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Introduction: Transcriptomic analyses from early human immunodeficiency virus (HIV) infection have the potential to reveal how HIV causes widespread and lasting damage to biological functions, especially in the immune system. Previous studies have been limited by difficulties in obtaining early specimens., Methods: A hospital symptom-based screening approach was applied in a rural Mozambican setting to enrol patients with suspected acute HIV infection (Fiebig stage I-IV). Blood samples were collected from all those recruited, so that acute cases and contemporaneously recruited, uninfected controls were included. PBMC were isolated and sequenced using RNA-seq. Sample cellular composition was estimated from gene expression data. Differential gene expression analysis was completed, and correlations were determined between viral load and differential gene expression. Biological implications were examined using Cytoscape, gene set enrichment analysis, and enrichment mapping., Results: Twenty-nine HIV infected subjects one month from presentation and 46 uninfected controls were included in this study. Subjects with acute HIV infection demonstrated profound gene dysregulation, with 6131 (almost 13% of the genome mapped in this study) significantly differentially expressed. Viral load was correlated with 1.6% of dysregulated genes, in particular, highly upregulated genes involved in key cell cycle functions, were correlated with viremia. The most profoundly upregulated biological functions related to cell cycle regulation, in particular, CDCA7 may drive aberrant cell division, promoted by overexpressed E2F family proteins. Also upregulated were DNA repair and replication, microtubule and spindle organization, and immune activation and response. The interferome of acute HIV was characterized by broad activation of interferon-stimulated genes with antiviral functions, most notably IFI27 and OTOF. BCL2 downregulation alongside upregulation of several apoptotic trigger genes and downstream effectors may contribute to cycle arrest and apoptosis. Transmembrane protein 155 (TMEM155) was consistently highly overexpressed during acute infection, with roles hitherto unknown., Discussion: Our study contributes to a better understanding of the mechanisms of early HIV-induced immune damage. These findings have the potential to lead to new earlier interventions that improve outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Parker, Judge, Pastor, Fuente-Soro, Jairoce, Carter, Anderson, Mandomando, Clifford, Naniche and Le Souëf.)

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6c cpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration., (© 2022. The Author(s).)

Competing Interests: None

Objective: To assess the risk of severe childhood infections within families, we conducted a sibling analysis in a population-based cohort study with genealogical linkage. We investigated the sibling risk of hospitalization with common infections, a marker of severity. We hypothesized that having siblings hospitalized for infection would increase the proband's risk of admission with infection., Study Design: We used population data on Western Australian live-born singletons and their siblings between 1980 and 2014. Measures of infection were infection-related hospitalizations from discharge diagnostic codes. Exposure was having a sibling who had an infection-related hospitalization. Outcomes were infection-related hospitalizations in the child/proband. Probands were followed until an infection-related hospitalization admission (up to the first three), death, 18th birthday, or end of 2014, whichever occurred first. Infection risks were estimated by adjusted Cox proportional hazard models for multiple events., Results: Of 512,279 probands, 142,915 (27.9%) had infection-related hospitalizations; 133,322 (26.0%) had a sibling with a previous infection-related hospitalization (i.e. exposed). Median interval between sibling and proband infection-related hospitalizations was 1.4 years (inter-quartile range 0.5-3.7). Probands had a dose-dependent increase in risk if sibling/s had 1, 2, or 3+ infection-related hospitalizations (adjusted hazard ratio, aHR 1.41, 95% CI 1.39-1.43; aHR 1.65, 1.61-1.69; aHR 1.83, 1.77-1.90, respectively). Among siblings with the same clinical infection type, highest sibling risks were for genitourinary (aHR 2.06, 1.68-2.53), gastrointestinal (aHR 2.07, 1.94-2.19), and skin/soft tissue infections (aHR 2.34, 2.15-2.54). Overall risk of infection-related hospitalization was higher in children with more siblings and with older siblings., Conclusion: In this population-based study, we observed an increased risk of infection-related hospitalization in children whose siblings were previously hospitalized for infection. Public health interventions may be particularly relevant in families of children hospitalized with infection., Competing Interests: The authors have declared that no competing interests exist.

Background: Maternal stressful life events during pregnancy have been associated with immune dysregulation and increased risk for asthma and atopy in offspring. Few studies have investigated whether prenatal stress is associated with increased overall or specific infectious diseases in childhood, nor explored sex differences. We sought to examine the relationship between the nature and timing of maternal stress in pregnancy and hospitalisation with infection in offspring., Methods: Between 1989 and 1992, exposure data on stressful life events were collected from pregnant women (Gen1) in the Raine Study at 18 and 34 weeks' gestation and linked to statutory state-wide hospital morbidity data. We examined associations between the number, category and timing of maternal prenatal stress events and overall and clinical groups of offspring (Gen2) infection-related hospitalisation until age 16 years, adjusting for maternal age, education, and smoking in pregnancy in addition to the presence of siblings at birth., Results: Of 2,141 offspring with complete stress in pregnancy data available, 1,089 had at least one infection-related hospitalisation, with upper respiratory tract infections the most common (n = 556). Each additional stressful life event during pregnancy was associated with increased risk in male offspring for hospitalisation with all infection types. There was little evidence of these associations in girls., Conclusions: Increased exposure to stressful life events in utero is associated with sex-specific infection-related hospitalisations in childhood. Prenatal stress may adversely affect early immune development for boys and increase the risk of more severe infections. Mechanistic understanding would inform preventative interventions., Competing Interests: The authors have declared that no competing interests exist.

