title: Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

authors: Lee, T. Verne

subjects: protein screening

abstract: Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

text: Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

year: 2010

title: Exploring the reliability of isometric benchmark tests and their relationship to performance characteristics in elite track sprint cyclists

authors: Thompson, Roné

subjects: isometrics

abstract: Benchmark tests in competitive cycling identify talent, individualise training, and monitor performance. However, varying protocols often produce conflicting results, reducing comparability. Isometric tests are prevalent, but reliability and performance correlation are underexplored. Determine the test–retest reliability of benchmark test metrics in elite track sprint cyclists and their relationship to a performance outcome. Nineteen elite track sprint cyclists (12 males, 7 females) completed seven benchmark tests across two days: modified sit-and-reach; on-bike rolling seated maximum 6-s sprints; 3-s bilateral on-bike isometrics at 90° crank angle; 3-s prone bench pull isometrics; 3-s lumbar extension isometrics; 3-s seated off-bike isometrics; and modified plank endurance. For the performance outcome, a third session within 7 days assessed peak power using an inertial load cycle ergometer. All tests showed excellent measurement consistency (ICC<inf>3,1</inf> ≥ 0.92), with low systematic bias (p ≥ 0.063), though confidence interval varied due to modest sample size. High test–retest reliability was supported by low typical errors (CV 2.0–5.5%; 9.6% for endurance). Nine benchmark metrics, including bilateral isometric measures, showed moderate to excellent correlation with peak power output (r = 0.52–0.94, p ≤ 0.023); six remained statistically significant after Bonferroni correction (p ≤ 0.005). All benchmark metrics were reliable, with six strongly and statistically significantly associated with performance.

text: Exploring the reliability of isometric benchmark tests and their relationship to performance characteristics in elite track sprint cyclists Benchmark tests in competitive cycling identify talent, individualise training, and monitor performance. However, varying protocols often produce conflicting results, reducing comparability. Isometric tests are prevalent, but reliability and performance correlation are underexplored. Determine the test–retest reliability of benchmark test metrics in elite track sprint cyclists and their relationship to a performance outcome. Nineteen elite track sprint cyclists (12 males, 7 females) completed seven benchmark tests across two days: modified sit-and-reach; on-bike rolling seated maximum 6-s sprints; 3-s bilateral on-bike isometrics at 90° crank angle; 3-s prone bench pull isometrics; 3-s lumbar extension isometrics; 3-s seated off-bike isometrics; and modified plank endurance. For the performance outcome, a third session within 7 days assessed peak power using an inertial load cycle ergometer. All tests showed excellent measurement consistency (ICC<inf>3,1</inf> ≥ 0.92), with low systematic bias (p ≥ 0.063), though confidence interval varied due to modest sample size. High test–retest reliability was supported by low typical errors (CV 2.0–5.5%; 9.6% for endurance). Nine benchmark metrics, including bilateral isometric measures, showed moderate to excellent correlation with peak power output (r = 0.52–0.94, p ≤ 0.023); six remained statistically significant after Bonferroni correction (p ≤ 0.005). All benchmark metrics were reliable, with six strongly and statistically significantly associated with performance.

year: 2025

title: Distributed agent-centric system for indigenous data sovereignty

authors: Noovao, Setephano

subjects: holochain

abstract: The sovereignty of indigenous data is a key issue facing indigenous communities. Holochain, a distributed ledger technology, has the potential to address some of the challenges indigenous communities face with respect to data sovereignty. The purpose of this thesis is to critically evaluate the feasibility of using holochain technology to support indigenous data sovereignty. This paper first explores the literature on holochain technology, examining its underlying architecture and features. The research employs a three-phase design science research methodology, conducting an experiment to explore the integration of a decentralized application, holochain, with a prominent cloud service provider. The first phase concentrates on emulating real data-sharing platforms that may utilize data at its disposal, following the principles of indigenous data sovereignty, where a centralized platform could be hosted on a cloud computing service with multiple services in operation. The second phase involves developing a holochain application guided by the principles of indigenous data sovereignty. The final phase seeks to combine the established centralized platform from the first phase with the outcomes of the holochain application from the second phase to experimentally assess whether both technologies can create a data sovereignty solution that aligns with the needs of indigenous data sovereignty. The results of the experiment will provide a solid foundation for understanding the current state of the field and identifying areas where holochain can potentially offer solutions. Furthermore, the results of our research indicate that holochain shows significant promise in tackling the issue of indigenous data sovereignty, although there are considerable limitations that must be addressed and resolved to achieve improved results.

text: Distributed agent-centric system for indigenous data sovereignty The sovereignty of indigenous data is a key issue facing indigenous communities. Holochain, a distributed ledger technology, has the potential to address some of the challenges indigenous communities face with respect to data sovereignty. The purpose of this thesis is to critically evaluate the feasibility of using holochain technology to support indigenous data sovereignty. This paper first explores the literature on holochain technology, examining its underlying architecture and features. The research employs a three-phase design science research methodology, conducting an experiment to explore the integration of a decentralized application, holochain, with a prominent cloud service provider. The first phase concentrates on emulating real data-sharing platforms that may utilize data at its disposal, following the principles of indigenous data sovereignty, where a centralized platform could be hosted on a cloud computing service with multiple services in operation. The second phase involves developing a holochain application guided by the principles of indigenous data sovereignty. The final phase seeks to combine the established centralized platform from the first phase with the outcomes of the holochain application from the second phase to experimentally assess whether both technologies can create a data sovereignty solution that aligns with the needs of indigenous data sovereignty. The results of the experiment will provide a solid foundation for understanding the current state of the field and identifying areas where holochain can potentially offer solutions. Furthermore, the results of our research indicate that holochain shows significant promise in tackling the issue of indigenous data sovereignty, although there are considerable limitations that must be addressed and resolved to achieve improved results.

year: 2024