Development of West Nile virus candidate vaccines in Nicotiana benthamiana

West Nile virus (WNV) is a widely disseminated flavivirus, with a geographical range that now includes Africa, America, Europe, the Middle East, West Asia and Australia. The virus is vectored by Culex mosquitoes and is maintained in a bird-mosquito transmission cycle with hundreds of bird species acting as reservoir hosts. In humans, infections can develop into febrile illness and severe meningoencephalitis and to date, there is no treatment or vaccine available. In horses, approximately 20% of infections are symptomatic, of which 90% of cases involve neurological disease, with 30-40% fatality rates. Several veterinary vaccines specific to the lineage 1 WNV strains are commercially produced in America and Europe, however, these vaccines are not easily obtainable for low and middle-income countries (LMIC) due to their high cost and that associated with importation as well as the need for annual vaccination. Due to continuous global disease outbreaks in birds, humans and horse populations with no preventative measures for humans, WNV poses a major public health threat, especially in naïve populations. The development of a vaccine that contributes to the ‘One Health' Initiative could be the answer to prevent the spread of the virus and control the disease. Current veterinary vaccines are produced in expensive cell culture systems that require sterile conditions, high-level biosafety facilities and trained personnel for their preparation. Transient plant-based expression systems have proven to be a very cost-effective means of making complex proteins. Plants can produce and modify proteins in a similar manner to mammalian cells and production does not require sterile conditions or specialised facilities. We propose that plants could be a viable means of making feasible, low-cost reagents for WNV, specifically virus-like particles (VLPs) for use as vaccines in South Africa and other LMIC. In this study, we set out to develop two particulate candidate vaccines based on a virulent South African WNV strain using Nicotiana benthamiana as the expression platform. We aimed to develop the first candidate vaccine by exploiting the virus's ability to form noninfectious VLPs by expressing only the WNV membrane (prM – precursor, M – matured) and envelope (E) proteins. Infiltration of these recombinant plasmids into plants yielded no protein expression unless co-expressed with the human chaperone protein calnexin (CNX), upon which expression of both M and E proteins were observed. We investigated the assembly of prM and E into VLPs by transmission electron microscopy (TEM), however, purification of these particles proved difficult with poor reproducibility and VLP yield. This led to the development of an alternative candidate vaccine making use of the antigendisplay technology based on the SpyTag (ST) and SpyCatcher (SC) peptides. The immunodominant epitope of the WNV E protein, domain III (EdIII), was selected for antigen display. Two constructs of the EdIII gene were generated, one with the SC peptide on the 5'- (SC-EdIII) and the other on the 3' end (EdIII-SC). Both SC-EdIII and EdIII-SC proteins were successfully expressed in the presence of the human chaperone protein calreticulin, and purified with yields of 9 mg/kg and 69 mg/kg fresh leaf weight (FLW), respectively. The VLP core selected for the display of the SC-linked EdIII proteins comprised the coat protein of the bacteriophage AP205 with the ST peptide linked to its N-terminus (ST-AP205). Spytagged-VLPs were purified by density gradient ultracentrifugation at a yield of approximately 50 mg/kg FLW. The purified SC-linked EdIII proteins and ST-AP205 VLPs were coupled in vitro, but successful complex formation of AP205:EdIII was only observed between ST-AP205 and EdIII-SC and not when the SC peptide was located on the N-terminus of EdIII. We further demonstrated the successful complex formation of AP205:EdIII in vivo by coinfiltration of the EdIII-SC and ST-AP205 constructs, as well as by extracting leaves of plants infiltrated individually with either of the constructs. Due to the ease of purification and the high yields of AP205:EdIII achieved, the co-extraction process was optimised to obtain the best coupling yield possible by evaluating different FLW extraction ratios and the formation of VLPs was confirmed by TEM. The optimal co-extraction process was established at a FLW ratio of 1:2 ST-AP205 to EdIII-SC yielding approximately 23 mg/kg AP205:EdIII/FLW processed. In this study, we describe the successful production of two particulate candidate vaccines. The first is based on the expression of the WNV prM and E genes in the presence of human CNX and the second is based on the ST/SC antigen-display technology. These outcomes exhibit the potential plants have of being used as biofactories for making significant pharmaceutical products for the ‘One Health' Initiative and could be used to address the need for their local production in LMIC

Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression

INTRODUCTION: In order to gain a better understanding of the molecular processes that underlie apoptosis and tissue regression in mammary gland, we undertook a large-scale analysis of transcriptional changes during the mouse mammary pregnancy cycle, with emphasis on the transition from lactation to involution. METHOD: Affymetrix microarrays, representing 8618 genes, were used to compare mammary tissue from 12 time points (one virgin, three gestation, three lactation and five involution stages). Six animals were used for each time point. Common patterns of gene expression across all time points were identified and related to biological function. RESULTS: The majority of significantly induced genes in involution were also differentially regulated at earlier stages in the pregnancy cycle. This included a marked increase in inflammatory mediators during involution and at parturition, which correlated with leukaemia inhibitory factor–Stat3 (signal transducer and activator of signalling-3) signalling. Before involution, expected increases in cell proliferation, biosynthesis and metabolism-related genes were observed. During involution, the first 24 hours after weaning was characterized by a transient increase in expression of components of the death receptor pathways of apoptosis, inflammatory cytokines and acute phase response genes. After 24 hours, regulators of intrinsic apoptosis were induced in conjunction with markers of phagocyte activity, matrix proteases, suppressors of neutrophils and soluble components of specific and innate immunity. CONCLUSION: We provide a resource of mouse mammary gene expression data for download or online analysis. Here we highlight the sequential induction of distinct apoptosis pathways in involution and the stimulation of immunomodulatory signals, which probably suppress the potentially damaging effects of a cellular inflammatory response while maintaining an appropriate antimicrobial and phagocytic environment

Understanding the ups and downs of living well: the voices of people experiencing early mental health recovery

Background: The aim of this study was to better understand early-stage mental health recovery experiences of people living with severe and persistent mental illness and complex needs.
 Methods: Semi-structured, in-depth interviews were conducted with 13 people engaged in an Australian program specifically designed for people facing complex barriers to their recovery. Interview data were analysed thematically using constant comparative methods.
 Results: Participants described engaging with seven interconnecting aspects of early recovery: (1) engaging with the challenge of recovery; (2) struggling for a secure and stable footing; (3) grieving for what was and what could have been; (4) seeking and finding hope; (5) navigating complex relationships; (6) connecting with formal and informal support, and finally, (7) juggling a complexity of health issues.
 Conclusions: This study illuminated the complexity of earlier-stage recovery which was characterised both by challenging personal circumstances and a hope for the future. It illustrated that even at an early point in their recovery journey, and amidst these challenging circumstances, people still actively engage with support, draw on inner strengths, source resources and find accomplishments. Stability and security was foundational to the ability of participants to draw on their own strengths and move forward. Stability came when material needs, including housing, were addressed, and an individual was able to connect with a supportive network of workers, carers, friends and family

Cardiomyocyte Deletion of \u3ci\u3eBmal1\u3c/i\u3e Exacerbates QT- and RR-Interval Prolongation in \u3ci\u3eScn5a\u3c/i\u3e\u3csup\u3e+/ΔKPQ\u3c/sup\u3e Mice