Background: The proportion of births via cesarean section (CS) varies worldwide and in many countries exceeds WHO-recommended rates. Long-term health outcomes for children born by CS are poorly understood, but limited data suggest that CS is associated with increased infection-related hospitalisation. We investigated the relationship between mode of birth and childhood infection-related hospitalisation in high-income countries with varying CS rates., Methods and Findings: We conducted a multicountry population-based cohort study of all recorded singleton live births from January 1, 1996 to December 31, 2015 using record-linked birth and hospitalisation data from Denmark, Scotland, England, and Australia (New South Wales and Western Australia). Birth years within the date range varied by site, but data were available from at least 2001 to 2010 for each site. Mode of birth was categorised as vaginal or CS (emergency/elective). Infection-related hospitalisations (overall and by clinical type) occurring after the birth-related discharge date were identified in children until 5 years of age by primary/secondary International Classification of Diseases, 10th Revision (ICD-10) diagnosis codes. Analysis used Cox regression models, adjusting for maternal factors, birth parameters, and socioeconomic status, with results pooled using meta-analysis. In total, 7,174,787 live recorded births were included. Of these, 1,681,966 (23%, range by jurisdiction 17%-29%) were by CS, of which 727,755 (43%, range 38%-57%) were elective. A total of 1,502,537 offspring (21%) had at least 1 infection-related hospitalisation. Compared to vaginally born children, risk of infection was greater among CS-born children (hazard ratio (HR) from random effects model, HR 1.10, 95% confidence interval (CI) 1.09-1.12, p < 0.001). The risk was higher following both elective (HR 1.13, 95% CI 1.12-1.13, p < 0.001) and emergency CS (HR 1.09, 95% CI 1.06-1.12, p < 0.001). Increased risks persisted to 5 years and were highest for respiratory, gastrointestinal, and viral infections. Findings were comparable in prespecified subanalyses of children born to mothers at low obstetric risk and unchanged in sensitivity analyses. Limitations include site-specific and longitudinal variations in clinical practice and in the definition and availability of some data. Data on postnatal factors were not available., Conclusions: In this study, we observed a consistent association between birth by CS and infection-related hospitalisation in early childhood. Notwithstanding the limitations of observational data, the associations may reflect differences in early microbial exposure by mode of birth, which should be investigated by mechanistic studies. If our findings are confirmed, they could inform efforts to reduce elective CS rates that are not clinically indicated., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Individuals with asthma have heightened antibody responses to rhinoviruses (RVs), although those specific for RV-C are lower than responses specific for RV-A, suggesting poor immunity to this species. Objectives: To ascertain and compare T-cell memory responses induced by RV-A and RV-C in children with and without asthma. Methods: Peripheral blood mononuclear cells from 17 children with asthma and 19 control subjects without asthma were stimulated in vitro with peptide formulations to induce representative species-specific responses to RV-A and RV-C. Molecular profiling (RNA sequencing) was used to identify enriched pathways and upstream regulators. Measurements and Main Results: Responses to RV-A showed higher expression of IFNG and STAT1 compared with RV-C, and significant expression of CXCL9, 10, and 11 was not found for RV-C. There was no reciprocal increase of T-helper cell type 2 (Th2) cytokine genes or the Th2 chemokine genes CCL11, CCL17, and CCL22. RV-C induced higher expression of CCL24 (eotaxin-2) than RV-A in the responses of children with and without asthma. Upstream regulator analysis showed both RV-A and, although to a lesser extent, RV-C induced predominant Th1 and inflammatory cytokine expression. The responses of children with asthma compared with those without asthma were lower for both RV-A and RV-C while retaining the pattern of gene expression and upstream regulators characteristic of each species. All groups showed activation of the IL-17A pathway. Conclusions: RV-C induced memory cells with a lower IFN-γ-type response than RV-A without T-helper cell type 2 (Th2) upregulation. Children with asthma had lower recall responses than those without asthma while largely retaining the same gene activation profile for each species. RV-A and RV-C, therefore, induce qualitatively different T-cell responses.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity., (Copyright © 2017 by The American Association of Immunologists, Inc.)

There are numerous reference equations available for the single-breath transfer factor of the lung for carbon monoxide ( T  LCO ); however, it is not always clear which reference set should be used in clinical practice. The aim of the study was to develop the Global Lung Function Initiative (GLI) all-age reference values for T  LCO Data from 19 centres in 14 countries were collected to define T  LCO reference values. Similar to the GLI spirometry project, reference values were derived using the LMS (lambda, mu, sigma) method and the GAMLSS (generalised additive models for location, scale and shape) programme in R.12 660 T  LCO measurements from asymptomatic, lifetime nonsmokers were submitted; 85% of the submitted data were from Caucasians. All data were uncorrected for haemoglobin concentration. Following adjustments for elevation above sea level, gas concentration and assumptions used for calculating the anatomic dead space volume, there was a high degree of overlap between the datasets. Reference values for Caucasians aged 5-85 years were derived for T  LCO , transfer coefficient of the lung for carbon monoxide and alveolar volume.This is the largest collection of normative T  LCO data, and the first global reference values available for T  LCO ., Competing Interests: Conflict of interest: None declared., (Copyright ©ERS 2017.)

The Global Lung Function Initiative (GLI) Network has become the largest resource for reference values for routine lung function testing ever assembled. This article addresses how the GLI Network came about, why it is important, and its current challenges and future directions. It is an extension of an article published in Breathe in 2013 [1], and summarises recent developments and the future of the GLI Network., Key Points: The Global Lung Function Initiative (GLI) Network was established as a result of international collaboration, and altruism between researchers, clinicians and industry partners. The ongoing success of the GLI relies on network members continuing to work together to further improve how lung function is reported and interpreted across all age groups around the world.The GLI Network has produced standardised lung function reference values for spirometry and gas transfer tests.GLI reference equations should be adopted immediately for spirometry and gas transfer by clinicians and physiologists worldwide.The recently established GLI data repository will allow ongoing development and evaluation of reference values, and will offer opportunities for novel research., Educational Aims: To highlight the advances made by the GLI Network during the past 5 years.To highlight the importance of using GLI reference values for routine lung function testing ( e.g.  spirometry and gas transfer tests).To discuss the challenges that remain for developing and improving reference values for lung function tests., Competing Interests: Conflict of interest None declared.

A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E 2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E 2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aim: To determine the incidence of hospitalisations and risk factors for vascular complications experienced during early adulthood in patients with childhood onset type 1 diabetes., Methods: A population-based childhood onset type 1 diabetes cohort was identified from a statewide register (1992-2012). Data linkage was used to identify a matched comparison cohort. Hospital admissions data were extracted to follow up both cohorts into early adulthood (1975-2012)., Results: The type 1 diabetes cohort (n=1316) had a mean age of diagnosis of 9.5years, 49.5% were women and mean age at the end of follow-up was 26.3years (range 18-38). Within the type 1 diabetes cohort 32 (2.4%) were hospitalised with a vascular complication during early adulthood. Poor glycaemic control during paediatric management was associated with a significant increase in risk for ophthalmic complication with 19.4% (n=12/62) of those with a mean HbA1c >12% (108mmol/mol) diagnosed compared to 0.72% (n=5/696) of those with mean HbA1c <9% (75mmol/mol), adjusted hazard ratio 8.4 (95% CI 2.0, 34.7)., Conclusion: Severe vascular complications requiring hospital admission continue to be observed during early adulthood. Both women and those with poor glycaemic control are at increased risk of requiring a hospital admission for these complications during early adulthood., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL., Competing Interests: RK Prinjha and N Smithers are employees and shareholders of GlaxoSmithKline, which is carrying out clinical development of epigenetic inhibitors. The remaining authors declare no conflict of interest.

The field of neuroimaging has embraced the need for sharing and collaboration. Data sharing mandates from public funding agencies and major journal publishers have spurred the development of data repositories and neuroinformatics consortia. However, efficient and effective data sharing still faces several hurdles. For example, open data sharing is on the rise but is not suitable for sensitive data that are not easily shared, such as genetics. Current approaches can be cumbersome (such as negotiating multiple data sharing agreements). There are also significant data transfer, organization and computational challenges. Centralized repositories only partially address the issues. We propose a dynamic, decentralized platform for large scale analyses called the Collaborative Informatics and Neuroimaging Suite Toolkit for Anonymous Computation (COINSTAC). The COINSTAC solution can include data missing from central repositories, allows pooling of both open and "closed" repositories by developing privacy-preserving versions of widely-used algorithms, and incorporates the tools within an easy-to-use platform enabling distributed computation. We present an initial prototype system which we demonstrate on two multi-site data sets, without aggregating the data. In addition, by iterating across sites, the COINSTAC model enables meta-analytic solutions to converge to "pooled-data" solutions (i.e., as if the entire data were in hand). More advanced approaches such as feature generation, matrix factorization models, and preprocessing can be incorporated into such a model. In sum, COINSTAC enables access to the many currently unavailable data sets, a user friendly privacy enabled interface for decentralized analysis, and a powerful solution that complements existing data sharing solutions.

Background: Reduced gestational age and low birthweight are associated with an increased risk of neonatal infections. However, the long-term risk of infection, especially in late preterm infants or those at near-normal birthweight, is unknown. We estimated whether rates of infection-related admissions to hospital for children in Western Australia were associated with age, gestational age, birthweight, and birth length., Methods: We did a population-based, data-linkage study using total-linked, registry data from the Western Australia Birth Register of all liveborn, non-Indigenous Australian singleton births recorded from Jan 1, 1980, to Dec 31, 2010. We followed up individuals from birth-related hospital discharge to age 18 years, death, or end of 2010, and linked to data about subsequent admissions to hospital or death registrations. Gestational age was assessed from both the last menstrual period and from estimates based on ultrasonography. We categorised birthweight by 500 g bands and birth length by 5 cm bands, and approximated the reference ranges for both to the 50th percentile. Because size at birth and gestational age are strongly associated, we calculated Z scores for gestational-specific and sex-specific birthweight, birth length, and ponderal index. Our primary outcomes were the number and type of infection-related admissions to hospital. We used multilevel negative binomial regression to generate rate ratios (RR) for such admissions, identified by codes from the International Classification of Diseases, versions 9 and 10-AM. We adjusted the RRs for maternal age at delivery, birth year, birth season, parity, sex, 5-min Apgar score, delivery method, socioeconomic status, and bronchopulmonary dysplasia., Findings: Of 719 311 liveborn singletons included in the analysis and followed up for 8 824 093 person-years, 365 867 infection-related admissions to hospital occurred for 213 683 (30%) children. Of the 719 311 children included in the analysis, 137 124 (19%) had one infection-related admission to hospital, 43 796 (6%) had two, 16 679 (2%) had three, and 16 084 (2%) had four or more. The 365 867 admissions to hospital included a diagnosis of infection of the upper respiratory tract for 174 653 (48%), the lower respiratory tract for 74 297 (20%), the gastrointestinal tract for 44 755 (12%), and a viral infection for 37 213 (10%). Infection-related rates of admissions to hospital increased by 12% for each week reduction in gestational age less than 39-40 weeks (RR 1·12, 95% CI 1·12-1·13), by 19% for each 500 g reduction in birthweight less than 3000-3500 g (1·19, 1·18-1·21), and by 41% for each 5 cm reduction in birth length less than 45-50 cm (1·41, 1·38-1·45). Gestational age-specific and sex-specific birthweight Z scores lower than the 25th to 50th percentile and birth length Z scores lower than the 10th to 25th percentile were associated with increased rates of infection-related admissions to hospital (eg, 1st-5th percentile RR 1·15, 95% CI 1·12-1·19, and 1·11, 1·07-1·14, respectively). Ponderal index Z scores lower than the 25th to 50th percentile were also associated with increased rates of infection-related admissions (eg, 1st-5th percentile RR 1·08, 95% CI 1·04-1·12). A gestational age of 41 weeks or later, a birthweight or birth length Z score above the 50th percentile, or a ponderal index Z score between the 75th and 95th percentile, were associated with modestly reduced rates of infection-related admissions to hospital., Interpretation: Children who were born with reduced gestational age, birthweight, and birth length have persistently increased rates of infection-related admissions to hospital until age 18 years. Pregnancy outcomes should be optimised to prevent infection occurring in this population, especially in resource-limited settings where suboptimum intrauterine growth and moderate prematurity are common., Funding: Australian National Health and Medical Research Council., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Research studies exploring the determinants of disease require sufficient statistical power to detect meaningful effects. Sample size is often increased through centralized pooling of disparately located datasets, though ethical, privacy and data ownership issues can often hamper this process. Methods that facilitate the sharing of research data that are sympathetic with these issues and which allow flexible and detailed statistical analyses are therefore in critical need. We have created a software platform for the Virtual Pooling and Analysis of Research data (ViPAR), which employs free and open source methods to provide researchers with a web-based platform to analyse datasets housed in disparate locations., Methods: Database federation permits controlled access to remotely located datasets from a central location. The Secure Shell protocol allows data to be securely exchanged between devices over an insecure network. ViPAR combines these free technologies into a solution that facilitates 'virtual pooling' where data can be temporarily pooled into computer memory and made available for analysis without the need for permanent central storage., Results: Within the ViPAR infrastructure, remote sites manage their own harmonized research dataset in a database hosted at their site, while a central server hosts the data federation component and a secure analysis portal. When an analysis is initiated, requested data are retrieved from each remote site and virtually pooled at the central site. The data are then analysed by statistical software and, on completion, results of the analysis are returned to the user and the virtually pooled data are removed from memory., Conclusions: ViPAR is a secure, flexible and powerful analysis platform built on open source technology that is currently in use by large international consortia, and is made publicly available at [http://bioinformatics.childhealthresearch.org.au/software/vipar/]., (© The Author 2015. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background/purpose: Falling from a low height is the most common history given by caregivers of pediatric nonaccidental trauma (NAT) victims evaluated for blunt abdominal trauma (BAT). The purpose of this study is to compare the patterns of injuries seen in children with BAT who are victims of NAT with those seen after a fall-related incident., Methods: Trauma database query from regional pediatric trauma centers identified patients were who were evaluated for BAT resulting from either NAT or a fall. Study groups included patients younger than five years who were (1) victims or NAT, or (2) fall casualties., Results: Sixty-five NATs and 115 fall casualties were identified. NAT victims had higher ISS, had more severe head injuries according to AIS scores, had more hollow viscus injuries, and had more pancreatic injuries. Fall casualties were more likely to have solid organ injuries., Conclusions: When evaluating children with a history of blunt abdominal trauma caused by a fall, suspicion for NAT is warranted if the child is younger than five years, has a hollow viscus, pancreatic, and/or severe head injury and has a high ISS. Likewise, isolated splenic or renal injury allays suspicion for NAT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Unlike mammals, zebrafish have the ability to regenerate damaged parts of their central nervous system (CNS) and regain functionality of the affected area. A better understanding of the molecular mechanisms involved in zebrafish regeneration may therefore provide insight into how CNS repair might be induced in mammals. Although many studies have described differences in gene expression in zebrafish during CNS regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined., Results: We used microarrays to analyse and integrate the mRNA and microRNA (miRNA) expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. Bioinformatic analysis identified 3 miRNAs and 657 mRNAs that were differentially expressed after injury. We then combined inverse correlations between our miRNA expression and mRNA expression, and integrated these findings with target predictions from TargetScan Fish to identify putative miRNA-gene target pairs. We focused on two over-expressed miRNAs (miR-29b and miR-223), and functionally validated seven of their predicted gene targets using RT-qPCR and luciferase assays to confirm miRNA-mRNA binding. Gene ontology analysis placed the miRNA-regulated genes (eva1a, layna, nefmb, ina, si:ch211-51a6.2, smoc1, sb:cb252) in key biological processes that included cell survival/apoptosis, ECM-cytoskeleton signaling, and heparan sulfate proteoglycan binding,, Conclusion: Our results suggest a key role for miR-29b and miR-223 in zebrafish regeneration. The identification of miRNA regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for CNS regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain.

Background: Pathogen-specific and overall infection burden may contribute to atherosclerosis and cardiovascular disease (CVD), but the effect of infection severity and timing is unknown. We investigated whether childhood infection-related hospitalisation (IRH, a marker of severity) was associated with subsequent adult CVD hospitalisation., Methods: Using longitudinal population-based statutorily-collected administrative health data from Western Australia (1970-2009), we identified adults hospitalised with CVD (ischaemic heart disease, ischaemic stroke, and peripheral vascular disease) and matched them (10:1) to population controls. We used Cox regression to assess relationships between number and type of childhood IRH and adulthood CVD hospitalisation, adjusting for sex, age, Indigenous status, socioeconomic status, and birth weight., Results: 631 subjects with CVD-related hospitalisation in adulthood (≥ 18 years) were matched with 6310 controls. One or more childhood (< 18 years) IRH was predictive of adult CVD-related hospitalisation (adjusted hazard ratio, 1.3; 95% CI 1.1-1.6; P < 0.001). The association showed a dose-response; ≥ 3 childhood IRH was associated with a 2.2 times increased risk of CVD-related hospitalisation in adulthood (adjusted hazard ratio, 2.2; 95% CI 1.7-2.9; P < 0.001). The association was observed across all clinical diagnostic groups of infection (upper respiratory tract infection, lower respiratory tract infection, infectious gastroenteritis, urinary tract infection, skin and soft tissue infection, and other viral infection), and individually with CVD diagnostic categories (ischaemic heart disease, ischaemic stroke and peripheral vascular disease)., Conclusions: Severe childhood infection is associated with CVD hospitalisations in adulthood in a dose-dependent manner, independent of population-level risk factors.

