Evaluation of driving posture for a competition wheelchair based on the relationship between physical load and manipulation force

Engineering of Sport 15 - Proceedings from the 15th International Conference on the Engineering of Sport (ISEA 2024) Competition wheelchairs are required to have various functions, such as quick turning, sudden starting and braking, and high speed driving. One of the previous studies on injuries in wheelchair athletes showed that the relationship between muscle weakness and shoulder pain was due to atrophy of the muscles around the scapula. If it would be possible to evaluate wheelchairs in view of the physical load, a suitable wheelchair may be individually selected for the athlete, and may lead to the reduction of injury risk. A method using the functional evaluation of effective muscle strength has been proposed as an example of evaluation for sports equipment and human exercise focusing on muscle characteristics. It was claimed that the method can evaluate output force of a limb and the mechanical properties of the muscles based on the functional role of the muscles. In this study, the method was applied to the wheelchair operation to calculate an effective muscle manipulative force (EMMF) using the muscle force estimated from the inverse dynamics analysis. This study also investigated the relationship between the EMMF and the physical force as an attempt to evaluate the driving posture which depends on the design parameters of the wheelchair. </p

Validation of the identity and purity of CoASH and acetyl CoA peaks detected in <i>Xenopus</i> embryo extracts.

<p>(A) 30 stage 8/9 embryos were extracted with 150 µl 5% PCA and the PCA soluble fraction was neutralised with TEA/K₂CO₃. CoA compounds in neutralised PCA extracts were analysed by HPLC before and after KOH treatment as described in the Materials and Methods section. KOH treatment caused conversion of peaks corresponding to succinyl CoA and acetyl CoA, to free CoASH. Retention times (in minutes): CoASH, 5.15; succinyl CoA, 12.13; acetyl CoA, 15.17. (B) 30 stage 8/9 embryos were extracted with 150 µl 5% PCA and the PCA soluble fraction was neutralised with TEA/K₂CO₃. The neutralised extract was analysed by HPLC without further treatment (top panel), or after incubation for 10 min at 30°C with 0.11 mM oxaloacetate (OA) and citrate synthase (CS) (middel panel), or with 60 mM KCl, 9.4 mM acetyl phosphate (AP), and phosphotransacetylase (PTA) (bottom panel). Incubation of <i>Xenopus</i> extract with citrate synthase, in the presence of oxaloacetate, caused complete disappearance of the peak corresponding to acetyl CoA and an increase in the size of the peak corresponding to CoASH (peak areas before treatment: CoASH, 9337.25; succinyl CoA, 11626; acetyl CoA, 6678. Peak areas after treatment: CoASH, 18217.5; succinyl CoA, 11252.25; acetyl CoA, no peak detected). Conversely, incubation with phosphotransacetylase, in the presence of acetyl phosphate, completely removed the peak corresponding to CoASH and increased the size of the acetyl CoA peak (peak areas after treatment: CoASH, not detected; succinyl CoA, 9278.5; acetyl CoA, 15213.5). These results confirm unequivocally the identity of the CoASH and acetyl CoA peaks and that there are no other compounds co-eluting with these peaks that absorb at 254 nm. Retention times in minutes: top panel; CoASH, 5.17; succinyl CoA, 12.26; acetyl CoA, 15.51; middle panel; CoASH, 5.24; succinyl CoA, 12.43; acetyl CoA, not detected; bottom panel; CoASH, not detected; succinyl CoA, 12.12; acetyl CoA, 15.36.</p

Chromatographic separation of CoA standards and a PCA extract of <i>Xenopus</i> embryos.

<p>(A) HPLC chromatogram illustrating separation of CoA compounds. Standards of CoASH, CoA thioesters and adenosine (20–50 pmol each) were separated on a C18 column (Kinetex C18 100 X 4.60 mm column with 2.6 µm particle size and 100 Å pore size) using a mobile phase consisting of 150 mM Na₂H₂PO₄ and 9% methanol and a flow rate of 0.8 ml/min (see Materials and Methods for details). Standards were made up in mobile phase, which additionally contained 5 mM EDTA and 10 mM TCEP before injection. CoA compounds were detected by absorbance at 254 nm. Retention times (in minutes): malonyl CoA, 3.21; adenosine, 4.16; CoASH, 5.06; methylmalonyl CoA, 7.26; dephospho CoA, 10.7; succinyl CoA, 11.87; HMG/acetoacetyl CoA, 13.96; acetyl CoA, 14.83. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097693#pone.0097693.s005" target="_blank">Table S1</a> for day-to-day variability in retention times. (B) Graphs showing linearity between the peak area and the amount of CoASH and acetyl CoA injected. Different amounts of CoASH and acetyl CoA standards were injected and the peak areas were determined by Borwin chromatography software. Each data point represents the mean of duplicate measurements. The limit of detection (LOD) for each compound was determined to be 5 pmol (100 nM). (C) Representative chromatogram showing separation of a PCA extract of stage 8/9 <i>Xenopus</i> embryos. CoA peaks were identified by comparison of retention times with those of authentic standards determined on the same day. Peaks corresponding to CoASH, succinyl CoA, HMG/acetoacetyl CoA and acetyl CoA were detected. Retention times (in minutes): CoASH, 5.21; succinyl CoA, 12.32; HMG/acetoacetyl CoA, 14.44; acetyl CoA, 15.43.</p

