Directed evolution of the non-ribosomal peptide synthetase BpsA to enable recognition by the human Sfp-like PPTase

Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that have an assembly line architecture and synthesise a diverse range of compounds such as antibiotics, siderophores and immunosuppressants. Within the assembly line, the peptidyl carrier protein (PCP) domain has a crucial role in shuttling substrates between the different catalytic domains. The PCP domain is a small four-helix bundle that requires a phosphopantetheinyl moiety to be attached to a conserved serine on the second alpha helix for functionality. This post-translational modification is catalysed by a family of enzymes called the phosphopantetheinyl transferases (PPTases). Due to their central role in activating enzymes involved in both primary (e.g., fatty acid synthetases) and secondary metabolism (e.g., NRPSs), PPTases have been identified as a promising antibiotic target in bacterial species such as Mycobacterium tuberculosis. We have previously developed a high-throughput enzymatic screen for PPTase inhibitors based on co-incubation of a target PPTase with a blue-pigment synthesising NRPS, BpsA. As part of the development of a complete screening platform, we also wanted to be able to rapidly counter-screen inhibitors for cross-inhibition of the endogenous human PPTase, as this is a potential source of toxicity. We found we were unable to use the native BpsA enzyme for this, as the human PPTase is incapable of recognising the PCP domain of BpsA. To improve with the ability of BpsA to be activated by the human PPTase, a directed evolution campaign was undertaken. Firstly, error-prone PCR was used to introduce mutations into the PCP domain of BpsA. Approximately 200,000 variants were screened using a high-throughput plate-based assay. Forty ‘hits’ were then characterized in a semi-quantitative liquid assay. Based on the pattern of amino acid substitutions in the most active variants, specific combinations of substitutions were rationally introduced into BpsA. The top variant identified was now capable of being rapidly phosphopantetheinylated by the human PPTase and we have shown this can be used to quickly screen bacterial PPTase inhibitors for cross-reactivity with the human PPTase. This work illustrates the flexible nature of the PCP domain and provides further evidence that only a few point mutations may be sufficient to dramatically change the specificity of PCP domains for different PPTases

Effects of a prophylactic knee sleeve on anterior cruciate ligament and lower extremity biomechanics: an examination using musculoskeletal simulation

The current study aimed using a two-experiment musculoskeletal simulation-based approach, measuring ACL biomechanics, knee joint kinematics and lower extremity joint loading to examine the effects of both a prophylactic knee sleeve on 1. a sport specific change of direction movement in female footballers and 2. a single leg landing in male footballers. Experiment 1 examined 12 female university first team level footballers (age 20.2 ± 1.34 years, height 1.61 ± 0.06 m, body mass 57.2 ± 5.6 kg) undertaking a 45° cutting movement in sleeve and no-sleeve conditions. Experiment 2 examined 10 male university first team level footballers (age 21.1 ± 1.13 years, height 1.77 ± 0.1 m, body mass 71.9 ± 8.6 kg) undertaking a single leg drop jump landing in sleeve and no-sleeve conditions. In each experiment, data was collected in a biomechanics laboratory and three-dimensional motion capture and ground reaction force information was collected. Three-dimensional kinematics, three-dimensional knee kinetics and ACL ligament forces/ strains were measured using musculoskeletal simulation, and participants were also asked to subjectively rate the knee sleeve in terms of both comfort and stability. Experiment 1 showed that the sleeve condition was associated with greater ACL strain (sleeve = 13.57% and no-sleeve = 10.26%) and forces (sleeve = 1.19BW and no-sleeve = 0.94BW). In addition, the brace condition also enhanced lateral compressive tibiofemoral (sleeve = 4.70BW and no-sleeve = 4.20BW) and total compressive tibiofemoral force (sleeve = 11.73BW and no-sleeve = 11.08BW). Finally, for the subjective ratings, participants indicated that the knee sleeve significantly improved perceived comfort and stability. Experiment 2 did not reveal and statistical differences between knee sleeve and no-sleeve conditions, nor any effects of the knee sleeve on subjective ratings of comfort or stability. Therefore, the findings from the current investigation suggest that the prophylactic knee sleeve examined in the current investigation does not appear to reduce the biomechanical parameters linked to the aetiology of knee pathologies in male/ female footballers

Effects Of A Prophylactic Knee Sleeve On The Anterior Cruciate Ligament And Lower Extremity Biomechanics: An Examination Using Musculoskeletal Simulation

