Advances in molecular genetic systems in malaria

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

Development of a professional practice audit questionnaire for mental health nursing in Aotearoa/New Zealand

This paper reports the three-stage development of a professional practice audit questionnaire for mental health nursing in Aotearoa/New Zealand. In Study 1, clinical indicator statements (n = 99) generated from focus group data, which were considered to be unobservable in the nursing documentation in consumer case notes, were included in a three-round Delphi process. Consensus of ratings occurred for the mental health nurse and academic participants (n = 7) on 83 clinical indicator statements. In Study 2, the clinical indicator statements (n = 67) that met importance and consensus criteria were incorporated into a questionnaire, which was piloted at a New Zealand mental health service. The questionnaire was then modified for use in a national field study. In Study 3, the national field study, registered mental health nurses (n = 422) from 11 New Zealand District Health Board mental health services completed the questionnaire. Five categories of nursing practice were identified: professional and evidence-based practice; consumer focus and reflective practice; professional development and integration; ethically and legally safe practice; and culturally safe practice. Analyses revealed little difference in the perceptions of nurses from different backgrounds regarding the regularity of the nursing practices. Further research is needed to calibrate the scores on each clinical indicator statement with behaviour in clinical practice.

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression
would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria
parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery
exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression
and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic
analysis if it is to be of use as a molecular genetic tool for malaria parasites.

PTEX is an essential nexus for protein export in malaria parasites

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

The PTEX component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium Translocon of Exported proteins (PTEX) and a cleavable Plasmodium Export Element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic P. berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection. This article is protected by copyright. All rights reserved.

Stage of perinatal development regulates skeletal muscle mitochondrial biogenesis and myogenic regulatory factor genes with little impact of growth restriction or cross-fostering

Foetal growth restriction impairs skeletal muscle development and adult muscle mitochondrial biogenesis. We hypothesized that key genes involved in muscle development and mitochondrial biogenesis would be altered following uteroplacental insufficiency in rat pups, and improving postnatal nutrition by cross-fostering would ameliorate these deficits. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed on day 18 of gestation. Males and females were investigated at day 20 of gestation (E20), 1 (PN1), 7 (PN7) and 35 (PN35) days postnatally. A separate cohort of Control and Restricted pups were cross-fostered onto a different Control or Restricted mother and examined at PN7. In both sexes, peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), cytochrome c oxidase subunits 3 and 4 (COX III and IV) and myogenic regulatory factor 4 expression increased from late gestation to postnatal life, whereas mitochondrial transcription factor A, myogenic differentiation 1 (MyoD), myogenin and insulin-like growth factor I (IGF-I) decreased. Foetal growth restriction increased MyoD mRNA in females at PN7, whereas in males IGF-I mRNA was higher at E20 and PN1. Cross-fostering Restricted pups onto a Control mother significantly increased COX III mRNA in males and COX IV mRNA in both sexes above controls with little effect on other genes. Developmental age appears to be a major factor regulating skeletal muscle mitochondrial and developmental genes, with growth restriction and cross-fostering having only subtle effects. It therefore appears that reductions in adult mitochondrial biogenesis markers likely develop after weaning.

Changes in the nutritional composition of captive early-mid stage Panulirus ornatus phyllosoma over ecdysis and larval development

