Identifying biomechanical gait parameters in adolescent boys with haemophilia using principal component analysis.

INTRODUCTION: Improvements in the medical management for those with haemophilia have resulted in improved clinical outcomes. However, current treatment regimens do not alleviate all joint haemarthroses with the potential for long-term joint deterioration remaining. The evaluation of functional activities such as gait, using standardized tools to monitor children with haemophilia is emerging. AIM: This study explored differences in sagittal plane biomechanics of walking in adolescent boys aged 11-18 years with haemophilia and an age-matched group of typically developing boys. METHODS: A motion capture system and 2 force platforms were used to collect sagittal plane kinematic, kinetic and temporal spatial data during level walking. Principal component analysis (PCA) was applied to kinematic and kinetic waveform variables. Group differences in temporal spatial and principal component scores for each kinematic and kinetic variable were evaluated using independent t tests. RESULTS: Significant alterations (P < .05) in temporal spatial and kinetic parameters were found in adolescent boys with haemophilia. Compared with typically developing adolescent boys, boys with haemophilia walked with reduced stance phase duration and altered pattern of external ankle joint moments during push off and the beginning of swing. CONCLUSION: The use of PCA rather than predetermined discriminatory variables provided additional insight into biomechanical alterations in adolescent boys with haemophilia, with adaptations occurring during terminal double support and early swing, affecting the ankle joint. This finding might be a key biomechanical marker that could be used to evaluate the joint function and the progression of early haemophilic arthropathy.

Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening.

Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.

Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development.

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.