Circadian rhythms are generated by cell autonomous circadian clocks that perform a ubiquitous cellular time-keeping function and cell type-specific functions important for normal physiology. Studies show inducing the deletion of the core circadian clock transcription factor Bmal1 in adult mouse cardiomyocytes disrupts cardiac circadian clock function, cardiac ion channel expression, slows heart rate, and prolongs the QT-interval at slow heart rates. This study determined how inducing the deletion of Bmal1 in adult cardiomyocytes impacted the in vivo electrophysiological phenotype of a knock-in mouse model for the arrhythmogenic long QT syndrome (Scn5a+/ΔKPQ). Electrocardiographic telemetry showed inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation increased the QT-interval at RR-intervals that were ≥130 ms. Inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation also increased the day/night rhythm-adjusted mean in the RR-interval, but it did not change the period, phase or amplitude. Compared to mice without the ΔKPQ-Scn5a mutation, mice with the ΔKPQ-Scn5a mutation had reduced heart rate variability (HRV) during the peak of the day/night rhythm in the RR-interval. Inducing the deletion of Bmal1 in cardiomyocytes did not affect HRV in mice without the ΔKPQ-Scn5a mutation, but it did increase HRV in mice with the ΔKPQ-Scn5a mutation. The data demonstrate that deleting Bmal1 in cardiomyocytes exacerbates QT- and RR-interval prolongation in mice with the ΔKPQ-Scn5a mutation

Sustained vs episodic mobilization among conflict-generated diasporas

There is increased interest in the connectivity of migrants with both their host-lands and their original homelands. This article brings a social movement perspective to bear on the issue of diaspora mobilization. Why do conflict-generated diasporas from the same original homeland and living in the same host-land mobilize in sustained versus episodic ways? This article focuses on the sustained mobilization of Bosnian Muslims versus the episodic mobilization of Croats and Serbs in the Netherlands in the early 2010s. I argue that a traumatic issue that binds three actors – diaspora, host-state, and home-state – is central to such mobilization. This issue is the failure of Dutch peace-keeping forces to protect the Srebrenica enclave in 1995. Migration integration regimes, threats from radical right parties, host-state foreign policy, and transnational influences can trigger episodic diaspora mobilization, but not sustain it

Development and optimization of a diagnostic system based on Illumina sequencing of genus wide PCR amplicons for the detection of viruses of grapevines

In this study we present a poly-specific PCR-based, high-throughput sequencing (HTS) diagnostic system together with an appropriate data analysis pipeline for the diagnosis of grapevine viruses. Poly-specific and virus-specific primers were established to be capable of detecting and identifying 37 grapevine infecting viruses from 11 genera. An analysis pipeline using CLC Genomics workbench was developed by utilising various defined artificial samples which were assembled and sequenced on the Illumina MiSeq platform. A threshold for percentage mapped reads of 0.4% during reference mapping was established to discriminate between presence or absence of viruses associated with reads. Various criteria for the evaluation of de novo assembled contigs and BLAST results were identified based on virus hits, E-value, percentage query overlap and percentage amplicon overlap. Various RT-PCR systems were used to screen 62 grapevine samples (field collected and candidate nuclear vines) for their virus populations. Seven samples were selected for Illumina MiSeq sequencing, and the data was analysed as per the optimized pipeline. The threshold established for reference mapping and the criteria for BLAST analysis was successfully implemented, proving the applicability of this PCR and HTS-based system in grapevine diagnostics. This system was compared to the standard ELISA system routinely utilised during certification. In our study, when samples evaluated by RT-PCR were tested using ELISA for the presence of GLRaV-1, -2 and -3, a false-negative rate in ELISA of 14.3% was observed, confirming that RT-PCR is the more sensitive test of the two. The capability of RTPCR to readily detect viruses present in low concentrations in woody plants, the availability of primers for virus identification, the ease and rapidity of the technique, together with constant improvement of HTS platforms especially in the area of cost makes this an extremely useful method for grapevine virus diagnostics.Dissertation (MSc)--University of Pretoria, 2017.WinetechMicrobiology and Plant PathologyMScUnrestricte

Understanding the ups and downs of living well: the voices of people experiencing early mental health recovery

