Investigating the relationship between glutamine deamidation and collagen breakdown in bone

Bone can survive in the burial environment for millions of years and can provide direct information about individuals, during their lifetime and post mortem. However it is not currently possible to directly date bone material beyond the limits of radiocarbon dating ~50,000 years before present(BP). It was suggested by van Doorn et al., (2012), that deamidation in bone collagen may be related to thermal age. However, a number of bones investigated from the same site resulted in a large range of glutamine deamidation measurements. This thesis therefore explores in detail the potential of glutamine deadmidation as a dating tool for bones older than 50,000 yeas BP. The work presented in this thesis investigates a number of potential causes for the observed variability. It was found that the pre-treatment of bone with HCl not only increased levels of deamidation, but also caused significant levels of swelling and breakdown of the collagen fibril structure. Areas of bone exhibiting localised macroscopic degradation were shown to be correlated with an increase in the levels of glutamine deamidation. This indicates that the measurement of deamidation may be used as a method by which to investigate the preservation state of bone. The analysis of synthetic peptide mixtures, containing glutamine or glutamic acid showed that an ionisation bias occurs when using MALDI, with product peptide E consistently underestimated. However, analysis of the same peptide mixtures using ESI showed no ionisation bias between the two peptide products and is therefore preferable for the measurement of glutamine containing peptides. Kinetic heating experiments performed at high temperatures (80 ᵒC -140 ᵒC), have proven unsuitable for the measurement of deamidation within the buffer soluble collagen fraction. Deamidation in the buffer soluble collagen fraction was successfully measured at low temperature (65 ᵒC), and resulted in an increase in deamidation over time. However levels of deamidation in the mineral bound fraction extracted from the same bone chips heated at 65 ᵒC did not appear to undergo deamidation over time; indicating that glutamine deamidation in this fraction is slower and therefore may be suitable for the analysis of preservation of older bone from the Late Pleistocene Period. Levels of deamidation in bones analysed from nine sites ranging in age from ~ 8000 - 30,000 years BP showed a correlation with levels of glutamine deamidation and thermal age. However, using the current methodology, it is not possible to identify the age of the sites using only the measured levels of deamidation. An adapted extraction method has been suggested to further investigate the relationship between deamidation and the thermal age of bone. The work carried out in this thesis has directly contributed to the broader knowledge of how collagen degrades in bone over time, and thus has wide reaching implications within the archaeological scientific community

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.</p

ATP-binding cassette family C member 1 constrains metabolic responses to high-fat diet in male mice

Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.</p

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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A positron emission tomography imaging study to confirm target engagement in the lungs of patients with idiopathic pulmonary fibrosis following a single dose of a novel inhaled αvβ6 integrin inhibitor

© 2020 The Author(s). Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with poor prognosis and a significant unmet medical need. This study evaluated the safety, pharmacokinetics (PK) and target engagement in the lungs, of GSK3008348, a novel inhaled alpha-v beta-6 (αvβ6) integrin inhibitor, in participants with IPF. Methods: This was a phase 1b, randomised, double-blind (sponsor unblind) study, conducted in the UK (two clinical sites, one imaging unit) between June 2017 and July 2018 (NCT03069989). Participants with a definite or probable diagnosis of IPF received a single nebulised dose of 1000 mcg GSK3008348 or placebo (ratio 5:2) in two dosing periods. In period 1, safety and PK assessments were performed up to 24 h post-dose; in period 2, after a 7-day to 28-day washout, participants underwent a total of three positron emission tomography (PET) scans: Baseline, Day 1 (~ 30 min post-dosing) and Day 2 (~ 24 h post-dosing), using a radiolabelled αvβ6-specific ligand, [18F]FB-A20FMDV2. The primary endpoint was whole lung volume of distribution (VT), not corrected for air volume, at ~ 30 min post-dose compared with pre-dose. The study success criterion, determined using Bayesian analysis, was a posterior probability (true % reduction in VT > 0%) of ≥80%. Results: Eight participants with IPF were enrolled and seven completed the study. Adjusted posterior median reduction in uncorrected VT at ~ 30 min after GSK3008348 inhalation was 20% (95% CrI:-9 to 42%). The posterior probability that the true % reduction in VT > 0% was 93%. GSK3008348 was well tolerated with no reports of serious adverse events or clinically significant abnormalities that were attributable to study treatment. PK was successfully characterised showing rapid absorption followed by a multiphasic elimination. Conclusions: This study demonstrated engagement of the αvβ6 integrin target in the lung following nebulised dosing with GSK3008348 to participants with IPF. To the best of our knowledge this is the first time a target-specific PET radioligand has been used to assess target engagement in the lung, not least for an inhaled drug. Trial registration: Clinicaltrials.gov: NCT03069989; date of registration: 3 March 2017

Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield

Improving photosynthesis is a major target for increasing crop yields and ensuring food security. Phenotyping of photosynthesis in the field is critical to understand the limits to crop performance in agricultural settings. Yet, detailed phenotyping of photosynthetic traits is relatively scarce in field-grown wheat, with previous studies focusing on narrow germplasm selections. Flag leaf photosynthetic traits, crop development, and yield traits were compared in 64 field-grown wheat cultivars in the UK. Pre-anthesis and post-anthesis photosynthetic traits correlated significantly and positively with grain yield and harvest index (HI). These traits included net CO2 assimilation measured at ambient CO2 concentrations and a range of photosynthetic photon flux densities, and traits associated with the light response of photosynthesis. In most cultivars, photosynthesis decreased post-anthesis compared with pre-anthesis, and this was associated with decreased Rubisco activity and abundance. Heritability of photosynthetic traits suggests that phenotypic variation can be used to inform breeding programmes. Specific cultivars were identified with traits relevant to breeding for increased crop yields in the UK: pre-anthesis photosynthesis, post-anthesis photosynthesis, light response of photosynthesis, and Rubisco amounts. The results indicate that flag leaf longevity and operating photosynthetic activity in the canopy can be further exploited to maximize grain filling in UK bread wheat

Plasma steroid concentrations reflect acute disease severity and normalise during recovery in people hospitalised with COVID-19

Objective: Endocrine systems are disrupted in acute illness, and symptoms reported following coronavirus disease 2019 (COVID-19) are similar to those found with clinical hormone deficiencies. We hypothesised that people with severe acute COVID-19 and with post-COVID symptoms have glucocorticoid and sex hormone deficiencies. Design/Patients: Samples were obtained for analysis from two UK multicentre cohorts during hospitalisation with COVID-19 (International Severe Acute Respiratory Infection Consortium/World Health Organisation [WHO] Clinical Characterization Protocol for Severe Emerging Infections in the UK study), and at follow-up 5 months after hospitalisation (Post-hospitalisation COVID-19 study). Measurements: Plasma steroids were quantified by liquid chromatography–mass spectrometry. Steroid concentrations were compared against disease severity (WHO ordinal scale) and validated symptom scores. Data are presented as geometric mean (SD). Results: In the acute cohort (n = 239, 66.5% male), plasma cortisol concentration increased with disease severity (cortisol 753.3 [1.6] vs. 429.2 [1.7] nmol/L in fatal vs. least severe, p < .001). In males, testosterone concentrations decreased with severity (testosterone 1.2 [2.2] vs. 6.9 [1.9] nmol/L in fatal vs. least severe, p < .001). In the follow-up cohort (n = 198, 62.1% male, 68.9% ongoing symptoms, 165 [121–192] days postdischarge), plasma cortisol concentrations (275.6 [1.5] nmol/L) did not differ with in-hospital severity, perception of recovery, or patient-reported symptoms. Male testosterone concentrations (12.6 [1.5] nmol/L) were not related to in-hospital severity, perception of recovery or symptom scores. Conclusions: Circulating glucocorticoids in patients hospitalised with COVID-19 reflect acute illness, with a marked rise in cortisol and fall in male testosterone. These findings are not observed 5 months from discharge. The lack of association between hormone concentrations and common post-COVID symptoms suggests steroid insufficiency does not play a causal role in this condition

Probing the hydrothermal system of the Chicxulub impact crater

The ~180-km-diameter Chicxulub peak-ring crater and ~240-km multiring basin, produced by the impact that terminated the Cretaceous, is the largest remaining intact impact basin on Earth. International Ocean Discovery Program (IODP) and International Continental Scientific Drilling Program (ICDP) Expedition 364 drilled to a depth of 1335 m below the sea floor into the peak ring, providing a unique opportunity to study the thermal and chemical modification of Earth’s crust caused by the impact. The recovered core shows the crater hosted a spatially extensive hydrothermal system that chemically and mineralogically modified ~1.4 × 105 km3 of Earth’s crust, a volume more than nine times that of the Yellowstone Caldera system. Initially, high temperatures of 300° to 400°C and an independent geomagnetic polarity clock indicate the hydrothermal system was long lived, in excess of 106 years