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1.  Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella 
Veterinary Research  2015;46:9.
Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.
doi:10.1186/s13567-014-0135-2
PMCID: PMC4333180
2.  A Health Impact Assessment Framework for Assessing Vulnerability and Adaptation Planning for Climate Change 
This paper presents a detailed description of an approach designed to investigate the application of the Health Impact Assessment (HIA) framework to assess the potential health impacts of climate change. A HIA framework has been combined with key climate change terminology and concepts. The fundamental premise of this framework is an understanding of the interactions between people, the environment and climate. The diversity and complexity of these interactions can hinder much needed action on the critical health issue of climate change. The objectives of the framework are to improve the methodology for understanding and assessing the risks associated with potential health impacts of climate change, and to provide decision-makers with information that can facilitate the development of effective adaptation plans. While the process presented here provides guidance with respect to this task it is not intended to be prescriptive. As such, aspects of the process can be amended to suit the scope and available resources of each project. A series of working tables has been developed to assist in the collation of evidence throughout the process. The framework has been tested in a number of locations including Western Australia, Solomon Islands, Vanuatu and Nauru.
doi:10.3390/ijerph111212896
PMCID: PMC4276652  PMID: 25514146
climate change; adaptation; health impact assessment; vulnerability
3.  Chicken Juice Enhances Surface Attachment and Biofilm Formation of Campylobacter jejuni 
Applied and Environmental Microbiology  2014;80(22):7053-7060.
The bacterial pathogen Campylobacter jejuni is primarily transmitted via the consumption of contaminated foodstuffs, especially poultry meat. In food processing environments, C. jejuni is required to survive a multitude of stresses and requires the use of specific survival mechanisms, such as biofilms. An initial step in biofilm formation is bacterial attachment to a surface. Here, we investigated the effects of a chicken meat exudate (chicken juice) on C. jejuni surface attachment and biofilm formation. Supplementation of brucella broth with ≥5% chicken juice resulted in increased biofilm formation on glass, polystyrene, and stainless steel surfaces with four C. jejuni isolates and one C. coli isolate in both microaerobic and aerobic conditions. When incubated with chicken juice, C. jejuni was both able to grow and form biofilms in static cultures in aerobic conditions. Electron microscopy showed that C. jejuni cells were associated with chicken juice particulates attached to the abiotic surface rather than the surface itself. This suggests that chicken juice contributes to C. jejuni biofilm formation by covering and conditioning the abiotic surface and is a source of nutrients. Chicken juice was able to complement the reduction in biofilm formation of an aflagellated mutant of C. jejuni, indicating that chicken juice may support food chain transmission of isolates with lowered motility. We provide here a useful model for studying the interaction of C. jejuni biofilms in food chain-relevant conditions and also show a possible mechanism for C. jejuni cell attachment and biofilm initiation on abiotic surfaces within the food chain.
doi:10.1128/AEM.02614-14
PMCID: PMC4249011  PMID: 25192991
4.  Evidence of a high incidence of subclinically affected calves in a herd of cattle with fatal cases of Bovine Neonatal Pancytopenia (BNP) 
BMC Veterinary Research  2014;10(1):245.
Background
Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by bone marrow trilineage hypoplasia, mediated by ingestion of alloantibodies in colostrum. Suspected subclinical forms of BNP have been reported, suggesting that observed clinical cases may not represent the full extent of the disease. However to date there are no objective data available on the incidence of subclinical disease or its temporal distribution. This study aimed to 1) ascertain whether subclinical BNP occurs and, if so, to determine the incidence on an affected farm and 2) determine whether there is evidence of temporal clustering of BNP cases on this farm. To achieve these aims, haematological screening of calves born on the farm during one calving season was carried out, utilising blood samples collected at defined ages. These data were then analysed in comparison to data from both known BNP-free control animals and histopathologically confirmed BNP cases. An ordinal logistic regression model was used to create a composite haematology score to predict the probabilities of calves being normal, based on their haematology measurements at 10–14 days old.
Results
This study revealed that 15% (21 of 139) of the clinically normal calves on this farm had profoundly abnormal haematology (<5% chance of being normal) and could be defined as affected by subclinical BNP. Together with clinical BNP cases, this gave the study farm a BNP incidence of 18%. Calves with BNP were found to be distributed throughout the calving period, with no clustering, and no significant differences in the date of birth of cases or subclinical cases were found compared to the rest of the calves. This study did not find any evidence of increased mortality or increased time from birth to sale in subclinical BNP calves but, as the study only involved a single farm and adverse effects may be determined by other inter-current diseases it remains possible that subclinical BNP has a detrimental impact on the health and productivity of calves under certain circumstances.
Conclusions
Subclinical BNP was found to occur at a high incidence in a herd of cattle with fatal cases of BNP.
doi:10.1186/s12917-014-0245-0
PMCID: PMC4216910  PMID: 25358526
Bovine Neonatal Pancytopenia (BNP); Haematology; Subclinical; Alloantibody
5.  HIV-1 DNA predicts disease progression and post-treatment virological control 
eLife  2014;3:e03821.
In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.
DOI: http://dx.doi.org/10.7554/eLife.03821.001
eLife digest
HIV is a virus that can hide in, and hijack, the cells of the immune system and force them to make new copies of the virus. This eventually destroys the infected cells and weakens the ability of a person with HIV to fight off infections and disease. If diagnosed early and treated, most people with HIV now live long and healthy lives and do not develop AIDS—the last stage of HIV infection when previously harmless, opportunistic infections can become life-threatening. However, there are still numerous hurdles and challenges that must be overcome before a cure for HIV/AIDS can be developed.
Treatment with drugs called antiretrovirals can reduce the amount of the HIV virus circulating in an infected person's bloodstream to undetectable levels. However, when HIV infects a cell, the virus inserts a copy of its genetic material into the cell's DNA—and, for most patients, antiretroviral treatment does not tackle these ‘hidden viruses’. As such, and in spite of their side-effects, antiretroviral drugs have to be taken for life in case the hidden viruses re-emerge.
As research into a cure for HIV/AIDS gathers momentum, patients who might be candidates for new experimental treatments will need to be identified. Although it is not recommended as part of standard clinical care, the only way to test if a patient's viral levels would remain suppressed without the drugs would be to temporarily stop the treatment under the close supervision of a physician. As such, a new method is needed to identify if there are patients who might benefit from stopping antiretroviral therapy, and more importantly, those who might not.
Williams, Hurst et al. have now tested whether measuring the levels of HIV DNA directly might help to predict if, and when, the virus might re-emerge (or rebound). In a group of HIV patients participating in a clinical trial, those with higher levels of HIV DNA at the point that the treatment was stopped were found to experience faster viral rebound than those with lower levels of HIV DNA. This method could therefore identify those patients who are at the greatest risk of HIV viral rebound, and are therefore unlikely to benefit if their treatment is interrupted.
Williams, Hurst et al. also found that measuring the levels of HIV DNA could help to predict how the disease would progress in treated and untreated patients. Furthermore, these predictions were more accurate than those based on measuring the amount of the virus circulating in a patient's body.
The next challenge is to identify other methods to distinguish patients who may remain ‘virus-free’ for a period without treatment, from those who would not. With this achieved, it might be possible to identify the mechanisms that determine why the virus comes back and so develop new treatments to stop this happening. This would make developing a cure for HIV/AIDS a much more tangible prospect.
DOI: http://dx.doi.org/10.7554/eLife.03821.002
doi:10.7554/eLife.03821
PMCID: PMC4199415  PMID: 25217531
HIV-1; reservoir; antiretroviral therapy; cure; primary infection; human
6.  Health Consequence Scales for Use in Health Impact Assessments of Climate Change 
While health impact assessment (HIA) has typically been applied to projects, plans or policies, it has significant potential with regard to strategic considerations of major health issues facing society such as climate change. Given the complexity of climate change, assessing health impacts presents new challenges that may require different approaches compared to traditional applications of HIA. This research focuses on the development of health consequence scales suited to assessing and comparing health effects associated with climate change and applied within a HIA framework. This assists in setting priorities for adaptation plans to minimize the public health impacts of climate change. The scales presented in this paper were initially developed for a HIA of climate change in Perth in 2050, but they can be applied across spatial and temporal scales. The design is based on a health effects pyramid with health measures expressed in orders of magnitude and linked to baseline population and health data. The health consequence measures are combined with a measure of likelihood to determine the level of risk associated with each health potential health impact. In addition, a simple visual framework that can be used to collate, compare and communicate the level of health risks associated with climate change has been developed.
doi:10.3390/ijerph110909607
PMCID: PMC4199038  PMID: 25229697
climate adaptation; health impact assessment; health consequence scales
7.  Family-based interventions to increase physical activity in children: a meta-analysis and realist synthesis protocol 
BMJ Open  2014;4(8):e005439.
Introduction
Despite the established relationship between physical activity and health, data suggest that many children are insufficiently active, and that levels decline into adolescence. Engaging the family in interventions may increase and maintain children's physical activity levels at the critical juncture before secondary school. Synthesis of existing evidence will inform future studies, but the heterogeneity in target populations recruited, behaviour change techniques and intervention strategies employed, and measurement conducted, may require a multifaceted review method. The primary objective of this work will therefore be to synthesis evidence from intervention studies that explicitly engage the family unit to increase children's physical activity using an innovative dual meta-analysis and realist approach.
Methods and analysis
Peer-reviewed studies will be independently screened by two authors for inclusion based on (1) including ‘healthy’ participants aged 5–12 years; (2) having a substantive intervention aim of increasing physical activity, by engaging the family and (3) reporting on physical activity. Duplicate data extraction and quality assessment will be conducted using a specially designed proforma and the Effective Public Health Practice Project Quality Assessment Tool respectively. STATA software will be used to compute effect sizes for meta-analyses, with subgroup analyses conducted to identify moderating characteristics. Realist syntheses will be conducted according to RAMESES quality and publication guidelines, including development of a programme theory and evidence mapping.
Dissemination
This review will be the first to use the framework of a traditional review to conduct a dual meta-analysis and realist synthesis, examining interventions that engage the family to increase physical activity in children. The results will be disseminated through peer-reviewed publications, conferences, formal presentations to policy makers and practitioners and informal meetings. Evidence generated from this synthesis will also be used to inform the development of theory-driven, evidence-based interventions aimed at engaging the family to increase physical activity levels in children.
Protocol registration
International Prospective Register for Systematic Reviews (PROSPERO): number CRD42013005780.
doi:10.1136/bmjopen-2014-005439
PMCID: PMC4127934  PMID: 25099934
PUBLIC HEALTH; EPIDEMIOLOGY; SPORTS MEDICINE
9.  An expression atlas of human primary cells: inference of gene function from coexpression networks 
BMC Genomics  2013;14:632.
Background
The specialisation of mammalian cells in time and space requires genes associated with specific pathways and functions to be co-ordinately expressed. Here we have combined a large number of publically available microarray datasets derived from human primary cells and analysed large correlation graphs of these data.
Results
Using the network analysis tool BioLayout Express3D we identify robust co-associations of genes expressed in a wide variety of cell lineages. We discuss the biological significance of a number of these associations, in particular the coexpression of key transcription factors with the genes that they are likely to control.
Conclusions
We consider the regulation of genes in human primary cells and specifically in the human mononuclear phagocyte system. Of particular note is the fact that these data do not support the identity of putative markers of antigen-presenting dendritic cells, nor classification of M1 and M2 activation states, a current subject of debate within immunological field. We have provided this data resource on the BioGPS web site (http://biogps.org/dataset/2429/primary-cell-atlas/) and on macrophages.com (http://www.macrophages.com/hu-cell-atlas).
doi:10.1186/1471-2164-14-632
PMCID: PMC3849585  PMID: 24053356
Clustering; Meta-analysis; Human; Primary cells; Dendritic cell; Macrophage; Microarray; Transcriptomics
10.  Myocardial infarction incidence and survival by ethnic group: Scottish Health and Ethnicity Linkage retrospective cohort study 
BMJ Open  2013;3(9):e003415.
Objective
Inequalities in coronary heart disease mortality by country of birth are large and poorly understood. However, these data misclassify UK-born minority ethnic groups and provide little detail on whether excess risk is due to increased incidence, poorer survival or both.
Design
Retrospective cohort study.
Setting
General resident population of Scotland.
Participants
All those residing in Scotland during the 2001 Census were eligible for inclusion: 2 972 120 people were included in the analysis. The number still residing in Scotland at the end of the study in 2008 is not known.
Primary and secondary outcome measures
As specified in the analysis plan, the primary outcome measures were first occurrence of admission or death due to myocardial infarction and time to event. There were no secondary outcome measures.
Results
Acute myocardial infarction (AMI) incidence risk ratios (95% CIs) relative to white Scottish populations (100) were highest among Pakistani men (164.1 (142.2 to 189.2)) and women (153.7 (120.5, 196.1)) and lowest for men and women of Chinese (39.5 (27.1 to 57.6) and 59.1 (38.6 to 90.7)), other white British (77 (74.2 to 79.8) and 72.2 (69.0 to 75.5)) and other white (83.1 (75.9 to 91.0) and 79.9 (71.5 to 89.3)) ethnic groups. Adjustment for educational qualification did not eliminate these differences. Cardiac intervention uptake was similar across most ethnic groups. Compared to white Scottish, 28-day survival did not differ by ethnicity, except in Pakistanis where it was better, particularly in women (0.44 (0.25 to 0.78)), a difference not removed by adjustment for education, travel time to hospital or cardiac intervention uptake.
Conclusions
Pakistanis have the highest incidence of AMI in Scotland, a country renowned for internationally high cardiovascular disease rates. In contrast, survival is similar or better in minority ethnic groups. Clinical care and policy should focus on reducing incidence among Pakistanis through more aggressive prevention.
doi:10.1136/bmjopen-2013-003415
PMCID: PMC3773657  PMID: 24038009
Epidemiology; Public Health
11.  RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib 
PLoS ONE  2013;8(6):e66003.
Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.
doi:10.1371/journal.pone.0066003
PMCID: PMC3686868  PMID: 23840389
12.  Examination of mid-intervention mediating effects on objectively assessed sedentary time among children in the Transform-Us! cluster-randomized controlled trial 
Background
The optimal targets and strategies for effectively reducing sedentary behavior among young people are unknown. Intervention research that explores changes in mediated effects as well as in outcome behaviors is needed to help inform more effective interventions. Therefore, the purpose of this study was to examine the mid-intervention mediating effects on children’s objectively assessed classroom and total weekday sedentary time in the Transform-Us! intervention.
Methods
The results are based on 293 children, aged 7- to 9-years-old at baseline, from 20 schools in Melbourne, Australia. Each school was randomly allocated to one of four groups, which targeted reducing sedentary time in the school and family settings (SB; n = 74), increasing or maintaining moderate- to vigorous-intensity physical activity in the school and family settings (PA; n = 75), combined SB and PA (SB + PA; n = 80), or the current practice control (C; n = 64). Baseline and mid-intervention data (5–9 months) were collected in 2010 and analyzed in 2012. Classroom and total weekday sedentary time was objectively assessed using ActiGraph accelerometers. The hypothesized mediators including, child enjoyment, parent and teacher outcome expectancies, and child perceived access to standing opportunities in the classroom environment, were assessed by questionnaire.
Results
The SB + PA group spent 13.3 min/day less in weekday sedentary time at mid-intervention compared to the control group. At mid-intervention, children in the SB group had higher enjoyment of standing in class (0.9 units; 5-unit scale) and all intervention groups had more positive perceptions of access to standing opportunities in the classroom environment (0.3-0.4 units; 3-unit scale), compared to the control group. However, none of the hypothesized mediator variables had an effect on sedentary time; thus, no mediating effects were observed.
Conclusions
While beneficial intervention effects were observed on some hypothesized mediating variables and total weekday sedentary time at mid-intervention, no significant mediating effects were found. Given the dearth of existing information, future intervention research is needed that explores mediated effects. More work is also needed on the development of reliable mediator measures that are sensitive to change overtime.
Trial registration
ACTRN12609000715279
ISRCTN83725066
doi:10.1186/1479-5868-10-62
PMCID: PMC3681598  PMID: 23688180
Sedentary behavior; Intervention; Mediators; Children
13.  A systematic review of intervention effects on potential mediators of children’s physical activity 
BMC Public Health  2013;13:165.
Background
Many interventions aiming to increase children’s physical activity have been developed and implemented in a variety of settings, and these interventions have previously been reviewed; however the focus of these reviews tends to be on the intervention effects on physical activity outcomes without consideration of the reasons and pathways leading to intervention success or otherwise.
To systematically review the efficacy of physical activity interventions targeting 5-12 year old children on potential mediators and, where possible, to calculate the size of the intervention effect on the potential mediator.
Methods
A systematic search identified intervention studies that reported outcomes on potential mediators of physical activity among 5-12 year old children. Original research articles published between 1985 and April 2012 were reviewed.
Results
Eighteen potential mediators were identified from 31 studies. Positive effects on cognitive/psychological potential mediators were reported in 15 out of 31 studies. Positive effects on social environmental potential mediators were reported in three out of seven studies, and no effects on the physical environment were reported. Although no studies were identified that performed a mediating analysis, 33 positive intervention effects were found on targeted potential mediators (with effect sizes ranging from small to large) and 73% of the time a positive effect on the physical activity outcome was reported.
Conclusions
Many studies have reported null intervention effects on potential mediators of children’s physical activity; however, it is important that intervention studies statistically examine the mediating effects of interventions so the most effective strategies can be implemented in future programs.
doi:10.1186/1471-2458-13-165
PMCID: PMC3585884  PMID: 23433143
Mediator; Child; Physical activity promotion; Theory
14.  Help bring back the celebration of life: A community-based participatory study of rural Aboriginal women’s maternity experiences and outcomes 
Background
Despite clear evidence regarding how social determinants of health and structural inequities shape health, Aboriginal women’s birth outcomes are not adequately understood as arising from the historical, economic and social circumstances of their lives. The purpose of this study was to understand rural Aboriginal women’s experiences of maternity care and factors shaping those experiences.
Methods
Aboriginal women from the Nuxalk, Haida and 'Namgis First Nations and academics from the University of British Columbia in nursing, medicine and counselling psychology used ethnographic methods within a participatory action research framework. We interviewed over 100 women, and involved additional community members through interviews and community meetings. Data were analyzed within each community and across communities.
Results
Most participants described distressing experiences during pregnancy and birthing as they grappled with diminishing local maternity care choices, racism and challenging economic circumstances. Rural Aboriginal women’s birthing experiences are shaped by the intersections among rural circumstances, the effects of historical and ongoing colonization, and concurrent efforts toward self-determination and more vibrant cultures and communities.
Conclusion
Women’s experiences and birth outcomes could be significantly improved if health care providers learned about and accounted for Aboriginal people’s varied encounters with historical and ongoing colonization that unequivocally shapes health and health care. Practitioners who better understand Aboriginal women’s birth outcomes in context can better care in every interaction, particularly by enhancing women’s power, choice, and control over their experiences. Efforts to improve maternity care that account for the social and historical production of health inequities are crucial.
doi:10.1186/1471-2393-13-26
PMCID: PMC3577503  PMID: 23360168
Aboriginal; Rural; Maternity care; Outcomes; Colonialism; Critical ethnography
15.  Paired Ductal Carcinoma In Situ and Invasive Breast Cancer Lesions in the D-Loop of the Mitochondrial Genome Indicate a Cancerization Field Effect 
BioMed Research International  2012;2013:379438.
Alterations in the mitochondrial genome have been chronicled in most solid tumors, including breast cancer. The intent of this paper is to compare and document somatic mitochondrial D-loop mutations in paired samples of ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) indicating a potential breast ductal epithelial cancerization field effect. Paired samples of these histopathologies were laser-captured microdissected (LCM) from biopsy, lumpectomy, and mastectomy tissues. Blood samples were collected as germplasm control references. For each patient, hypervariable region 1 (HV1) in the D-loop portion of the mitochondrial genome (mtGenome) was sequenced for all 3 clinical samples. Specific parallel somatic heteroplasmic alterations between these histopathologies, particularly at sites 16189, 16223, 16224, 16270, and 16291, suggest the presence of an epithelial, mitochondrial cancerization field effect. These results indicate that further characterization of the mutational pathway of DCIS and IBC may help establish the invasive potential of DCIS. Moreover, this paper indicates that biofluids with low cellularity, such as nipple aspirate fluid and/or ductal lavage, warrant further investigation as early and minimally invasive detection mediums of a cancerization field effect within breast tissue.
doi:10.1155/2013/379438
PMCID: PMC3591154  PMID: 23509716
16.  Macrophage Differentiation from Embryoid Bodies Derived from Human Embryonic Stem Cells 
Journal of stem cells  2009;4(1):29-45.
