Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

The adsorption of chelating reagents on oxide minerals

This work constitutes a fundamental study of the interaction between chelating reagents and oxide minerals . The adsorption mechanisms have been elucidated for most of the systems generated by the oxides of copper(II) or iron(III) and chelating reagents octyl hydroxamate, N-phenylbenzohydroxamate, salicylaldoxime, 5-nitrosalicylaldoxime or 8-hydroxyquinoline. The results of the preliminary work on one of the systems, viz. the oxide-hydroxamate system, indicated that the classical type adsorption process, in which the reagent forms a uniform layer of chelate over the oxide surface was not applicable. Rather, the adsorption occurred via the formation of a discrete metal-chelate precipitate at the oxide surface. In order to better understand the associated with copper (II) oxide, adsorption process the oxide was recrystallized to produce a coarser material with a more uniform surface. This allowed the oxide surface to be viewed under the scanning electron microscope and also enabled the relative concentration of "surface" and "bulk" chelate to be assessed. A detailed investigation of the effect of the system variables; pH, conditioning period, concentration, temperature, surface area and dispersing reagent on the rate of precipitation of the copper chelate species of general form, Cu(chel)2' was made. In addition the chemical nature of the adsorbed species and the structural form of the precipitates were determined with the aid of infra-red spectroscopy and the scanning electron microscope. On the basis of these results a model has been formulated for the adsorption processes. In this model the adsorption dissolution, is considered to occur in stages: 1. Oxide dissolution, 2. metal complex formation, 3. Metal chelate precipitation at the oxide surface and 4. “bulk” chelate formation by post-precipitation processes. The precipitation process was examined in more detail by the study of the adsorption of chelate on copper metal. The results of this study showed that it was possible to relate the structural type of precipitate formed, ie. fibrous or platelike, to the degree of supersaturation of the metal complex in solution. Furthermore, it was found that the precipitate structure determined whether it remained attached to the surface or detached. Contact angle measurements of air bubbles on copper metal conditioned with chelate were related to the adsorption results in an attempt to isolate the optimum conditions for flotation of oxide minerals

Separation and characterisation of chromium (III) carboxylate solutions

From Summary: The complexes present in chromium(III) carboxylate solutions (acetate or formate) were separated using ion exchange chromatography, electrophoresis and gel filtration. Eight cationic species and one non-ionic species with charges from 0 to +3 and nuclearities ranging from mononuclear to quadranuclear were isolated from chromium(III) acetate solutions. Six cationic species with charges from +1 to +3 and nuclearities ranging from mononuclear to trinuclear were isolated from chromium(III) formate solutions. The empirical formulae for these complexes were determined by analytical methods. Infra- red spectroscopy was used to distinguish between monodentate and bidentate bridged carboxylate ligands. An immediate change in the visible spectrum of a complex on its acidification was used as evidence that a terminal hydroxo group was present

A Comparative Analysis of Factors Influencing Spectatorship of Disability Sport: A Qualitative Inquiry and Next Steps

To effectively market a sport event, it is necessary to determine the factors that influence spectator attendance. While a developed line of inquiry exists on the marketing of able-bodied sports, a gap in the literature exists in the disability sport context. Thus, there is a need to examine the synergy between existing motives that have been confirmed in traditional sport versus those that are unique to disability sport. This line of inquiry will help future researchers determine whether additional factors should be considered when marketing disability sport. The purpose of this study was to qualitatively explore the explicit and tacit motives of spectator attendance at disability sport. Findings from the Quad Rugby Nationals indicate that while some of the traditional sport spectator motives in the literature are the same, there are also unique motives, which should be used to accurately measure spectator interest in this emerging context

Scaling-up an evidence-based intervention for osteoarthritis in real-world settings : a pragmatic evaluation using the RE-AIM framework

Scaling-up and sustaining effective healthcare interventions is essential for improving healthcare; however, relatively little is known about these processes. In addition to quantitative experimental designs, we need approaches that use embedded, observational studies on practice-led, naturally occurring scale-up processes. There are also tensions between having adequately rigorous systems to monitor and evaluate scale-up well that are proportionate and pragmatic in practice. The study investigated the scale-up of an evidence-based complex intervention for knee and hip osteoarthritis (ESCAPE-pain) within 'real-world' settings by England's 15 Academic Health Science Networks (AHSNs). A pragmatic evaluation of the scale-up of ESCAPE-pain using the RE-AIM framework to measure Reach, Effectiveness, Adoption, Implementation and Maintenance. The evaluation used routine monitoring data collected from April 2014 to December 2018 as part of a national scale-up programme. Between 2014 and 2018, ESCAPE-pain was adopted by over 110 clinical and non-clinical sites reaching over 9000 people with osteoarthritis. The programme showed sustained clinical effectiveness (pain, function and quality of life) and high levels of adherence (78.5% completing 75% of the programme) within a range of real-world settings. Seven hundred seventy people (physiotherapists and exercise professionals) have been trained to deliver ESCAPE-pain, and 84.1% of sites have continued to deliver the programme post-implementation. ESCAPE-pain successfully moved from being an efficacious "research intervention" into an effective intervention within 'real-world' clinical and non-clinical community settings. However, scale-up has been a gradual process requiring on-going, dedicated resources over 5 years by a national network of Academic Health Science Networks (AHSNs). Whilst the collection of monitoring and evaluation data is critical in understanding implementation and scale-up, there remain significant challenges in developing systems sufficiently rigorous, proportionate and locally acceptable

Does repetitive task training improve functional activity after stroke? A Cochrane systematic review and meta-analysis.

Repetitive task training resulted in modest improvement across a range of lower limb outcome measures, but not upper limb outcome measures. Training may be sufficient to have a small impact on activities of daily living. Interventions involving elements of repetition and task training are diverse and difficult to classify: the results presented are specific to trials where both elements are clearly present in the intervention, without major confounding by other potential mechanisms of action

Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation

ACKNOWLEDGMENTS We acknowledge Jennifer Lodge, Woei Lam, and Rajendra Upadhya for developing and sharing the chitin and chitosan MTBH assay. We thank Todd Brennan of Duke University for providing MyD88-deficient mice. We acknowledge Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We also acknowledge Connie Nichols for critical reading of the manuscript. These experiments were supported by an NIH grant to J.A.A. and F.L.W., Jr. (R01 AI074677). C.M.L.W. was supported by a fellowship provided through the Army Research Office of the Department of Defense (no. W911NF-11-1-0136 f) (F.L.W., Jr.). J.W., L.W., and C.M. were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the MRC, Centre for Medical Mycology (MR/N006364/1). FUNDING INFORMATION MRC Centre for Medical MycologyMR/N006364/1 Carol A. Munro HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060R01 AI074677J. Andrew Alspaugh Wellcome https://doi.org/10.13039/100010269097377 Carol A. Munro DOD | United States Army | RDECOM | Army Research Office (ARO) https://doi.org/10.13039/100000183W911NF-11-1-0136 f Chrissy M. Leopold WagerPeer reviewedPublisher PD

Genome selection in fruit breeding: application to table grapes

Genomic selection (GS) has recently been proposed as a new selection strategy which represents an innovative paradigm in crop improvement, now widely adopted in animal breeding. Genomic selection relies on phenotyping and high-density genotyping of a sufficiently large and representative sample of the target breeding population, so that the majority of loci that regulate a quantitative trait are in linkage disequilibrium with one or more molecular markers and can thus be captured by selection. In this study we address genomic selection in a practical fruit breeding context applying it to a breeding population of table grape obtained from a cross between the hybrid genotype D8909-15 (Vitis rupestris × Vitis arizonica/girdiana), which is resistant to dagger nematode and Pierce’s disease (PD), and ‘B90-116’, a susceptible Vitis vinifera cultivar with desirable fruit characteristics. Our aim was to enhance the knowledge on the genomic variation of agronomical traits in table grape populations for future use in marker-assisted selection (MAS) and GS, by discovering a set of molecular markers associated with genomic regions involved in this variation. A number of Quantitative Trait Loci (QTL) were discovered but this method is inaccurate and the genetic architecture of the studied population was better captured by the BLasso method of genomic selection, which allowed for efficient inference about the genetic contribution of the various marker loci. The technology of genomic selection afforded greater efficiency than QTL analysis and can be very useful in speeding up the selection procedures for agronomic traits in table grapes

