Multidisciplinary management of inducible laryngeal obstruction and breathing pattern disorder.

We provide an overview of the assessment and management of inducible laryngeal obstruction and breathing pattern disorder. We highlight the multidisciplinary team members involved and their essential roles within a complex breathlessness service. We discuss treatments initiated by physiotherapy and speech and language therapy, the importance of joint working, and discuss the high incidence of comorbidities and the association with other respiratory disorders. Educational aims: Inducible laryngeal obstruction and breathing pattern disorder are common causes of breathlessness.Inducible laryngeal obstruction is an inappropriate, transient, reversible narrowing of the laryngeal area that causes breathlessness and laryngeal symptoms.Breathing pattern disorder is an alteration in the normal biomechanical patterns of breathing that results in intermittent or chronic symptoms, which may be respiratory and/or non-respiratory.People with inducible laryngeal obstruction or breathing pattern disorder often have other comorbidities that will also need addressing.Multidisciplinary assessment and treatment is essential for comprehensive workup and holistic care.Timely assessment and diagnosis can prevent unnecessary medication use and hospital admissions and facilitate effective management of the condition using reassurance, advice, education, breathing retraining and vocal exercises.

APOLLO: A genome-scale metabolic reconstruction resource of 247,092 diverse human microbes spanning multiple continents, age groups, and body sites.

Computational modelling of microbiome metabolism has proved instrumental to catalyse our understanding of diet-host-microbiome-disease interactions through the interrogation of mechanistic, strain- and molecule-resolved metabolic models. We present APOLLO, a resource of 247,092 human microbial genome-scale metabolic reconstructions spanning 19 phyla and accounting for microbial genomes from 34 countries, all age groups, and five body sites. We explored the metabolic potential of the reconstructed strains and developed a machine learning classifier able to predict with high accuracy the taxonomic strain assignments. We also built 14,451 sample-specific microbial community models, which could be stratified by body site, age, and disease states. Finally, we predicted faecal metabolites enriched or depleted in gut microbiomes of people with Crohn's disease, Parkinson disease, and undernourished children. APOLLO is compatible with the human whole-body models, and thus, provide unprecedented opportunities for systems-level modelling of personalised host-microbiome co-metabolism. APOLLO will be freely available under https://www.vmh.life/.

Shared decision-making for people living with dementia in extended care settings: a systematic review.

BACKGROUND: Shared decision-making is recognised as an important element of person-centred dementia care. OBJECTIVES: The aim of this review was to explore how people living with dementia and cognitive impairment can be included in day-to-day decisions about their health and care in extended care settings. DESIGN: A systematic review including primary research relating to shared decision-making, with cognitively impaired adults in (or transferrable to) extended care settings. Databases searched were: CINAHL, PubMed, the Cochrane Library, NICE Evidence, OpenGrey, Autism Data, Google Scholar, Scopus and Medicines Complete (June to October 2016 and updated 2018) for studies published in the last 20 years. RESULTS: Of the 19 included studies 15 involved people with living dementia, seven in extended care settings. People living with cognitive impairment often have the desire and ability to participate in decision-making about their everyday care, although this is regularly underestimated by their staff and family care partners. Shared decision-making has the potential to improve quality of life for both the person living with dementia and those who support them. How resources to support shared decision-making are implemented in extended care settings is less well understood. CONCLUSIONS: Evidence suggests that people living with cognitive impairment value opportunities to be involved in everyday decision-making about their care. How these opportunities are created, understood, supported and sustained in extended care settings remains to be determined. TRIAL REGISTRATION NUMBER: CRD42016035919.

Shared decision-making for people living with dementia in extended care settings: protocol for a systematic review.

INTRODUCTION: Approximately 450 000 people in the UK are living in care homes, 70% of whom are thought to have dementia or significant memory problems. This means that they may need support with day-to-day decisions about their health and care. Shared decision-making interventions can have a positive impact on patient outcomes. They recognise an individual's rights to make decisions about their care or treatment and support person-centred approaches to care delivery. METHODS: A systematic review of studies designed to assess, implement, measure and/or explore shared decision-making with cognitively impaired adults in (or transferrable to) an extended care setting, with a view to answering the research question: How can people living with dementia and cognitive impairment be included in day-to-day decisions about their health and care in extended care settings? The systematic review will be started in May 2016. Studies are excluded that focus on advance decision-making. The search strategy is limited to a 20-year timeframe and English language and includes electronic databases; CINAHL, PubMed, the Cochrane Library, NICE Evidence, OpenGrey, Autism Data, Google Scholar, Scopus and MedicinesComplete. ETHICS AND DISSEMINATION: Ethical approval not required. Planned dissemination routes for protocol and systematic review through conference presentations, peer-reviewed journals and research networks including the East of England CLAHRC, INTERDEM, and the National Care Homes Research and Development Forum. DISCUSSION: The review will explore how shared decision-making is characterised and constructed in extended care settings for people living with cognitive impairment and their staff and family carers, in relation to their preferences and desires, the roles people play, facilitators, barriers, risk and benefits. The findings will inform an intervention study facilitating shared decision-making for people living with dementia in care homes and have the potential to inform future policy and practice. TRIAL REGISTRATION NUMBER: CRD42016035919.

Design, synthesis and evaluation of pyridine-based chromatographic adsorbents for antibody purification.

The structure-based design and synthesis of four series of adsorbents for antibody purification by affinity chromatography has been investigated. The structures of 10 ligands were based on pyridine compounds that possessed thioalkyl substituents containing a primary amine, which was required for immobilisation of the ligands onto an epoxy-activated matrix (epoxy-Sepharose Fast Flow(®)). These new adsorbents were screened in monoclonal antibody binding assays in order to determine optimal buffer conditions for capture and elution under static and dynamic adsorption conditions. From batch binding measurements, the binding affinities, KD's, were found to be in the range of 3-5µM and the maximum capacities, qm's were between 12 and 30mgmAb/mL resin, depending on the substitution pattern of the thioalkylamine in the N-heterocyclic ring structure of the ligands. The amount of monoclonal antibody bound and eluted under overload conditions was influenced by the concentration of the sample loaded, the flow rate at which the sample was applied and the loading/volume. Further, the ability of these new adsorbents to selectively capture monoclonal antibodies of the class IgG1 from supernatants derived from genetically engineered CHO cells cultured in chemically defined media was investigated, documenting efficient capture and recovery of the mAb.

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure.

A new set of ligands based on substituted pyridine and other N-heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy-activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants KD 's and binding capacities Qmax 's. Typically, the KD values were in the range of 2-5 µM and the Qmax values between 20 and 75 mg mAb/ml resin, depending on the stereo-electronic properties of the substituent in the N-heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established.

Wnt3a regulates survival, expansion, and maintenance of neural progenitors derived from human embryonic stem cells.

Many reports describe the efficient derivation and expansion of neural progenitors (NP) from human embryonic stem cells (hESC). However, little is known about the signaling factors found within the neurosphere microenvironment that regulate NP maintenance and differentiation. We show that Wnt ligand and receptor transcripts are endogenously upregulated within neurospheres derived from noggin-primed hESC. In addition, neurosphere formation and size were significantly greater in the presence of exogenous Wnt3a compared to control conditions. Inhibition of endogenous Wnt signaling resulted in a significant reduction in the efficiency of neurosphere formation and overall size, due to effects on both NP proliferation and apoptosis. These findings demonstrate a requirement of Wnt signaling for maintenance, proliferation, and survival of NP when cultured in neurosphere conditions.

Expression of the human activin type I and II receptor extracellular domains in Pichia pastoris.

Methods for the expression in Pichia pastoris and purification of the human activin receptor type I and II extracellular domains (ARIa/ARIb-ECDs, ARIIA/ARIIB-ECDs) are described. Key experimental aspects are also documented of the vector transformation methodology and the binding characteristics of these ECDs with activin A and inhibin. The cDNA constructs for these ECDs contained a C-terminal His6-tag with either the native signal (N) or the yeast alpha mating factor (alphaMF) sequence and were introduced into the pPICZ expression vector either as a single-copy or as a four-copy expression cassette. Hyper-resistant transformants (zeo(R): 500 microg/mL) generated from the cassette containing a single copy of the expression vector gave the stronger signal intensity with a DNA dot-blot screening assay. These transformants also produced higher quantities of the corresponding recombinant protein compared to transformants using the four-copy cassette vector. All receptor-ECD proteins expressed were found to be heterogeneously glycosylated, whereby the ARIIA-ECD and ARIIB-ECD had undergone two Asn-linked glycosylation events and the ARIb-ECD a single event. By SDS-PAGE, the de-glycosylated proteins migrated larger than the expected core size, indicating that they may have undergone O-linked glycosylation. Biacore-based procedures with the glycosylated and de-glycosylated ARIIA-ECD were employed to determine the kinetic and equilibrium binding parameters for the interaction with activin A and inhibin. The glycosylated ARIIA-ECD bound to activin A with a KD of 11.9 nM and inhibin with a KD of 21.1 nM. Although glycosylation of ARIIA-ECD was not strictly required for high affinity interactions with activin A or inhibin, it markedly improved the overall stability of the ARIIA-ECD.

Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production.

The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and mut(s) phenotypes, intracellular expression and folding pathways is examined. The roles of pre-pro signal sequences such as the alpha mating factor (alpha-MF) and the Glu-Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed.