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1.  Tracking Genomic Cancer Evolution for Precision Medicine: The Lung TRACERx Study 
PLoS Biology  2014;12(7):e1001906.
TRACERx, a prospective study of patients with primary non-small cell lung cancer, aims to map the genomic landscape of lung cancer by tracking clonal heterogeneity and tumour evolution from diagnosis to relapse.
The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types.
doi:10.1371/journal.pbio.1001906
PMCID: PMC4086714  PMID: 25003521
2.  Risk Factors for Low Receptive Vocabulary Abilities in the Preschool and Early School Years in the Longitudinal Study of Australian Children 
PLoS ONE  2014;9(7):e101476.
Receptive vocabulary development is a component of the human language system that emerges in the first year of life and is characterised by onward expansion throughout life. Beginning in infancy, children's receptive vocabulary knowledge builds the foundation for oral language and reading skills. The foundations for success at school are built early, hence the public health policy focus on reducing developmental inequalities before children start formal school. The underlying assumption is that children's development is stable, and therefore predictable, over time. This study investigated this assumption in relation to children's receptive vocabulary ability. We investigated the extent to which low receptive vocabulary ability at 4 years was associated with low receptive vocabulary ability at 8 years, and the predictive utility of a multivariate model that included child, maternal and family risk factors measured at 4 years. The study sample comprised 3,847 children from the first nationally representative Longitudinal Study of Australian Children (LSAC). Multivariate logistic regression was used to investigate risks for low receptive vocabulary ability from 4–8 years and sensitivity-specificity analysis was used to examine the predictive utility of the multivariate model. In the multivariate model, substantial risk factors for receptive vocabulary delay from 4–8 years, in order of descending magnitude, were low receptive vocabulary ability at 4 years, low maternal education, and low school readiness. Moderate risk factors, in order of descending magnitude, were low maternal parenting consistency, socio-economic area disadvantage, low temperamental persistence, and NESB status. The following risk factors were not significant: One or more siblings, low family income, not reading to the child, high maternal work hours, and Aboriginal or Torres Strait Islander ethnicity. The results of the sensitivity-specificity analysis showed that a well-fitted multivariate model featuring risks of substantive magnitude does not do particularly well in predicting low receptive vocabulary ability from 4–8 years.
doi:10.1371/journal.pone.0101476
PMCID: PMC4079336  PMID: 24988308
3.  Probes of the Mitochondrial cAMP-dependent Protein Kinase 
Biochimica et biophysica acta  2013;1834(7):1359-1363.
The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R2C2) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN).
doi:10.1016/j.bbapap.2013.02.002
PMCID: PMC3665737  PMID: 23410952
signal transduction; sensor; fluorescent peptide; cAMP-dependent protein kinase (PKA); calpain
4.  Is The Effect of Compulsory Community Treatment on Preventable Deaths From Physical Disorders Mediated by Better Access to Specialized Medical Procedures? 
Objectives
Compulsory community treatment has been shown to reduce preventable deaths from physical disorders—these causes being up to 10 times more common than suicide in psychiatric patients. We investigated whether this was mediated by better access to specialized medical procedures.
Method:
All patients on compulsory community treatment for over 11 years were compared with matched control subjects using linked administrative health data from Western Australia (state population of about 2.24 million). Outcomes were access to revascularization and other specialized procedures at 1-, 2-, and 3-year follow-up. Logistic regression was used to adjust for demographics, prior health service use, diagnosis, and length of psychiatric history.
Results:
There were 2757 patients and 2687 control subjects (total n = 5444). Sixty-five per cent were males (n = 3522), and the average age was 36 years (SD 13.2). Most had schizophrenia or other nonaffective psychoses (74%), followed by affective disorders (26%). At 2-year follow-up, 2% (n = 53) of patients and 2.6% (n = 69) of control subjects had undergone a specialized intervention. Compulsory community treatment did not result in greater access to specialized procedures at all 3 time points even after adjusting for potential confounders.
Conclusions:
Greater access to specialized procedures does not explain the reduced mortality from preventable physical illness that had been reported in patients on community treatment orders. There must be other explanations for this finding, such as mental health staff facilitating access to chronic disease management in primary care. This warrants further research.