Background: Repetitive transcranial magnetic stimulation is increasingly used as a treatment for neurological dysfunction. Therapeutic effects have been reported for low intensity rTMS (LI-rTMS) although these remain poorly understood., Objective: Our study describes for the first time a systematic comparison of the cellular and molecular changes in neurons in vitro induced by low intensity magnetic stimulation at different frequencies., Methods: We applied 5 different low intensity repetitive magnetic stimulation (LI-rMS) protocols to neuron-enriched primary cortical cultures for 4 days and assessed survival, and morphological and biochemical change., Results: We show pattern-specific effects of LI-rMS: simple frequency pulse trains (10 Hz and 100 Hz) impaired cell survival, while more complex stimulation patterns (theta-burst and a biomimetic frequency) did not. Moreover, only 1 Hz stimulation modified neuronal morphology, inhibiting neurite outgrowth. To understand mechanisms underlying these differential effects, we measured intracellular calcium concentration during LI-rMS and subsequent changes in gene expression. All LI-rMS frequencies increased intracellular calcium, but rather than influx from the extracellular milieu typical of depolarization, all frequencies induced calcium release from neuronal intracellular stores. Furthermore, we observed pattern-specific changes in expression of genes related to apoptosis and neurite outgrowth, consistent with our morphological data on cell survival and neurite branching., Conclusions: Thus, in addition to the known effects on cortical excitability and synaptic plasticity, our data demonstrate that LI-rMS can change the survival and structural complexity of neurons. These findings provide a cellular and molecular framework for understanding what low intensity magnetic stimulation may contribute to human rTMS outcomes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Research in modern biomedicine and social science requires sample sizes so large that they can often only be achieved through a pooled co-analysis of data from several studies. But the pooling of information from individuals in a central database that may be queried by researchers raises important ethico-legal questions and can be controversial. In the UK this has been highlighted by recent debate and controversy relating to the UK's proposed 'care.data' initiative, and these issues reflect important societal and professional concerns about privacy, confidentiality and intellectual property. DataSHIELD provides a novel technological solution that can circumvent some of the most basic challenges in facilitating the access of researchers and other healthcare professionals to individual-level data., Methods: Commands are sent from a central analysis computer (AC) to several data computers (DCs) storing the data to be co-analysed. The data sets are analysed simultaneously but in parallel. The separate parallelized analyses are linked by non-disclosive summary statistics and commands transmitted back and forth between the DCs and the AC. This paper describes the technical implementation of DataSHIELD using a modified R statistical environment linked to an Opal database deployed behind the computer firewall of each DC. Analysis is controlled through a standard R environment at the AC., Results: Based on this Opal/R implementation, DataSHIELD is currently used by the Healthy Obese Project and the Environmental Core Project (BioSHaRE-EU) for the federated analysis of 10 data sets across eight European countries, and this illustrates the opportunities and challenges presented by the DataSHIELD approach., Conclusions: DataSHIELD facilitates important research in settings where: (i) a co-analysis of individual-level data from several studies is scientifically necessary but governance restrictions prohibit the release or sharing of some of the required data, and/or render data access unacceptably slow; (ii) a research group (e.g. in a developing nation) is particularly vulnerable to loss of intellectual property-the researchers want to fully share the information held in their data with national and international collaborators, but do not wish to hand over the physical data themselves; and (iii) a data set is to be included in an individual-level co-analysis but the physical size of the data precludes direct transfer to a new site for analysis., (© The Author 2014; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

The International Collaboration for Autism Registry Epidemiology (iCARE) is the first multinational research consortium (Australia, Denmark, Finland, Israel, Norway, Sweden, USA) to promote research in autism geographical and temporal heterogeneity, phenotype, family and life course patterns, and etiology. iCARE devised solutions to challenges in multinational collaboration concerning data access security, confidentiality and management. Data are obtained by integrating existing national or state-wide, population-based, individual-level data systems and undergo rigorous harmonization and quality control processes. Analyses are performed using database federation via a computational infrastructure with a secure, web-based, interface. iCARE provides a unique, unprecedented resource in autism research that will significantly enhance the ability to detect environmental and genetic contributions to the causes and life course of autism.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Background: Exposure to arsenic via drinking water is a global environmental health problem. In utero exposure to arsenic via drinking water increases the risk of lower respiratory tract infections during infancy and mortality from bronchiectasis in early adulthood., Objectives: We aimed to investigate how arsenic exposure in early life alters lung development and pathways involved in innate immunity., Methods: Pregnant BALB/c, C57BL/6, and C3H/HeARC mice were exposed to 0 (control) or 100 μg/L arsenic via drinking water from gestation day 8 until the birth of their offspring. We measured somatic growth, lung volume, and lung mechanics of mice at 2 weeks of age. We used fixed lungs for structural analysis and collected lung tissue for gene expression analysis by microarray., Results: The response to arsenic was genetically determined, and C57BL/6 mice were the most susceptible. Arsenic-exposed C57BL/6 mice were smaller in size, had smaller lungs, and had impaired lung mechanics compared with controls. Exposure to arsenic in utero up-regulated the expression of genes in the lung involved in mucus production (Clca3, Muc5b, Scgb3a1), innate immunity (Reg3γ, Tff2, Dynlrb2, Lplunc1), and lung morphogenesis (Sox2). Arsenic exposure also induced mucous cell metaplasia and increased expression of CLCA3 protein in the large airways., Conclusions: Alterations in somatic growth, lung development, and the expression of genes involved in mucociliary clearance and innate immunity in the lung are potential mechanisms through which early life arsenic exposure impacts respiratory health.

In a previous study, we detected a 6p25-p24 region linked to schizophrenia in families with high composite cognitive deficit (CD) scores, a quantitative trait integrating multiple cognitive measures. Association mapping of a 10 Mb interval identified a 260 kb region with a cluster of single-nucleotide polymorphisms (SNPs) significantly associated with CD scores and memory performance. The region contains two colocalising genes, LYRM4 and FARS2, both encoding mitochondrial proteins. The two tagging SNPs with strongest evidence of association were located around the overlapping putative promoters, with rs2224391 predicted to alter a transcription factor binding site (TFBS). Sequencing the promoter region identified 22 SNPs, many predicted to affect TFBSs, in a tight linkage disequilibrium block. Luciferase reporter assays confirmed promoter activity in the predicted promoter region, and demonstrated marked downregulation of expression in the LYRM4 direction under the haplotype comprising the minor alleles of promoter SNPs, which however is not driven by rs2224391. Experimental evidence from LYRM4 expression in lymphoblasts, gel-shift assays and modelling of DNA breathing dynamics pointed to two adjacent promoter SNPs, rs7752203-rs4141761, as the functional variants affecting expression. Their C-G alleles were associated with higher transcriptional activity and preferential binding of nuclear proteins, whereas the G-A combination had opposite effects and was associated with poor memory and high CD scores. LYRM4 is a eukaryote-specific component of the mitochondrial biogenesis of Fe-S clusters, essential cofactors in multiple processes, including oxidative phosphorylation. LYRM4 downregulation may be one of the mechanisms involved in inefficient oxidative phosphorylation and oxidative stress, increasingly recognised as contributors to schizophrenia pathogenesis.

Background: Medulloblastoma (MB) is the most common type of malignant childhood brain tumour. Although deregulated microRNA (miRNA) expression has been linked to MB pathogenesis, the selection of appropriate candidate endogenous control (EC) reference genes for MB miRNA expression profiling studies has not been systematically addressed. In this study we utilised reverse transcriptase quantitative PCR (RT-qPCR) to identify the most appropriate EC reference genes for the accurate normalisation of miRNA expression data in primary human MB specimens and neural stem cells., Results: Expression profiling of 662 miRNAs and six small nuclear/ nucleolar RNAs in primary human MB specimens, two CD133+ neural stem cell (NSC) populations and two CD133- neural progenitor cell (NPC) populations was performed using TaqMan low-density array (TLDA) cards. Minimal intra-card variability for candidate EC reference gene replicates was observed, however significant inter-card variability was identified between replicates present on both TLDA cards A and B. A panel of 18 potentially suitable EC reference genes was identified for the normalisation of miRNA expression on TLDA cards. These candidates were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary MB specimens. Of the six sn/snoRNA EC reference genes recommended by the manufacturer, only RNU44 was uniformly expressed between primary MB specimens and CD133+ NSC/CD133- NPC populations (P = 0.709; FC = 1.02). The suitability of candidate EC reference genes was assessed using geNorm and NormFinder software, with hsa-miR-301a and hsa-miR-339-5p found to be the most uniformly expressed EC reference genes on TLDA card A and hsa-miR-425* and RNU24 for TLDA card B., Conclusions: A panel of 18 potential EC reference genes that were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary human MB specimens was identified. The top ranked EC reference genes described here should be validated in a larger cohort of specimens to verify their utility as controls for the normalisation of RT-qPCR data generated in MB miRNA expression studies. Importantly, inter-card variability observed between replicates of certain candidate EC reference genes has major implications for the accurate normalisation of miRNA expression data obtained using the miRNA TLDA platform.

Introduction: We sought to determine the perceptions of physicians staffing rural emergency departments (EDs) in southwestern Ontario with respect to factors affecting patient care in the domains of physical resources, available support and education., Methods: A confidential 30-item survey was distributed through ED chiefs to physicians working in rural EDs in southwestern Ontario. Using a 5-point Likert scale, physicians were asked to rate their perception of factors that affect patient care in their ED. Demographic and practice characteristics were collected to accurately represent the participating centres and physicians., Results: Twenty-seven of the 164 surveys distributed were completed (16% response rate). Responses were received from 13 (81.3%) of the 16 surveyed EDs. Most of the respondents (78%) held CCFP (Certificant of the College of Family Physicians) credentials, with no additional emergency medicine training. Crowding from inpatient boarding, and inadequate physician staffing or coverage in EDs were identified as having a negative impact on patient care. Information sharing within the hospital, access to emergent laboratory studies and physician access to medications in the ED were identified as having the greatest positive impact on patient care. Respondents viewed all questions in the domain of education as either positive or neutral., Conclusion: Our survey results reveal that physicians practising emergency medicine in southwestern Ontario perceive crowding as the greatest barrier to providing patient care. Conversely, the survey identified that rural ED physicians perceive information sharing within the hospital, the availability of emergent laboratory studies and access to medications within the ED as having a strongly positive impact on patient care. Interestingly, our findings suggest that physicians in rural EDs view their access to education as adequate, as responses were either positive or neutral in regard to access to training and ability to maintain relevant skills.

The cure rate for pediatric patients with B precursor acute lymphoblastic leukemia (pre-B ALL) is steadily improving, however relapses do occur despite initial response to therapy. To identify links between drug resistance and gene deregulation we used oligonucleotide microarray technology and determined in 184 pre-B ALL specimen genes differentially expressed compared to normal CD34(+) specimens. We identified 20 signature genes including CTGF, BMP-2, CXCR4 and IL7R, documented to regulate interactions in the bone marrow. We recorded remarkably similar levels of expression in three independent patient cohorts, and found distinct patterns in cytogenetically defined subgroups of pre-B ALL. The canonical pathways that were affected are involved in inter- and intra-cellular communication, regulating signaling within the microenvironment. We tested experimentally whether interaction with stromal cells conferred protection to four drugs used in current ALL therapy, and demonstrated that bone marrow stromal cells significantly influenced resistance to vincristine and cytosine arabinoside. Compounds designed to block the identified cellular interactions within the bone marrow microenvironment are expected to mobilise the leukemic cells and make them more accessible to contemporary antileukemic agents. The data provide novel insight into the pathobiology of ALL and indicate new therapeutic targets for patients with ALL., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Endocrine disruptor chemicals elicit adverse health effects by perturbing nuclear receptor signalling systems. It has been speculated that these compounds may also perturb epigenetic mechanisms and thus contribute to the early origin of adult onset disease. We hypothesised that histone methylation may be a component of the epigenome that is susceptible to perturbation. We used coexpression analysis of publicly available data to investigate the combinatorial actions of nuclear receptors and genes involved in histone methylation in normal testis and when faced with endocrine disruptor compounds., Methodology/principal Findings: The expression patterns of a set of genes were profiled across testis tissue in human, rat and mouse, plus control and exposed samples from four toxicity experiments in the rat. Our results indicate that histone methylation events are a more general component of nuclear receptor mediated transcriptional regulation in the testis than previously appreciated. Coexpression patterns support the role of a gatekeeper mechanism involving the histone methylation modifiers Kdm1, Prdm2, and Ehmt1 and indicate that this mechanism is a common determinant of transcriptional integrity for genes critical to diverse physiological endpoints relevant to endocrine disruption. Coexpression patterns following exposure to vinclozolin and dibutyl phthalate suggest that coactivity of the demethylase Kdm1 in particular warrants further investigation in relation to endocrine disruptor mode of action., Conclusions/significance: This study provides proof of concept that a bioinformatics approach that profiles genes related to a specific hypothesis across multiple biological settings can provide powerful insight into coregulatory activity that would be difficult to discern at an individual experiment level or by traditional differential expression analysis methods.

The hallmarks of many haematological malignancies and solid tumours are chromosomal translocations, which may lead to gene fusions. Recently, next-generation sequencing techniques at the transcriptome level (RNA-Seq) have been used to verify known and discover novel transcribed gene fusions. We present FusionFinder, a Perl-based software designed to automate the discovery of candidate gene fusion partners from single-end (SE) or paired-end (PE) RNA-Seq read data. FusionFinder was applied to data from a previously published analysis of the K562 chronic myeloid leukaemia (CML) cell line. Using FusionFinder we successfully replicated the findings of this study and detected additional previously unreported fusion genes in their dataset, which were confirmed experimentally. These included two isoforms of a fusion involving the genes BRK1 and VHL, whose co-deletion has previously been associated with the prevalence and severity of renal-cell carcinoma. FusionFinder is made freely available for non-commercial use and can be downloaded from the project website (http://bioinformatics.childhealthresearch.org.au/software/fusionfinder/).