Elucidation of the key nutritional requirements for complete larval development of the tropical spiny rock lobster, Panulirus ornatus, presents a major challenge for the development of robust commercial aquaculture for this crustacean. As a foundation study in this area, the chemical composition of early-mid stage P. ornatus phyllosoma (Stages I-VI) receiving a novel formulated diet was analysed immediately prior and post-ecdysis to provide insight into the crude nutritional trends during the larval development cycle. From the onset of moulting, cyclical patterns were evident in the proximate composition of phyllosoma, resulting in substantial restructuring between the pre- and post-moult stages of the moult cycle. Proportions of protein, lipid and ash were high at the premoult stage, reflecting growth and nutrient accumulation over the intermoult period, and reduced at the post-moult stage, reflecting the large uptake of water to facilitate subsequent growth. Polar lipid was the dominant lipid class, accounting for >. 90% of the total lipid content. Conversely, triacylglycerol concentrations were low (<. 5%), despite being the principal lipid class available in the formulated diet. Likewise, despite receiving high concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (9.2 and 7.6% of the dietary lipid source, respectively), levels of these fatty acids were comparatively low in phyllosoma (3.4 and 4.7%, respectively). In contrast, there is selective deposition of these fatty acids in wild caught phyllosoma. This finding suggests a poor assimilation of triacylglycerols by captive larvae and highlights the importance of future investigations into alternative sources of EPA and DHA. Ultimately, this study provides insight into the nutritional requirements of phyllosoma, providing valuable knowledge on diet formulation for commercially viable hatchery production of spiny rock lobsters. © 2014 Elsevier B.V.

Tensions and dilemmas in community development: new discourses, new Trojans?

This paper begins by noting the range of contradictions and dilemmas facing those involved in community development today. It then draws on research into the operating frameworks that set the stage for much current community development activity. It discusses four key operating frameworks and how each framework can affect community development practice. The final section deals with the ways in which the frameworks, and the discourses associated with them, come together.

Repositioning drama to centre stage: drama, English, text and literacy

A version of this article was first presented at the Drama Australia Conference, Fremantle, July 2002. It draws upon Freebody and Luke's four resources literacy framework, where they describe four kinds of literacy  practices. It shows how this model is used within the literacy community and argues that this model is useful to describe the contribution that drama can make to literacy development. Freebody and Luke's model is used and  promoted throughout Australia and the author argues that it is politically astute for drama teachers to reclaim and promote their links to the English/Literacy curriculum.

Removal of directors of public companies takes centre stage in Australia : an exploration of the corporate law and governance issues

Increasing attention is being given to the legal and governance issues relating to the removal of directors in Australian public companies. This has been due mainly to the difficulties experienced by the board of National Australia Bank in attempting to remove one of its fellow directors, and the subsequent development of public companies entering into so-called 'prenuptial agreements' with new directors, requiring that the director 'resign' if the board pass a vote of no-confidence in the director. In this article, the author revisits the area of director removal in Australian public companies for two reasons. The first reason, which covers the majority of the article, is to engage in a detailed analysis of whether the pre-nuptial agreements which some public companies have indicated that they support using to remove directors, are in fact enforceable under Australia's Corporations Act The second reason is to outline a law reform proposal to enable public companies to remove directors without requiring the vote of shareholders at a general meeting. The proposal involves providing Australia' corporate  regulator, the Australian Securities and Investments Commission (ASIC) with the power to grant relief from the statutory removal provisions to public companies, but in a way which balances the competing objectives of commercial efficiency and shareholder participation and, very importantly, encourages good corporate governance practices by companies in relation to the performance assessment  of directors.

It is in the interests of both shareholders and directors to agree on a set of ground rules for the effective supervision of companies that reconciles the rights of the owners to overall control with the much tougher demands on modern directors

Case studies in the development towards a daylighting roadmap

Through the International Energy Agency (lEA) Task-31 Daylighting in the 21 st Century a subtask has been organized to undertake the development of a Daylighting Design Roadmap. This project will attempt to unfold a path as to how we may approach and provide support for daylighting at the various project stages of a design. Although this roadmap is in its preliminary stages, the authors have examined several case studies of daylighting projects. This paper outlines an initial overview of a daylighting design roadmap by reflecting on several aspects of the case studies. An attempt is made to provide reasonable guidance of parameters which could be analyzed at various stages in a project. The roadmap gives reference to several design tools and simulation programs acknowledging that the process can be selective in targeting results to particular circumstances. Voids of information and methodology in the construction of the daylighting roadmap, at this stage in the project, are also acknowledged. It is expected that this initial paper on the subject, will provide awareness and hopefully generate a contribution from other experts in the field.