Changes in Acetyl CoA Levels during the Early Embryonic Development of <i>Xenopus laevis</i>

<div><p>Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change <i>in vivo</i> during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of <i>Xenopus laevis</i> using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis.</p></div

Whole-embryo acetylation levels of core histones during early <i>Xenopus</i> embryonic development.

<p>20 embryos collected at different stages of development were lysed in 80 µl lysis buffer and approximately 40 µg of soluble protein was separated by SDS-PAGE, transferred to PVDF membranes and probed with either site-specific anti-acetyl histone antibodies or total anti-histone antibodies. Total and acetylated histones were determined on separate membranes. Representative blots are shown. The graphs show means +/− SEM of normalised band intensity/size obtained from 3 independent fertilisations. The band intensity/size of total and acetylated histones at each stage is expressed relative to the mean of all the stages analysed.</p

Microinjection of acetyl CoA to stage one embryo increases protein acetylation levels.

<p>Immediately after dejellying (30–45 minutes after fertilisation), stage one embryos were microinjected with increasing amounts of acetyl CoA, as described in the Materials and Methods section. Control embryos were injected with water. Injected embryos were allowed to progress to stage 3 and collected for HPLC analysis of CoA compounds or Western blot analysis with anti-acetyl-lysine antibody. For both analyses, 10 injected embryos were used per condition. In addition, embryos were collected after reaching stage 9. Microinjection of acetyl CoA dose dependently increased acetylation levels of several proteins (labelled 1–4). Equal protein loading was confirmed by Ponceau staining of the blot. A section of the Ponceau stained membrane is shown (the complete membrane is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097693#pone.0097693.s004" target="_blank">Figure S4</a>). For the Western blot shown, linear contrast and brightness adjustment was applied uniformly to the whole blot for clarity. The intensity/size of each of the bands numbered 1–4 was quantified and expressed relative to control embryos.</p

<i>PANK2</i> mutations result in increased rate of ROS production, depletion of cellular GSH and higher oxidative stress.

<p>A) Representative images of glutathione levels in control and patient-derived neurons measured by monochlorobimane (MCB) fluorescence in live cells. B) Quantification of MCB fluorescence in iPSCs and neuronal cells showed reduced glutathione levels in patient-derived cells and lower levels in iPSCs, (Bⁱⁱ shows pooled data). C) Basal rates of ROS production were quantified in neurons using the dihydroethidium assay. D) Quantification showed an increased rate of ROS production in patient cells compared to controls and in all cell lines in response to 30 minutes pre-incubation with iron chelator DFO (Dⁱⁱ depicts pooled data from Dⁱ). E) The level of lipid peroxidation was quantified in the neuronal cells using the ratiometric dye BODIPY C11 and two of the patient-derived neuronal cells displayed significantly higher levels of lipid peroxidation (pooled data shown in Eⁱⁱ). Scale bar represents 20 μm. Significance was calculated via one way ANOVA with post-hoc Tukey’s HSD correction for multiple comparisons *p<0.05, **p<0.01, ***p<0.001, ns not significant.</p

Primer sequences, amplicon size and TD-PCR programmes for gDNA Sanger sequencing of PANK2-mutations.

<p>Detailed Sanger sequencing protocol and cycling conditions upon request.</p

Overview of clinical and genetic features of atypical PKAN cases.

<p>PANK2 transcript: ENST00000316562.</p

Analysis of the neuronal iron content.

<p>Neuronal pellets were analyzed for total iron content by ICP-MS in basal conditions and after 16 hours of FAC media supplementation. A) Individual data shown for each biological replicate and B) shows mean results for control lines versus patient-derived neuronal pellets. Control and patient-derived neurons displayed similar levels of basal iron content as well as similar storage of excess iron after iron stress. Numbers in histograms represent experimental replicates.</p