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Publication Fiber optic Raman spectroscopy for the evaluation of disease state in Duchenne muscular dystrophy: An assessment using the mdx model and human muscle(Wiley, 2022-07-15) Alix, James; Plesia, Maria; Hool, Sarah; Coldicott, Ian; Kendall, Catherine; Shaw Dbe, Pamela; Mead, Richard; Day, John; Kendall, Catherine; Healthcare ScientistsIntroduction/aims: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients. Methods: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity. Results: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice. Discussion: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.Publication Non-negative matrix factorisation of Raman spectra finds common patterns relating to neuromuscular disease across differing equipment configurations, preclinical models and human tissue(Wiley, 2022-12-22) Alix, James; Plesia, Maria; Schooling, Chloe; Dudgeon, Alexander; Kendall, Catherine; Kadirkamanathan, Visakan; McDermott, Christopher; Gorman, Grainne; Taylor, Robert; Mead, Richard; Shaw, Pamela; Day, John; Dudgeon, Alexander; Kendall, Catherine; Healthcare ScientistsRaman spectroscopy shows promise as a biomarker for complex nerve and muscle (neuromuscular) diseases. To maximise its potential, several challenges remain. These include the sensitivity to different instrument configurations, translation across preclinical/human tissues and the development of multivariate analytics that can derive interpretable spectral outputs for disease identification. Nonnegative matrix factorisation (NMF) can extract features from high-dimensional data sets and the nonnegative constraint results in physically realistic outputs. In this study, we have undertaken NMF on Raman spectra of muscle obtained from different clinical and preclinical settings. First, we obtained and combined Raman spectra from human patients with mitochondrial disease and healthy volunteers, using both a commercial microscope and in-house fibre optic probe. NMF was applied across all data, and spectral patterns common to both equipment configurations were identified. Linear discriminant models utilising these patterns were able to accurately classify disease states (accuracy 70.2-84.5%). Next, we applied NMF to spectra obtained from the mdx mouse model of a Duchenne muscular dystrophy and patients with dystrophic muscle conditions. Spectral fingerprints common to mouse/human were obtained and able to accurately identify disease (accuracy 79.5-98.8%). We conclude that NMF can be used to analyse Raman data across different equipment configurations and the preclinical/clinical divide. Thus, the application of NMF decomposition methods could enhance the potential of Raman spectroscopy for the study of fatal neuromuscular diseases.Publication A review of kilovoltage radiotherapy treatment in the United Kingdom: quality control, radiation dosimetry, treatment equipment, and workload(British Institute of Radiology Oxford University Press, 2025-01-10) Palmer, Antony; Brimelow, Jason; Downes, Patrick; Munshi, Mayur; Nash, David; Rai, Bhupinder; White, Andrew; Brimelow, Jason; Additional Professional Scientific and TechnicalAbstract Objectives To survey kilovoltage (kV) radiotherapy in the United Kingdom, updating a 2016 study, focussing on radiotherapy physics, including equipment quality control (QC) and radiation dosimetry, with information on installed equipment and clinical activity. Methods All UK radiotherapy physics departments (n = 68) were invited to complete a comprehensive survey. An analysis of the installed equipment base, patient numbers, clinical activity, QC testing, and radiation dosimetry processes were undertaken. Results 91% of centres (n = 62) responded to the survey. kV radiotherapy was available in 70% of UK radiotherapy departments, with a wide variation in workload; 7-436 patients/centre annually. There has been an increase in centres using treatment calculation software rather than manual methods, up from 36% in 2016 to 50% currently. Only 50% of centres use an independent calculation check method. There was an increase in the use of the addendum to the UK dosimetry code of practice, enabling medium energy calibration in-air rather than at depth in phantom, citing “clinical relevance.” Appropriate levels of QC testing were being conducted at UK centres, with Institute of Physics and Engineering in Medicine (IPEM) Report 81 cited as a primary source of guidance. Good consensus for the frequency and tolerance values used for QC was seen across UK centres. Conclusions A comprehensive review of consensus practice for QC and dosimetry in kV radiotherapy across the United Kingdom is presented, with supporting information on equipment installation and clinical use. Advances in Knowledge Updated data are presented on kV radiotherapy treatment in the United Kingdom, with focus on physics aspects of QC and dosimetry.Publication Optimising Shifted Excitation Raman Difference Spectroscopy (SERDS) for application in highly fluorescent biological samples, using fibre optic probes(Royal Society of Chemistry, 2024-11-19) Sheridan, Hannah; Dudgeon, Alexander; Day, John; Kendall, Catherine; Hall, Charlie; Stone, Nick; Dudgeon, Alexander; Kendall, Catherine; Hall, Charlie; Stone, Nick; Healthcare Scientists; Medical and DentalFibre optic probe based Raman spectroscopy can deliver in vivo molecular compositional analysis of a range of diseases. However, some biological tissues exhibit high levels of fluorescence which limit the utility of the technique, particularly when the fluorescence induces CCD etaloning, which can be particulalry hard to remove in subsequent analysis. Furthermore, use of fibre probes can result in silica signals superimposed on the biological Raman signals. Shifted excitation Raman difference spectroscopy (SERDS) utilises a small seperation in excitation wavelengths to remove signals from fluorescence, room lights, optical components and etaloning contributions, while retaining chemical signals from the sample. In this study, we sought to measure the optimum SERDS spectra enabling reconstruction of a range a narrow and broad peaks found in biological samples. A original wavelength of 830 nm was utilised with 7 different shifts between 0.4 and 3.9 nm to determine which gave the best performance. This range roughly corresponds to the typical range of peak widths within biological Raman spectra at 830 nm excitation; 0.41 – 3.25 nm or 6 – 47 cm−1. An wavelength shift of 2.4 nm was identified as optimal. Finally, a fibre optic Raman probe was used to measure 2 human lymph nodes ex vivo to demonstrate the feasibility of the approach with real-world examples.Publication Conformational fingerprinting with Raman spectroscopy reveals protein structure as a translational biomarker of muscle pathology(Royal Society of Chemistry, 2024-03-27) Alix, James; Plesia, Maria; Dudgeon, Alexander; Kendall, Catherine; Hewamadduma, Channa; Hadjivassiliou, Marios; Gorman, Gráinne; Taylor, Robert; McDermott, Christopher; Shaw, Pamela; Mead, Richard; Day, John; Dudgeon, Alexander; Kendall, Catherine; Healthcare ScientistsNeuromuscular disorders are a group of conditions that can result in weakness of skeletal muscles. Examples include fatal diseases such as amyotrophic lateral sclerosis and conditions associated with high morbidity such as myopathies (muscle diseases). Many of these disorders are known to have abnormal protein folding and protein aggregates. Thus, easy to apply methods for the detection of such changes may prove useful diagnostic biomarkers. Raman spectroscopy has shown early promise in the detection of muscle pathology in neuromuscular disorders and is well suited to characterising the conformational profiles relating to protein secondary structure. In this work, we assess if Raman spectroscopy can detect differences in protein structure in muscle in the setting of neuromuscular disease. We utilise in vivo Raman spectroscopy measurements from preclinical models of amyotrophic lateral sclerosis and the myopathy Duchenne muscular dystrophy, together with ex vivo measurements of human muscle samples from individuals with and without myopathy. Using quantitative conformation profiling and matrix factorisation we demonstrate that quantitative 'conformational fingerprinting' can be used to identify changes in protein folding in muscle. Notably, myopathic conditions in both preclinical models and human samples manifested a significant reduction in α-helix structures, with concomitant increases in β-sheet and, to a lesser extent, nonregular configurations. Spectral patterns derived through non-negative matrix factorisation were able to identify myopathy with a high accuracy (79% in mouse, 78% in human tissue). This work demonstrates the potential of conformational fingerprinting as an interpretable biomarker for neuromuscular disorders.