Ranking metabolite sets by their activity levels

Related metabolites can be grouped into sets in many ways, e.g., by their participation in series of chemical reactions (forming metabolic pathways), or based on fragmentation spectral similarities or shared chemical substructures. Understanding how such metabolite sets change in relation to experimental factors can be incredibly useful in the interpretation and understanding of complex metabolomics data sets. However, many of the available tools that are used to perform this analysis are not entirely suitable for the analysis of untargeted metabolomics measurements. Here, we present PALS (Pathway Activity Level Scoring), a Python library, command line tool, and Web application that performs the ranking of significantly changing metabolite sets over different experimental conditions. The main algorithm in PALS is based on the pathway level analysis of gene expression (PLAGE) factorisation method and is denoted as mPLAGE (PLAGE for metabolomics). As an example of an application, PALS is used to analyse metabolites grouped as metabolic pathways and by shared tandem mass spectrometry fragmentation patterns. A comparison of mPLAGE with two other commonly used methods (overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA)) is also given and reveals that mPLAGE is more robust to missing features and noisy data than the alternatives. As further examples, PALS is also applied to human African trypanosomiasis, Rhamnaceae, and American Gut Project data. In addition, normalisation can have a significant impact on pathway analysis results, and PALS offers a framework to further investigate this. PALS is freely available from our project Web site

Characterising the evidence base for advanced clinical practice in the UK: a scoping review protocol

Copyright © Author(s) (or their employer(s)) 2020. Introduction A global health workforce crisis, coupled with ageing populations, wars and the rise of non-communicable diseases is prompting all countries to consider the optimal skill mix within their health workforce. The development of advanced clinical practice (ACP) roles for existing non-medical cadres is one potential strategy that is being pursued. In the UK, National Health Service (NHS) workforce transformation programmes are actively promoting the development of ACP roles across a wide range of non-medical professions. These efforts are currently hampered by a high level of variation in ACP role development, deployment, nomenclature, definition, governance and educational preparation across the professions and across different settings. This scoping review aims to support a more consistent approach to workforce development in the UK, by identifying and mapping the current evidence base underpinning multiprofessional advanced level practice in the UK from a workforce, clinical, service and patient perspective. Methods and analysis This scoping review is registered with the Open Science Framework (https://osf.io/tzpe5). The review will follow Joanna Briggs Institute guidance and involves a multidisciplinary and multiprofessional team, including a public representative. A wide range of electronic databases and grey literature sources will be searched from 2005 to the present. The review will include primary data from any relevant research, audit or evaluation studies. All review steps will involve two or more reviewers. Data extraction, charting and summary will be guided by a template derived from an established framework used internationally to evaluate ACP (the Participatory Evidence-Informed Patient-Centred Process-Plus framework). Dissemination The review will produce important new information on existing activity, outcomes, implementation challenges and key areas for future research around ACP in the UK, which, in the context of global workforce transformations, will be of international, as well as local, significance. The findings will be disseminated through professional and NHS bodies, employer organisations, conferences and research papers.Health Education England (DN384826— Evaluation for HEE ACP Programme—Current Evidence Based for Advanced Level Practice within Health and Related Environments)

CFTR founder mutation causes protein trafficking defects in Chinese patients with cystic fibrosis

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Characterising the outcomes, impacts and implementation challenges of advanced clinical practice roles in the UK: A scoping review

Copyright © Author(s) (or their employer(s)) 2021. Objectives In response to demographic and health system pressures, the development of non-medical advanced clinical practice (ACP) roles is a key component of National Health Service workforce transformation policy in the UK. This review was undertaken to establish a baseline of evidence on ACP roles and their outcomes, impacts and implementation challenges across the UK. Design A scoping review was undertaken following JBI methodological guidance. Methods 13 online databases (Medline, CINAHL, ASSIA, Embase, HMIC, AMED, Amber, OT seeker, PsycINFO, PEDro, SportDiscus, Osteopathic Research and PenNutrition) and grey literature sources were searched from 2005 to 2020. Data extraction, charting and summary was guided by the PEPPA-Plus framework. The review was undertaken by a multi-professional team that included an expert lay representative. Results 191 papers met the inclusion criteria (any type of UK evidence, any sector/setting and any profession meeting the Health Education England definition of ACP). Most papers were small-scale descriptive studies, service evaluations or audits. The papers reported mainly on clinical aspects of the ACP role. Most papers related to nursing, pharmacy, physiotherapy and radiography roles and these were referred to by a plethora of different titles. ACP roles were reported to be achieving beneficial impacts across a range of clinical and health system outcomes. They were highly acceptable to patients and staff. No significant adverse events were reported. There was a lack of cost-effectiveness evidence. Implementation challenges included a lack of role clarity and an ambivalent role identity, lack of mentorship, lack of continuing professional development and an unclear career pathway. Conclusion This review suggests a need for educational and role standardisation and a supported career pathway for advanced clinical practitioners (ACPs) in the UK. Future research should: (i) adopt more robust study designs, (ii) investigate the full scope of the ACP role and (iii) include a wider range of professions and sectors.Health Education England (DN384826— Evaluation for HEE ACP Programme—Current Evidence Based for Advanced Level Practice within Health and Related Environments)

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity

The Trypanosoma cruzi Virulence Factor Oligopeptidase B (OPBTc) Assembles into an Active and Stable Dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca2+-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein