Locomotor adaptation to a powered ankle-foot orthosis depends on control method

<p>Abstract</p> <p>Background</p> <p>We studied human locomotor adaptation to powered ankle-foot orthoses with the intent of identifying differences between two different orthosis control methods. The first orthosis control method used a footswitch to provide bang-bang control (a kinematic control) and the second orthosis control method used a proportional myoelectric signal from the soleus (a physiological control). Both controllers activated an artificial pneumatic muscle providing plantar flexion torque.</p> <p>Methods</p> <p>Subjects walked on a treadmill for two thirty-minute sessions spaced three days apart under either footswitch control (n = 6) or myoelectric control (n = 6). We recorded lower limb electromyography (EMG), joint kinematics, and orthosis kinetics. We compared stance phase EMG amplitudes, correlation of joint angle patterns, and mechanical work performed by the powered orthosis between the two controllers over time.</p> <p>Results</p> <p>During steady state at the end of the second session, subjects using proportional myoelectric control had much lower soleus and gastrocnemius activation than the subjects using footswitch control. The substantial decrease in triceps surae recruitment allowed the proportional myoelectric control subjects to walk with ankle kinematics close to normal and reduce negative work performed by the orthosis. The footswitch control subjects walked with substantially perturbed ankle kinematics and performed more negative work with the orthosis.</p> <p>Conclusion</p> <p>These results provide evidence that the choice of orthosis control method can greatly alter how humans adapt to powered orthosis assistance during walking. Specifically, proportional myoelectric control results in larger reductions in muscle activation and gait kinematics more similar to normal compared to footswitch control.</p

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle

An Early Health Economic Analysis of the Potential Cost Effectiveness of an Adherence Intervention to Improve Outcomes for Patients with Cystic Fibrosis

Background Cystic fibrosis (CF) negatively impacts upon health-related quality of life and survival. Adherence to nebulised treatments is low; improving adherence is hypothesised to reduce rates of exacerbation requiring intravenous antibiotics and lung function decline. Objective A state transition model was developed to assess the cost effectiveness of an intervention aimed at increasing patient adherence to nebulised and inhaled antibiotics compared with current CF care, in advance of the forthcoming CFHealthHub randomised controlled trial (RCT). Methods The model estimated the costs and health outcomes for each option from the perspective of the UK National Health Service and Personal Social Services over a lifetime horizon. Health gains were valued in terms of quality-adjusted life-years (QALYs) gained. Forced expiratory volume in 1 second (FEV1) trajectories were predicted over three lung function strata: (1) FEV1 ≥70%, (2) FEV1 40–69% and (3) FEV1 <40%. Additional states were included to represent ‘post-lung transplantation’ and ‘dead’. The model was populated using CF Registry data, literature and expert opinion. Costs were presented at 2016 values. Uncertainty was assessed using deterministic and probabilistic sensitivity analyses. Results If effective, the adherence intervention is expected to produce an additional 0.19 QALYs and cost savings of £64,078 per patient. Across all analyses, the intervention dominated current care. Over a 5-year period, the intervention is expected to generate cost savings of £49.5 million for the estimated 2979 patients with CF with Pseudomonas aeruginosa currently aged ≥16 years in the UK. If applied to a broader population of adult patients with CF receiving any nebulised therapy, the expected savings could be considerably greater. Conclusions If effective, the adherence intervention is expected to produce additional health gains at a lower cost than current CF care. However, the economic analysis should be revisited upon completion of the full RCT. More generally, the analysis suggests that considerable gains could be accrued through the implementation of adherence interventions that shift care from expensive hospital-based rescue to community-based prevention

ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin−Sca-1+CD49fhigh (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells

Feasibility and acceptance of electronic quality of life assessment in general practice: an implementation study

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