Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly.

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

Perceived benefits, rationale and preferences of exercises utilized within Pilates group exercise programmes for people with chronic musculoskeletal conditions: A questionnaire of Pilates-trained physiotherapists.

INTRODUCTION: An estimated 29% of the population, around 17.8 million people, live with a musculoskeletal condition in the UK. Pilates exercise has positive benefits, including reducing pain and disability for people with musculoskeletal conditions. The aim of the present study was to explore the views of Pilates-trained physiotherapists in relation to the perceived benefits of, and clinical reasoning for, exercise prescription in Pilates classes. METHODS: A qualitative approach was taken to both data collection and analysis, using a thematic framework. Data were collected via electronic questionnaires using open-ended questions. Participants included 15 Pilates-trained physiotherapists who regularly ran Pilates exercise classes within seven private physiotherapy clinics for people with a range of chronic musculoskeletal conditions. RESULTS: The results were organized into six main themes: Perceived benefits 1: Improved function and increased levels of activity. Perceived benefits 2: Improved ability to manage their musculoskeletal condition autonomously; Efficacy of group Pilates exercise; Optimum combination of exercises used within a class; Physiotherapist rationale for the most effective exercises; Precautions with specific exercises. CONCLUSIONS: The study was the first to investigate perceived benefits, rationale and preference for choice of exercise among Pilates-trained physiotherapists. Physiotherapists felt that the Pilates classes facilitated an active lifestyle and self-management approach. They used a combination of exercises in each class to address all the main muscle groups, but some had specific exercise preferences related to the patients' needs. Exercises were linked to evidence around neuromuscular control, direction preference and biomechanical principles.

Early observations of the effects of lateral wedge orthoses on lower limb muscle length and potential for exacerbating spasticity.

Lateral wedge orthoses are often prescribed to correct varus deformity after stroke. Spasticity is implicated in varus deformity and is caused by velocity-related muscle length changes, so a lateral wedge may affect spasticity by manipulating foot and ankle kinematics which, in turn, may alter the length of these muscles. We sought to test this theory in healthy participants. Eight volunteers walked with no wedge and with lateral wedges of 5 and 8.5 degrees in both shoes. Qualysis motion capture system collected kinematic data and SIMM musculoskeletal modeling software calculated muscle tendon length of plantarflexor and peroneal muscle groups using 3-D ankle and knee joint angle data. The wedges increased ankle eversion (p < 0.0001) and total excursion of tibialis posterior, peroneus longus and brevis by 13-29% (p < 0.05). Muscle length of peroneus longus and brevis increased by <1% (p < 0.005). Potentially clinical meaningful effects were found for tibialis posterior (15%), peroneus brevis (23%) and peroneus tertius (13%). Further research is required to be conclusive and to explore the effects of lateral wedge orthoses in patients with stroke. If such changes are seen in people with stroke, a change in orthotic prescription practice could be indicated as lateral wedge orthoses may exacerbate spasticity.

Magnetic resonance imaging overestimates ferumoxide-labeled stem cell survival after transplantation in the heart.

BACKGROUND: Stem cell labeling with iron oxide (ferumoxide) particles allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment. However, the validity of MRI for distinguishing surviving ferumoxide-labeled cells from other sources of MRI signal, for example, macrophages containing ferumoxides released from nonsurviving cells, has not been thoroughly investigated. We sought to determine the relationship between the persistence of iron-dependent MRI signals and cell survival 3 weeks after injection of syngeneic or xenogeneic ferumoxides-labeled stem cells (cardiac-derived stem cells) in rats. METHODS AND RESULTS: We studied nonimmunoprivileged human and rat cardiac-derived stem cells and human mesenchymal stem cells doubly labeled with ferumoxides and beta-galactosidase and injected intramyocardially into immunocompetent Wistar-Kyoto rats. Animals were imaged at 2 days and 3 weeks after stem cell injection in a clinical 3-T MRI scanner. At 2 days, injection sites of xenogeneic and syngeneic cells (cardiac-derived stem cells and mesenchymal stem cells) were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks (50% to 90% of initial signal). Histology (at 3 weeks) revealed the presence of iron-containing macrophages at the injection site, identified by CD68 staining, but very few or no beta-galactosidase-positive stem cells in the animals transplanted with syngeneic or xenogeneic cells, respectively. CONCLUSIONS: The persistence of significant iron-dependent MRI signal derived from ferumoxide-containing macrophages despite few or no viable stem cells 3 weeks after transplantation indicates that MRI of ferumoxide-labeled cells does not reliably report long-term stem cell engraftment in the heart.

An inverse method for the recovery of tissue parameters from colour images.

The interpretation of colour images is presented as an inverse problem in which a mapping is sought between image colour vectors and the physiological parameters characterizing a tissue. To ensure the necessary one-to-one correspondence between the image colours and the parameters, the mapping must be unique. This can be established through testing the sign of the determinant of the Jacobian matrix, a multi-dimensional equivalent of a discrete derivative, over the space of all parameter values. Furthermore, an optimisation procedure is employed to find the set of filters for image capture which generate image vectors minimizing the mapping error. This methodology applied to interpretation of skin images shows that the standard RGB system of filters provides for a unique mapping between image values and parameters characterizing the normal skin. It is further shown that an optimal set of filters reduces the error of quantification by a factor of 2, on average.

Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock tissues.

Ractopamine HCl is a beta-adrenergic agonist (beta-agonist) recently approved by the U.S. Food and Drug Administration, but not other governmental agencies, for use in finishing swine. For these reasons, it was important to develop and validate mass spectrometric methods for the detection and confirmation of ractopamine residues in livestock marker tissues. Incurred tissues in cattle, sheep, turkeys, and ducks were generated during 7-day ractopamine feeding (20 ppm in diets) periods. Disposition of ractopamine residues in liver and pigmented retinal epithelium was determined in animals slaughtered with withdrawal periods of 0, 3, and 7 days. Ractopamine residues, purified using solid-phase extraction, were measured using liquid chromatography (LC) and electrospray with detection by tandem mass spectrometry (MS/MS) in the multiple reaction-monitoring (MRM) mode. Total ractopamine residues (parent ractopamine + hydrolyzed conjugates) in liver were detected in all species on withdrawal day 0 (2-97 ppb) and were greatly diminished in all species by withdrawal day 7 (<1 ppb). Bovine and ovine retina had lower levels of ractopamine (0.5-3 ppb) than liver, and occular residues increased with withdrawal time, suggesting redistribution into this tissue. Lower limits of quantification were found to be approximately 0.1 ppb in liver and retina. Incurred ractopamine residues were confirmed by the precise and accurate agreement of MRM intensity ratios of diagnostic fragment ions (m/z 284, 164, and 121) from the protonated molecule between ractopamine residues in incurred samples and an authentic ractopamine standard. The limits of confirmation in liver and retina using recognized acceptance criteria were below 1 ppb. The high sensitivity and specificity for measurement and confirmation of ractopamine residues suggests this method will be applicable for regulatory residue surveillance programs.