Understanding how volunteer companionship impacts those during the end of life: A realist evaluation.

Volunteers are a popular unpaid support role in end of life care yet how accompaniment influences the dying is underdeveloped. This study examined how companionship works, for whom, in what circumstances and why. Initial realist ideas were developed through participant observation (14 months), document analysis, and realist interviews with companionship trainers (n = 6). Theory testing involved volunteer interviews (n = 7), accounts from the dying, proxy accounts for the dying, and written reflections from companionship training. Companionship helps people live well until they die, prepare for death, and experience a good death. Four areas of volunteering explain these outcomes namely a loving friend, a holistic presence, a non-judgmental intermediary, and wrap around care. The four areas activate mechanisms related to reminiscing, preserving dignity/personhood, and easing suffering, contingent on specific contexts. The findings unpack how volunteering exerts its influence and what contextual factors facilitate outcomes, advancing the knowledge in this area.

Combining realist evaluation and transformative evaluation to advance research in palliative care: The case of end of life companionship.

BACKGROUND: Palliative care requires innovative methods to understand what works, for whom, in what circumstances and why. Realist evaluation has become one prominent approach due to its preoccupation with building, and testing, causal theories to explain the influence of contextual factors on outcomes. Undertaking realist evaluation is not without challenges and may amplify issues of underrepresentation, disempower those working in palliative care, and produce results with poor ecological validity. Complementary approaches are needed which mitigate these challenges, whilst producing credible findings that advances knowledge. PURPOSE: In this article it is outlined how realist evaluation provides a toolkit to advance research to explain, and empirically test, the complex contours of palliative care. Moreover, it is proposed that transformative evaluation can provide a catalyst to engage and empower those within palliative care, create the opportunity for care transformation, and produce more informed and authentic theories. DISCUSSION: Contemporary issues in palliative care pertain to the complexity of palliative care, the insufficiency of experimental designs alone, and the challenges of achieving inclusive research participation. In this article it is argued that theory led, participatory, opportunistic and naturalistic approaches can provide an antidote to the issues in the literature. The combination also mitigates many methodological critiques of the individual approaches, by increasing the transformative potential of realist evaluation, and explanatory potential of transformative evaluation.

Replication of 15 loci involved in human plasma protein N-glycosylation in 4802 samples from four cohorts.

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the 16 loci reported previously, 15 were replicated in our study. For the remaining locus (near the KREMEN1 gene), the replication power was low, and hence, replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The 15 replicated loci present a good target for further functional studies. Among these, eight loci contain genes encoding glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4 and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo.

Defining the genetic control of human blood plasma N-glycome using genome-wide association study.

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MSE workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C6 and 13 C6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C6 /13 C6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.

Resisting the (re-)medicalisation of dying and grief in the post-digital age: Natural language processing and qualitative analysis of data from internet support forums.

In the mid-twentieth century, the social movement of death revivalism sought to resist the medicalisation of dying and grief through promotion of the dying person retaining autonomy, and societal openness toward death and bereavement. Despite this advocacy, present-day dying in high income countries is largely institutionalised, with value placed on control over the body and emotions. These phenomena are at odds with the ambitions of death revivalism, and demonstrate the re-medicalisation of dying and grief. Furthermore, contemporary society is continually advancing into the post-digital age, reflected in digital technologies being a tacit part of human existence. Within this framework, this study aims to investigate how people living with life-limiting illness and their loved ones experience, negotiate, and resist medicalisation of dying and grief through online internet forums. We collected posts through web-scraping and utilised Natural Language Processing techniques to select 7048 forum posts from 2003 to 2020, and initially categorise data, before utilising Inductive Thematic Analysis, which generated two major themes. The theme of 'Comfort' describes online forums facilitating psychosocial support which was often used to compensate for systemic deficiencies, especially during the Covid-19 pandemic. Common sources of comfort included animal companions and spirituality, in stark contrast with the medicalised model. The theme of 'Capability' describes online forums acting as solutions for people facing disempowering care systems, including providing information on legal rights and benefits which may not be otherwise easily available, and facilitating collective advocacy. Our findings indicate that community-led online forums can play an effective and sustainable role in democratising care and retaining agency when facing life-limiting illness and grief. Future palliative and bereavement care research must focus on how online forums can be integrated into existing systems, made transparent and accessible, be adequately funded and structured, and be optimised, including compensating for service disruption encountered during future pandemics.

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.

Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides.

Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other 'omics platforms in a systems biology context.

Deathbed Etiquette - The Guide: A Qualitative Study Exploring the Views of Practitioners on its Introduction into End-of-Life Care Settings.

Objectives: It is common for relatives to feel uncertain about what to expect at the bedside of a dying loved one. The Centre for the Art of Dying Well together with clinical, academic and communications experts created a 'Deathbed Etiquette' guide offering information and reassurance to relatives. This study explores the views of practitioners with experience in end-of-life care on the guide and how it might be used. Methods: Three online focus groups and nine individual interviews were conducted with a purposive sample of 21 participants involved in end-of-life care. Participants were recruited through hospices and social media. Data were analysed using thematic analysis. Results: Discussions highlighted the importance of effective communication that normalises experiences of being by the bedside of a dying loved one. Tensions around the use of the words 'death' and 'dying' were identified. Most participants also expressed reservations about the title, with the word 'deathbed' found to be old-fashioned and the word 'etiquette' not capturing the varied experiences of being by the bedside. Overall, however, participants agreed that the guide is useful for 'mythbusting' death and dying. Conclusion: There is a need for communication resources that can support practitioners in having honest and compassionate conversations with relatives in end-of-life care. The 'Deathbed Etiquette' guide is a promising resource to support relatives and healthcare practitioners by providing them with suitable information and helpful phrases. More research is needed on how to implement the guide in healthcare settings.

Antipsychotic treatment of acute paranoid schizophrenia patients with olanzapine results in altered glycosylation of serum glycoproteins.

Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.

Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation.

N-glycans attached to the C(H)2 domains of the Fc or the antigen binding regions of IgG play an important role in stabilizing and modulating antibody activity. Exhaustive elucidation of 32 IgG N-glycans using a combination of weak anion exchange enrichment and exoglycosidase array digestion with subsequent profiling exceeded 48 h. Pursuing increased throughput and associated structural annotation confidence, we compared the 1.7 µm hydrophilic interaction phase for UPLC with CE-LIF for the rapid and comprehensive characterization of N-glycans released from healthy human serum polyclonal IgG. Combination of the data individually generated using each technique demonstrated that complete structural annotation was possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms. The parallel use of both analytical techniques provides a powerful platform for rapid and comprehensive analysis of IgG N-glycosylation present on therapeutic antibodies or on antibodies of biomedical or pathological significance.

Discovering new clinical markers in the field of glycomics.

Glycosylation modifications have been reported in a number of disease states and, as a result, there is significant focus on the discovery and development of glycan-based biomarkers. Glyco-biomarkers have the potential to enhance the efficacy and efficiency of the diagnostic procedures for these diseases.

Early Stage Glycosylation Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is of great cause for concern in our ageing population, which currently lacks diagnostic tools to permit accurate and timely diagnosis for affected individuals. The development of such tools could enable therapeutic interventions earlier in the disease course and thus potentially reducing the debilitating effects of AD. Glycosylation is a common, and important, post translational modification of proteins implicated in a host of disease states resulting in a complex array of glycans being incorporated into biomolecules. Recent investigations of glycan profiles, in a wide range of conditions, has been made possible due to technological advances in the field enabling accurate glycoanalyses. Amyloid beta (Aß) peptides, tau protein, and other important proteins involved in AD pathogenesis, have altered glycosylation profiles. Crucially, these abnormalities present early in the disease state, are present in the peripheral blood, and help to distinguish AD from other dementias. This review describes the aberrant glycome in AD, focusing on proteins implicated in development and progression, and elucidates the potential of glycome aberrations as early stage biomarkers of AD.

Supportive interventions to improve retention on ART in people with HIV in low- and middle-income countries: A systematic review.

OBJECTIVES: To determine whether supportive interventions can increase retention in care for patients on antiretroviral therapy (ART) in low- and middle-income countries (LMIC). DESIGN: Systematic review and meta-analysis. METHODS: We used Cochrane Collaboration methods. We included randomised controlled trials (RCT) and observational studies with comparators conducted in LMIC. Our principal outcomes were retention, mortality and the combined outcome of lost-to-follow-up (LTFU) or death. RESULTS: We identified seven studies (published in nine articles); six of the studies were from Sub-Saharan Africa. We found four types of interventions: 1) directly observed therapy plus extra support ("DOT-plus"), 2) community-based adherence support, 3) adherence clubs and 4) extra care for patients with low CD4 count. One RCT of a community-based intervention showed significantly improved retention at 12 months (RR 1.14, 95% CI 1.02 to 1.27), and three observational studies found significantly improved retention for paediatric patients followed for 12 to 36 months (RR 1.07, 95 CI 1.03 to 1.11), and for adult patients at 12 (RR 1.38, 95% CI 1.13 to 1.70) and 60 months (RR 1.07, 95% CI 1.07 to 1.08). One observational study of adherence clubs showed significantly reduced LTFU or mortality (RR 0.20, 95% CI 0.12 to 0.33). A cluster RCT of an extra-care intervention for high-risk patients also showed a significant increase in retention (RR 1.06, 95% CI 1.01 to 1.10), and an observational study of extra nursing care found a significant decrease in LTFU or mortality (RR 0.76, 95% CI 0.66 to 0.87). CONCLUSIONS: Supportive interventions are associated with increased ART programme retention, but evidence quality is generally low to moderate. The data from this review suggest that programmes addressing psychosocial needs can significantly help retain patients in care.

Plasma N-glycans in colorectal cancer risk.

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.

Drug metabolism by tumours: its nature, relevance and therapeutic implications.

Drug-metabolising enzymes (DMEs) are present in tumours and are capable of biotransforming a variety of antineoplastics. Tumoural drug metabolism is both a potential mechanism of resistance and a means of achieving optimal therapy. This review addresses the classes of DMEs, their cytotoxic substrates and distribution in specific malignancies. The limitations of preclinical and clinical studies are highlighted. Their role in predicting therapeutic response, the activation of prodrugs and the potential for their modulation for gain is also addressed. The contribution of tumoural DMEs to cancer therapy can only be ascertained through large prospective studies and supported by new technologies. Only then can efforts be concentrated in the design of better prodrugs or combination therapy to optimise individual therapy.

The mucosa of the small intestine: how clinically relevant as an organ of drug metabolism?

The intestinal mucosa is capable of metabolising drugs via phase I and II reactions. Increasingly, as a result of in vitro and in vivo (animal and human) data, the intestinal mucosa is being implicated as a major metabolic organ for some drugs. This has been supported by clinical studies of orally administered drugs (well-known examples include cyclosporin, midazolam, nifedipine and tacrolimus) where intestinal drug metabolism has significantly reduced oral bioavailability. This review discusses the intestinal properties and processes that contribute to drug metabolism. An understanding of the interplay between the processes controlling absorption, metabolism and P-glycoprotein-mediated efflux from the intestinal mucosa into the intestinal lumen facilitates determination of the extent of the intestinal contribution to first-pass metabolism. The clinical relevance of intestinal metabolism, however, depends on the relative importance of the metabolic pathway involved, the therapeutic index of the drug and the inherent inter- and intra-individual variability. This variability can stem from genetic (metabolising enzyme polymorphisms) and/or non-genetic (including concomitant drug and food intake, route of administration) sources. An overwhelming proportion of clinically relevant drug interactions where the intestine has been implicated as a major contributor to first-pass metabolism involve drugs that undergo cytochrome P450 (CYP) 3A4-mediated biotransformation and are substrates for the efflux transporter P-glycoprotein. Much work is yet to be done in characterising the clinical impact of other enzyme systems on drug therapy. In order to achieve this, the first-pass contributions of the intestine and liver must be successfully decoupled.

High-throughput quantitative N-glycan analysis of glycoproteins.

N-linked oligosaccharides are complex non-template-derived structures that are attached to the side chains of asparagine, via the nitrogen atom. Specific changes in the N-glycans of serum glycoproteins have been associated with the pathogenesis of many diseases. The oligosaccharides present on the C(H)2 domain of immunoglobulins are known to modulate the effector functions of the molecule. These glycans provoke various biological effects, necessitating the development of robust high-throughput technology in order to fully characterize the N-glycosylation profile. This chapter describes in detail four methods to release N-glycans from the glycoprotein of interest. Two of these protocols, referred to as the "In-Gel Block" and "1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis" methods, require immobilization of the glycoprotein prior to analysis. An automated method is also described, involving the purification of immunoglobulins directly from fermentation media, and, finally, an "In-solution method" is detailed, which directly releases the N-glycans into solution. HILIC and WAX-HPLC are used to analyze the N-glycan profile. Exoglycosidase enzymes digestion arrays, in combination with computer-assisted data analysis, are used to determine both the sequence and linkage of the N-glycans present.

Transport is not rate-limiting in morphine glucuronidation in the single-pass perfused rat liver preparation.

Binding, transport, and metabolism are factors that influence morphine (M) removal in the rat liver. For M and the morphine 3beta-glucuronide metabolite (M3G), modest binding existed with 4% bovine serum albumin (unbound fractions of 0.89 +/- 0.07 and 0.98 +/- 0.09, respectively), and there was partitioning of M into red blood cells. Transport studies of M (<750 microM) showed similar, concentration-independent uptake clearances (CLs) of 1.5 ml min(-1) g(-1) among zonal and homogeneous, isolated rat hepatocytes. Transport of M3G, ascertained in multiple indicator dilution studies at various steady-state M3G concentrations (10-262 microM), uncovered a low and concentration-independent influx clearance (<10% of flow rate). The outflow dilution curve of [(3)H]M3G was superimposable onto that of [(14)C]sucrose, the extracellular reference, displaying similarity in transit times (23.5 and 22.2 s), negligible biliary excretion, and almost complete dose recovery from perfusate. In contrast, M3G occurred abundantly in both perfusate and bile in single-pass perfusion studies of the precursor, M, and revealed a biliary clearance of formed M3G that was 12.3-fold that of preformed M3G, suggesting a sinusoidal, diffusional barrier for M3G. With increasing concentrations of M (9-474 microM), clearance decreased, and metabolism and biliary excretion displayed concentration-dependent kinetics. Fitting of the data to a physiologically based liver model revealed that M removal mechanisms were saturable, with a K(m,met) of 52.2 microM and V(max,met) of 58.8 nmol min(-1) g(-1) for metabolism, and a K(m,ex) of 41.2 microM and V(max,ex) of 8.1 nmol min(-1) g(-1) for excretion. Sinusoidal transport was not rate-limiting for M removal.