Closing the Feedback Loop: Collaborative Design of a Musculoskeletal Revision Course

Clinical Teaching Fellows (CTFs) at Leicester Medical School decided to supplement the learning needs of first year students on the musculoskeletal (MSK) module. Mid-unit evaluation had demonstrated that students had remarked positively on CTF teaching and many requested further anatomy teaching and demonstrations using prosections. It is increasingly important to provide students with evidence that their feedback is being acted upon, therefore CTFs collaborated in providing additional learning resources in the form of CTF-led revision courses while the MSK module was still ongoing. A survey was designed which aimed to engage students and to further explore their learning needs when developing the course. Based on these responses, two half-day CTF-led revision courses were designed, which included educational methods and topics the students themselves had suggested. CTFs collaboratively developed eight different stations, with one CTF designing and delivering the teaching material. Attendance was high and feedback indicated this was a valuable learning experience for students, with particularly positive responses about the interactive nature and high quality of the teaching. This experience demonstrates the benefits of working in partnership with students when developing learning activities, closing the feedback loop to improve student satisfaction, and collaborative planning when designing revision resources

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. © 2016 The Author(s)

Applying chemical genetic tools to the study of phospho-signalling pathways in malaria parasites

Until very recently there has been very little information about the phospho-signalling pathways in apicomplexan parasites including the most virulent species of human malaria parasite, Plasmodium falciparum. With the advancement of mass spectrometry-based phosphoproteomics and the development of chemical genetic approaches to target specific parasite protein kinases, the complexity of the essential role played by phosphorylation in maintaining the viability of apicomplexan parasites is now being revealed. This review will describe these recent advances and will discuss how these approaches can be used to validate parasite protein kinases as drug targets and to determine the on- and off-target action of protein kinases inhibitors

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the; Plasmodium falciparum; protein kinase; Pf; CLK3, which we used in combination with chemogenetics to validate; Pf; CLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for; Pf; CLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of; Pf; CLK3 mediated rapid killing of asexual liver- and blood-stage; P. falciparum; and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against; P. berghei; and; P. knowlesi; Hence, our data establish; Pf; CLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria