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1.  Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I 
Open Biology  2016;6(9):160114.
Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.
doi:10.1098/rsob.160114
PMCID: PMC5043576  PMID: 27683155
cardiomyopathy; missense single-nucleotide polymorphism; titin protein structure; transgenic muscle; transgenic mouse model
2.  Proteomic Identification of Immunodiagnostic Antigens for Trypanosoma vivax Infections in Cattle and Generation of a Proof-of-Concept Lateral Flow Test Diagnostic Device 
PLoS Neglected Tropical Diseases  2016;10(9):e0004977.
Trypanosoma vivax is one of the causative agents of Animal African Trypanosomosis in cattle, which is endemic in sub-Saharan Africa and transmitted primarily by the bite of the tsetse fly vector. The parasite can also be mechanically transmitted, and this has allowed its spread to South America. Diagnostics are limited for this parasite and in farm settings diagnosis is mainly symptom-based. We set out to identify, using a proteomic approach, candidate diagnostic antigens to develop into an easy to use pen-side lateral flow test device. Two related members the invariant surface glycoprotein family, TvY486_0045500 and TvY486_0019690, were selected. Segments of these antigens, lacking N-terminal signal peptides and C-terminal transmembrane domains, were expressed in E. coli. Both were developed into ELISA tests and one of them, TvY486_0045500, was developed into a lateral flow test prototype. The tests were all evaluated blind with 113 randomised serum samples, taken from 37 calves before and after infection with T. vivax or T. congolense. The TvY486_0045500 and TvY486_0019690 ELISA tests gave identical sensitivity and specificity values for T. vivax infection of 94.5% (95% CI, 86.5% to 98.5%) and 88.0% (95% CI, 75.7% to 95.5%), respectively, and the TvY486_0045500 lateral flow test prototype a sensitivity and specificity of 92.0% (95% CI, 83.4% to 97.0%) and 89.8% (95% CI, 77.8% to 96.6%), respectively. These data suggest that recombinant TvY486_0045500 shows promise for the development of a pen-side lateral flow test for the diagnosis of T. vivax animal African trypanosomosis.
Author Summary
African Animal Trypanosomosis presents a significant problem for agricultural development in sub-Saharan Africa and leads to large economic losses. One of the main parasites responsible is Trypanosoma vivax. Current diagnostic methods are either symptom-based or too costly and technologically demanding for use in endemic regions. Here, we identified T. vivax proteins selectively recognized by infected cattle sera and developed two related proteins into ELISA tests and one of these into a lateral flow test prototype. All three tests performed well when tested against randomised calf sera, suggesting good potential for the development of a pen-side T. vivax animal African trypanosomosis diagnostic device for use in endemic regions.
doi:10.1371/journal.pntd.0004977
PMCID: PMC5015970  PMID: 27606593
3.  Proteomic Selection of Immunodiagnostic Antigens for Trypanosoma congolense 
Animal African Trypanosomosis (AAT) presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG) family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.
Author Summary
Animal African Trypanosomosis (AAT) is a set of diseases whereby animals are infected with single-cell parasites that replicate in their bloodstream. The disease in cattle results in weight-loss and death, and AAT is a significant veterinary problem for sub-Saharan Africa. One of the principal trypanosome species responsible for AAT in cattle is Trypanosoma congolense and, although there are drug-treatments for these infections, current diagnostic methods are impractical for field use. Our aim was to discover protein molecules from the parasite to which infected animals make antibodies, to then make these proteins in bacteria and to subsequently demonstrate that they can be used to detect antibodies in cattle serum, thus diagnosing AAT. To discover the diagnostic proteins, we dissolved parasites in a detergent solution and applied them to beads coated with antibodies from infected cattle and to beads coated with antibodies from un-infected cattle. We then compared the proteins bound to each and selected those proteins that were at least 100-fold enriched by the infected cattle antibodies. We refined this list, according to practical and performance considerations, and settled on one protein, called Tc38630. Testing Tc38630 with cattle sera showed that it can detect about nine out of ten AAT infections.
doi:10.1371/journal.pntd.0002936
PMCID: PMC4055490  PMID: 24922510
4.  Crystal structure of Leishmania major ADP ribosylation factor-like 1 and a classification of related GTPase family members in this Kinetoplastid 
ADP ribosylation factor-like (ARL) proteins are small GTPases that undergo conformational changes upon nucleotide binding, and which regulate the affinity of ARLs for binding other proteins, lipids or membranes. There is a paucity of structural data on this family of proteins in the Kinetoplastida, despite studies implicating them in key events related to vesicular transport and regulation of microtubule dependent processes. The crystal structure of Leishmania major ARL1 in complex with GDP has been determined to 2.1 Å resolution and reveals a high degree of structural conservation with human ADP ribosylation factor 1 (ARF1). Putative L. major and Trypanosoma brucei ARF/ARL family members have been classified based on structural considerations, amino acid sequence conservation combined with functional data on Kinetoplastid and human orthologues. This classification may guide future studies designed to elucidate the function of specific family members.
doi:10.1016/j.molbiopara.2010.08.002
PMCID: PMC3065712  PMID: 20801163
ADP ribosylation factor-like; GTPase; Leishmania; protein structure

Results 1-4 (4)