Abstract Background The aim of this study was to better understand early-stage mental health recovery experiences of people living with severe and persistent mental illness and complex needs. Methods Semi-structured, in-depth interviews were conducted with 13 people engaged in an Australian program specifically designed for people facing complex barriers to their recovery. Interview data were analysed thematically using constant comparative methods. Results Participants described engaging with seven interconnecting aspects of early recovery: (1) engaging with the challenge of recovery; (2) struggling for a secure and stable footing; (3) grieving for what was and what could have been; (4) seeking and finding hope; (5) navigating complex relationships; (6) connecting with formal and informal support, and finally, (7) juggling a complexity of health issues. Conclusions This study illuminated the complexity of earlier-stage recovery which was characterised both by challenging personal circumstances and a hope for the future. It illustrated that even at an early point in their recovery journey, and amidst these challenging circumstances, people still actively engage with support, draw on inner strengths, source resources and find accomplishments. Stability and security was foundational to the ability of participants to draw on their own strengths and move forward. Stability came when material needs, including housing, were addressed, and an individual was able to connect with a supportive network of workers, carers, friends and family

Disrupting the Circadian Clock Mechanism in Cardiomyocytes Exacerbates the LQT3-related phenotype in Scn5a(ΔKPQ/+) Mice

Introduction: The pro-arrhythmic LQTS type 3 (LQT3) is caused by gain-of-function mutations in the cardiac Na+ channel Scn5a. LQT3 patients typically have an abnormally long heart rate-corrected QT-interval (QTc), but even patients with the same disease-causing mutation show a wide range of clinical phenotypes. This suggests additional factors influence the LQT3-related phenotype. Hypothesis: Many LQT3 patients show an increased incidence in life-threatening arrhythmias at night. We tested the hypothesis that disruption of the cardiomyocyte molecular clock that underlies circadian rhythms modifies the LQT3-related phenotype. Methods: We used in vivo ECG telemetry of control mice and mice that harbor an LQT3-causing mutation (Scn5aΔKPQ/+). All animals were genetically engineered to enable us to induce the deletion of Bmal1, a key component of the molecular clock, in adult cardiomyocytes. We calculated the RR-interval, QT-interval, and the QTc-interval using the correction formula from Mitchell et al. AJP 1998. Results: Before Bmal1 deletion, Scn5aΔKPQ/+ mice showed a prolongation in the RR, QT and QTc-intervals compared to control animals. Bmal1 deletion slowed the RR and QT-intervals in both groups, but the QTc-interval remained unchanged. Linear regression analysis revealed that the slope of the QT-RR relation in Scn5aΔKPQ/+ mice was double that of control animals and Bmal1 deletion increased the slope in both groups. Additionally, Bmal1 deletion lengthened the QT-interval at a lower RR-interval in Scn5aΔKPQ/+ animals compared to control. Conclusion: Inducing Bmal1 deletion in control and Scn5aΔKPQ/+ mice did not change the QTc interval, but increased the slope of the QT-RR relation so at slower RR-intervals there is a greater change in the QT-interval. Scn5aΔKPQ/+ mice showed the greatest QT prolongation at slow RR-intervals. We conclude that disruption in the molecular clock mechanism exacerbates the LQT3-related phenotype, especially at slow heart rates

Ableism

People living with disability may face significant barriers in work, study, sport and joining in everyday activities. Community attitudes and experiences of discrimination can further impact on a person’s wellbeing, with the resultant ableism leading to perceptions of the disabled as weak and needy, and experiences of rejection and oppression. Allied health professionals are constantly called on to recognise the multi-layered impact of ableism on those who seek their help, while simultaneously challenging their own perceptions and stereotypes. The chapter starts with definitions and statistics to contextualise the concept of disability within the contemporary Australian environment. The link between ableism and mental health is critically examined, and poignantly brought to life in the personal experiences of three individuals as they navigate living with invisible and visible disabilities. Experiential activities that encourage the learner to test and challenge societal stereotypes, their own perceptions and gain a greater understanding of environmental, social and institutional barriers faced by people living with disability conclude this chapter