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Item Open Access Recommendations for embedding simulation in health services(BioMed Central, 2023) Davies, E.; Montagu, A.; Brazil, V.Aspirations to achieve quality and safety goals in health services through simulation have led to significant investments in simulation equipment, space and faculty. However, the optimal governance and operational models through which these resources are expertly applied in health services are not known. There is growing evidence supporting ‘service’ models for simulation. In these models, simulation activities are co-designed and delivered by a team of simulation experts in partnership with health service units, specifically targeting quality and safety goals. Embedded simulation specialist teams working within these programs offer benefits not fully captured by traditional models of health education or by traditional systems for quality and safety. In this article, we explore broad and specific recommendations for establishing a simulation consultancy service within an Australian metropolitan health service. We base these recommendations on a review of current Australian practice and healthcare simulation literature, and on a specific example within a large outer metropolitan health service. The broad domains discussed include (1) governance and leadership; (2) human resources; (3) principles and planning; (4) operationalise and evaluate and (5) look to the future. The recommendations recognise that healthcare simulation is moving beyond solely addressing individual learning outcomes. The value of simulation addressing organisation and system objectives through various simulation modalities is increasingly being explored and demonstrating value. There is a growing demand for translational simulation in these contexts, and a consequent requirement for organisations to consider how simulation services can be successfully operationalised. Recommendations included in this paper are discussed and described with the intent of facilitating a deeper appreciation of the complexities associated with, and opportunities afforded by, a well-integrated simulation service.Item Open Access Safety of BCG vaccination and revaccination in healthcare workers.(Taylor & Francis, 2023) Villanueva, P.; Crawford, N.W.; Garcia Croda, M.; Collopy, S.; Araújo Jardim, B.; de Almeida Pinto Jardim, T.; Marshall, H.; Prat-Aymerich, C.; Sawka, A.; Sharma, K.; Troeman, D.; Wadia, U.; Warris, A.; Wood, N.; Messina, N.L.; Curtis, N.; Pittet, L.F.BCG vaccination and revaccination are increasingly being considered for the protection of adolescents and adults against tuberculosis and, more broadly, for the off-target protective immunological effects against other infectious and noninfectious diseases. Within an international randomized controlled trial of BCG vaccination in healthcare workers (the BRACE trial), we evaluated the incidence of local and serious adverse events, as well as the impact of previous BCG vaccination on local injection site reactions (BCG revaccination). Prospectively collected data from 99% (5351/5393) of participants in Australia, Brazil, Spain, The Netherlands and the UK was available for analysis. Most BCG recipients experienced the expected self-limiting local injection site reactions (pain, tenderness, erythema, swelling). BCG injection site itch was an additional common initial local symptom reported in 49% of BCG recipients. Compared to BCG vaccination in BCG-naïve individuals, BCG revaccination was associated with increased frequency of mild injection site reactions, as well as earlier onset and shorter duration of erythema and swelling, which were generally self-limiting. Injection site abscess and regional lymphadenopathy were the most common adverse events and had a benign course. Self-resolution occurred within a month in 80% of abscess cases and 100% of lymphadenopathy cases. At a time when BCG is being increasingly considered for its off-target effects, our findings indicate that BCG vaccination and revaccination have an acceptable safety profile in adults.Item Open Access The effect of energy poverty on mental health, cardiovascular disease and respiratory health: a longitudinal analysis(Elsevier BV, 2023) Bentley, R.; Daniel, L.; Li, Y.; Baker, E.; Li, A.Background Houses in mild-climate countries, such as Australia, are often ill-equipped to provide occupants protection during cold weather due to their design. As a result, we rely on energy to warm homes, however, energy is becoming increasingly expensive, and evidence is emerging of a sizable burden to population health of being unable to afford to warm homes causing exposure to cold indoor temperatures. Methods We use a large longitudinal sample of adult Australians (N = 32,729, Obs = 288,073) collected annually between 2000 and 2019 to estimate the relationship between exposure to energy poverty and mental health (SF-36 mental health score), and a smaller sample from waves collected in 2008–9, 2012–13, and 2016–17 (N = 22,378, Obs = 48,371) to estimate the relationship between energy poverty and onset of asthma, chronic bronchitis or emphysema, hypertension, coronary heart disease, and depression/anxiety. Fixed effects and correlated randomeffects regression was used in models. As exposure and outcomes were self-reported, we tested alternative specifications of each to examine bias from measurement error. Findings When people can no longer afford to warm their homes, their mental health declines significantly (by 4.6- points on the SF-36 mental health scale, 95% CI −4.93 to −4.24), their odds of reporting depression/anxiety or hypertension increases by 49% (OR 1.49, 95% CI 1.09 to 2.02) and 71% (OR 1.71, 95% CI 1.13 to 2.58) respectively. The findings for the decline in mental health were supported in additional analyses that tested alternative specifications of the exposure measure, including co-resident verification of respondent reporting of being able to afford to warm the home. Support for an effect of energy poverty on hypertension was less clearly supported in these same sensitivity models. There was little evidence of an effect of energy poverty on asthma or chronic bronchitis onset in this adult population noting, however, that we could not examine exacerbation of symptoms. Interpretation Reducing exposure to energy poverty should be considered as an intervention with clear benefits for mental health and potential benefits for cardiovascular health.Item Open Access Readout of histone methylation by Trim24 locally restricts chromatin opening by p53.(Springer Science and Business Media LLC, 2023) Isbel, L.; Iskar, M.; Durdu, S.; Weiss, J.; Grand, R.S.; Hietter-Pfeiffer, E.; Kozicka, Z.; Michael, A.K.; Burger, L.; Thomä, N.H.; Schübeler, D.The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.Item Metadata only Progesterone receptor mediates ovulatory transcription through RUNX transcription factor interactions and chromatin remodelling.(Oxford University Press, 2023) Dinh, D.T.; Breen, J.; Nicol, B.; Foot, N.J.; Bersten, D.C.; Emery, A.; Smith, K.M.; Wong, Y.Y.; Barry, S.C.; Yao, H.H.C.; Robker, R.L.; Russell, D.L.Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.Item Open Access Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity(Public Library of Science (PLoS), 2021) Wang, J.; Farkas, C.; Benyoucef, A.; Carmichael, C.; Haigh, K.; Wong, N.; Huylebroeck, D.; Stemmler, M.P.; Brabletz, S.; Brabletz, T.; Nefzger, C.M.; Goossens, S.; Berx, G.; Polo, J.M.; Haigh, J.J.; Eaves, C.J.The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.Item Metadata only Efficient non-viral CAR-T cell generation via silicon-nanotube-mediated transfection(ELSEVIER SCI LTD, 2023) Chen, Y.; Mach, M.; Shokouhi, A.R.; Yoh, H.Z.; Bishop, D.C.; Murayama, T.; Suu, K.; Morikawa, Y.; Barry, S.C.; Micklethwaite, K.; Elnathan, R.; Voelcker, N.H.Cell-based immunotherapy such as chimeric antigen receptor (CAR)-T therapy holds great promise in treating cancer and other diseases; but the current viral-based method represents a significant cost and safety hurdle. Here, we show for the first time successful CAR transfection into primary T cells via vertically aligned silicon nanotube (SiNT) arrays. SiNT-mediated transfection achieves comparable or even higher delivery efficiency (20–37%) and expression efficiency (18–24%) to that achieved by electroporation. Scanning electron microscopy imaging after focused ion beam milling demonstrated the tight T cell–SiNT interface. The induced membrane invaginations and the proximity between individual SiNTs and the nucleus might enhance endocytic pathways, and enable direct delivery of CAR construct into the nucleus, thus resulting in higher CAR expression efficiency. SiNT-interfacing also results in faster proliferation of T cells compared to cells transfected by electroporation; nonactivated T (N_SiNT) cells undergo higher numbers of cell division than pre-activated ones (A_SiNT). By co-culturing with target lymphoma Raji cells, we prove that SiNT-transfected CAR-T cells can suppress Raji cell growth, indicated by significant increase in effector:target (E:T) ratio (by up to 30.7-fold). While SiNTs induce an overall upregulation of cytokine production in T cells, N_SiNT T cells exhibited high increase in secretion of IFNc and IL-6, and relatively high in TNFa, which could contribute to their enhanced killing ability (∼96% cytotoxicity), demonstrated by their stronger inhibition on target Raji cells through luciferase assay. The results demonstrate the capacity of SiNT-mediated transfection of generating effective anti-lymphoma CAR-T cells. Considering the growing potential of cell-based therapies, we expect that a non-viral nanoinjection platform such as ours will facilitate the full realization of their therapeutic promise.Item Open Access A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.(Springer Science and Business Media LLC, 2023) Mayoh, C.; Gifford, A.J.; Terry, R.; Lau, L.M.S.; Wong, M.; Rao, P.; Shai-Hee, T.; Saletta, F.; Khuong-Quang, D.-A.; Qin, V.; Mateos, M.K.; Meyran, D.; Miller, K.E.; Yuksel, A.; Mould, E.V.A.; Bowen-James, R.; Govender, D.; Senapati, A.; Zhukova, N.; Omer, N.; et al.BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.Item Metadata only Hypotension-Avoidance Versus Hypertension-Avoidance Strategies in Noncardiac Surgery : An International Randomized Controlled Trial(American College of Physicians, 2023) Marcucci, M.; Painter, T.W.; Conen, D.; Lomivorotov, V.; Sessler, D.I.; Chan, M.T.V.; Borges, F.K.; Leslie, K.; Duceppe, E.; Martínez-Zapata, M.J.; Wang, C.Y.; Xavier, D.; Ofori, S.N.; Wang, M.K.; Efremov, S.; Landoni, G.; Kleinlugtenbelt, Y.V.; Szczeklik, W.; Schmartz, D.; Garg, A.X.; et al.Background: Among patients having noncardiac surgery, perioperative hemodynamic abnormalities are associated with vascular complications. Uncertainty remains about what intraoperative blood pressure to target and how to manage long-term antihypertensive medications perioperatively. Objective: To compare the effects of a hypotension-avoidance and a hypertension-avoidance strategy on major vascular complications after noncardiac surgery. Design: Partial factorial randomized trial of 2 perioperative blood pressure management strategies (reported here) and tranexamic acid versus placebo. (ClinicalTrials.gov: NCT03505723) Setting: 110 hospitals in 22 countries. Patients: 7490 patients having noncardiac surgery who were at risk for vascular complications and were receiving 1 or more long-term antihypertensive medications. Intervention: In the hypotension-avoidance strategy group, the intraoperative mean arterial pressure target was 80mm Hg or greater; before and for 2 days after surgery, renin– angiotensin–aldosterone system inhibitors were withheld and the other long-term antihypertensive medications were administered only for systolic blood pressures 130mm Hg or greater, following an algorithm. In the hypertension-avoidance strategy group, the intraoperative mean arterial pressure target was 60mm Hg or greater; all antihypertensive medications were continued before and after surgery. Measurements: The primary outcome was a composite of vascular death and nonfatal myocardial injury after noncardiac surgery, stroke, and cardiac arrest at 30 days. Outcome adjudicators were masked to treatment assignment. Results: The primary outcome occurred in 520 of 3742 patients (13.9%) in the hypotension-avoidance group and in 524 of 3748 patients (14.0%) in the hypertension-avoidance group (hazard ratio, 0.99 [95% CI, 0.88 to 1.12]; P =0.92). Results were consistent for patients who used 1 or more than 1 antihypertensive medication in the long term. Limitation: Adherence to the assigned strategies was suboptimal; however, results were consistent across different adherence levels. Conclusion: In patients having noncardiac surgery, our hypotension-avoidance and hypertension-avoidance strategies resulted in a similar incidence of major vascular complications.Item Open Access INTEGRATE II: randomised phase III controlled trials of regorafenib containing regimens versus standard of care in refractory Advanced Gastro-Oesophageal Cancer (AGOC): a study by the Australasian Gastro-Intestinal Trials Group (AGITG)(Springer Science and Business Media LLC, 2023) Lam, L.L.; Pavlakis, N.; Shitara, K.; Sjoquist, K.M.; Martin, A.J.; Yip, S.; Kang, Y.K.; Bang, Y.J.; Chen, L.T.; Moehler, M.; Bekaii-Saab, T.; Alcindor, T.; O’Callaghan, C.J.; Tebbutt, N.C.; Hague, W.; Chan, H.; Rha, S.Y.; Lee, K.W.; Gebski, V.; Jaworski, A.; et al.BACKGROUND: Advanced gastro-oesophageal cancer (AGOC) carries a poor prognosis. No standard of care treatment options are available after first and second-line therapies. Regorafenib is an oral multi-targeted tyrosine kinase inhibitor targeting angiogenic, stromal, and oncogenic receptor tyrosine kinases. Regorafenib 160 mg daily prolonged progression free survival compared to placebo (INTEGRATE, phase 2). Regorafenib 80 mg daily in combination with nivolumab 3 mg/kg showed promising objective response rates (REGONIVO). METHODS/DESIGN: INTEGRATE II (INTEGRATE IIa and IIb) platform comprises two international phase III randomised controlled trials (RCT) with 2:1 randomisation in favor of experimental intervention. INTEGRATE IIa (double-blind) compares regorafenib 160 mg daily on days 1 to 21 of each 28-day cycle to placebo. INTEGRATE IIb (open label) compares REGONIVO, regorafenib 90 mg days 1 to 21 in combination with intravenous nivolumab 240 mg days 1 and 15 each 28-day cycle with investigator's choice of chemotherapy (control). Treatment continues until disease progression or intolerable adverse events as per protocol. Eligible participants include adults with AGOC who have failed two or more lines of treatment. Stratification is by location of tumour (INTEGRATE IIa only), geographic region, prior VEGF inhibitor and prior immunotherapy use (INTEGRATE IIb only). Primary endpoint is overall survival. Secondary endpoints are progression free survival, objective response rate, quality of life, and safety. Tertiary/correlative objectives include biomarker and pharmacokinetic evaluation. DISCUSSION: INTEGRATE II provides a platform to evaluate the clinical utility of regorafenib alone, as well as regorafenib in combination with nivolumab in treatment of participants with refractory AGOC. TRIAL REGISTRATION: INTEGRATE IIa prospectively registered 1 April 2016 Australia New Zealand Clinical Trial Registry: ACTRN12616000420448 (ClinicalTrials.gov NCT02773524). INTEGRATE IIb prospectively registered 10 May 2021 ClinicalTrials.gov: NCT04879368.Item Open Access Regulatory T cells are paramount effectors in progesterone regulation of embryo implantation and fetal growth(American Society for Clinical Investigation, 2023) Green, E.S.; Moldenhauer, L.M.; Groome, H.M.; Sharkey, D.J.; Chin, P.Y.; Care, A.S.; Robker, R.L.; McColl, S.R.; Robertson, S.A.Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 0.5 and 2.5 post coitum (dpc) to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in mid-gestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells - but not T conventional (Tconv) cells - alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure, and restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation, and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.Item Metadata only Neonatal Group B Streptococcal Infection in Australia: A Case-control Study.(Lippincott, Williams & Wilkins, 2023) Yanni, M.; Stark, M.; Francis, L.; Francis, J.R.; McMillan, M.; Baird, R.; Heath, P.T.; Gordon, A.; Riccardione, J.; Wilson, A.; Lee, R.; Chooi, K.; Quinn, O.-P.; Marshall, H.S.BACKGROUND: To determine maternal and neonatal risk factors for, and incidence of, neonatal early-onset group B streptococcus (EOGBS) and late-onset (LOGBS) infection in South Australia (SA) and the Northern Territory (NT). METHODS: A case-control study with 2:1 matched controls to cases. The study included tertiary hospitals in South Australia and the Northern Territory, Australia. Retrospective data were collected from a 16-year epoch (2000-2015). RESULTS: Of a total of 188 clinically suspected or confirmed cases, 139 were confirmed, of which 56.1% (n = 78) were EOGBS and 43.9% (n = 61) were LOGBS. The incidence of clinically suspected and confirmed cases of EOGBS was 0.26/1000 live births in SA and 0.73/1000 live births in the NT, and the incidence of confirmed cases was 0.19/1000 for SA and 0.36/1000 for the NT. The incidence of clinically suspected or confirmed LOGBS was 0.18/1000 live births in SA and 0.16/1000 for the NT. The majority of infants with GBS presented with sepsis, pneumonia, or meningitis. Developmental delay was the most commonly recorded long-term complication at 1 year old. Risk factors for EOGBS included maternal GBS carriage, previous fetal death, identifying as Aboriginal and/or Torres Strait Islander, and maternal fever in labor/chorioamnionitis. CONCLUSIONS: GBS remains a leading cause of neonatal morbidity and mortality. Adding previous fetal death to GBS screening guidelines would improve GBS prevention. The introduction of maternal GBS vaccination programs should be guided by country-specific disease epidemiology.Item Open Access Movement of cerebrospinal fluid tracer into brain parenchyma and outflow to nasal mucosa is reduced at 24 h but not 2 weeks post-stroke in mice(Springer Science and Business Media LLC, 2023) Warren, K.E.; Coupland, K.G.; Hood, R.J.; Kang, L.; Walker, F.R.; Spratt, N.J.Background Recent data indicates that cerebrospinal fluid (CSF) dynamics are disturbed after stroke. Our lab has previously shown that intracranial pressure rises dramatically 24 h after experimental stroke and that this reduces blood flow to ischaemic tissue. CSF outflow resistance is increased at this time point. We hypothesised that reduced transit of CSF through brain parenchyma and reduced outflow of CSF via the cribriform plate at 24 h after stroke may contribute to the previously identified post-stroke intracranial pressure elevation. Methods Using a photothrombotic permanent occlusion model of stroke in C57BL/6 adult male mice, we examined the movement of an intracisternally infused 0.5% Texas Red dextran throughout the brain and measured tracer efflux into the nasal mucosa via the cribriform plate at 24 h or two weeks after stroke. Brain tissue and nasal mucosa were collected ex vivo and imaged using fluorescent microscopy to determine the change in CSF tracer intensity in these tissues. Results At 24 h after stroke, we found that CSF tracer load was significantly reduced in brain tissue from stroke animals in both the ipsilateral and contralateral hemispheres when compared to sham. CSF tracer load was also reduced in the lateral region of the ipsilateral hemisphere when compared to the contralateral hemisphere in stroke brains. In addition, we identified an 81% reduction in CSF tracer load in the nasal mucosa in stroke animals compared to sham. These alterations to the movement of CSF-borne tracer were not present at two weeks after stroke. Conclusions Our data indicates that influx of CSF into the brain tissue and efflux via the cribriform plate are reduced 24 h after stroke. This may contribute to reported increases in intracranial pressure at 24 h after stroke and thus worsen stroke outcomes.Item Open Access CRISPR screens identify gene targets at breast cancer risk loci(Springer Science and Business Media LLC, 2023) Tuano, N.K.; Beesley, J.; Manning, M.; Shi, W.; Perlaza-Jimenez, L.; Malaver-Ortega, L.F.; Paynter, J.M.; Black, D.; Civitarese, A.; McCue, K.; Hatzipantelis, A.; Hillman, K.; Kaufmann, S.; Sivakumaran, H.; Polo, J.M.; Reddel, R.R.; Band, V.; French, J.D.; Edwards, S.L.; Powell, D.R.; et al.Background: Genome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in noncoding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS. Results: Here, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants. Conclusions: We demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.Item Metadata only Association between dairy intake, lipids and vascular structure and function in diabetes(BAISHIDENG PUBLISHING GROUP INC, 2017) Petersen, K.S.; Keogh, J.B.; Lister, N.; Weir, J.M.; Meikle, P.J.; Clifton, P.M.AIM to determine lipid species that change in response to a change in dairy consumption. In addition, to investigate whether dairy associated lipid species are correlated with changes in measures of vascular structure and function. METHODS A 12-mo randomised controlled trial was conducted to determine the effect of increased consumption of fruit, vegetables and dairy, compared to usual diet, on measures of vascular structure and function in adults with type 1 and type 2 diabetes (n = 108). This paper comprises post-hoc analyses investigating the relationship between dairy intake, serum lipid species and vascular health. Central and peripheral blood pressure, carotid femoral pulse wave velocity, augmentation index, serum lipid species and dietary intake were measured at baseline and 3-mo. Common carotid artery intima media thickness was measured at baseline and 12-mo. RESULTS serum lipid species [lysophosphatidylcholine (LPC) 14:0, LPC 15:0, LPC 16:1, phosphatidylcholine (PC) 29:0 PC 30:0, PC 31:0 and cholesterol ester (CE) 14:0] were associated with the change in full fat dairy consumption (rho 0.19-0.25; p < 0.05). The 3-mo change in some lipids was positively associated with the 3-mo change in central systolic [LPC 14:0 (rho 0.30; p = 0.007), PC 30:0 (rho 0.28; p = 0.010)] and diastolic blood pressure [LPC 14:0 (rho 0.32; p = 0.004), LPC 15:0 (rho 0.23; p = 0.04), LPC 16:1 (rho 0.23; p = 0.035), PC 29:0 (rho 0.28; p = 0.01), PC 30:0 (rho 0.36; p = 0.001), PC 31:0 (rho 0.30; p = 0.007)] and 12-mo change in common carotid artery intimal medial thickness [CE 14:0 (rho 0.22; p = 0.02)]. Pulse wave velocity and augmentation index were unrelated to dairy and lipid species. CONCLUSION an increase in dairy associated lipids appears to be associated with an increase in blood pressure and common carotid intimal medial thickness.Item Metadata only Sparse spike trains and the limitation of rate codes underlying rapid behaviours.(Royal Society, The, 2023) Fabian, J.M.; O'Carrol, D.C.; Wiederman, S.D.Animals live in dynamic worlds where they use sensorimotor circuits to rapidly process information and drive behaviours. For example, dragonflies are aerial predators that react to movements of prey within tens of milliseconds. These pursuits are likely controlled by identified neurons in the dragonfly, which have well-characterized physiological responses to moving targets. Predominantly, neural activity in these circuits is interpreted in context of a rate code, where information is conveyed by changes in the number of spikes over a time period. However, such a description of neuronal activity is difficult to achieve in real-world, real-time scenarios. Here, we contrast a neuroscientists' post-hoc view of spiking activity with the information available to the animal in real-time. We describe how performance of a rate code is readily overestimated and outline a rate code's significant limitations in driving rapid behaviours.Item Open Access Statement in Support of: "Virology under the Microscope-a Call for Rational Discourse"(American Society for Microbiology, 2023) Speck, P.; Mackenzie, J.; Bull, R.A.; Slobedman, B.; Drummer, H.; Fraser, J.; Herrero, L.; Helbig, K.; Londrigan, S.; Moseley, G.; Prow, N.; Hansman, G.; Edwards, R.; Ahlenstiel, C.; Abendroth, A.; Tscharke, D.; Hobson-Peters, J.; Kriiger-Loterio, R.; Parry, R.; Marsh, G.; et al.Item Open Access Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes(Frontiers Media SA, 2022) Agnew, H.N.; Brazel, E.B.; Tikhomirova, A.; van der Linden, M.; McLean, K.T.; Paton, J.C.; Trappetti, C.Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infectedmice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.Item Open Access Termination of STING responses is mediated via ESCRT-dependent degradation(EMBO, 2023) Balka, K.R.; Venkatraman, R.; Saunders, T.L.; Shoppee, A.; Pang, E.S.; Magill, Z.; Homman-Ludiye, J.; Huang, C.; Lane, R.M.; York, H.M.; Tan, P.; Schittenhelm, R.B.; Arumugam, S.; Kile, B.T.; O'Keeffe, M.; De Nardo, D.cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.Item Metadata only One immune system plays many parts: The dynamic role of the immune system in chronic pain and opioid pharmacology.(Elsevier BV, 2023) Mustafa, S.; Bajic, J.E.; Barry, B.; Evans, S.; Siemens, K.R.; Hutchinson, M.R.; Grace, P.M.The transition from acute to chronic pain is an ongoing major problem for individuals, society and healthcare systems around the world. It is clear chronic pain is a complex multidimensional biological challenge plagued with difficulties in pain management, specifically opioid use. In recent years the role of the immune system in chronic pain and opioid pharmacology has come to the forefront. As a highly dynamic and versatile network of cells, tissues and organs, the immune system is perfectly positioned at the microscale level to alter nociception and drive structural adaptations that underpin chronic pain and opioid use. In this review, we highlight the need to understand the dynamic and adaptable characteristics of the immune system and their role in the transition, maintenance and resolution of chronic pain. The complex multidimensional interplay of the immune system with multiple physiological systems may provide new transformative insight for novel targets for clinical management and treatment of chronic pain.