The current study aimed using a two-experiment musculoskeletal simulation-based approach, measuring ACL biomechanics, knee joint kinematics and lower extremity joint loading to examine the effects of both a prophylactic knee sleeve on 1. a sport specific change of direction movement in female footballers and 2. a single leg landing in male footballers. Experiment 1 examined 12 female university first team level footballers (age 20.2 ± 1.34 years, height 1.61 ± 0.06 m, body mass 57.2 ± 5.6 kg) undertaking a 45° cutting movement in sleeve and no-sleeve conditions. Experiment 2 examined 10 male university first team level footballers (age 21.1 ± 1.13 years, height 1.77 ± 0.1 m, body mass 71.9 ± 8.6 kg) undertaking a single leg drop jump landing in sleeve and no-sleeve conditions. In each experiment, data was collected in a biomechanics laboratory and three-dimensional motion capture and ground reaction force information was collected. Three-dimensional kinematics, three-dimensional knee kinetics and ACL ligament forces/ strains were measured using musculoskeletal simulation, and participants were also asked to subjectively rate the knee sleeve in terms of both comfort and stability. Experiment 1 showed that the sleeve condition was associated with greater ACL strain (sleeve = 13.57% and no-sleeve = 10.26%) and forces (sleeve = 1.19BW and no-sleeve = 0.94BW). In addition, the brace condition also enhanced lateral compressive tibiofemoral (sleeve = 4.70BW and no-sleeve = 4.20BW) and total compressive tibiofemoral force (sleeve = 11.73BW and no-sleeve = 11.08BW). Finally, for the subjective ratings, participants indicated that the knee sleeve significantly improved perceived comfort and stability. Experiment 2 did not reveal and statistical differences between knee sleeve and no-sleeve conditions, nor any effects of the knee sleeve on subjective ratings of comfort or stability. Therefore, the findings from the current investigation suggest that the prophylactic knee sleeve examined in the current investigation does not appear to reduce the biomechanical parameters linked to the aetiology of knee pathologies in male/ female footballers

Adenosine-mono-phosphate-activated protein kinase-independent effects of metformin in T cells

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK

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https://place.asburyseminary.edu/freemethodistminutesyearbooks/1016/thumbnail.jp

Predicting the impacts of anthropogenic disturbances on marine populations

Marine ecosystems are increasingly exposed to anthropogenic disturbances that cause animals to change behavior and move away from potential foraging grounds. Here we present a process-based modeling framework for assessing population consequences of such sub-lethal behavioral effects. It builds directly on how disturbances influence animalmovements, foraging and energetics, and is therefore applicable to a wide range of species. To demonstrate the model we assess the impact of wind farm construction noise on the North Sea harbor porpoise population. Subsequently, we demonstrate how the model can be used to minimize population impacts of disturbances through spatial planning. Population models that build on the fundamental processes that determine animal fitness have a high predictive power in novel environments, making them ideal for marine management

Resource partitioning between ungulate populations in arid environments

Herbivores are major drivers of ecosystem structure, diversity, and function. Resilient ecosystems therefore require viable herbivore populations in a sustainable balance with environmental resource availability. This balance is becoming harder to achieve, with increasingly threatened species reliant on small protected areas in increasingly harsh and unpredictable environments. Arid environments in North Africa exemplify this situation, featuring a biologically distinct species assemblage exposed to extreme and volatile conditions, including habitat loss and climate change-associated threats. Here, we implement an integrated likelihood approach to relate scimitar-horned oryx (Oryx dammah) and dorcas gazelle (Gazella dorcas) density, via dung distance sampling, to habitat, predator, and geographic correlates in Dghoumes National Park, Tunisia. We show how two threatened sympatric ungulates partition resources on the habitat axis, exhibiting nonuniform responses to the same vegetation gradient. Scimitar-horned oryx were positively associated with plant species richness, selecting for vegetated ephemeral watercourses (wadis) dominated by herbaceous cover. Conversely, dorcas gazelle were negatively associated with vegetation density (herbaceous height, litter cover, and herbaceous cover), selecting instead for rocky plains with sparse vegetation. We suggest that adequate plant species richness should be a prerequisite for areas proposed for future ungulate reintroductions in arid and semi-arid environments. This evidence will inform adaptive management of reintroduced ungulates in protected environments, helping managers and planners design sustainable ecosystems and effective conservation programs

Using social network analysis of mixed-species groups in African savanna herbivores to assess how community structure responds to environmental change

The dynamics of wildlife populations often depend heavily on interspecific interactions and understanding the underlying principles can be an important step in designing conservation strategies. Behavioural ecological studies can here provide useful insights into the structure and function of communities and their likely response to environmental changes. In this study of the Masai Mara herbivore community, we use a social network approach to investigate social affinities between species and how these change over the year in response to seasonal changes in ecological conditions. We find that even though social networks were correlated across different ecological conditions, for half the species dyads in the community, the strength of social affinities responded to changes in rainfall and/or the presence of migratory wildebeest. Several species consequentially adopted more or less central positions in the network depending on ecological conditions. The findings point out interspecific social links that are likely to be attenuated or strengthened as a consequence of human-induced environmental changes, and therefore call for particular attention of conservation managers. The eco-evolutionary ramifications of the perturbations of social affinities still require further stud