Effect of insults associated with preterm birth on mouse brain development

Preterm birth (PTB) is associated with an increased incidence of many neurodevelopmental disorders. The mechanisms underlying this association are unclear, but insults associated with PTB such as hypoxia, inflammation and stress can affect brain development. In this thesis I examine the effects of these insults on the mouse brain at times with neurodevelopmental relevance to PTB. Mice when born are roughly equivalent in terms of brain development to a human at 24 weeks of gestation and mature to term equivalence by postnatal day (P) 10. As such, this course of postnatal brain development in mice provides an opportunity to study insults associated with PTB at timepoints neurodevelopmentally relevant to PTB in humans. DNA methylation is crucial to the fine spatial and temporal control of gene expression associated with normal brain development. Altered DNA methylation (5-methylcytosine; 5mC) has been shown in the brains of individuals with autism spectrum disorders, schizophrenia and other neurodevelopmental disorders. Additionally, preterm birth is associated with altered DNA methylation at several key genes associated with neurodevelopment. However, many of these studies cannot distinguish 5mC and 5- hydroxymethylation (5hmC), which is generated from 5mC by the Ten-eleven translocation (TET) enzymes. The TET enzymes are highly susceptible to environmental perturbations and are therefore a good candidate for epigenetic dysregulation associated with PTB related insults. I hypothesised that common PTB-related insults such as inflammation and/or hypoxia would affect the expression of the TET enzymes and this would be associated with altered DNA hydroxymethylation. To test this hypothesis, I used a forebrain slice culture model and showed that hypoxia results in increased expression of the TET enzymes. Previous studies have shown that DNA hydroxymethylation accumulates at classic hypoxic response genes following hypoxia in the cancer environment. I tested the applicability of this to brain development and found significant accumulation of 5hmC in these areas following hypoxia, demonstrating the relevance of this mechanism to neonatal hypoxia. Early life stress is also thought to be important in PTB. To test effects of stress on brain development, I developed a novel paradigm of early life stress in mice (modified maternal separation or MMS). MMS consists of separating a pup from its mother for 1.5 hours/ day from P4-P6. During this separation period the pup is moved into a supine position, from where it will struggle to return to the prone position and the process is repeated. I hypothesised that MMS would affect the transcriptome and DNA methylome in the perinatal period and result in altered behaviour in adulthood. Using 3’ mRNA sequencing and DNA methylation immunoprecipitation-sequencing I found subtle alterations in gene expression and profound alterations in DNA methylation in the hypothalamus immediately following MMS. In adulthood, I demonstrated that pups exposed to MMS have a hyperactivity phenotype in the elevated plus and open field mazes but do not display hyperactivity under habitual conditions. At 4 months of age, there were no changes in candidate gene expression in the hypothalamus, but there were changes in the expression of genes associated with stress signalling in the hippocampus following MMS. Finally, there were no persistent changes in DNA methylation in candidate regions in the hypothalamus. I also showed an NFkB signature in the hypothalamic transcriptome following MMS. NFkB signalling is classically associated with inflammation, I therefore hypothesised that inflammation, induced by LPS, would potentiate subsequent MMS. Using immunohistochemistry, I found no difference in specific cell populations in the cortex or hippocampus. I then performed 3’ mRNA sequencing to assess the transcriptome wide effect of LPS and MMS in the cortex. I showed enrichment of NFkB binding sites among differentially expressed genes in MMS but there was no additional effect of prior LPS exposure. Finally, I sought to explore whether LPS and/or MMS affected synaptic pruning in the hippocampus, however there was no evidence for altered expression of genes associated with synaptic pruning. In summary, in this thesis I show that LPS, hypoxia and early life stress can affect mouse brain development. These results further the knowledge around how insults associated with PTB may affect brain development and neurodevelopmental outcome

Early life stress and LPS interact to modify the mouse cortical transcriptome in the neonatal period

Introduction: Preterm birth (PTB) is closely associated with atypical cerebral cortical development and cognitive impairment. Early exposure to extrauterine life often results in atypical environmental and biological experiences that co-occur, including early life stress (ELS) and systemic inflammation. Understanding how these experiences interact to shape cortical development is an essential prerequisite to developing therapeutic interventions that will work in the complex postnatal environment of the preterm infant. Here, we studied the effects of a murine model of infection and ELS on the neonatal cortex transcriptome. Methods: We used a mouse model of infection (1 ​mg/kg LPS at postnatal day (P)3) +/− ELS (modified maternal separation; MMS on days P4–P6) at timepoints with neurodevelopmental relevance to PTB. We used 4 groups: control, LPS, MMS and LPS ​+ ​MMS. Cortices were dissected at P6 for 3′RNA sequencing. Results: LPS exposure resulted in reduced weight gain and increased expression of inflammation-associated genes in the brain. More genes were differentially expressed following LPS (15) and MMS (29) than with LPS ​+ ​MMS (8). There was significant overlap between the LPS and MMS datasets, particularly amongst upregulated genes, and when comparing LPS and MMS datasets with LPS ​+ ​MMS. Gene Ontology terms related to the extracellular matrix and cytokine response were enriched following MMS, but not following LPS or LPS ​+ ​MMS. 26 Reactome pathways were enriched in the LPS group, none of which were enriched in the LPS ​+ ​MMS group. Finally, a rank-rank hypergeometric overlap test showed similarities, particularly in upregulated genes, in the LPS and MMS conditions, indicating shared mechanisms. Conclusion: LPS and MMS interact to modify the cortical transcriptome in the neonatal period. This has important implications for understanding the neural basis of atypical cortical development associated with early exposure to extrauterine life

