Utility of animal models for studying the human neuromuscular junction in health and disease

The neuromuscular junction (NMJ) is a synapse formed between a lower motor neuron (LMN) and a skeletal muscle fibre. It comprises three major components: a motor nerve terminal, acetylcholine receptors (AChRs) at the motor endplate, and one or more terminal Schwann cells (tSCs). Structural and functional disruption of the NMJ is one of the earliest signs of neurodegeneration in motor neuron diseases (MND) such as amyotrophic lateral sclerosis (ALS), a fatal condition with no effective cure. ALS is characterized by progressive muscle weakness that eventually leads to paralysis and death from respiratory failure. Study of the NMJ is therefore critical to develop an understanding of both the disease process and the development of novel therapeutic interventions. For many decades, research has relied heavily on rodent models to study the NMJ. However, recent studies have revealed fundamental differences between rodents and humans in the cellular and molecular organization of the neuronal and muscular parts of the NMJ, as well as in the response to pathological events and therapeutic agents. The first aim of this project was to extend our knowledge of human NMJ morphology by presenting the first detailed structural analysis of human tSCs. The findings demonstrated notable morphological differences between mouse and human tSCs. Despite having a similar number of tSCs per NMJ, human tSCs were significantly smaller in size and had more extended cytoplasm compared to murine tSCs. Furthermore, the positioning of the cell bodies relative to the endplate, which has clinical implications, was different between the two species: while mouse tSCs had significantly more synaptic nuclei, human nuclei were more widely distributed. These results provide further evidence for the stark morphological differences between mouse and human NMJs, encouraging the identification of alternative models that better represent the morphology of the human NMJ. Based on these findings, the second part of the project explored NMJ morphology across several large mammalian species. The NMJs of the cat, dog, sheep, and pig were examined in a range of pelvic limb muscles, and baseline inter-species reference data were generated. These observations revealed that the morphology of sheep and pig NMJs more closely resembles that of the human NMJ compared with those of mice. As such, sheep and pigs may represent better models for translational work than either mice or rats. Finally, to emphasize the critical role of the NMJ as an early target in MND, a thorough analysis of NMJ morphology was performed in an ALS mouse model (Thy1-hTDP-43). This part of the study aimed to characterize the morphological changes and the time course of denervation along with the differential vulnerability of NMJs in distinct body regions. Morphological assessment of NMJs was performed at three disease stages: pre-symptomatic, early symptomatic and end-stage; and in pelvic limb, cranial, thoracic limb muscles and abdominal muscles. Characteristically, muscle denervation was present in the pelvic limb from an early symptomatic stage. The disease progressed rapidly, and by the end-stage, all pelvic limb muscles showed severe denervation and loss of tSCs. In contrast, cranial muscles were only mildly denervated, whilst thoracic limb and abdominal muscles were remarkably preserved. Pathological features were mainly apparent in the pre-synaptic structures (nerve terminals and tSCs), whereas the post-synaptic structures (endplates and muscle fibres) were relatively unaffected. This comprehensive analysis and time course of NMJ pathology can be built-on to determine the sequence of pathological events occurring at the NMJ in ALS. Overall, this project significantly deepens our understanding of differences between human NMJs and NMJs in other mammals, particularly mice. These differences must be considered when attempting to translate animal research to the human context. The project also emphasises the necessity of finding an appropriate animal model to accurately replicate the human NMJ in health and disease, encouraging further exploration of sheep and pig for future NMJ studies. And finally, it further emphasises the importance of the NMJ in MND and its early involvement in the pathogenesis of ALS

Optimal Novel Taxiing Navigation of a BOEING-747 Aircraft Using Artificial Intelligence

Aircraft ground movement coordination plays a key role in improving airport efficiency, as it acts as a link to all other ground operations. Finding novel approaches to coordinate the movements of a fleet of aircraft at an airport in order to improve system resilience to disruptions with increasing autonomy is at the centre of many key studies for airport airside operations. Moreover, autonomous taxiing is envisioned as a key component in future digitalized airports. However, state-of-the-art routing and scheduling algorithms for airport ground movements do not consider high-fidelity aircraft models at both the proactive and reactive planning phases. The majority of such algorithms do not actively seek to optimize fuel efficiency and reduce harmful greenhouse gas emissions. This thesis proposes new approaches using Artificial Intelligence (AI) for optimal taxiing navigation of a high-fidelity aircraft model, working in conjunction with a routing and scheduling algorithm that determines the taxi route, waypoints, and time deadlines. The proposed approaches used in this thesis are: PID controller, artificial neural networks controller, Fuzzy Inference System (FIS) model and an online controller using reinforcement learning. The proposed approaches integrate a MATLAB-Simulink model of the BOEING-747 aircraft with artificial intelligence based control that successfully generate fuel-efficient four- dimensional trajectories 4DTs in real time, while taking constraints on operations into account. The proposed methodologies are realistic and simple to implement. Moreover, simulation studies show that the proposed approaches are capable of providing a reduction in the fuel consumed during the taxiing of a large Boeing 747-100 jumbo jet

Vehicle Routing Optimization for Non-Profit Organization Systems

The distributor management system has long been a challenge for many organizations and companies. Overall, successful distribution involves several moving entities and methods, requiring a resilient distribution management strategy powered by data analysis. For nonprofit organizations, the distribution system requires efficient distribution and management. This includes minimizing time, distance, and cost. As a consequence, service quality and financial efficiency can be achieved. This paper proposes a methodology to tackle the vehicle routing problems (VRP) faced by nonprofit organizations. The methodology consists of four subsequent approaches—greedy, intraroute, interroute, and tabu search—to improve the functionality and performance of nonprofit organizations. The methodology was validated by applying it to a real nonprofit organization. Furthermore, the proposed system was compared to another state-of-the-art system; the achieved results were satisfactory and suggest that this methodology is capable of handling the VRP accordingly, improving the functionality and performance of nonprofit organizations

Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

Neuromuscular junction (NMJ) dysfunction underlies several diseases, including congenital myasthenic syndromes (CMSs) and motor neuron disease (MND). Molecular pathways governing NMJ stability are therefore of interest from both biological and therapeutic perspectives. Muscle-specific kinase (MuSK) is necessary for the formation and maintenance of post-synaptic elements of the NMJ, and downstream of tyrosine kinases 7 (DOK7) is crucial for activation of the MuSK pathway. Overexpression of DOK7 using AAV9 has been shown to ameliorate neuromuscular pathology in pre-clinical disease models of CMS and MND. However, long-term consequences of DOK7 expression have been sparsely investigated and targeted overexpression of DOK7 in skeletal muscle yet to be established. Here, we developed and characterized a novel AAV9-DOK7 facilitating forced expression of DOK7 under a skeletal muscle-specific promoter. AAV9-tMCK-DOK7 was systemically delivered to newborn mice that were monitored over 6 months. DOK7 overexpression was restricted to skeletal muscles. Body weight, blood biochemistry, and histopathological assessments were unaffected by AAV9-tMCK-DOK7 treatment. In contrast, forced expression of DOK7 resulted in enlargement of both the pre- and post-synaptic components of the NMJ, without causing denervation. We conclude that muscle-specific DOK7 overexpression can be achieved in a safe manner, with the capacity to target NMJs in vivo

aNMJ-morph macro

# Contents # 1. aNMJ-morph macro; 2. aNMJ-morph tutorial video; 3. Reference images (20 sample NMJ images for use with aNMJ-morph); 4. Reference spreadsheet (data generated from aNMJ-morph analysis of the 20 sample NMJ images).It is recommended that users of the aNMJ-morph macro are familiar with the original NMJ-morph workflow in a practical setting; please refer to the following publication for background details: "NMJ-morph reveals principal components of synaptic morphology influencing structure-function relationships at the neuromuscular junction" (Jones RA et al, Open Biology, 2016, doi.org/10.1098/rsob.160240).Minty, Gavin; Hoppen, Alex; Boehm, Ines; Alhindi, Abrar; Gibb, Larissa; Potter, Ellie; Wagner, Boris; Miller, Janice; Skipworth, Richard; Gillingwater, Tom; Jones, Ross. (2019). aNMJ-morph macro, [dataset]. University of Edinburgh. https://doi.org/10.7488/ds/2625

SARS-CoV-2 vaccination modelling for safe surgery to save lives: data from an international prospective cohort study

Background: Preoperative SARS-CoV-2 vaccination could support safer elective surgery. Vaccine numbers are limited so this study aimed to inform their prioritization by modelling. Methods: The primary outcome was the number needed to vaccinate (NNV) to prevent one COVID-19-related death in 1 year. NNVs were based on postoperative SARS-CoV-2 rates and mortality in an international cohort study (surgical patients), and community SARS-CoV-2 incidence and case fatality data (general population). NNV estimates were stratified by age (18-49, 50-69, 70 or more years) and type of surgery. Best- and worst-case scenarios were used to describe uncertainty. Results: NNVs were more favourable in surgical patients than the general population. The most favourable NNVs were in patients aged 70 years or more needing cancer surgery (351; best case 196, worst case 816) or non-cancer surgery (733; best case 407, worst case 1664). Both exceeded the NNV in the general population (1840; best case 1196, worst case 3066). NNVs for surgical patients remained favourable at a range of SARS-CoV-2 incidence rates in sensitivity analysis modelling. Globally, prioritizing preoperative vaccination of patients needing elective surgery ahead of the general population could prevent an additional 58 687 (best case 115 007, worst case 20 177) COVID-19-related deaths in 1 year. Conclusion: As global roll out of SARS-CoV-2 vaccination proceeds, patients needing elective surgery should be prioritized ahead of the general population