17β-Estradiol is important in maintaining bone structure, and regulation of its synthesis plays an important role in the development of postmenopausal osteoporosis. We and others have demonstrated associations between variation in the CYP19A1 gene (encoding aromatase) and areal bone mineral density (aBMD) phenotypes in women. In the present study 33 tag polymorphisms were genotyped across the CYP19A1 gene in a population of 1,185 Caucasian postmenopausal women to test the association between sequence variations, total DXA hip aBMD, and circulating 17β-estradiol levels. An in silico bioinformatics analysis was performed for single nucleotide polymorphisms (SNPs) associated with aBMD to identify putative functional effects, while linkage disequilibrium analysis of these SNPs was undertaken with previously published sequence variants. Five SNPs located in the central third of the gene were strongly associated with total-hip aBMD after adjustment for age (P = 0.006-0.013). A haplotype analysis of these five SNPs revealed an association between the haplotype C-G-G-G-C and increased aBMD (P = 0.008) and the haplotype A-A-A-A-A and a decreased aBMD (P = 0.021). The haplotype frequency was 9.0% for C-G-G-G-C and 15.4% for A-A-A-A-A, with the variation in mean total-hip aBMD explained by the haplotype analyses being 5% and 7%, respectively. None of these polymorphisms was significantly associated with circulating 17β-estradiol levels. In conclusion, common genetic variations within the CYP19A1 gene are significantly associated with aBMD in postmenopausal Caucasian women.

CMT4D disease is a severe autosomal recessive demyelinating neuropathy with extensive axonal loss leading to early disability, caused by mutations in the N-myc downstream regulated gene 1 (NDRG1). NDRG1 is expressed at particularly high levels in the Schwann cell (SC), but its physiological function(s) are unknown. To help with their understanding, we characterise the phenotype of a new mouse model, stretcher (str), with total Ndrg1 deficiency, in comparison with the hypomorphic Ndrg1 knock-out (KO) mouse. While both models display normal initial myelination and a transition to overt pathology between weeks 3 and 5, the markedly more severe str phenotype suggests that even low Ndrg1 expression results in significant phenotype rescue. Neither model replicates fully the features of CMT4D: although axon damage is present, regenerative capacity is unimpaired and the mice do not display the early severe axonal loss typical of the human disease. The widespread large fibre demyelination coincides precisely with the period of rapid growth of the animals and the dramatic (160-500-fold) increase in myelin volume and length in large fibres. This is followed by stabilisation after week 10, while small fibres remain unaffected. Gene expression profiling of str peripheral nerve reveals non-specific secondary changes at weeks 5 and 10 and preliminary data point to normal proteasomal function. Our findings do not support the proposed roles of NDRG1 in growth arrest, terminal differentiation, gene expression regulation and proteasomal degradation. Impaired SC trafficking failing to meet the considerable demands of nerve growth, emerges as the likely pathogenetic mechanism in NDRG1 deficiency., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.

Background: Kawasaki disease results from an abnormal immunological response to one or more infectious triggers. We hypothesised that heritable differences in immune responses in Kawasaki disease-affected children and their families would result in different epidemiological patterns of other immune-related conditions. We investigated whether hospitalisation for infection and asthma/allergy were different in Kawasaki disease-affected children and their relatives., Methods/major Findings: We used Western Australian population-linked health data from live births (1970-2006) to compare patterns of hospital admissions in Kawasaki disease cases, age- and sex-matched controls, and their relatives. There were 295 Kawasaki disease cases and 598 age- and sex-matched controls, with 1,636 and 3,780 relatives, respectively. Compared to controls, cases were more likely to have been admitted at least once with an infection (cases, 150 admissions (50.8%) vs controls, 210 admissions (35.1%); odds ratio (OR) = 1.9, 95% confidence interval (CI) 1.4-2.6, P = 7.2×10⁻⁶), and with asthma/allergy (cases, 49 admissions (16.6%) vs controls, 42 admissions (7.0%); OR = 2.6, 95% CI 1.7-4.2, P = 1.3×10⁻⁵). Cases also had more admissions per person with infection (cases, median 2 admissions, 95% CI 1-5, vs controls, median 1 admission, 95% CI 1-4, P = 1.09×10⁻⁵). The risk of admission with infection was higher in the first degree relatives of Kawasaki disease cases compared to those of controls, but the differences were not significant., Conclusion: Differences in the immune phenotype of children who develop Kawasaki disease may influence the severity of other immune-related conditions, with some similar patterns observed in relatives. These data suggest the influence of shared heritable factors in these families.

Unlabelled: Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up-regulated with increasing hepatic iron (R(2) between 0.602 and 0.164), including those of the rate-limiting enzyme, 3-hydroxy-3-methylglutarate-coenzyme A reductase (Hmgcr; R(2) = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R(2) = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R(2) = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol-regulatory element binding factor 2 (Srebf2; R(2) = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron., Conclusion: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity.

Background: Researchers wishing to conduct genetic association analysis involving single nucleotide polymorphisms (SNPs) or haplotypes are often confronted with the lack of user-friendly graphical analysis tools, requiring sophisticated statistical and informatics expertise to perform relatively straightforward tasks. Tools, such as the SimHap package for the R statistics language, provide the necessary statistical operations to conduct sophisticated genetic analysis, but lacks a graphical user interface that allows anyone but a professional statistician to effectively utilise the tool., Results: We have developed SimHap GUI, a cross-platform integrated graphical analysis tool for conducting epidemiological, single SNP and haplotype-based association analysis. SimHap GUI features a novel workflow interface that guides the user through each logical step of the analysis process, making it accessible to both novice and advanced users. This tool provides a seamless interface to the SimHap R package, while providing enhanced functionality such as sophisticated data checking, automated data conversion, and real-time estimations of haplotype simulation progress., Conclusion: SimHap GUI provides a novel, easy-to-use, cross-platform solution for conducting a range of genetic and non-genetic association analyses. This provides a free alternative to commercial statistics packages that is specifically designed for genetic association analysis.

The objective of this study was to determine whether single nucleotide polymorphisms (SNPs) in the Interleukin-1 (IL-1) gene family are associated with central obesity and metabolic syndrome in a coronary heart disease population. The IL-1 alpha C-889T (rs1800587) and IL-1 beta +3954 (rs1143634) SNPs were studied in a Western Australian coronary heart disease (CHD) population (N = 556). Subjects who were TT homozygous at either SNP had larger waist circumference (IL-1 alpha: 1.8 cm greater, P = 0.04; IL-1 beta: 4 cm greater, P = 0.0004) compared with major allele homozygotes. Individuals with two copies of the IL-1 alpha:IL-1 beta T:T haplotype had greater waist circumference (4.7 cm greater, P = 0.0001) compared to other haplotypes. There was a significant interaction between the IL-1 beta SNP and BMI level on waist circumference (P = 0.01). When the cohort was stratified by median BMI, TT carriers for IL-1 beta with above median BMI had greater waist circumference (6.1 cm greater, P = 0.007) compared to baseline carriers, whilst no significant association was seen in the below median group. Similarly, when the cohort was stratified by median fibrinogen level (IL-1 alpha interaction P = 0.01; IL-1 beta interaction P = 0.04), TT carriers for both SNPs in the above median fibrinogen group had greater waist circumference (IL-1 alpha 2.7 cm greater, P = 0.007; IL-1 beta 3.3 cm greater, P = 0.003) compared with major allele homozygotes. This association was not seen in the below median group. Also, we found a trend of increased metabolic syndrome for IL-1 beta TT homozygotes (P = 0.07). In conclusion, our findings suggest that in a CHD population IL-1 gene polymorphisms may be involved in increased central obesity, and the genetic influences are more evident among patients who have a higher level of obesity or inflammatory markers.

PARL (presenilin-associated rhomboid-like) is a mitochondrial protein involved in mitochondrial membrane remodelling, and maps to a quantitative trait locus (3q27) associated with metabolic traits. Recently the rs3732581 (Leu262Val) variant was found to be associated with increased levels of plasma insulin, a finding not replicated in a larger cohort. The aim of the current study was to investigate the associations between rs3732581 and levels of plasma insulin, metabolic syndrome (MetS) and its components, and cardiovascular disease. The CUPID population consisted of 556 subjects with angiographically proven CAD and the CUDAS cohort consisted of 1,109 randomly selected individuals from Perth, Western Australia. Samples were genotyped using mutation-specific PCR. No significant associations were observed between rs3732581 and levels of plasma insulin, glucose, BMI or MetS in either population. However, carriers of the minor allele had significantly lower mean intima-media thickness (IMT) [0.69 mm, 95% CI (0.69, 0.70 mm); P = 0.004], compared with major allele homozygotes [mean IMT = 0.71 mm, 95% CI (0.70, 0.72 mm)] in the CUDAS population. Further analysis using a recessive model showed homozygous carriers of the minor allele were predisposed to CAD [OR 1.55, 95% CI (1.11, 2.16); P = 0.01]. Despite the functional evidence for a role of PARL in regulating insulin levels, no association with rs3732581 was found in the current study. Additionally, there were no associations with glucose levels, BMI or MetS. There were significant effects of the variant on mean IMT and risk of CAD. A role for PARL in metabolic conditions cannot be excluded and more comprehensive genetic studies are warranted.

Serum high density lipoprotein (HDL) levels are inversely related to the development of coronary artery disease (CAD). Apolipoproteins AI and AII are the major protein constituents of HDL particles. APOAI and APOAII genetic polymorphisms have been proposed to affect transcriptional efficiency of their respective genes, thereby altering serum lipid levels and influencing atherosclerotic disease risk. 556 subjects with angiographically proven CAD (>50% stenosis) and 1109 randomly selected individuals from metropolitan Perth, Western Australia, were included in an association study. APOAI -75G/A (rs670) and APOAII -256T/C (rs5082) polymorphisms were both found to be not associated with plasma HDL levels. In a case-control analysis of 484 male CAD patients and 498 male controls, individuals carrying the 'CC' genotype for the APOAII rs5082 polymorphism had significantly lower risk of CAD than the 'T' allele carriers (OR=0.57, 95% CI 0.39-0.84, p=0.004). The minor 'A' allele of the APOAI rs670 polymorphism was found to be not associated with CAD, contrary to previous reports. We conclude that the APOAII rs5082 polymorphism appears to be cardioprotective in this representative Caucasian Australian population.

Summary: Traditional two-dimensional (2D) software programs for drawing pedigrees are limited when dealing with extended pedigrees. In successive generations, the number of individuals grows exponentially, leading to an unworkable amount of space required in the horizontal direction for 2D displays. In addition, it is not always possible to place closely related individuals near each other due to the lack of space in 2Ds. To address these issues we have developed three-dimensional (3D) pedigree drawing techniques to enable clearer visualization of extended pedigrees. Currently no other methods are available for displaying extended pedigrees in 3Ds. We have made freely available a software tool--'Celestial3D'--that implements these novel techniques., Availability: Freely available to non-commercial users.

Endothelin-1 is a potent vasoconstrictor in the body. Previous studies have identified associations between the coding polymorphism K198N and hypertension, systolic blood pressure and HDL levels. We sought to examine the evidence for these associations and, additionally, the association between K198N, insulin resistance, metabolic syndrome and coronary artery disease (CAD). We used generalised linear modelling to test K198N for association with hypertension and systolic blood pressure, lipid levels, insulin resistance scores and metabolic syndrome in a general cross-sectional community sample. Mean carotid intima media thickness and risk of carotid plaque were examined in the general population sample, and Gensini score was examined in a sample of patients with CAD. A case/control sample was used to examine the association of K198N with risk of CAD. There was no significant evidence for association between K198N and hypertension, systolic blood pressure, lipid levels, insulin resistance or metabolic syndrome in either population. The minor allele was marginally associated with increased mean IMT levels (P = 0.02) in the general population sample, although not with CAD in the case/control study or with the severity of disease in patients with CAD. In conclusion, we found no robust evidence for the associations between K198N and hypertension, systolic blood pressure or HDL levels seen in previous studies.