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.
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.
Human lineage tracing; mtDNA mutations; lung basal progenitor stem cells; stochastic homeostasis; airways; Human
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.
Behavior; Brain; Cytokines; Gene Expression; Inflammation; Lead; Microarray
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.
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.
transcription factors; gene expression; single-molecule; fluorescence; Human
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.
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.
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.
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.
saliva; biomarker; proteomics; surface plasmon resonance; emission microarray; SPRI; SPCE; personalized medicine; biofluid; biomolecule
Photolysis; wavelength; cobalamin; fluorescent; selective
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.
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.
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.
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.
Public health; Sugar; Obesity; Food supply
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.
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.
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.
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).
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.
The environmental heavy metal toxicant, lead (Pb), has been shown to be more harmful to the central nervous system (CNS) of children than to adults, given that Pb exposure affects the neural system during development. Because growth factors and cytokines play very important roles in development of the CNS, we have examined the impact of Pb exposure on the expression of cytokines during CNS development. Cytokine expression was studied in post-natal-day 21(pnd21) mice by microarray, real-time RT-PCR, Luminex, and ELISA methodologies. BALB/c mouse pups were exposed to Pb through the dam’s drinking water (0.1 mM Pb acetate), from gestation-day 8 (gd8) to pnd21. Two cytokines, interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), displayed significantly changed transcript levels in the presence of Pb. IL-6 and TGF-β1 both have signal transduction cascades that can cooperatively turn on the gene for the astrocyte marker glial-fibrillary acidic protein (GFAP). Microarray results indicated that Pb exposure significantly increased expression of GFAP. Pb also modulated IL-6, TGF-β1, and IL-18 protein expression in select brain regions. The deleterious effects of Pb on learning and long-term memory are posited to result from excessive astrocyte growth and/or activation with concomitant interference with neural connections. Differential neural expression of cytokines in brain regions needs to be further investigated to mechanistically associate Pb and neuroinflammation with behavioral and cognitive changes.
astrocyte; brain; central nervous system; cytokine; lead; neuron; signal transduction; transcription
The frequency and potential long-term effects of sport-related traumatic brain injuries (TBI) make it a major public health concern. The culture within contact sports, such as ice hockey, encourages aggression that puts youth at risk of TBI such as concussion. Newspaper reports play an important role in conveying and shaping the culture around health-related behaviors. We qualitatively studied reports about sport-related TBI in four major North American newspapers over the last quarter-century. We used the grounded-theory approach to identify major themes and then did a content analysis to compare the frequency of key themes between 1998–2000 and 2009–2011. The major themes were: perceptions of brain injury, aggression, equipment, rules and regulations, and youth hockey. Across the full study period, newspaper articles from Canada and America portrayed violence and aggression that leads to TBI both as integral to hockey and as an unavoidable risk associated with playing the game. They also condemned violence in ice hockey, criticized the administrative response to TBI, and recognized the significance of TBI. In Canada, aggression was reported more often recently and there was a distinctive shift in portraying protective equipment as a solution to TBI in earlier years to a potential contributing factor to TBI later in the study period. American newspapers gave a greater attention to ‘perception of risks’ and the role of protective equipment, and discussed TBI in a broader context in the recent time period. Newspapers from both countries showed similar recent trends in regards to a need for rule changes to curb youth sport-related TBI. This study provides a rich description of the reporting around TBI in contact sport. Understanding this reporting is important for evaluating whether the dangers of sport-related TBI are being appropriately communicated by the media.
Routinely used biomarkers of bacterial etiology of infection, such as C-reactive protein and procalcitonin, have limited usefulness for evaluation of infections since their expression is enhanced by a number of different conditions. Therefore, several inflammatory cytokines and chemokines were analyzed with sera from patients hospitalized for moderate bacterial and viral infectious diseases. In total, 57 subjects were enrolled: 21 patients with community-acquired bacterial infections, 26 patients with viral infections, and 10 healthy subjects (control cohorts). The laboratory analyses were performed using Luminex technology, and the following molecules were examined: IL-1Ra, IL-2, IL-4, IL-6, IL-8, TNF-α, INF-γ, MIP-1β, and MCP-1. Bacterial etiology of infection was associated with significantly (P < 0.001) elevated serum concentrations of IL-1Ra, IL-2, IL-6, and TNF-α in comparison to levels observed in the sera of patients with viral infections. In the patients with bacterial infections, IL-1Ra and IL-8 demonstrated positive correlation with C-reactive protein, whereas, IL-1Ra, TNF-α, and MCP-1 correlated with procalcitonin. Furthermore, elevated levels of IL-1Ra, IL-6, and TNF-α decreased within 3 days of antibiotic therapy to levels observed in control subjects. The results show IL-1Ra as a potential useful biomarker of community-acquired bacterial infection.
The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R2C2), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via western blot analysis and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.
Despite the increasing understanding of the mechanisms relating to weight loss and maintenance, there are currently no validated public health interventions that are able to achieve sustained long-term weight loss or to stem the increasing prevalence of obesity in the population. We aimed to examine the models of energy balance underpinning current research about weight-loss intervention from the field of public health, and to determine whether they are consistent with the model provided by basic science. EMBASE was searched for papers published in 2011 on weight-loss interventions. We extracted details of the population, nature of the intervention, and key findings for 27 articles.
Most public health interventions identified were based on a simple model of energy balance, and thus attempted to reduce caloric consumption and/or increase physical activity in order to create a negative energy balance. There appeared to be little consideration of homeostatic feedback mechanisms and their effect on weight-loss success. It seems that there has been a lack of translation between recent advances in understanding of the basic science behind weight loss, and the concepts underpinning the increasingly urgent efforts to reduce excess weight in the population.
Public health weight-loss interventions seem to be based on an outdated understanding of the science. Their continued failure to achieve any meaningful, long-term results reflects the need to develop intervention science that is integrated with knowledge from basic science. Instead of asking why people persist in eating too much and exercising too little, the key questions of obesity research should address those factors (environmental, behavioral or otherwise) that lead to dysregulation of the homeostatic mechanism of energy regulation. There is a need for a multidisciplinary approach in the design of future weight-loss interventions in order to improve long-term weight-loss success.
Energy balance; obesity; public health; weight-loss intervention
Rationale: Patients with isolated mediastinal lymphadenopathy (IML) are a common presentation to physicians, and mediastinoscopy is traditionally considered the “gold standard” investigation when a pathological diagnosis is required. Endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) is established as an alternative to mediastinoscopy in patients with lung cancer.
Objective: To determine the efficacy and health care costs of EBUS-TBNA as an alternative initial investigation to mediastinoscopy in patients with isolated IML.
Methods: Prospective multicenter single-arm clinical trial of 77 consecutive patients with IML from 5 centers between April 2009 and March 2011. All patients underwent EBUS-TBNA. If EBUS-TBNA did not provide a diagnosis, then participants underwent mediastinoscopy.
Measurements and Main Results: EBUS-TBNA prevented 87% of mediastinoscopies (95% confidence interval [CI], 77–94%; P < 0.001) but failed to provide a diagnosis in 10 patients (13%), all of whom underwent mediastinoscopy. The sensitivity and negative predictive value of EBUS-TBNA in patients with IML were 92% (95% CI, 83–95%) and 40% (95% CI, 12–74%), respectively. One patient developed a lower respiratory tract infection after EBUS-TBNA, requiring inpatient admission. The cost of the EBUS-TBNA procedure per patient was £1,382 ($2,190). The mean cost of the EBUS-TBNA strategy was £1,892 ($2,998) per patient, whereas a strategy of mediastinoscopy alone was significantly more costly at £3,228 ($5,115) per patient (P < 0.001). The EBUS-TBNA strategy is less costly than mediastinoscopy if the cost per EBUS-TBNA procedure is less than £2,718 ($4,307) per patient.
Conclusions: EBUS-TBNA is a safe, highly sensitive, and cost-saving initial investigation in patients with IML.
Clinical trial registered with ClinicalTrials.gov (NCT00932854).
endobronchial ultrasound; mediastinal lymphadenopathy; sarcoidosis; tuberculosis; lymphoma
The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities was used to detect and characterize CD4+ T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabelled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.
T cell microarray; SPR; SPCE
Among patients with psychiatric disorders, there are 10 times as many preventable deaths from physical disorders as there are from suicide. We investigated whether compulsory community treatment, such as community treatment orders, could reduce all-cause mortality among patients with psychiatric disorders.
We conducted a population-based survival analysis of an inception cohort using record linking. The study period extended from November 1997 to December 2008. The cohort included patients from all community-based and inpatient psychiatric services in Western Australia (state population 1.8 million). We used a 2-stage design of matching and Cox regression to adjust for demographic characteristics, previous use of health services, diagnosis and length of psychiatric history. We collected data on successive cohorts for each year for which community treatment orders were used to measure changes in numbers of patients, their characteristics and outcomes. Our primary outcome was 2-year all-cause mortality. Our secondary outcomes were 1-and 3-year all-cause mortality.
The study population included 2958 patients with community treatment orders (cases) and 2958 matched controls (i.e., patients with psychiatric disorders who had not received a community treatment order). The average age for cases and controls was 36.7 years, and 63.7% (3771) of participants were men. Schizophrenia and other nonaffective psychoses were the most common diagnoses (73.4%) among participants. A total of 492 patients (8.3%) died during the study. Cox regression showed that, compared with controls, patients with community treatment orders had significantly lower all-cause mortality at 1, 2 and 3 years, with an adjusted hazard ratio of 0.62 (95% confidence interval 0.45–0.86) at 2 years. The greatest effect was on death from physical illnesses such as cancer, cardiovascular disease or diseases of the central nervous system. This association disappeared when we adjusted for increased outpatient and community contacts with psychiatric services.
Community treatment orders might reduce mortality among patients with psychiatric disorders. This may be partly explained by increased contact with health services in the community. However, the effects of uncontrolled confounders cannot be excluded.
Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, spores, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a non-toxic form of Pseudomonas aeruginosa exotoxin A (ntPE), Bacillus globigii, Mycoplasma hyopneumoniae, Listeria monocytogenes, Escherichia coli, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free and highly multiplexed assay requiring nanoliter amounts of capture reagents.
multi-analyte detection; pathogen detection; cell and protein microarray; high-throughput SPR biosensor; grating-coupled surface plasmon resonance imaging; multiplexed immunoassay
Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.
Danshen, the root and rhizome of Salvia miltiorrhiza Bge, a Traditional Chinese Medicine, especially for cardiovascular and cerebrovascular diseases, has unique immunomodulatory effects. Danshen is capable of anti-inflammation and antiallergy, which are immunosuppressive activities, whereas it is also able to promote immunity against cancer, viruses, and bacteria. Most previous reports were performed with use of a purified compound or compounds of Danshen. Since there are more than twenty active compounds in Danshen, it is very difficult to predict that one compound will act the same way when it is combined with other compounds. In order to overcome this limitation, we used the crude form of Danshen to study its immunomodulatory effects in a mouse model. The mice were fed daily diet supplements of Danshen for three months and then tested for their immunity, including leukocyte subsets in peripheral blood, humoral and cell-mediated immune responses, and host defenses against a Listeria monocytogenes (LM) infection. Different doses of Danshen caused different immunomodulatory effects. Danshen at 0.5% decreased serum IgE production in BALB/c mice; 1% Danshen promoted cell-mediated immunity; Danshen at 0.5 and 1% inhibited the production of oxygen free radicals in liver and spleen and NO production in liver; 2% Danshen enhanced the host resistance against LM with increased numbers of peripheral monocytes and natural killer (NK) cells and decreased production of IL-1β and NO.
Statistical time series derived from administrative data sets form key indicators in measuring progress in addressing disadvantage in Aboriginal and Torres Strait Islander populations in Australia. However, inconsistencies in the reporting of Indigenous status can cause difficulties in producing reliable indicators. External data sources, such as survey data, provide a means of assessing the consistency of administrative data and may be used to adjust statistics based on administrative data sources.
We used record linkage between a large-scale survey (the Western Australian Aboriginal Child Health Survey), and two administrative data sources (the Western Australia (WA) Register of Births and the WA Midwives’ Notification System) to compare the degree of consistency in determining Indigenous status of children between the two sources. We then used a logistic regression model predicting probability of consistency between the two sources to estimate the probability of each record on the two administrative data sources being identified as being of Aboriginal and/or Torres Strait Islander origin in a survey. By summing these probabilities we produced model-adjusted time series of neonatal outcomes for Aboriginal and/or Torres Strait Islander births.
Compared to survey data, information based only on the two administrative data sources identified substantially fewer Aboriginal and/or Torres Strait Islander births. However, these births were not randomly distributed. Births of children identified as being of Aboriginal and/or Torres Strait Islander origin in the survey only were more likely to be living in urban areas, in less disadvantaged areas, and to have only one parent who identifies as being of Aboriginal and/or Torres Strait Islander origin, particularly the father. They were also more likely to have better health and wellbeing outcomes. Applying an adjustment model based on the linked survey data increased the estimated number of Aboriginal and/or Torres Strait Islander births in WA by around 25%, however this increase was accompanied by lower overall proportions of low birth weight and low gestational age babies.
Record linkage of survey data to administrative data sets is useful to validate the quality of recording of demographic information in administrative data sources, and such information can be used to adjust for differential identification in administrative data.
The aim of this study was to validate a model for optimal birth weight derived from neonatal records, and to test the assumption that preterm births may be considered optimally grown if they are not exposed to common factors that perturb fetal growth.
Weights of fetuses were estimated from serial biometric ultrasound scans (N = 2,848) and combined with neonatal weights for a prospective pregnancy cohort (N = 691). Non-Caucasians, fetuses subsequently born preterm and those with diagnosed or suspected determinants of aberrant growth were excluded leaving fetuses assumed to have experienced normal growth. A generalised linear longitudinal growth model for optimal weight was derived, including terms for gestational duration, infant sex, maternal height and birth order. This model was compared to a published model derived solely from birth weights.
Prior to 30 weeks gestation, the published model yielded systematically lower weights than the model derived from both fetal weight and neonatal weight. From 30 weeks gestation the two models were indistinguishable.
The model for optimal birth weight was valid for births that have attained at least 30 weeks gestation. The model derived from both fetal and neonatal weights is recommended prior to this gestation.
Fetal growth; Preterm delivery; Proportion of optimal birth weight