Foreground Detection Analysis of Ultrasound Image Sequences Identifies Markers of Motor Neurone Disease across Diagnostically Relevant Skeletal Muscles.

Diagnosis of motor neurone disease (MND) includes detection of small, involuntary muscle excitations, termed fasciculations. There is need to improve diagnosis and monitoring of MND through provision of objective markers of change. Fasciculations are visible in ultrasound image sequences. However, few approaches that objectively measure their occurrence have been proposed; their performance has been evaluated in only a few muscles; and their agreement with the clinical gold standard for fasciculation detection, intramuscular electromyography, has not been tested. We present a new application of adaptive foreground detection using a Gaussian mixture model (GMM), evaluating its accuracy across five skeletal muscles in healthy and MND-affected participants. The GMM provided good to excellent accuracy with the electromyography ground truth (80.17%-92.01%) and was robust to different ultrasound probe orientations. The GMM provides objective measurement of fasciculations in each of the body segments necessary for MND diagnosis and hence could provide a new, clinically relevant disease marker.

Real-Time Ultrasound Segmentation, Analysis and Visualisation of Deep Cervical Muscle Structure.

Despite widespread availability of ultrasound and a need for personalised muscle diagnosis (neck/back pain-injury, work related disorder, myopathies, neuropathies), robust, online segmentation of muscles within complex groups remains unsolved by existing methods. For example, Cervical Dystonia (CD) is a prevalent neurological condition causing painful spasticity in one or multiple muscles in the cervical muscle system. Clinicians currently have no method for targeting/monitoring treatment of deep muscles. Automated methods of muscle segmentation would enable clinicians to study, target, and monitor the deep cervical muscles via ultrasound. We have developed a method for segmenting five bilateral cervical muscles and the spine via ultrasound alone, in real-time. Magnetic Resonance Imaging (MRI) and ultrasound data were collected from 22 participants (age: 29.0±6.6, male: 12). To acquire ultrasound muscle segment labels, a novel multimodal registration method was developed, involving MRI image annotation, and shape registration to MRI-matched ultrasound images, via approximation of the tissue deformation. We then applied polynomial regression to transform our annotations and textures into a mean space, before using shape statistics to generate a texture-to-shape dictionary. For segmentation, test images were compared to dictionary textures giving an initial segmentation, and then we used a customized Active Shape Model to refine the fit. Using ultrasound alone, on unseen participants, our technique currently segments a single image in [Formula: see text] to over 86% accuracy (Jaccard index). We propose this approach is applicable generally to segment, extrapolate and visualise deep muscle structure, and analyse statistical features online.

Blunted angiogenesis and hypertrophy are associated with increased fatigue resistance and unchanged aerobic capacity in old overloaded mouse muscle.

We hypothesize that the attenuated hypertrophic response in old mouse muscle is (1) partly due to a reduced capillarization and angiogenesis, which is (2) accompanied by a reduced oxidative capacity and fatigue resistance in old control and overloaded muscles, that (3) can be rescued by the antioxidant resveratrol. To investigate this, the hypertrophic response, capillarization, oxidative capacity, and fatigue resistance of m. plantaris were compared in 9- and 25-month-old non-treated and 25-month-old resveratrol-treated mice. Overload increased the local capillary-to-fiber ratio less in old (15 %) than in adult (59 %) muscle (P < 0.05). Although muscles of old mice had a higher succinate dehydrogenase (SDH) activity (P < 0.05) and a slower fiber type profile (P < 0.05), the isometric fatigue resistance was similar in 9- and 25-month-old mice. In both age groups, the fatigue resistance was increased to the same extent after overload (P < 0.01), without a significant change in SDH activity, but an increased capillary density (P < 0.05). Attenuated angiogenesis during overload may contribute to the attenuated hypertrophic response in old age. Neither was rescued by resveratrol supplementation. Changes in fatigue resistance with overload and aging were dissociated from changes in SDH activity, but paralleled those in capillarization. This suggests that capillarization plays a more important role in fatigue resistance than oxidative capacity.

Validation of a New Semi-Automated Technique to Evaluate Muscle Capillarization.

The method of capillary domains has often been used to study capillarization of skeletal and heart muscle. However, the conventional data processing method using a digitizing tablet is an arduous and time-consuming task. Here we compare a new semi-automated capillary domain data collection and analysis in muscle tissue with the standard capillary domain method. The capillary density (1481±59 vs. 1447±54 caps mm(-2); R2:0.99; P<0.01) and heterogeneity of capillary spacing (0.085±0.002 vs. 0.085±0.002; R2:0.95; P<0.01) were similar in both methods. The fiber cross-sectional area correlated well between the methods (R2:0.84; P<0.01) and did not differ significantly (~8% larger in the old than new method at P=0.08). The latter was likely due to differences in outlining the contours between the two methods. In conclusion, the semi-automated method gives quantitatively and qualitatively similar data as the conventional method and saves a considerable amount of time.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Improved yield of a ligand-binding GPCR expressed in E. coli for structural studies.

The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in Escherichia coli as a functional, ligand-binding receptor, but yields of purified receptor are still low for comprehensive structural studies. Here, several approaches have been examined to optimize the yields of active, ligand-binding receptor. Optimisation of E. coli strain and induction protocol yielded a significant improvement in expression of active receptor. Expression of the receptor in BL21(DE3) cells, in combination with autoinduction improved expression 10-fold when compared with previously reported expression protocols using IPTG-mediated induction in DH5alpha cells. Optimization of the purification protocol revealed that supplementation of buffers with phospholipids enhanced recovery of active receptor. The methods examined are potentially applicable to other GPCRs expressed in E. coli.

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance.

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.