PMCID: PMC4079220  PMID: 24444325
community treatment order; mortality; psychiatric services
5.  Higher Maternal Protectiveness Is Associated with Higher Odds of Child Overweight and Obesity: A Longitudinal Australian Study 
PLoS ONE  2014;9(6):e100686.
In recent years there has been an increasing interest in overprotective parenting and the potential role it plays in child development. While some have argued that a trend towards increased parental fear and reduced opportunity for independent mobility may be linked to increasing rates of child overweight and obesity, there is limited empirical information available to support this claim. Using data from the Longitudinal Study of Australian Children, this study aimed to examine the longitudinal relationships between maternal protectiveness and child overweight and obesity. A cohort of 4–5 year old children was followed up at 6–7, 8–9 and 10–11 years of age (n  =  2596). Measures included a protective parenting scale administered when children were 6–7 and 8–9 years of age, child body mass index (BMI), family characteristics including household income, neighbourhood disadvantage, child's position amongst siblings, and maternal BMI, education, employment, mental health and age at first birth. International Obesity Taskforce age- and sex-specific BMI cut points were used to determine if children were in the normal, overweight or obese BMI range. There was no association between maternal protectiveness and the odds of children being overweight or obese at age 4–5, 6–7 or 8–9 years. However at age 10–11 years, a 1 standard deviation increase in maternal protectiveness was associated with a 13% increase in the odds of children being overweight or obese. The results provide evidence of a relationship between maternal protectiveness and child overweight and obesity, however further research is required to understand the mechanism(s) that links the two concepts.
doi:10.1371/journal.pone.0100686
PMCID: PMC4067382  PMID: 24955586
6.  Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis 
Analytical chemistry  2013;85(12):6136-6142.
A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.
doi:10.1021/ac401106e
PMCID: PMC3704224  PMID: 23682679
7.  Premature return to play and return to learn after a sport-related concussion 
Canadian Family Physician  2014;60(6):e310-e315.
Abstract
Objective
To determine what proportion of patients experience an exacerbation of their symptoms as a result of premature return to play (RTP) and return to learn (RTL) following sport-related concussions.
Design
Retrospective study of electronic medical records from the office-based practice of one family and sport medicine physician who had systematically provided recommendations for cognitive and physical rest based on existing consensus recommendations. Two blinded authors independently reviewed each chart, which included Sport Concussion Assessment Tool (SCAT) and SCAT2 symptom self-report forms to determine whether an athlete had returned to play or learn prematurely. If there was a discrepancy between the 2 reviewers then a third author reviewed the charts.
Setting
A sport medicine and family practice in Ontario. The physician assessed sport-related concussions after self-referral or referral from other primary care physicians, teams, and schools.
Participants
A total of 170 charts of 159 patients were assessed for sport-related concussion during a 5-year period (April 2006 to March 2011). All participants were students who were participating in sports at the time of injury. There were 41 concussions in elementary students, 95 concussions in high school students, and 34 concussions in college or university students.
Main outcome measures
Premature RTP and RTL were defined as chart records documenting the recurrence or worsening of symptoms that accompanied the patients’ RTP or RTL. Measures were compared using the earliest available SCAT forms and self-reporting.
Results
In 43.5% of concussion cases, the patient returned to sport too soon and in 44.7% of concussion cases, the patient returned to school too soon. Patients with a history of previous concussion required more days of rest before being permitted to participate in any physical activity than those patients without a previous history of concussion. Elementary school students required fewer days of rest before being permitted to return to any physical activity compared with high school students and college or university students.
Conclusion
Currently, physicians recommend restrictions on mental and physical activity following sport-related concussion. This is done without clear guidelines as to what cognitive rest entails for students. Further research is required to determine how to implement a management plan for student athletes to facilitate complete recovery after concussion.
PMCID: PMC4055342  PMID: 24925965
8.  Maternal Exposure to Mercury Chloride During Pregnancy and Lactation Affects the Immunity and Social Behavior of Offspring 
Toxicological Sciences  2013;133(1):101-111.
Developmental HgCl2 exposures of F1 offspring (H-2q/s) from unsociable SJL/J (H-2s) dams with high susceptibility to Hg-induced autoimmunity (SFvF1) and from highly sociable FVB/NJ (FVB; H-2q) dams with lower susceptibility to Hg-induced autoimmunity (FvSF1) were investigated. Hg exposure increased the serum IgG levels of all offspring at postnatal day 21 (pnd21) and of SJL/J dams but not of FVB dams. Serum IgG anti-brain antibody (Ab) levels of pnd21 SFvF1 offspring and SJL dams were higher than those of the FvSF1 offspring and FVB dams, but Hg only increased the titers of the FVB dams and their offspring. Hg significantly elevated the presence of IgG in all brain regions of the pnd21 SFvF1 offspring, and the SFvF1 offspring had greater amounts of IgG in the brain than the FvSF1 offspring, which had Hg-induced increases in only two brain regions. Cytokine levels were elevated in the brain regions of Hg-treated pnd21 SFvF1 but not of FvSF1 offspring, and SFvF1 females had more brain regions expressing cytokines than the males. At pnd70, the serum IgG, serum antibrain Abs, amounts of brain IgG, and brain cytokine levels of all of the Hg-treated offspring were equivalent to those of their appropriate controls, suggesting that developmental Hg exposure did not induce chronic immunological effects. However, the social behaviors of Hg-exposed SFvF1 offspring at pnd70 were significantly impaired, and SFvF1 females displayed greater decline in social behaviors than males, suggesting that the higher neuroinflammation of SFvF1 females earlier in life is associated with the altered behavior. Thus, developmental Hg exposure induces long-lasting effects on social behavior of offspring, which is dependent on sex and genetics and the induction of neuroinflammation.
doi:10.1093/toxsci/kft023
PMCID: PMC3693512  PMID: 23392568
mercury; mouse social behavior; IgG antibrain antibodies; maternal influences.
9.  Gaps in Indigenous disadvantage not closing: a census cohort study of social determinants of health in Australia, Canada, and New Zealand from 1981–2006 
BMC Public Health  2014;14:201.
Background
Australia, Canada, and New Zealand are all developed nations that are home to Indigenous populations which have historically faced poorer outcomes than their non-Indigenous counterparts on a range of health, social, and economic measures. The past several decades have seen major efforts made to close gaps in health and social determinants of health for Indigenous persons. We ask whether relative progress toward these goals has been achieved.
Methods
We used census data for each country to compare outcomes for the cohort aged 25–29 years at each census year 1981–2006 in the domains of education, employment, and income.
Results
The percentage-point gaps between Indigenous and non-Indigenous persons holding a bachelor degree or higher qualification ranged from 6.6% (New Zealand) to 10.9% (Canada) in 1981, and grew wider over the period to range from 19.5% (New Zealand) to 25.2% (Australia) in 2006. The unemployment rate gap ranged from 5.4% (Canada) to 16.9% (Australia) in 1981, and fluctuated over the period to range from 6.6% (Canada) to 11.0% (Australia) in 2006. Median Indigenous income as a proportion of non-Indigenous median income (whereby parity = 100%) ranged from 77.2% (New Zealand) to 45.2% (Australia) in 1981, and improved slightly over the period to range from 80.9% (Canada) to 54.4% (Australia) in 2006.
Conclusions
Australia, Canada, and New Zealand represent nations with some of the highest levels of human development in the world. Relative to their non-Indigenous populations, their Indigenous populations were almost as disadvantaged in 2006 as they were in 1981 in the employment and income domains, and more disadvantaged in the education domain. New approaches for closing gaps in social determinants of health are required if progress on achieving equity is to improve.
doi:10.1186/1471-2458-14-201
PMCID: PMC3937433  PMID: 24568143
Indigenous; Inequality; Social determinants of health; Education; Unemployment; Income
10.  “Deeply Quenched” Monitoring of Protein Arginine Deiminase Activity: Multicolor Visualization of Citrullination** 
doi:10.1002/anie.201208464
PMCID: PMC3752692  PMID: 23339123
protein arginine deiminase; citrullination; fluorescent sensor; multicolor visualization; epigenetics
12.  A three generation study of the mental health relationships between grandparents, parents and children 
BMC Psychiatry  2013;13:299.
Background
It is well known that children of parents with mental illness are at greater risk of mental illness themselves. However the patterns of familial mental health problems across multiple generations in families are less clear. This study aimed to examine mental health relationships across three generations of Australian families.
Methods
Mental health data, along with a range of family demographic information, were collected from over 4600 families in Growing Up in Australia: The Longitudinal Study of Australian Children, a nationally representative cohort study. The social and emotional wellbeing of two cohorts of children aged 4–5 years and 8–9 years was measured using the parent-rated Strengths and Difficulties Questionnaire (SDQ). The mental health of mothers and fathers was measured using the Kessler 6-item K6 scale, and the mental health history of maternal and paternal grandmothers and grandfathers was measured using a dichotomous parent-report item. Multivariate linear regression analyses were used assess the relationships between grandparent and parent mental health and child social and emotional wellbeing at ages 4–5 years and 8–9 years.
Results
Both cohorts of children had greater mental health distress with higher SDQ scores on average if their mother or father had a mental health problem. For children aged 8–9 years, a history of mental health problems in maternal grandmothers and grandfathers was associated with higher SDQ scores in grandchildren, after controlling for maternal and paternal mental health and other family characteristics. For children aged 4–5 years, only a mental health history in paternal grandfathers was associated with higher SDQ scores.
Conclusions
The mental health histories of both parents and grandparents play an important role in the social and emotional wellbeing of young children.
doi:10.1186/1471-244X-13-299
PMCID: PMC3829660  PMID: 24206921
Intergenerational transfer; Mental health; Children and families
13.  Correction: Risk Factors for Children's Receptive Vocabulary Development from Four to Eight Years in the Longitudinal Study of Australian Children 
PLoS ONE  2013;8(11):10.1371/annotation/a730446c-0150-4079-98ea-95b12e1e3c28.
doi:10.1371/annotation/a730446c-0150-4079-98ea-95b12e1e3c28
PMCID: PMC3821768  PMID: 24250762
14.  Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking 
Cell Stress & Chaperones  2012;18(3):279-291.
Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3+ (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19+ B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.
doi:10.1007/s12192-012-0380-0
PMCID: PMC3631090  PMID: 23111563
Nitric oxide; Norepinephrine; Sympathetic nervous system; Neutrophils; Cold restraint stress
15.  Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors 
eLife  2013;2:e00966.
Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease.
DOI: http://dx.doi.org/10.7554/eLife.00966.001
eLife digest
As air flows into our lungs, the lining of the nasal cavity, the throat and the rest of the respiratory tract prevents microbes, bacteria, dust and other small particles from entering the lungs. The lining of these airways is made up of many different types of cells, which must be continuously replaced as they become damaged. Experiments in mice have shown that cells called basal cells act as progenitor cells to keep the lining supplied with new cells. Progenitor cells are similar to stem cells: they divide to make, on average, one copy of themselves and one mature cell of another type (such as a secretory cell). This ensures that healthy supply of progenitor cells is maintained for the future. However, it is not clear whether this process takes place at the level of individual progenitor cells or as an average for a population of cells.
Teixeira et al. have now performed a study which shows that basal cells achieve this balance as a result of averaging. The study took advantage of the fact that cellular organelles called mitochondria have their own DNA, which gradually accumulates mutations over time. This makes it possible to identify groups of cells that are descended from a single progenitor cell because they will all contain the same mitochondrial mutation.
By studying lung tissue from seven individuals, Teixeira et al. were able to identify clusters of related cells and found that, as expected, the size of the clusters increased with age. And by applying a mathematical model across all the cells in the study, it was discovered that whenever one basal progenitor cell committed to a particular fate, another progenitor cell duplicated itself: however, this balancing process happened in a random manner across a large number of cells, and not at the level of individual progenitor cells. Interestingly, it was found random cell division happened among smokers too, but was accelerated. This leads to clusters of identical cells forming more quickly in smokers than in non-smokers. In addition to providing further insights into the origins of lung cancer, the statistical methods developed by Teixeira et al. could be used to analyse the behaviour of many other types of stem or progenitor cells.
DOI: http://dx.doi.org/10.7554/eLife.00966.002
doi:10.7554/eLife.00966
PMCID: PMC3804062  PMID: 24151545
Human lineage tracing; mtDNA mutations; lung basal progenitor stem cells; stochastic homeostasis; airways; Human
16.  Developmental lead effects on behavior and brain gene expression in male and female BALB/cAnNTac mice 
Neurotoxicology  2012;33(5):1005-1020.
Lead (Pb) was one of the first poisons identified, and the developing nervous system is particularly vulnerable to its toxic effects. Relatively low, subclinical doses, of Pb that produce no overt signs of encephalopathy can affect cognitive, emotional, and motor functions. In the present study, the effects of developmental Pb-exposure on behavioral performance and gene expression in BALB/cAnNTac mice were evaluated. Pups were exposed to Pb from gestational-day (gd) 8 to postnatal-day (pnd) 21 and later evaluated in exploratory behavior, rotarod, Morris water maze, and resident-intruder assays as adults. Pb-exposure caused significant alterations in exploratory behavior and water maze performance during the probe trial, but rotarod performance was not affected. Pb-exposed males displayed violent behavior towards their cage mates, but not to a stranger in the resident-intruder assay. Gene expression analysis at pnd21 by microarray and qRT-PCR was performed to provide a molecular link to the behavior changes that were observed. Pb strongly up-regulated gene expression within the signaling pathways of mitogen activated protein kinases (MAPKs), extra-cellular matrix (ECM) receptor, focal adhesion, and vascular endothelial growth-factor (VEGF), but Pb down-regulated gene expression within the pathways for glycan structures-biosynthesis 1, purine metabolism, and N-glycan biosynthesis. Pb increased transcription of genes for major histocompatibility (MHC) proteins, the chemokine Ccl28, chemokine receptors, IL-7, IL7R, and proteases. The qRT-PCR analysis indicated an increase of gene expression in the whole brain for caspase 1 and NOS2. Analysis of IL-1β, caspase 1, NOS2, Trail, IL-18 and IL-33 gene expression of brain regions indicated that Pb perturbed the inter-regional expression pattern of pro-inflammatory genes. Brain region protein concentrations for IL-10, an anti-inflammatory cytokine, showed a significant decrease only within the cortex region. Results indicate that Pb differentially affects the behavior of male and female mice in that females did less exploration and the males were selectively more aggressive. Gene expression data pointed to evidence of neuroinflammation in the brain of both female and male mice. Pb had more of an effect in the males on expression of vomeronasal receptor genes associated with odor detection and social behavior.
doi:10.1016/j.neuro.2012.04.017
PMCID: PMC3463649  PMID: 22609695
Behavior; Brain; Cytokines; Gene Expression; Inflammation; Lead; Microarray
17.  Direct observation of frequency modulated transcription in single cells using light activation 
eLife  2013;2:e00750.
Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.
DOI: http://dx.doi.org/10.7554/eLife.00750.001
eLife digest
The process by which a gene is expressed as a protein consists of two stages: transcription, which involves the DNA of the gene being copied into messenger RNA (mRNA); and translation, in which the mRNA is used as a template to assemble amino acids into a protein. Transcription and translation are controlled by many interlinked pathways, which ensures that genes are expressed when and where required.
One of these regulatory pathways involves steroid receptors. The binding of a steroid molecule to its receptor causes the receptor to move into the nucleus and interact with a specific gene, triggering transcription of that gene. When measured at the level of the whole organism, this transcriptional response is dose-dependent—the more steroid molecules that are present, the greater the amount of transcription. However, this is not the case in single cells, in which transcription is either activated or not. This ‘on/off’ behaviour is also seen over time: steroid-activated transcription occurs in bursts, separated by periods of inactivity.
To unravel the molecular mechanism behind this phenomenon, Larson et al. created a light-activated form of the ligand that activates a specific steroid receptor. Using this molecule, they were able to switch transcription of the gene controlled by that receptor on and off. They then used fluorescent proteins to label the mRNA and protein molecules that were produced as a result.
They found that activating the steroid receptor increases the likelihood of transcription occurring inside a cell, but not the duration of individual bursts of transcriptional activity, nor the amount of mRNA produced during each burst. Activation of a steroid receptor seems to control transcription by reducing the length of time each cell spends in the ‘off’ state between bursts.
Larson et al. incorporated their findings into a model that also takes into account the natural variability in levels of transcription between cells, and found that this could explain how the digital (on/off) control of transcription at the cellular level leads to analogue, dose-dependent control at the level of a whole organism. These findings should lead to further insights into how transcription is controlled at the molecular level.
DOI: http://dx.doi.org/10.7554/eLife.00750.002
doi:10.7554/eLife.00750
PMCID: PMC3780543  PMID: 24069527
transcription factors; gene expression; single-molecule; fluorescence; Human
18.  Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells 
PLoS ONE  2013;8(9):e75517.
The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.
doi:10.1371/journal.pone.0075517
PMCID: PMC3783398  PMID: 24086551
19.  Risk Factors for Children's Receptive Vocabulary Development from Four to Eight Years in the Longitudinal Study of Australian Children 
PLoS ONE  2013;8(9):e73046.
Receptive vocabulary develops rapidly in early childhood and builds the foundation for language acquisition and literacy. Variation in receptive vocabulary ability is associated with variation in children's school achievement, and low receptive vocabulary ability is a risk factor for under-achievement at school. In this study, bivariate and multivariate growth curve modelling was used to estimate trajectories of receptive vocabulary development in relation to a wide range of candidate child, maternal and family level influences on receptive vocabulary development from 4–8 years. The study sample comprised 4332 children from the first nationally representative Longitudinal Study of Australian Children (LSAC). Predictors were modeled as risk variables with the lowest level of risk as the reference category. In the multivariate model, risks for receptive vocabulary delay at 4 years, in order of magnitude, were: Maternal Non- English Speaking Background (NESB), low school readiness, child not read to at home, four or more siblings, low family income, low birthweight, low maternal education, maternal mental health distress, low maternal parenting consistency, and high child temperament reactivity. None of these risks were associated with a lower rate of growth from 4–8 years. Instead, maternal NESB, low school readiness and maternal mental health distress were associated with a higher rate of growth, although not sufficient to close the receptive vocabulary gap for children with and without these risks at 8 years. Socio-economic area disadvantage, was not a risk for low receptive vocabulary ability at 4 years but was the only risk associated with a lower rate of growth in receptive vocabulary ability. At 8 years, the gap between children with and without socio-economic area disadvantage was equivalent to eight months of receptive vocabulary growth. These results are consistent with other studies that have shown that social gradients in children's developmental outcomes increase over time.
doi:10.1371/journal.pone.0073046
PMCID: PMC3770643  PMID: 24039856
20.  Development of a Peptidase-Resistant Substrate for Single-Cell Measurement of Protein Kinase B Activation 
Analytical chemistry  2012;84(16):7195-7202.
An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg−1 s−1 and 0.04 ± 0.08 zmol pg−1 s−1, respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find wide-spread utility in both lysate-based and single-cell assays of kinase activity.
doi:10.1021/ac301489d
PMCID: PMC3428732  PMID: 22881604
21.  Mining the Salivary Proteome with Grating-Coupled Surface Plasmon Resonance Imaging and Surface Plasmon Coupled Emission Microarrays 
Biological indicators have numerous and widespread utility in personalized medicine, but the measurement of these indicators also pose many technological and practical challenges. Blood/plasma has typically been used as the sample source with which to measure these indicators, but the invasiveness associated with procurement of samples has led to increased interest in saliva as an attractive alternative. However, there are unique issues associated with the measurement of saliva biomarkers. These issues are compounded by the imperfect correlation between saliva and plasma with respect to biomarker profiles. In this manuscript, we address the technical challenges associated with saliva biomarker quantification describe a high-content microarray assay that employs both grating-coupled surface plasmon resonance imaging surface plasmon coupled emission modalities in a highly sensitive assay that has a large dynamic range. This powerful approach provides the tools to map the proteome of saliva, which in turn should greatly enhance the utility of salivary biomarker profiles in personalized medicine.
doi:10.1002/0471140856.tx1816s53
PMCID: PMC3461276  PMID: 22896008
saliva; biomarker; proteomics; surface plasmon resonance; emission microarray; SPRI; SPCE; personalized medicine; biofluid; biomolecule
22.  Merging of Confocal and Caging Technologies: Selective Three Color Communication with Profluorescent Reporters 
doi:10.1002/anie.201202820
PMCID: PMC3523745  PMID: 22740297
Photolysis; wavelength; cobalamin; fluorescent; selective
23.  Trends in sugar supply and consumption in Australia: is there an Australian Paradox? 
BMC Public Health  2013;13:668.
Background
High consumption of refined carbohydrate, in particular sugar, has been identified as a possible contributory factor in greater risk of excess weight gain. In spite of data limitations, one recent paper suggests that Australian sugar consumption has decreased over the same time period that obesity has increased, a so called ‘Australian Paradox’. Given the significant public health focus on nutrition, we aimed to estimate Australian sugar supply and consumption over recent decades, to determine whether these data could be used to make any conclusions about sugar’s role in obesity.
Methods
Foods high in sugar were identified. Data relating to sugar supply and consumption from 1988 to 2010 were obtained from multiple sources. Using these data we attempted to generate a time series estimate of sugar in Australia’s food supply.
Results
Australia produces and exports sugar from sugar cane and the sugar in imported foods has received little attention. We were unable to produce a reliable and robust estimate of total sugars in the Australian diet due to data limitations and a lack of current data sources. However, available Import data showed large increases in the volume and value of imported sweetened products between 1988 and 2010 to over 30 grams of sugar per person per day. Value estimates of local production of sweetened products also show substantial increases in this period.
Conclusion
The Australian Paradox assertion is based on incomplete data, as it excludes sugar contained in imported processed foods, which have increased markedly. A major Australian public health target is to improve the quality of the food supply, and actions have been set in terms of achieving broader environmental changes. However, evaluation of progress is hampered by lack of high quality data relating to supply and consumption. We recommend the regular collection of comprehensive food supply statistics, which include both local production and imports. This would provide an inexpensive addition to survey data and could assist in monitoring sugar consumption trends in food supply. Such information would also help inform public health policy.
doi:10.1186/1471-2458-13-668
PMCID: PMC3726354  PMID: 23866719
Public health; Sugar; Obesity; Food supply
24.  Metabolism of Peptide Reporters in Cell Lysates and Single Cells 
The Analyst  2012;137(13):3028-3038.
The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg−;1 s−;1 with a t1/2 of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an (N-methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in kcat as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells.
doi:10.1039/c2an16162a
PMCID: PMC3697743  PMID: 22314840
25.  Smoking, mental illness and socioeconomic disadvantage: analysis of the Australian National Survey of Mental Health and Wellbeing 
BMC Public Health  2013;13:462.
Background
High rates of smoking and lower rates of smoking cessation are known to be associated with common mental disorders such as anxiety and depression, and with individual and community measures of socioeconomic status. It is not known to what extent mental illness and socioeconomic status might be jointly associated with smoking behaviour. We set out to examine the relationship between mental illness, measures of socioeconomic disadvantage and both current smoking and smoking cessation rates.
Methods
We used data from the 2007 Australian National Survey of Mental Health and Wellbeing to examine the relationship between mental illness, socioeconomic status and both current smoking and smoking cessation. We used cross-classified tables and logistic regression to examine the relationship between psychosocial and sociodemographic predictors and current smoking. We also used proportional hazards regression to examine the relationship between the factors and smoking cessation.
Results
Both mental illness and socioeconomic status were independently associated with current smoking and with lower likelihood of smoking cessation, with gradients in smoking by mental health status being observed within levels of socioeconomic indicators and vice versa. Having a mental illness in the past 12 months was the most prevalent factor strongly associated with smoking, affecting 20.0% of the population, associated with increased current smoking (OR 2.43; 95% CI: 1.97-3.01) and reduced likelihood of smoking cessation (HR: 0.77; 95% CI: 0.65-0.91).
Conclusions
The association between mental illness and smoking is not explained by the association between mental illness and socioeconomic status. There are strong socioeconomic and psychosocial gradients in both current smoking and smoking cessation. Incorporating knowledge of the other adverse factors in smokers’ lives may increase the penetration of tobacco control interventions in population groups that have historically benefitted less from these activities.
doi:10.1186/1471-2458-13-462
PMCID: PMC3660247  PMID: 23663362

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