Characterization of two putative potassium channels in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Potassium channels are essential for cell survival and participate in the regulation of cell membrane potential and electrochemical gradients. During its lifecycle, <it>Plasmodium falciparum </it>parasites must successfully traverse widely diverse environmental milieus, in which K⁺channel function is likely to be critical. Dramatically differing conditions will be presented to the parasite in the mosquito mid-gut, red blood cell (RBC) cytosol and the human circulatory system.</p> <p>Methods</p> <p><it>In silico </it>sequence analyses identified two open-reading frames in the <it>P. falciparum </it>genome that are predicted to encode for proteins with high homology to K⁺channels. To further analyse these putative channels, specific antisera were generated and used in immunoblot and immunofluorescence analyses of <it>P. falciparum</it>-infected RBCs. Recombinant genome methods in cultured <it>P. falciparum </it>were used to create genetic knock outs of each K⁺channel gene to asses the importance of their expression.</p> <p>Results</p> <p>Immunoblot and IFA analyses confirmed the expression of the two putative <it>P. falciparum </it>K⁺channels (PfK1 and PfK2). PfK1 is expressed in all asexual stage parasites, predominantly in late stages and localizes to the RBC membrane. Conversely, PfK2 is predominantly expressed in late schizont and merozoite stage parasites and remains primarily localized to the parasite. Repeated attempts to knockout PfK1 and PfK2 expression by targeted gene disruption proved unsuccessful despite evidence of recombinant gene integration, indicating that <it>pfk1 </it>and <it>pfk2 </it>are apparently refractory to genetic disruption.</p> <p>Conclusion</p> <p>Putative K⁺channel proteins PfK1 and PfK2 are expressed in cultured <it>P. falciparum </it>parasites with differing spatial and temporal patterns. Eventual functional characterization of these channels may reveal future pharmacological targets.</p

A Developmental Framework for Mentorship in SoTL Illustrated by Three Examples of Unseen Opportunities for Mentoring

Mentoring relationships that form between scholars of teaching and learning occur formally and informally, across varied pathways and programs. In order to better understand such relationships, this paper proposes an adapted version of a three-stage model of mentoring (McKinsey 2016), using three examples of unseen opportunities for mentoring in the Scholarship of Teaching and Learning (SoTL) to illustrate how this framework might be operationalized. We discuss how the adapted framework might be useful to SoTL scholars in the future to examine mentorship and how unseen opportunities for mentoring might shape how we consider this subset of mentorship going forward

Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

C1 - Journal Articles Referee

Plasmodium falciparum Purine Nucleoside Phosphorylase Is Critical for Viability of Malaria Parasites*S⃞

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Δpfpnp). Lysates of the Δpfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Δpfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Δpfpnp parasites. The Δpfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

AbstractIntra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (Kd=0.60µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin