Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients.

RATIONALE: Meal ingestion triggers secretion of a variety of gut and endocrine peptides important in diabetes research which are routinely measured by immunoassays. However, similarities between some peptides (glucagon, oxyntomodulin and glicentin) can cause specificity issues with immunoassays. We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to unambiguously monitor multiple gut peptides in human plasma. METHODS: A simple acetonitrile-based protein precipitation step, followed by evaporation and solid-phase extraction, removed high-abundance proteins from samples prior to nano-LC/MS/MS analysis on an Orbitrap Q-Exactive Plus mass spectrometer using a data-dependent methodology. Database searching using PEAKS identified multiple gut-derived peptides, including peptides in the mid-pg/mL range. The relative levels of these and previously characterised peptides were assessed in plasma samples from gastrectomised and control subjects during an oral glucose tolerance test. RESULTS: Analysis of plasma extracts revealed significantly elevated levels of a number of peptides following glucose ingestion in subjects who had undergone gastrectomy compared with controls. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide. Motilin levels decreased following glucose ingestion. Results showed good correlation with immunoassay-derived concentrations of some peptides in the same samples. The gastrectomy group also had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1. CONCLUSIONS: Overall, the approach showed that a fast, generic and reproducible LC/MS/MS methodology requiring only a small volume of plasma was capable of the multiplexed detection of a variety of diabetes-related peptides.Wellcome Trust and MR

'A life in a day' simulation experience : perceptions of oncology nurses and pharmacy staff

BACKGROUND: Due to an increase in patient numbers, more cancer patients are being reviewed by non-medical healthcare professionals (HCPs), and it is essential that they can empathise with patients and care for them holistically. ‘A Life in a Day’ is a role reversal simulation (RRS) which demonstrates the challenges, choices and impacts that cancer patients face every day, facilitated by a Smartphone application (app). This study focused on renal cell carcinoma (RCC) and was designed to evaluate the impact of RRS on participants from the British Oncology Pharmacy Association (BOPA) and the UK Oncology Nursing Society (UKONS), and identify any changes made to clinical practice as a result. METHOD: A survey was conducted via the app before and after the experience. Individual semi-structured interviews were conducted with participants over Microsoft Teams. RESULTS: Data from the survey showed that after the experience 97% of all participants strongly agreed that they ‘feel empathy for RCC patients’ and 90% strongly agreed that they ‘feel inspired to place patients at the centre of their work’. There were 5 themes extrapolated from the qualitative data: Holistic understanding of Patients, Reflections on Practice, Changes in Practice, Outreach to Colleagues, Education & Training. CONCLUSION: Participants reported an increase in empathy for their patients which inspired them to make changes to their practice. This involved being more holistic in their care and taking on more responsibility. They recommended use of RRS for HCP training and continued professional development. They also suggested incorporation of RRS into the pharmacy undergraduate curriculum

Investigation of humans individual differences as predictors of their animal interaction styles, focused on the domestic cat

Humans' individual differences including their demographics, personality, attitudes and experiences are often associated with important outcomes for the animals they interact with. This is pertinent to companion animals such as cats and dogs, given their social and emotional importance to humans and degree of integration into human society. However, the mechanistic underpinnings and causal relationships that characterise links between human individual differences and companion animal behaviour and wellbeing are not well understood. In this exploratory investigation, we firstly quantified the underlying structure of, and variation in, human's styles of behaviour during typical human-cat interactions (HCI), focusing on aspects of handling and interaction known to be preferred by cats (i.e. 'best practice'), and their variation. We then explored the potential significance of various human individual differences as predictors of these HCI styles. Seven separate HCI styles were identified via Principal Component Analysis (PCA) from averaged observations for 119 participants, interacting with sociable domestic cats within a rehoming context. Using General Linear Models (GLMs) and an Information Theoretic (IT) approach, we found these HCI PC components were weakly to strongly predicted by factors including cat-ownership history, participant personality (measured via the Big Five Inventory, or BFI), age, work experience with animals and participants' subjective ratings of their cat behaviour knowledge. Paradoxically, greater cat ownership experiences and self-assessed cat knowledge were not positively associated with 'best practice' styles of HCI, but were instead generally predictive of HCI styles known to be less preferred by cats, as was greater participant age and Neuroticism. These findings have important implications regarding the quality of human-companion animal relationships and dyadic compatibility, in addition to the role of educational interventions and their targeting for optimal efficacy. In the context of animal adoption, these results strengthen the (limited) evidence base for decision making associated with cat-adopter screening and matching. In particular, our results suggest that greater cat ownership experiences and self-reports of cat knowledge might not necessarily convey advantages for cats in the context of HCI

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing

Organoid Sample Preparation and Extraction for LC-MS Peptidomics.

This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020)

Providing humans with practical, best practice handling guidelines during human-cat interactions increases cats' affiliative behaviour and reduces aggression and signs of conflict

The importance of animals' experiences and associated comfort during Human-Animal Interactions (HAI), and particularly Animal Assisted Interventions (AAI), are increasingly recognised. However, there remains a paucity of published research, particularly concerning less formal but frequent HAIs to which companion animals are typically exposed, such as stroking or petting. Additionally, few practical evidence-based guides to facilitate humans' optimal animal handling and interaction in these contexts exist. A simple set of Human-Cat Interaction (HCI) guidelines were therefore created, with the aim to enhance domestic cats' comfort during generic HCI contexts. Based around a “CAT” acronym, guidelines focused on providing the cat with choice and control (“C”), paying attention (“A”) to the cats' behaviour and body language and limiting touch (“T”), primarily to their temporal regions. Guidelines were presented to human participants during a brief training intervention, and guideline efficacy was subsequently assessed. Domestic cats available for rehoming at Battersea Dogs and Cats Home, UK (n = 100) were filmed during interactions with novel members of the public (n = 120). Cats were exposed to a maximum of six, 5-min interaction sessions, balanced across “control” (interactions with humans pre-training) and “intervention” conditions (interactions with humans post-training). For each observation, cat behaviour and posture were coded and humans' cat-directed behaviour rated on the degree to which it reflected best practice principles. Data were extracted from a total of 535 observations and average human interaction ratings and cat behaviour values compared between control and intervention conditions via paired Wilcoxon tests. Compared to the control, humans' interaction styles were rated as significantly more closely aligned with best practice principles in the intervention condition. Cats also displayed significantly greater frequencies and/or durations of affiliative and positively-valenced behaviours in the intervention. In contrast, cats in the control displayed significantly greater frequencies of human-directed aggression, in addition to greater frequencies and/or durations of behaviours associated with conflict and negative valence. Results demonstrate the positive impact of practical interaction guidelines on cats' social behaviour and comfort during HCI, with the potential to improve cats' general experiences during interactions, reduce human-directed aggression and ultimately improve cat-human relationships

Nivolumab With or Without Ipilimumab in Pediatric Patients With High-Grade CNS Malignancies: Safety, Efficacy, Biomarker, and Pharmacokinetics: CheckMate 908

BACKGROUND: Therapeutic options are limited in pediatric CNS malignancies. CheckMate 908 (NCT03130959) is an open-label, sequential-arm, phase 1b/2 study investigating nivolumab (NIVO) and NIVO+ipilimumab (IPI) in pediatric patients with high-grade CNS malignancies. METHODS: Patients (N=166) in 5 cohorts received NIVO 3 mg/kg every 2 weeks (Q2W) or NIVO 3 mg/kg+IPI 1 mg/kg every 3 weeks (4 doses) followed by NIVO 3 mg/kg Q2W. Primary endpoints included overall survival (OS; newly diagnosed diffuse intrinsic pontine glioma [DIPG]) and progression-free survival (PFS; other recurrent/progressive or relapsed/resistant CNS cohorts). Secondary endpoints included other efficacy metrics and safety. Exploratory endpoints included pharmacokinetics and biomarker analyses. RESULTS: As of January 13, 2021, median OS (80% CI) was 11.7 (10.3-16.5) and 10.8 (9.1-15.8) months with NIVO and NIVO+IPI, respectively, in newly diagnosed DIPG. Median PFS (80% CI) with NIVO and NIVO+IPI was 1.7 (1.4-2.7) and 1.3 (1.2-1.5) months, respectively, in recurrent/progressive high-grade glioma; 1.4 (1.2-1.4) and 2.8 (1.5-4.5) months in relapsed/resistant medulloblastoma; and 1.4 (1.4-2.6) and 4.6 (1.4-5.4) months in relapsed/resistant ependymoma. In patients with other recurrent/progressive CNS tumors, median PFS (95% CI) was 1.2 (1.1-1.3) and 1.6 (1.3-3.5) months, respectively. Grade 3/4 treatment-related adverse-event rates were 14.1% (NIVO) and 27.2% (NIVO+IPI). NIVO and IPI first-dose trough concentrations were lower in youngest and lowest-weight patients. Baseline tumor programmed death ligand 1 expression was not associated with survival. CONCLUSIONS: NIVO±IPI did not demonstrate clinical benefit relative to historical data. The overall safety profiles were manageable with no new safety signals

Selective stimulation of colonic L cells improves metabolic outcomes in mice

Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268Abstract: Aims/hypothesis: Insulin-like peptide-5 (INSL5) is found only in distal colonic L cells, which co-express glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). GLP-1 is a well-known insulin secretagogue, and GLP-1 and PYY are anorexigenic, whereas INSL5 is considered orexigenic. We aimed to clarify the metabolic impact of selective stimulation of distal colonic L cells in mice. Methods: Insl5 promoter-driven expression of Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) was employed to activate distal colonic L cells (LdistalDq). IPGTT and food intake were assessed with and without DREADD activation. Results: LdistalDq cell stimulation with clozapine N-oxide (CNO; 0.3 mg/kg i.p.) increased plasma GLP-1 and PYY (2.67- and 3.31-fold, respectively); INSL5 was not measurable in plasma but was co-secreted with GLP-1 and PYY in vitro. IPGTT (2 g/kg body weight) revealed significantly improved glucose tolerance following CNO injection. CNO-treated mice also exhibited reduced food intake and body weight after 24 h, and increased defecation, the latter being sensitive to 5-hydroxytryptamine (5-HT) receptor 3 inhibition. Pre-treatment with a GLP1 receptor-blocking antibody neutralised the CNO-dependent improvement in glucose tolerance but did not affect the reduction in food intake, and an independent group of animals pair-fed to the CNO-treatment group demonstrated attenuated weight loss. Pre-treatment with JNJ-31020028, a neuropeptide Y receptor type 2 antagonist, abolished the CNO-dependent effect on food intake. Assessment of whole body physiology in metabolic cages revealed LdistalDq cell stimulation increased energy expenditure and increased activity. Acute CNO-induced food intake and glucose homeostasis outcomes were maintained after 2 weeks on a high-fat diet. Conclusions/interpretation: This proof-of-concept study demonstrates that selective distal colonic L cell stimulation has beneficial metabolic outcomes. Graphical abstrac