Influence of commissioning arrangements on implementing and sustaining a complex healthcare intervention (ESCAPE-pain) for osteoarthritis : a qualitative case study

Funding in health care has a critical impact on the implementation and sustainability of evidence-based interventions. This study explored the perspectives of physiotherapists on the influence of commissioning arrangements on the implementation and sustainability of a group rehabilitation programme for osteoarthritis (ESCAPE-pain). A qualitative case study approach using in-depth interviews. National Health Service (NHS) musculoskeletal (MSK) outpatient departments in England. Thirty physiotherapists in clinical and senior management roles from 11 NHS MSK providers. Five themes were identified: (1) clinical perspectives of ESCAPE-pain - MSK services wanted to implement and sustain ESCAPE-pain because it provided evidence-based, quality care; (2) focusing on clinical activity over outcomes - commissioners were perceived as prioritising activity-based performance over delivering clinical outcomes; (3) rationing availability - patient access to ESCAPE-pain could be limited due to rationing resources; (4) absorbing costs - contracts did not always cover the activities associated with delivering ESCAPE-pain meaning that providers bore the costs; and (5) relationship between commissioners and providers - physiotherapists perceived a disconnect with commissioners and had little power to influence decisions. Commissioning arrangements for MSK physiotherapy services can impede providers from implementing and sustaining a clinically and cost-effective intervention. To be implemented and sustained, an intervention needs to integrate into clinical practice and the wider healthcare system. Commissioning arrangements for MSK physiotherapy need to allow providers the flexibility to deliver interventions that best meet the needs of their patients. The move to more strategic, integrated, outcome-based commissioning has the potential to facilitate the spread and sustainability of interventions. [Abstract copyright: Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

The role of leadership in implementing and sustaining an evidence-based intervention for osteoarthritis (ESCAPE-pain) in NHS physiotherapy services : a qualitative case study

To explore the role of leadership by physiotherapists in implementing and sustaining an evidence-based complex intervention (ESCAPE-pain) for osteoarthritis. A qualitative case study approach using in-depth interviews with 23 clinicians and managers from 4 National Health Service (NHS) physiotherapy providers in England between 2016 and 2017. Data were analysed using thematic analysis. Different leadership roles and actions were characterised with four themes: (1) Clinical champions - clinicians driving the sustainability of ESCAPE-pain; (2) Supporters - junior clinicians directly supporting clinical champions' efforts to sustain ESCAPE-pain; (3) Senior Manager - clinical champions' senior managers influence on sustainability; (4) Decision-making - (in)formal processes underpinning decisions to (not) sustain the programme. The study characterises the role of leadership in physiotherapy to sustain an evidence-based intervention for osteoarthritis (OA) within the NHS. Sustaining the intervention required on-going leadership, it did not stop at implementation. Senior specialist physiotherapists (as Champions) had a critical leadership role in driving sustainability. Their structural position (bridging the operational and strategic) and personal attributes allowed them to integrate different levels of leadership (i.e., senior managers and operational staff) to mobilise the collective, on-going work required for sustaining the programme. IMPLICATIONS FOR REHABILITATION Senior managers and clinicians in practice settings need to be aware that sustaining an intervention is an on-going, collective effort that continues post-implementation. Senior managers need to enable senior clinicians (who straddle strategic and operational functions) to have sufficient autonomy to access and mobilise resources and scope to restructure local systems and practice to support intervention sustainability. Operational staff need to be supported to have the practical know-how to deliver evidence-based intervention, which includes instilling the value of and a commitment for the interventions. Managers need to utilise dispersed leadership to empower and enthuse frontline clinicians to participate fully in the work to refine and sustain interventions, because it cannot be achieved by lone individuals