Mass spectrometry imaging of 3D tissue models

A 3D cell culture is an artificially created environment in which cells are permitted to grow/interact with their surroundings in all three dimensions. Derived from 3D cell culture, organoids are generally small‐scale constructs of cells that are fabricated in the laboratory to serve as 3D representations of in vivo tissues and organs. Due to regulatory, economic and societal issues concerning the use of animals in scientific research it seems clear that the use of 3D cell culture and organoids in for example early stage studies of drug efficacy and toxicity will increase. The combination of such 3D tissue models with mass spectrometry imaging provides a label free methodology for the study of drug absorption/penetration, drug efficacy/toxicity and drug biotransformation. In this article, some of the successes achieved to date and challenges to be overcome before this methodology is more widely adopted are discussed

Exploring the lived experiences of pastoral staff identifying and supporting pupil mental health needs in mainstream secondary schools

The prevalence of children and young people with mental health needs is of national and local concern. Changes in legislation have explicitly made the identification and support of pupil mental health needs a role for schools. Research indicates the identification of pupil mental health needs has a multitude of barriers meaning said needs are frequently unidentified. This is of concern given it is estimated that half of mental health needs have their onset during adolescence and are related to negative outcomes such as poor educational attainment and in some cases, suicide. School staff are tasked with performing a Tier 1 (Universal Services) mental health professional role. However, there is considerable variation in the support school staff receive when performing this pastoral role and routine pastoral care has received little attention in the literature. The purpose of this research was to explore the lived experiences of lead pastoral staff identifying and supporting adolescent pupil mental health needs in mainstream secondary schools. Semi-structured interviews were conducted with four purposively selected members of secondary school staff performing lead pastoral roles. The interview transcripts were analysed using Interpretative Phenomenological Analysis (IPA) and the following four overarching themes were found to be relevant for all participants: Multiple Conceptualisations of Role, A Myriad of Emotion, Constraints and Conflict, and Remit. These findings are discussed in the context of existing literature and the limitations of this research are considered. Implications for educational psychology practice are proposed, relating to the different levels at which educational psychologists work: individual, group and organisational. Suggestions for future research related to pastoral leads identifying and supporting adolescent pupil mental health needs are provided

Narrowing the gap: Literature review

This review supports a research programme that is exploring how technology can narrow the gap for under- and low-achieving learners in secondary schools. Technology can support learning for underachievers by improving results and increasing engagement. Research shows that it can do this by: •monitoring behaviour and targeting learners who need special attention •making the curriculum relevant to learners' everyday experiences •encouraging learners' self-assessment and developing educators' reflective practices •offering targeted online support to learners need it •improving the literacy skills of Non-English Mother Tongue learners •developing learners' executive attention and improving their intelligence scores. Research shows that many different approaches using technology are effective. For instance, playing electronic games can encourage learners to practice skills such as language development. Games can also engage learners with special educational needs, such as ADHD. In one study, boys aged 9-11 with ADHD played a computer driving game. They were more engaged and had a tiny dropout rate compared to boys with ADHD who did not play. Using presentation software in the classroom can also produce results. Researchers found that learners rate educators who use this technology more highly than educators who don't

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 μm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development

Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17β-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol

Appetite and Energy Intake Responses to Acute Energy Deficits in Females versus Males.

Purpose: To explore whether compensatory responses to acute energy deficits induced by exercise or diet differ by sex. Methods: In experiment one, twelve healthy women completed three 9 h trials (control, exercise-induced (Ex-Def) and food restriction induced energy deficit (Food-Def)) with identical energy deficits being imposed in the Ex-Def (90 min run, ~70% of VO2 max) and Food-Def trials. In experiment two, 10 men and 10 women completed two 7 h trials (control and exercise). Sixty min of running (~70% of VO2 max) was performed at the beginning of the exercise trial. Participants rested throughout the remainder of the exercise trial and during the control trial. Appetite ratings, plasma concentrations of gut hormones and ad libitum energy intake were assessed during main trials. Results: In experiment one, an energy deficit of ~3500 kJ induced via food restriction increased appetite and food intake. These changes corresponded with heightened concentrations of plasma acylated ghrelin and lower peptide YY3-36. None of these compensatory responses were apparent when an equivalent energy deficit was induced by exercise. In experiment two, appetite ratings and plasma acylated ghrelin concentrations were lower in exercise than control but energy intake did not differ between trials. The appetite, acylated ghrelin and energy intake response to exercise did not differ between men and women. Conclusions: Women exhibit compensatory appetite, gut hormone and food intake responses to acute energy restriction but not in response to an acute bout of exercise. Additionally, men and women appear to exhibit similar acylated ghrelin and PYY3-36 responses to exercise-induced energy deficits. These findings advance understanding regarding the interaction between exercise and energy homeostasis in women

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade

Individual variation in hunger, energy intake and ghrelin responses to acute exercise

Purpose This study aimed to characterize the immediate and extended effect of acute exercise on hunger, energy intake, and circulating acylated ghrelin concentrations using a large data set of homogenous experimental trials and to describe the variation in responses between individuals. Methods Data from 17 of our group's experimental crossover trials were aggregated yielding a total sample of 192 young, healthy males. In these studies, single bouts of moderate to high-intensity aerobic exercise (69% ± 5% V˙O2 peak; mean ± SD) were completed with detailed participant assessments occurring during and for several hours postexercise. Mean hunger ratings were determined during (n = 178) and after (n = 118) exercise from visual analog scales completed at 30-min intervals, whereas ad libitum energy intake was measured within the first hour after exercise (n = 60) and at multiple meals (n = 128) during the remainder of trials. Venous concentrations of acylated ghrelin were determined at strategic time points during (n = 118) and after (n = 89) exercise. Results At group level, exercise transiently suppressed hunger (P < 0.010, Cohen's d = 0.77) but did not affect energy intake. Acylated ghrelin was suppressed during exercise (P < 0.001, Cohen's d = 0.10) and remained significantly lower than control (no exercise) afterward (P < 0.024, Cohen's d = 0.61). Between participants, there were notable differences in responses; however, a large proportion of this spread lay within the boundaries of normal variation associated with biological and technical assessment error. Conclusion In young men, acute exercise suppresses hunger and circulating acylated ghrelin concentrations with notable diversity between individuals. Care must be taken to distinguish true interindividual variation from random differences within normal limits

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707