Development of a test methodology for the assessment of human impacts in sport

The study described in this paper aims to develop a suitable method for the measurement of contact forces, pressures and velocities of simulated human-on-human impacts typical of those experienced within American Football. A thin-film pressure sensor system was chosen to enable the impacts to be quantified, however, initial testing suggested that the measured impact forces were underestimated by circa 30% with the system calibrated in the standard, static pressure manner. A two-stage, dynamic calibration was therefore developed, in which the sensors were subsequently dynamically loaded in a manner more representative of the impacts, allowing an appropriate dynamic calibration factor to be derived. To determine the typical impact force levels experienced in a shoulder-on-thigh impact event, eight subjects were required to perform three "good" tackles at two different velocities. The processed results identified a peak, transmitted force of 1.1 (0.4) and 1.7 (0.5) kN for "low" and "medium" velocities respectively, with corresponding effective areas of application of 70 (22) cm2 and 85 (25) cm2 and contact times of 0.257 (0.098) s and 0.245 (0.112) s respectively. © 2012 Published by Elsevier Ltd

Angelman Syndrome: From Mouse Models to Therapy

The UBE3A gene is part of the chromosome 15q11-q13 region that is frequently deleted or duplicated, leading to several neurodevelopmental disorders (NDD). Angelman syndrome (AS) is caused by the absence of functional maternally derived UBE3A protein, while the paternal UBE3A gene is present but silenced specifically in neurons. Patients with AS present with severe neurodevelopmental delay, with pronounced motor deficits, absence of speech, intellectual disability, epilepsy, and sleep problems. The pathophysiology of AS is still unclear and a treatment is lacking. Animal models of AS recapitulate the genotypic and phenotypic features observed in AS patients, and have been invaluable for understanding the disease process as well as identifying apropriate drug targets. Using these AS mouse models we have learned that loss of UBE3A probably affects many areas of the brain, leading to increased neuronal excitability and a loss of synaptic spines, along with changes in a number of distinct behaviours. Inducible AS mouse models have helped to identify the critical treatment windows for the behavioral and physiological phenotypes. Additionally, AS mouse models indicate an important role for the predominantly nuclear UBE3A isoform in generating the characteristic AS pathology. Last, but not least, the AS mice have been crucial in guiding Ube3a gene reactivation treatments, which present a very promising therapy to treat AS

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance: A K-ε-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events

A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community (https://www.angelman-proteome-project.org/)

Assessing the requirements of prenatal UBE3A expression for rescue of behavioral phenotypes in a mouse model for Angelman syndrome

Background: Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of functional ubiquitin protein ligase E3A (UBE3A). In neurons, UBE3A expression is tightly regulated by a mechanism of imprinting which suppresses the expression of the paternal UBE3A allele. Promising treatment strategies for AS are directed at activating paternal UBE3A gene expression. However, for such strategies to be successful, it is important to know when such a treatment should start, and how much UBE3A expression is needed for normal embryonic brain development. Methods: Using a conditional mouse model of AS, we further delineated the critical period for UBE3A expression during early brain development. Ube3a gene expression was induced around the second week of gestation and mouse phenotypes were assessed using a behavioral test battery. To investigate the requirements of embryonic UBE3A expression, we made use of mice in which the paternal Ube3a allele was deleted. Results: We observed a full behavioral rescue of the AS mouse model phenotypes when Ube3a gene reactivation was induced around the start of the last week of mouse embryonic development. We found that full silencing of the paternal Ube3a allele was not completed till the first week after birth but that deletion of the paternal Ube3a allele had no significant effect on the assessed phenotypes. Limitations: Direct translation to human is limited, as we do not precisely know how human and mouse brain development aligns over gestational time. Moreover, many of the assessed phenotypes have limited translational value, as the underlying brain regions involved in these tasks are largely unknown. Conclusions: Our findings provide further important insights in the requirement of UBE3A expression during brain development. We found that loss o

Comparison of standing postural control and gait parameters in people with and without chronic low back pain: A cross-sectional case-control study

Objective Differences in postural control and gait have been identified between people with and without chronic low back pain (CLBP); however, many previous studies present data from small samples, or have used methodologies with questionable reliability. This study, employing robust methodology, hypothesised that there would be a difference in postural control, and spatiotemporal parameters of gait in people with CLBP compared with asymptomatic individuals. Methods This cross-sectional case-control study age-matched and gender-matched 16 CLBP and 16 asymptomatic participants. Participants were assessed barefoot (1) standing, over three 40 s trials, under four posture challenging conditions (2) during gait. Primary outcome was postural stability (assessed by root mean squared error of centre of pressure (CoP) displacement (CoP RMSEAP) and mean CoP velocity (CoP VELAP), both in the anteroposterior direction); gait outcomes were hip range of movement and peak moments, walking speed, cadence and stride length, assessed using force plates and a motion analysis system. Results There were no differences between groups in CoP RMSEAP (P=0.26), or CoP VELAP (P=0.60) for any standing condition. During gait, no differences were observed between groups for spatiotemporal parameters, maximum, minimum and total ranges of hip movement, or peak hip flexor or extensor moments in the sagittal plane. Conclusions In contrast to previous research, this study suggests that people with mild to moderate CLBP present with similar standing postural control, and parameters of gait to asymptomatic individuals. Treatments directed at influencing postural stability (eg, standing on a wobble board) or specific parameters of gait may be an unnecessary addition to a treatment programme.Masai G