Resource allocation decision-making in dementia care with and without budget constraints: a qualitative analysis

Introduction: Health systems in many different countries have increasingly been reorienting the delivery of dementia care to home and community care settings. This paper provides information on how health and social care professionals (HSCPs) in Ireland make decisions on resource allocation for people with dementia living at home and how resource constraints affect their decisions and choices.Methods: A balance of care approach was used to assess resource allocation across six dementia case types, from low to high needs. Workshops were held with 24 HSCPs from multiple disciplines. Participants allocated services in two scenarios: allocation with and without a budget constraint. Nominal group technique was used to structure discussions around resource allocation in both scenarios. Thematic analysis was applied to analyse the qualitative data using a general inductive approach.Results: The following themes influenced allocative deliberations: whose needs are being met; what needs are identified; decision making context; decision making process; and allocation outcomes. Participants were proficient in making decisions, using ‘decision rules’ or heuristics to help them make decisions under fixed budget rules and sticking to conventional provision when constraints were in place.Conclusions: Freedom from a budget constraint allowed HSCPs to consider a broader range of services and to take a more expansive view on what needs should be considered, with a particular emphasis on adopting a proactive, preventative approach to the allocation of resources. The effect of the budget constraint overall was to narrow all considerations, using heuristics to limit the type of needs addressed and the range of services and supports provided. The consequences were a largely reactive, less personalised system of care. The findings emphasise the need for an integrated and comprehensive assessment process that is more concerned with individualised responses rather than relying on existing models of care alone

Innovating Pedagogy 2020: Open University Innovation Report 8

This series of reports explores new forms of teaching, learning and assessment for an interactive world, to guide teachers and policy makers in productive innovation. This eighth report, produced by The Open University in collaboration with the National Institute for Digital Learning (NIDL) in Ireland, describes ten innovations that have the potential to influence education in the coming years

HIV outbreaks among people who inject drugs in Europe, North America and Israel

During 2011–16, HIV outbreaks occurred among people who inject drugs (PWID) in Canada (southeastern Saskatchewan), Greece (Athens), Ireland (Dublin), Israel (Tel Aviv), Luxembourg, Romania (Bucharest), Scotland (Glasgow), and USA (Scott County, Indiana). Factors common to many of these outbreaks included community economic problems, homelessness, and changes in drug injection patterns. The outbreaks differed in size (from under 100 to over 1000 newly reported HIV cases among PWID) and in the extent to which combined prevention had been implemented before, during, and after the outbreaks. Countries need to ensure high coverage of HIV prevention services and coverage higher than the current UNAIDS recommendation might be needed in areas in which short acting drugs are injected. In addition, monitoring of PWID with special attention for changing drug use patterns, risk behaviours, and susceptible subgroups (eg, PWID experiencing homelessness) needs to be in place to prevent or rapidly detect and contain new HIV outbreaks

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.Chemistry and Chemical Biolog

Immune activation by DNA damage predicts response to chemotherapy and survival in oesophageal adenocarcinoma.

OBJECTIVE: Current strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC. DESIGN: Transcriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS). RESULTS: A total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: p<0.001; stromal: p=0.026) as well as PD-L1 expression (intratumoural: p=0.047; stromal: p=0.025). CONCLUSION: The DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.This work was supported by the Gastrointestinal Cancer Research Charitable Fund administered by the Belfast Health and Social Care Trust, the Cancer Research UK Experimental Cancer Medicine Centre Initiative, Invest Northern Ireland and Almac Diagnostics. Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) was funded by a programme grant from Cancer Research UK (RG66287). We would like to thank the Human Research Tissue Bank, which is supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre from Addenbrooke’s Hospital. Additional infrastructure support was provided from the CRUK funded Experimental Cancer Medicine Centre. RF has programmatic funding from the Medical Research Council and infrastructure support from the NIHR Biomedical Research Centre and the Cambridge Experimental Medicine Centre. Tissue samples used in this research were received from the Northern Ireland Biobank, which is funded by HSC Research and Development Division of the Public Health Agency in Northern Ireland and Cancer Research UK through the Belfast Cancer Research UK Centre and the Northern Ireland Experimental Cancer Medicine Centre; additional support was received from the Friends of the Cancer Centre. The Northern Ireland Molecular Pathology Laboratory has received funding from Cancer Research UK, the Friends of the Cancer Centre and the Sean Crummey Foundation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no 721906. The OCCAMS Study Group is a multicentre UK collaboration

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