Sports stars and heroes have always been a product of the times in which they have existed and, as such, athlete stars, largely constructed by and through the media, have evolved over time. In ...particular, the meanings of characteristics that have defined stars of different generations have changed as society’s expectations and social norms have evolved. This can be understood through Richard Dyer’s (1998) star theory, which states that to cultivate their stardom a celebrity must resonate with the ideals, values, and spirit of the time.
This theoretical paper aims to highlight how Dyer’s star theory – originally developed as a means of understanding the construction of movie stars by mainstream media – can be used to understand the ways in which athlete stars can promote star attributes to cultivate their stardom by displaying modern values and presenting themselves as both “ordinary” and “extraordinary.” By creating a framework for understanding the process of creating athlete stardom, we are also able to begin to understand how athletes’ social media activities and stardom will evolve in the future, thus creating an important tool for athletes and their managers responsible for enhancing their brand. To expound and enrich the theory a survey of sports fans is included. The survey responses provide insights into how the expectations of fans can influence social athlete social media strategies and the cultivation of athlete stars.
The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required ...for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.
Summary
Leishmania mexicana has a large family of cyclin‐dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies ...indicated that Cdc2‐related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co‐expression of a CRK3 transgene during rapamycin‐induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3T178E mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.
Many protein kinases are essential post‐transcriptional regulators of cell cycle and life cycle progression in Leishmania. To investigate their function, we have developed a conditional method for deletion of essential genes, based on diCre‐lox mediated excision, which we have applied to the cdc2‐related protein kinase CRK3. We demonstrate that CRK3 activity is essential for cell cycle progression through mitosis, and show that conditional deletion of the gene in mammalian infectious L. mexicana promastigotes prevents proliferation in a murine model of infection.
In this review we describe the expanding repertoire of molecular tools with which to study gene function in Leishmania. Specifically we review the tools available for studying functions of essential ...genes, such as plasmid shuffle and DiCre, as well as the rapidly expanding portfolio of available CRISPR/Cas9 approaches for large scale gene knockout and endogenous tagging. We include detail on approaches that allow the direct manipulation of RNA using RNAi and protein levels via Tet or DiCre induced overexpression and destabilization domain mediated degradation. The utilisation of current methods and the development of more advanced molecular tools will lead to greater understanding of the role of essential genes in the parasite and thereby more robust drug target validation, thereby paving the way for the development of novel therapeutics to treat this important disease.
The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in the parasitic protozoan Trypanosoma brucei involves fatty acid remodeling of the GPI precursor molecules before ...they are transferred to protein in the endoplasmic reticulum. The genes encoding the requisite phospholipase A2 and A1 activities for this remodeling have thus far been elusive. Here, we identify a gene, Tb927.7.6110, that encodes a protein that is both necessary and sufficient for GPI-phospholipase A2 (GPI-PLA2) activity in the procyclic form of the parasite. The predicted protein product belongs to the alkaline ceramidase, PAQR receptor, Per1, SID-1, and TMEM8 (CREST) superfamily of transmembrane hydrolase proteins and shows sequence similarity to Post-GPI-Attachment to Protein 6 (PGAP6), a GPI-PLA2 that acts after transfer of GPI precursors to protein in mammalian cells. We show the trypanosome Tb927.7.6110 GPI-PLA2 gene resides in a locus with two closely related genes Tb927.7.6150 and Tb927.7.6170, one of which (Tb927.7.6150) most likely encodes a catalytically inactive protein. The absence of GPI-PLA2 in the null mutant procyclic cells not only affected fatty acid remodeling but also reduced GPI anchor sidechain size on mature GPI-anchored procyclin glycoproteins. This reduction in GPI anchor sidechain size was reversed upon the re-addition of Tb927.7.6110 and of Tb927.7.6170, despite the latter not encoding GPI precursor GPI-PLA2 activity. Taken together, we conclude that Tb927.7.6110 encodes the GPI-PLA2 of GPI precursor fatty acid remodeling and that more work is required to assess the roles and essentiality of Tb927.7.6170 and the presumably enzymatically inactive Tb927.7.6150.
The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and ...glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.
Abstract
Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by ...Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.
Using a combined Huizingian-Bourdieu framework, this paper analyses the significance of sport’s transformation into a business and how the prevailing business structure that defines professional ...sport has influenced the ‘lived experience’ of those playing at sport’s elite level. Furthermore, this paper highlights how the actions of players, coaches and other participants serve to reinforce, legitimise and normalise the business characteristics of sport’s dominant business structure.
Importantly, this paper illuminates how the professionalization of sport corrupts the act of playing and indeed gives rise to play tactics, such as ‘sledging’, which both reflects the increased seriousness of sport and, in its very execution, further reinforces the dominant business structures of professional sport, all the while corrupting the essence of sport – play.
In doing so we are challenged to consider how society’s fields could be different in structure, and in the ‘lived experience’ within the field.
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