An unbiased immunization strategy results in the identification of enolase as a potential marker for nanobody-based detection of Trypanosoma evansi

Trypanosoma evansi is a widely spread parasite that causes the debilitating disease “surra” in several types of ungulates. This severely challenges livestock rearing and heavily weighs on the socio-economic development in the affected areas, which include countries on five continents. Active case finding requires a sensitive and specific diagnostic test. In this paper, we describe the application of an unbiased immunization strategy to identify potential biomarkers for Nanobody (Nb)-based detection of T. evansi infections. Alpaca immunization with soluble lysates from different T. evansi strains followed by panning against T. evansi secretome resulted in the selection of a single Nb (Nb11). By combining Nb11-mediated immuno-capturing with mass spectrometry, the T. evansi target antigen was identified as the glycolytic enzyme enolase. Four additional anti-enolase binders were subsequently generated by immunizing another alpaca with the recombinant target enzyme. Together with Nb11, these binders were evaluated for their potential use in a heterologous sandwich detection format. Three Nb pairs were identified as candidates for the further development of an antigen-based assay for Nb-mediated diagnosis of T. evansi infection

Telling adults about it: children’s experience of disclosing interpersonal violence in community sport

A challenge in safeguarding children from interpersonal violence (IV) in sport is the reliance on self-disclosures and a limited understanding of the frequency, barriers to and process of disclosures of IV. Through a mixed-methods design, combining survey and interviews, we explored the frequencies of childhood disclosures of experiences of IV in Australian community sport as well as who children disclosed to and how the interaction unfolded. Those who experienced peer violence disclosed at the highest frequency (35%), followed by coach (27%) or parent (13%) perpetrated IV. A parent/carer was most often the adult that the child disclosed to. Interviews highlighted how the normalisation of violence influenced all aspects of the disclosure and elements of stress buffering (normalising or rationalising) particularly underpinned the disclosure interaction. Policies and practices should explicitly identify all forms of IV in sport as prohibited conduct; education and intervention initiatives should target parents as first responders to disclosures

Nurture commodified? An investigation into commercial human milk supply chains

The material conditions in which women provide breast milk range widely, on the basis of their class and geographical provenance. The commercialisation of breast milk provision throws up questions related to debates on the transnational reconfiguration of social reproduction as they intersect with discourses on motherhood and healthy child development as well as contemporary processes of commodification of the body and the emergence of new gendered forms of atypical work in the global economy (Bakker 2007; LeBaron 2010; Steans & Tepe 2010). This article presents a study of the first commercial human milk processor in India, NeoLacta Lifesciences that obtained an export license for the Australian market in 2017. These practices may be seen to be part of a wider Reproductive Industrial Complex (Vertommen 2016), in which women’s reproductive bodily capacities are enrolled in wider economic and financial processes, instantiating new relations between gender, race, economies and care. This article employs a feminist political economy framework that places into dialogue analyses of social reproduction and commodification with feminist science/technology studies and medical/political anthropology in order to analyse the social, political, and technical processes that transform breast milk into a commodity that is internationally traded and the implications of this for contemporary understandings of work and gender

Cross-cultural adaptation of the VISA-A questionnaire, an index of clinical severity for patients with Achilles tendinopathy, with reliability, validity and structure evaluations

BACKGROUND: Achilles tendinopathy is considered to be one of the most common overuse injuries in elite and recreational athletes and the recommended treatment varies. One factor that has been stressed in the literature is the lack of standardized outcome measures that can be used in all countries. One such standardized outcome measure is the Victorian Institute of Sports Assessment – Achilles (VISA-A) questionnaire, which is designed to evaluate the clinical severity for patients with Achilles tendinopathy. The purpose of this study was to cross-culturally adapt the VISA-A questionnaire to Swedish, and to perform reliability, validity and structure evaluations. METHODS: Cross-cultural adaptation was performed in several steps including translations, synthesis of translations, back translations, expert committee review and pre-testing. The final Swedish version, the VISA-A Swedish version (VISA-A-S) was tested for reliability on healthy individuals (n = 15), and patients (n = 22). Tests for internal consistency, validity and structure were performed on 51 patients. RESULTS: The VISA-A-S had good reliability for patients (r = 0.89, ICC = 0.89) and healthy individuals (r = 0.89–0.99, ICC = 0.88–0.99). The internal consistency was 0.77 (Cronbach's alpha). The mean [95% confidence interval] VISA-A-S score in the 51 patients (50 [44–56]) was significantly lower than in the healthy individuals (96 [94–99]). The VISA-A-S score correlated significantly (Spearman's r = -0.68) with another tendon grading system. Criterion validity was considered good when comparing the scores of the Swedish version with the English version in both healthy individuals and patients. The factor analysis gave the factors pain/symptoms and physical activity CONCLUSION: The VISA-A-S questionnaire is a reliable and valid instrument and comparable to the original version. It measures two factors: pain/symptoms and physical activity, and can be used in both research and the clinical setting

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins

High-confidence glycosome proteome for procyclic form <em>Trypanosoma brucei</em> by epitope-tag organelle enrichment and SILAC proteomics

The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed

Entry and Exit Strategies in Migration Dynamics

This work is devoted to study the role of combined entry and exit strategies in the migration process. We develop a real option model in which the community of immigrants in the host country is described as a club and the immigrants benefits is a U-shaped function, depending on the dimension of the district. There exist two threshold levels: the first one triggers the migration choice, while the second triggers the return to the country of origin. The theoretical results show that the phenomenon of hysteresis is amplified by the existence of a community both in the entry case and in the exit case. Furthermore, the community can reduce the minimum wage level required to trigger both exit and entry: this fact could explain why in some cases we observe migration inflows with a low wage differential and also with underunemployment. We show also some possible further extensions of the model: in one case we introduce a possible way to select the entrants skills and in another case we show some theoretical implementations to include possible policy shocks in the migrants choice