Introduction: We frequently associate microbes with infection, rarely expounding on their usefulness and importance to healthy development. For humanity to leverage these microbial “super powers”, learners from all backgrounds need to appreciate their utility and consider how microbes could help solve some of the most critical problems we face. However, learners are frequently uninterested or intimidated by microbiology. The card game “No Guts No Glory” was created to engage students by piquing curiosity and encouraging informal learning to change perceptions and advocate the value of microbes to good health. Methods: Undergraduates from various faculties in the National University of Singapore were invited to play and give feedback on accessibility, engagement and self-reported learning gains. Results: The game was well-received across disciplinary backgrounds with positive feedback (5-point scale) on game mechanics being fun (4.17±0.63), attractive artwork (3.83±1.00) and scientific snippets (3.79±1.04), positive re-playability (3.46±0.84), player engagement for those without foundational knowledge in microbiology (3.63±1.04), and usefulness of knowledge taught (3.54±1.10). Areas for improvement evidenced from feedback included unclear instructions (2.74±0.98), limited content taught (2.76±0.93), not generating interest to attend formal microbiome classes (2.88±1.17) and not prompting lifestyle changes (1.98±1.11). Conclusion: This pilot study provided valuable insights from the target demographic, with concrete ideas on how to improve the educational potential of “No Guts No Glory”. Findings further lay the groundwork for the design of future instruments to objectively quantify learning gains from gameplay. Keywords: Game-based Learning, Microbiome, Microbiology, Card Game, No Guts No Glory
Abstract The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality.
Abstract The total syntheses of antimalarial agent Falcitidin (I) as well as related N‐acyl analogues (II) are outlined and the efficiency of the latter compounds against chloroquine‐sensitive Plasmodium falciparum 3D7 is tested.
An alternative antimalarial pathway of an ‘outdated’ drug, chloroquine (CQ), may facilitate its return to the shrinking list of effective antimalarials. Conventionally, CQ is believed to interfere with hemozoin formation at nanomolar concentrations, but resistant parasites are able to efflux this drug from the digestive vacuole (DV). However, we show that the DV membrane of both resistant and sensitive laboratory and field parasites is compromised after exposure to micromolar concentrations of CQ, leading to an extrusion of DV proteases. Furthermore, only a short period of exposure is required to compromise the viability of late-stage parasites. To study the feasibility of this strategy, mice malaria models were used to demonstrate that high doses of CQ also triggered DV permeabilization in vivo and reduced reinvasion efficiency. We suggest that a time-release oral formulation of CQ may sustain elevated blood CQ levels sufficiently to clear even CQ-resistant parasites.
Abstract Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.
Due to the widespread prevalence of resistant parasites, chloroquine (CQ) was removed from front-line antimalarial chemotherapy in the 1990s despite its initial promise of disease eradication. Since then, resistance-conferring mutations have been identified in transporters such as the PfCRT, that allow for the efflux of CQ from its primary site of action, the parasite digestive vacuole. Chemosensitizing/chemoreversing compounds interfere with the function of these transporters thereby sensitizing parasites to CQ once again. However, compounds identified thus far have disappointing in vivo efficacy and screening for alternative candidates is required to revive this strategy. In this study, we propose a simple and direct means to rapidly screen for such compounds using a fluorescent-tagged CQ molecule. When this screen was applied to a small library, seven novel chemosensitizers (octoclothepin, methiothepin, metergoline, loperamide, chlorprothixene, L-703,606 and mibefradil) were quickly elucidated, including two which showed greater potency than the classical chemosensitizers verapamil and desipramine.
The ABO blood group antigens are expressed on erythrocytes but also on endothelial cells, platelets and serum proteins. Notably, the ABO blood group of a malaria patient determines the development of the disease given that blood group O reduces the probability to succumb in severe malaria, compared to individuals of groups A, B or AB. P. falciparum rosetting and sequestration are mediated by PfEMP1, RIFIN and STEVOR, expressed at the surface of the parasitized red blood cell (pRBC). Antibodies to these antigens consequently modify the course of a malaria infection by preventing sequestration and promoting phagocytosis of pRBC. Here we have studied rosetting P. falciparum and present evidence of an immune evasion mechanism not previously recognized. We find the accessibility of antibodies to PfEMP1 at the surface of the pRBC to be reduced when P. falciparum forms rosettes in blood group A RBC, as compared to group O RBC. The pRBC surrounds itself with tightly bound normal RBC that makes PfEMP1 inaccessible to antibodies and clearance by the immune system. Accordingly, pRBC of in vitro cloned P. falciparum devoid of ABO blood group dependent rosetting were equally well detected by anti-PfEMP1 antibodies, independent of the blood group utilized for their propagation. The pathogenic mechanisms underlying the severe forms of malaria may in patients of blood group A depend on the ability of the parasite to mask PfEMP1 from antibody recognition, in so doing evading immune clearance.
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