Human embryonic stem cells can differentiate into CD34+ hematopoietic progenitors by co-culture on murine feeders such as OP9 and S17. These CD34+ progenitors can be further differentiated into several cells of the hematopoietic lineage including macrophages. However, co-culture on murine feeders is time consuming and involves extensive manipulations. Furthermore, CD45 expression is low on hematopoietic cultures derived from stromal co-cultures. In this study we describe a novel and highly efficient system of generating differentiated macrophages from hematopoietic progenitors generated from embryoid body cultures of human embryonic stem cells. The hematopoietic progenitors generated from these embryoid bodies express higher numbers of CD45+ cells and are able to differentiate to macrophages when cultured in presence of cytokines. Using this system we were able to generate higher yields of CD14+ macrophages compared to traditional stromal cell culture methods. The embryoid body derived macrophages are phagocytic, respond to Toll-like receptor stimulation and express phenotypic markers of mature macrophages. Importantly, the embryoid body system generates hematopoietic progenitors suitable for clinical use by eliminating the need for murine feeder cells. Furthermore, this system is amenable to genetic manipulation and may thus be used to study important mechanisms of macrophage differentiation and function.
PMCID: PMC3476843  PMID: 20498689
17.  A prospective study of effects of psychological factors and sleep on obstetric interventions, mode of birth, and neonatal outcomes among low-risk British Columbian women 
Background
Obstetrical interventions, including caesarean sections, are increasing in Canada. Canadian women’s psychological states, fatigue, and sleep have not been examined prospectively for contributions to obstetric interventions and adverse neonatal outcomes.
Context and purpose of the study: The prospective study was conducted in British Columbia (BC), Canada with 650 low-risk pregnant women. Of those women, 624 were included in this study. Women were recruited through providers’ offices, media, posters, and pregnancy fairs. We examined associations between pregnant women’s fatigue, sleep deprivation, and psychological states (anxiety and childbirth fear) and women’s exposure to obstetrical interventions and adverse neonatal outcomes (preterm, admission to NICU, low APGARS, and low birth weight).
Methods
Data from our cross-sectional survey were linked, using women’s personal health numbers, to birth outcomes from the Perinatal Services BC database. After stratifying for parity, we used Pearson’s Chi-square to examine associations between psychological states, fatigue, sleep deprivation and maternal characteristics. We used hierarchical logistic regression modeling to test 9 hypotheses comparing women with high and low childbirth fear and anxiety on likelihood of having epidural anaesthetic, a caesarean section (stratified for parity), assisted vaginal delivery, and adverse neonatal outcomes and women with and without sleep deprivation and high levels of fatigue on likelihood of giving birth by caesarean section, while controlling for maternal, obstetrical (e.g., infant macrosomia), and psychological variables.
Results
Significantly higher proportions of multiparas, reporting difficult and upsetting labours and births, expectations of childbirth interventions, and health stressors, reported high levels of childbirth fear. Women who reported antenatal relationship, housing, financial, and health stressors and multiparas reporting low family incomes were significantly more likely to report high anxiety levels. The hypothesis that high childbirth fear significantly increased the risk of using epidural anaesthesia was supported.
Conclusions
Controlling for some psychological states and sleep quality while examining other contributors to outcomes decreases the likelihood of linking childbirth fear anxiety, sleep deprivation, and fatigue to increased odds of caesarean section. Ameliorating women’s childbirth fear to reduce their exposure to epidural anaesthesia can occur through developing effective interventions. These include helping multiparous women process previous experiences of difficult and upsetting labour and birth.
doi:10.1186/1471-2393-12-78
PMCID: PMC3449197  PMID: 22862846
Childbirth fear; Sleep deprivation; Fatigue; Anxiety; Obstetrical interventions; Neonatal outcomes
18.  Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment 
The ISME Journal  2011;5(8):1253-1261.
The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 1019 bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.
doi:10.1038/ismej.2011.15
PMCID: PMC3146270  PMID: 21368907
integron; pollution; sewage; agriculture; horizontal gene transfer; antibiotic resistance
19.  Behavioural management of migraine 
Annals of Indian Academy of Neurology  2012;15(Suppl 1):S78-S82.
