The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria

Evaluating the effectiveness of styles of play in elite soccer

The aim of this study was to evaluate the effectiveness of styles of play in soccer and the influence of contextual variables (i.e. match status, venue and quality of opposition). Team possessions (n = 68,766) from the 380 matches of the 2015–2016 English Premier League season were collected for this study. The Possession Effectiveness Index, based on Expected Goals and Ball Movement Points metrics, was used to measure the effectiveness of team possessions. Linear mixed models were applied to analyse the influence of contextual variables on the effectiveness score for each style. Results showed that the effectiveness of Direct Play, Counterattack, Maintenance and Crossing significantly increased when teams were winning by two or more goals. Counterattack increased its effectiveness when teams were winning by one goal and reduced its effectiveness when losing by one goal. The effectiveness of Direct Play increased when losing by two goals or more. Playing away negatively affected the effectiveness of Direct Play, Maintenance and High Pressure. In addition, playing against a stronger opposition reduced the effectiveness of all styles of play. The results suggest that the effectiveness of styles of play changes under specific circumstances and that not all contextual variables affect them in the same way

Improved synchronous production of Plasmodium falciparum gametocytes in vitro.

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis

Contextual information influences diagnosis accuracy and decision making in simulated emergency medicine emergencies

Background: It is well documented that adaptations in cognitive processes with increasing skill levels support decision making in multiple domains. We examined skill-based differences in cognitive processes in emergency medicine physicians, and whether performance was significantly influenced by the removal of contextual information related to a patient's medical history. Method: Skilled (n=9) and less skilled (n=9) emergency medicine physicians responded to high-fidelity simulated scenarios under high- and low-context information conditions. Results: Skilled physicians demonstrated higher diagnostic accuracy irrespective of condition, and were less affected by the removal of context-specific information compared with less skilled physicians. The skilled physicians generated more options, and selected better quality options during diagnostic reasoning compared with less skilled counterparts. These cognitive processes were active irrespective of the level of context-specific information presented, although high-context information enhanced understanding of the patients' symptoms resulting in higher diagnostic accuracy. Conclusions: Our findings have implications for scenario design and the manipulation of contextual information during simulation training

Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle

The half-time talk: A mixed-method examination of youth-elite football coaches’ behaviours and team-management strategies

Football, unlike some other team sports, includes limited game interruptions for coaches to easily communicate with players and affect their performance. However, a reduced number of studies have explored how coaches attempt to influence players during half-time. This study examined football coaches’ behaviours during half-time and their perceptions underpinning their talks’ delivery. Five Spanish coaches (Mage = 32.2, SD = 8.8) working for a La Liga academy were systematically observed during half-time talks (n = 20) and participated in a semi-structured interview each. Half-time talks were coded using a modified version of the Coach Analysis and Intervention System, and semi-structured interviews were analysed following thematic analysis procedures. Instruction and feedback were the most employed behaviours for four coaches, with younger age-group coaches employing greater divergent questioning and in-talk player participation. Furthermore, data suggested that coaches conferred with their staff, before entering the changing room and rapidly progressed from divergent to convergent questions and feedback and instruction. The team's ‘level of play’ was the most perceived relevant factor affecting the verbal and vocal strategies of coaches’ messages, albeit the score gained importance for coaches of older age-groups. This study is pioneering, examining how coaches attempt to influence their players during half-time talks of competitive youth football

30 days wild: development and evaluation of a large-scale nature engagement campaign to improve well-being

There is a need to increase people’s engagement with and connection to nature, both for human well-being and the conservation of nature itself. In order to suggest ways for people to engage with nature and create a wider social context to normalise nature engagement, The Wildlife Trusts developed a mass engagement campaign, 30 Days Wild. The campaign asked people to engage with nature every day for a month. 12,400 people signed up for 30 Days Wild via an online sign-up with an estimated 18,500 taking part overall, resulting in an estimated 300,000 engagements with nature by participants. Samples of those taking part were found to have sustained increases in happiness, health, connection to nature and pro-nature behaviours. With the improvement in health being predicted by the improvement in happiness, this relationship was mediated by the change in connection to nature

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>STEVOR proteins, encoded by the multicopy <it>stevor </it>gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.</p> <p>Methods</p> <p>Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.</p> <p>Results</p> <p>Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.</p> <p>Conclusion</p> <p>The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.</p

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites