A peptidomic investigation into enteroendocrine cells and islets of humans and mice

Enteroendocrine cells (EECs) of the gastrointestinal (GI) tract and islets of Langerhans (islets) of the pancreas are endocrine cells capable of sensing nutrient intake or the nutrient content of the blood and responding by secreting peptide hormones to control various physiological functions. This thesis focuses on characterising the peptidome of EECs and islets by utilising liquid-chromatography coupled to mass spectrometry (LC-MS). Due to their ability to control food intake and glucose tolerance, hormones from EECs and islets are of great therapeutic interest for the treatment of type 2 diabetes (T2D) and obesity. I hypothesised that there may be undiscovered peptide hormones derived from EECs or islets that could also be of therapeutic interest for the treatment of T2D. In order to identify potentially novel hormone candidates, peptidomic analyses of mouse EECs, mouse islets and human GI tissue were carried out utilising LC-MS. 13 interesting peptide candidates identified in the peptidomic analyses were picked out to be synthesised for in vivo and/or in vitro characterisation. All 13 peptides were screened for activity at Gs or Gi signalling pathways in various cell lines although unfortunately no peptide seemed to exhibit activity through these signalling mechanisms in the cell lines tested. Despite this, studies were carried out to assess the ability of the 2 top peptide candidates to control food intake and glucose tolerance in vivo. 1 of these peptides, a peptide derived from progastrin, seemed to mildly improve glucose tolerance in lean mice, an effect that was seen in across several studies. I hypothesised that this progastrin derived peptide may be altering peptide secretions from islets to modulate glucose tolerance but no effect of this novel peptide was seen on insulin, somatostatin-14 or glucagon secretion from islets. The progastrin derived peptide was then administered to diet-induced obese mice to assess its therapeutic potential but unfortunately no effect was seen on glucose tolerance. More work needs to be performed to verifying my findings and investigating a possible mechanism of action before this novel progastrin derived peptide may be considered a gut hormone. In regard to the peptidome of islets, there are some gaps in the literature that our initial peptidomic analysis did not answer. Questions such as do islets produce GLP1 (glucagon-like peptide 1) receptor agonists and how is the islet peptidome altered in response to metabolic stress. I therefore carried out an investigation to address these questions. Targeted LC-MS methodology was used to search for intra-islet GLP1 receptor agonists. No peptides derived from glucose-dependent insulinotropic polypeptide (GIP) were found in mouse islets but active GLP-1 was found in both human and mouse islets. Comparisons were made between the abundance of active GLP-1 and glucagon to assess which of these peptides might be responsible for the intra-islet “incretin” effect. My peptidomic analysis of islets from T2D humans and obese mice may not have revealed new major findings but it has provided some clarity over how metabolic stress alters the peptidome of islets in T2D an obesity. In summary; a comprehensive analysis of the peptidome of EECs and islets was carried out, a number of novel peptide hormone candidates were synthesised and one of which exhibited a mild improvement in the glucose tolerance of lean mice, and clarity was provided on how metabolic stress alters the islet peptidome in mice and humans. I hope that the data produced in this thesis can contribute to a better understanding of the peptidome of the EECs and islets and may contribute to advances in therapeutic options for patients suffering from obesity or T2D

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)

Long-period Radio Pulsars: Population Study in the Neutron Star and White Dwarf Rotating Dipole Scenarios

© 2024 The Author(s). Published by the American Astronomical Society. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The nature of two recently discovered radio emitters with unusually long periods of 18min (GLEAM-X J1627-52) and 21min (GPM J1839-10) is highly debated. Their bright radio emission resembles that of radio magnetars, but their long periodicities and lack of detection at other wavelengths challenge the neutron-star interpretation. In contrast, long rotational periods are common in white dwarfs but, although predicted, dipolar radio emission from isolated magnetic white dwarfs has never been unambiguously observed. In this work, we investigate these long-period objects as potential isolated neutron-star or white-dwarf dipolar radio emitters and find that both scenarios pose significant challenges to our understanding of radio emission via pair production in dipolar magnetospheres. We also perform population-synthesis simulations based on dipolar spin-down in both pictures, assuming different initial-period distributions, masses, radii, beaming fractions, and magnetic-field prescriptions, to assess their impact on the ultra-long pulsar population. In the neutron-star scenario, we do not expect a large number of ultra-long period pulsars under any physically motivated (or even extreme) assumptions for the period evolution. On the other hand, in the white-dwarf scenario, we can easily accommodate a large population of long-period radio emitters. However, no mechanism can easily explain the production of such bright coherent radio emission in either scenarios.Peer reviewe

A long-period radio transient active for three decades: population study in the neutron star and white dwarf rotating dipole scenarios

The nature of two recently discovered radio emitters with unusually long periods of 18min (GLEAM-X J1627-52) and 21min (GPM J1839-10) is highly debated. Their bright radio emission resembles that of radio magnetars, but their long periodicities and lack of detection at other wavelengths challenge the neutron-star interpretation. In contrast, long rotational periods are common in white dwarfs but, although predicted, dipolar radio emission from isolated magnetic white dwarfs has never been unambiguously observed. In this work, we investigate these long-period objects as potential isolated neutron-star or white-dwarf dipolar radio emitters and find that both scenarios pose significant challenges to our understanding of radio emission via pair production in dipolar magnetospheres. We also perform population-synthesis simulations based on dipolar spin-down in both pictures, assuming different initial-period distributions, masses, radii, beaming fractions, and magnetic-field prescriptions, to assess their impact on the ultra-long pulsar population. In the neutron-star scenario, we cannot reproduce the large number of expected ultra-long period pulsars under any physically motivated (or even extreme) assumptions. Thus, if GLEAM-X J1627-52 and GPM J1839-10 are confirmed as neutron-star pulsars (even if they are magnetars), this would necessarily call for a significant revision of our understanding of birth parameters at the population level. On the other hand, in the white-dwarf scenario, no mechanism can explain the production of such a bright coherent radio emission in isolated magnetic white dwarf systems (binaries with low mass companions are still viable), although we can easily accommodate a large population of long-period radio emitters.Comment: 8 pages, 4 figures; ApJ Letters submitte

The alarms should no longer be ignored: survey of the demand, capacity and provision of adult community eating disorder services in England and Scotland before COVID-19.

This national pre-pandemic survey compared demand and capacity of adult community eating disorder services (ACEDS) with NHS England (NHSE) commissioning guidance. Thirteen services in England and Scotland responded (covering 10.7 million population). Between 2016-2017 and 2019-2020 mean referral rates increased by 18.8%, from 378 to 449/million population. Only 3.7% of referrals were from child and adolescent eating disorder services (CEDS-CYP), but 46% of patients were aged 18-25 and 54% were aged >25. Most ACEDS had waiting lists and rationed access. Many could not provide full medical monitoring, adapt treatment for comorbidities, offer assertive outreach or provide seamless transitions. For patient volume, the ACEDS workforce budget was 15%, compared with the NHSE workforce calculator recommendations for CEDS-CYP. Parity required £7 million investment/million population for the ACEDS. This study highlights the severe pressure in ACEDS, which has increased since the COVID-19 pandemic. Substantial investment is required to ensure NHS ACEDS meet national guidance, offer evidence-based treatment, reduce risk and preventable deaths, and achieve parity with CEDS-CYP

Enterovirus D68 outbreak detection through a syndromic disease epidemiology network

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories

Soluble guanylate cyclase signalling mediates etoposide resistance in progressing small cell lung cancer

From Springer Nature via Jisc Publications RouterHistory: received 2020-12-10, accepted 2021-10-19, registration 2021-10-26, pub-electronic 2021-11-17, online 2021-11-17, collection 2021-12Publication status: PublishedFunder: CRUK Manchester Institute (grant no. A27412) CRUK Manchester Centre (grant no. A25254) CRUK Manchester Experimental Cancer Medicines Centre (grant no. A20465) CRUK Lung Cancer Centre of Excellence (grant no. A25146) NIHR Manchester Biomedical Research CentreAbstract: Small cell lung cancer (SCLC) has a 5-year survival rate of <7%. Rapid emergence of acquired resistance to standard platinum-etoposide chemotherapy is common and improved therapies are required for this recalcitrant tumour. We exploit six paired pre-treatment and post-chemotherapy circulating tumour cell patient-derived explant (CDX) models from donors with extensive stage SCLC to investigate changes at disease progression after chemotherapy. Soluble guanylate cyclase (sGC) is recurrently upregulated in post-chemotherapy progression CDX models, which correlates with acquired chemoresistance. Expression and activation of sGC is regulated by Notch and nitric oxide (NO) signalling with downstream activation of protein kinase G. Genetic targeting of sGC or pharmacological inhibition of NO synthase re-sensitizes a chemoresistant CDX progression model in vivo, revealing this pathway as a mediator of chemoresistance and potential vulnerability of relapsed SCLC

Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets

Funder: W. D. Armstrong Fund (School of Technology, University of Cambridge)Abstract: Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues