Characterization of the ATP4 ion pump in Toxoplasma gondii

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+. Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.This work was supported by National Health and Medical Research Council Grant 1042272 (to K. K.) and Australian Research Council Discovery Project Grant DP150102883 (to K. K. and G. G. v. D.), Linkage Project Grant LP150101226 (to K. K.), Discovery Early Career Researcher Award DE160101035 (to A. M. L.), QEII Fellowship DP110103144 (to G. G. v. D.), and Future Fellowship FT120100164 (to C. J. T.). C. J. T. is grateful for institutional support from the Victorian State Government Operational Infrastructure Support Program and the National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme

The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

In this study the `Malaria Box' chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite's formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target.Aspects of this work were supported by a Project Grant (1042272 to KK) from the Australian National Health and Medical Research Council (NHMRC). AML is the recipient of an Australian Research Council Discovery Early Career Researcher Award (DE160101035), REM was supported by a NHMRC Career Development Fellowship (1053082) and MJM is a NHMRC Principal Research Fello

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce i

A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

PfATP4 and the biochemical signature of PfATP4-associated compounds

In 2016 more than 200 million cases of malaria were reported and nearly 500 000 people died from the disease. Although there are antimalarial drugs available, the most virulent species of the disease-causing parasite, Plasmodium falciparum, has evolved some level of resistance to many of them. A global effort to accelerate antimalarial drug discovery has led to the finding that one particular parasite protein, the P-type ATPase PfATP4, appears to be the target of a number of potent novel antimalarial compounds. Two of these ‘PfATP4-associated compounds’ have entered the clinical pipeline; one of these is the spiroindolone KAE609 (also known as cipargamin). PfATP4 is located on the plasma membrane of the parasite. Although an early study yielded evidence consistent with PfATP4 being a Ca2+ transporter, more recent studies have provided evidence that PfATP4 extrudes Na+ ions from the parasite while importing H+ ions, allowing the parasite to maintain a large inward Na+ concentration gradient. When parasites are exposed to PfATP4-associated compounds, there is an increase in the Na+ concentration and the pH inside the parasite. In the work reported in this Thesis I have investigated the nature and function of PfATP4, extended the characterisation of the effects of PfATP4-associated compounds on mature asexual-stage P. falciparum parasites, and identified a number of new compounds of this class. A phylogenetic analysis revealed that PfATP4 belongs to a unique subgroup of Type II P-type ATPases, specific to apicomplexan parasites and their closest relatives. Previous studies have reported that PfATP4-associated compounds cause parasite swelling. The effect of KAE609 on the volume of isolated parasites and parasitised erythrocytes was characterised using a Coulter Multisizer. KAE609 caused isolated parasites to swell in a Na+-dependent manner. KAE609 also caused intact infected erythrocytes to swell and thereby increase in osmotic fragility. Another six PfATP4-associated compounds were also shown to induce parasite swelling. Protecting parasitised erythrocytes from excessive swelling by growing them in a hyperosmotic medium increased the concentration of KAE609 that was required to kill the parasite, consistent with cell swelling playing a role in the mechanism of action of KAE609. The ‘biochemical signature’ of PfATP4-associated compounds was used to identify novel PfATP4-associated compounds from a set of 400 diverse drug-like compounds. Experiments investigating the effects of these compounds on parasite Na+, pH and volume, as well as cross-resistance studies using KAE609-resistant parasites, provided evidence that eleven compounds act in a manner consistent with inhibition of PfATP4. These compounds add to the chemical diversity of known PfATP4-associated compounds. PfATP4 has previously been proposed to be a Ca2+ transporter. The role of Ca2+ in the mechanism of action of PfATP4-associated compounds was explored using a physiological approach. The results support the prevailing view that PfATP4-associated compounds disrupt Na+ homeostasis directly, and are inconsistent with a role for Ca2+ in this phenomenon. This study furthers our understanding of the effects of PfATP4-associated compounds on parasite physiology and, in extending our knowledge of the characteristic biochemical signature of these compounds, provides a basis for identifying further such compounds

Using Experience Sampling to Investigate Affect at Encoding and Episodic Memory

Intensive longitudinal data was collected through theconcurrent use of a passive experience sampling (ES)smartphone application collecting objective measures ofexperience, and an ecological momentary assessment (EMA)app collecting self-reported affect. After a week-longretention interval, participants completed a memory testgenerated from paired ES and EMA data. Participants wereasked to select the GPS location at the time of a paired targetevent from four alternatives. Correct retrieval was notpredicted by self-reports grouped by negative valence/higharousal or negative valence/low arousal. Positivevalence/high arousal reported at encoding predicted greaterprobability of incorrect responses. Conversely, positivevalence/low arousal predicted greater probability of correctidentification of target. At retrieval, choice was predicted bydissimilarities in discrete emotions between target anddistractors, suggesting the use of affect as a contextualmechanism

What is sexual liberation in the age of AIDS?

Adelaide Festival of Ideas session, Brookman Hall, 12:00pm, Sunday 13 July, 2003. Chaired by Ian Purcell.http://adelaidefestivalofideas.com.au

Diverse antimalarials from wholecell phenotypic screens disrupt malaria parasite ion and volume homeostasis

Four hundred structurally diverse drug-like compounds comprising the Medicines for Malaria Venture’s ‘Pathogen Box’ were screened for their effect on a range of physiological parameters in asexual blood-stage malaria (Plasmodium falciparum) parasites. Eleven of these compounds were found to perturb parasite Na+, pH and volume in a manner consistent with inhibition of the putative Na+ efflux P-type ATPase PfATP4. All eleven compounds fell within the subset of 125 compounds included in the Pathogen Box on the basis of their having been identified as potent inhibitors of the growth of asexual blood-stage P. falciparum parasites. All eleven compounds inhibited the Na+-dependent ATPase activity of parasite membranes and showed reduced efficacy against parasites carrying mutations in PfATP4. This study increases the number of chemically diverse structures known to show a ‘PfATP4- associated’ phenotype, and adds to emerging evidence that a high proportion (7–9%) of the structurally diverse antimalarial compounds identified in whole cell phenotypic screens share the same mechanism of action, exerting their antimalarial effect via an interaction with PfATP4This work was supported by a Project Grant (1042272) from the Australian National Health and Medical Research Council and by Australian Research Council Linkage Project Grant LP150101226. A.M.L. is the recipient of an Australian Research Council Discovery Early Career Researcher Award (DE160101035

'Power to empower': conceptions of teaching and learning in a pedagogical co-design partnership

This study is an autoethnographic reflection on power and expertise in an evolving student/staff partnership. The partnership was initiated as pedagogical co-design in the development and implementation of a peer-assisted learning programme. Through a process of critical reflection that linked our partnership experience with themes from relevant literature, we (the staff and student authors) became co-researchers of our practice. The evolution of the partnership provided a unique perspective from which to compare our experiences of power and expertise across both contexts. We characterised our pedagogical co-design partnership as a shift from the more traditional ‘power over’ model of delivery towards ‘power to empower’ where both student and staff partners had agency and voice. Key to this important transition was a shared philosophy of student-centred teaching. As the partnership transformed from co-teaching to coresearching we needed to re-negotiate power dynamics; while our different pathways had converged on a common view of studentcentred learning, our research expertise remained disparate. We were able to negotiate this challenge by drawing on our existing relationship based on respect, reciprocity and responsibility, reinforcing views of partnership that value equality of opportunity and a focus on learning and process, rather than equality of contributions and outcomes