It is important to recognise that migraine is a ‘biological’ and not a ‘psychological’ entity. However, psychological factors can be involved in migraine in 4 different ways:- 1) Migraines can be triggered by psychological stressors; 2) Severe migraine can itself be a cause of significant psychological stress which can, in turn, exacerbate the problem; 3) Even if psychological stress is not significantly involved in the genesis of the headache, pain management techniques can help people cope with their pain more effectively; 4) Longitudinal data demonstrate a complex bidirectional association between mood disorders and migraine. Treatment of a co-existing mood disorder, for example with cognitive behavioural techniques, may therefore reduce the impact of migraine. It would thus appear logical to view medical and psychological approaches as potentially synergistic rather than mutually exclusive. Functional imaging indicates that cognition, emotions, and pain experiences change the way the brain processes pain inputs. This may provide a physiological rationale for psychological interventions in pain management. As most studies of psychological management of migraine have been relatively small and the approach often varies between clinicians, the magnitude of benefit, optimum method of delivery, and the length of intervention are uncertain.
doi:10.4103/0972-2327.100018
PMCID: PMC3444212  PMID: 23024569
Cognitive behavioural therapy; migraine; psychological; psychology
20.  Human Health and Climate Change: Leverage Points for Adaptation in Urban Environments 
The design of adaptation strategies that promote urban health and well-being in the face of climate change requires an understanding of the feedback interactions that take place between the dynamical state of a city, the health of its people, and the state of the planet. Complexity, contingency and uncertainty combine to impede the growth of such systemic understandings. In this paper we suggest that the collaborative development of conceptual models can help a group to identify potential leverage points for effective adaptation. We describe a three-step procedure that leads from the development of a high-level system template, through the selection of a problem space that contains one or more of the group’s adaptive challenges, to a specific conceptual model of a sub-system of importance to the group. This procedure is illustrated by a case study of urban dwellers’ maladaptive dependence on private motor vehicles. We conclude that a system dynamics approach, revolving around the collaborative construction of a set of conceptual models, can help communities to improve their adaptive capacity, and so better meet the challenge of maintaining, and even improving, urban health in the face of climate change.
doi:10.3390/ijerph9062134
PMCID: PMC3397369  PMID: 22829795
cities; urban health; climate adaptation; systems thinking; system dynamics; conceptual models; co-effects; leverage points
21.  A cluster-randomized controlled trial to reduce sedentary behavior and promote physical activity and health of 8-9 year olds: The Transform-Us! Study 
BMC Public Health  2011;11:759.
Background
Physical activity (PA) is associated with positive cardio-metabolic health and emerging evidence suggests sedentary behavior (SB) may be detrimental to children's health independent of PA. The primary aim of the Transform-Us! study is to determine whether an 18-month, behavioral and environmental intervention in the school and family settings results in higher levels of PA and lower rates of SB among 8-9 year old children compared with usual practice (post-intervention and 12-months follow-up). The secondary aims are to determine the independent and combined effects of PA and SB on children's cardio-metabolic health risk factors; identify the factors that mediate the success of the intervention; and determine whether the intervention is cost-effective.
Methods/design
A four-arm cluster-randomized controlled trial (RCT) with a 2 × 2 factorial design, with schools as the unit of randomization. Twenty schools will be allocated to one of four intervention groups, sedentary behavior (SB-I), physical activity (PA-I), combined SB and PA (SB+PA-I) or current practice control (C), which will be evaluated among approximately 600 children aged 8-9 years in school year 3 living in Melbourne, Australia. All children in year 3 at intervention schools in 2010 (8-9 years) will receive the intervention over an 18-month period with a maintenance 'booster' delivered in 2012 and children at all schools will be invited to participate in the evaluation assessments. To maximize the sample and to capture new students arriving at intervention and control schools, recruitment will be on-going up to the post-intervention time point. Primary outcomes are time spent sitting and in PA assessed via accelerometers and inclinometers and survey.
Discussion
To our knowledge, Transform-Us! is the first RCT to examine the effectiveness of intervention strategies for reducing children's overall sedentary time, promoting PA and optimizing health outcomes. The integration of consistent strategies and messages to children from teachers and parents in both school and family settings is a critical component of this study, and if shown to be effective, may have a significant impact on educational policies as well as on pedagogical and parenting practices.
Trial registration
ACTRN12609000715279; Current Controlled Trials ISRCTN83725066
doi:10.1186/1471-2458-11-759
PMCID: PMC3213038  PMID: 21970511
22.  Deciphering c-MYC-regulated genes in two distinct tissues 
BMC Genomics  2011;12:476.
Background
The transcription factor MYC is a critical regulator of diverse cellular processes, including both replication and apoptosis. Differences in MYC-regulated gene expression responsible for such opposing outcomes in vivo remain obscure. To address this we have examined time-dependent changes in global gene expression in two transgenic mouse models in which MYC activation, in either skin suprabasal keratinocytes or pancreatic islet β-cells, promotes tissue expansion or involution, respectively.
Results
Consistent with observed phenotypes, expression of cell cycle genes is increased in both models (albeit enriched in β-cells), as are those involved in cell growth and metabolism, while expression of genes involved in cell differentiation is down-regulated. However, in β-cells, which unlike suprabasal keratinocytes undergo prominent apoptosis from 24 hours, there is up-regulation of genes associated with DNA-damage response and intrinsic apoptotic pathways, including Atr, Arf, Bax and Cycs. In striking contrast, this is not the case for suprabasal keratinocytes, where pro-apoptotic genes such as Noxa are down-regulated and key anti-apoptotic pathways (such as Igf1-Akt) and those promoting angiogenesis are up-regulated. Moreover, dramatic up-regulation of steroid hormone-regulated Kallikrein serine protease family members in suprabasal keratinocytes alone could further enhance local Igf1 actions, such as through proteolysis of Igf1 binding proteins.
Conclusions
Activation of MYC causes cell growth, loss of differentiation and cell cycle entry in both β-cells and suprabasal keratinocytes in vivo. Apoptosis, which is confined to β-cells, may involve a combination of a DNA-damage response and downstream activation of pro-apoptotic signalling pathways, including Cdc2a and p19Arf/p53, and downstream targets. Conversely, avoidance of apoptosis in suprabasal keratinocytes may result primarily from the activation of key anti-apoptotic signalling pathways, particularly Igf1-Akt, and induction of an angiogenic response, though intrinsic resistance to induction of p19Arf by MYC in suprabasal keratinocytes may contribute.
doi:10.1186/1471-2164-12-476
PMCID: PMC3206520  PMID: 21961992
23.  Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244) 
Cancer research  2010;70(6):2264-2273.
Selumetinib (AZD6244, ARRY-142886) is a selective, non–ATP-competitive inhibitor of mitogen-activated protein/extracellular signal–regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR–based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.
doi:10.1158/0008-5472.CAN-09-1577
PMCID: PMC3166660  PMID: 20215513
24.  Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency 
The Journal of General Virology  2010;91(Pt 2):463-469.
Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.
doi:10.1099/vir.0.015073-0
PMCID: PMC2888311  PMID: 19864499
25.  Whole-Genome SNP Association in the Horse: Identification of a Deletion in Myosin Va Responsible for Lavender Foal Syndrome 
PLoS Genetics  2010;6(4):e1000909.
Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A). Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.
Author Summary
Genetic disorders affect many domesticated species, including the horse. In this study we have focused on Lavender Foal Syndrome, a seizure disorder that leads to suffering and death in foals soon after birth. A recessively inherited disorder, its occurrence is often unpredictable and difficult for horse breeders to avoid without a diagnostic test for carrier status. The recent completion of the horse genome sequence has provided new tools for mapping traits with unprecedented resolution and power. We have applied one such tool, the Equine SNP50 genotyping chip, to a small sample set from horses affected with Lavender Foal Syndrome. A single genetic location associated with the disorder was rapidly identified using this approach. Subsequent sequencing of functional candidate genes in this location revealed a single base deletion that likely causes Lavender Foal Syndrome. From a practical standpoint, this discovery and the development of a diagnostic test for the LFS allele provides a valuable new tool for breeders seeking to avoid the disease in their foal crop. However, this work also illustrates the utility of whole-genome association studies in the horse.
doi:10.1371/journal.pgen.1000909
PMCID: PMC2855325  PMID: 20419149

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