Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional “bulk” analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling.
SIGNIFICANCE STATEMENT
High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.
To investigate pedometer-measured physical activity (PA) in 2000 and change in PA over 5 years with subsequent risk of dysglycemia by 2005.This prospective cohort study in Tasmania, Australia, analyzed 458 adults with normal glucose tolerance and a mean (SD) age of 49.7 (12.1) years in 2000. Variables assessed in 2000 and 2005 included PA, by pedometer and questionnaire, nutrient intake, and other lifestyle factors. Incident dysglycemia was defined as the development of impaired fasting glucose or impaired glucose tolerance revealed by oral glucose tolerance testing in 2005, without type 2 diabetes.Incident dysglycemia developed in 26 participants during the 5-year period. Higher daily steps in 2000 were independently associated with a lower 5-year risk of incident dysglycemia (adjusted odds ratio [AOR] 0.87 [95% CI 0.77-0.97] per 1,000-step increment). Higher daily steps in 2005, after controlling for baseline steps in 2000 (thus reflecting change in steps over 5 years), were not associated with incident dysglycemia (AOR 1.02 [0.92-1.14]). Higher daily steps in 2000 were also associated with lower fasting blood glucose, but not 2-h plasma glucose by 2005. Further adjustment for BMI or waist circumference did not remove these associations.Among community-dwelling adults, a higher rate of daily steps is associated with a reduced risk of incident dysglycemia. This effect appears to be not fully mediated through reduced adiposity.
We aimed to (i) determine the relative importance of childhood gain in upper body adiposity for insulin resistance (IR) and triglyceridemia (TG); (ii) examine whether the associations between adiposity and metabolic indices were more evident in those with the ACE DD genotype. We examined a birth cohort study of 292 children with measures in the neonatal period (day 4) including subscapular and triceps skinfolds; repeat skinfold measures at age 8, cardiorespiratory (CR) fitness, IR by the homeostasis model assessment (HOMA) equation (HOMA‐IR) and serum triglyceride (TG) concentrations and measures of ACE I/D gene variants. A multiple linear regression analysis incorporating a life course approach was undertaken. Childhood gain in upper body adiposity was positively associated with HOMA‐IR and TG independently of neonatal skinfolds ( P ≤ 0.02). The magnitude of these associations was higher among those of the ACE DD genotype. For example, subscapular skinfold gain was not strongly associated with HOMA‐IR or TG among those with II or ID genotype ( b = 0.03, P = 0.05; b = 0.02, P = 0.18 respectively) but was positively associated among those with the DD genotype ( b = 0.11, P = 0.001; b = 0.08, P = 0.003); difference in effect P = 0.05; P = 0.01 respectively. Upper body fat accumulation during childhood was positively associated with HOMA‐IR and TG independently of neonatal skinfolds. Further, the stronger associations for those with the ACE DD genotype is consistent with randomised controlled trial findings that ACE inhibition is associated with a reduced risk of developing type 2 diabetes. Further work is required to confirm and extend these findings.
Background. An intervention to reduce the prevalence of the prone sleeping position during infancy was implemented in Tasmania particularly from 1991 onward. The purpose of this report is to assess the impact of public health activities in promoting avoidance of the prone infant sleeping position among cohort study participants. Methods. The prospective cohort study involved the one-fifth of Tasmanian live births who are assessed perinatally as being at higher risk for Sudden Infant Death Syndrome (SIDS). From 1 January 1988 until 30 April 1992, 5,403 infants participated in the hospital (4 days postnatal age) and home interviews (5 weeks postnatal age) (88% of eligible infants). After the finding that cohort infants who usually slept prone were at significantly greater risk for SIDS, additional questions on awareness and choice of infant sleep position were asked. Results. The proportion of infants usually sleeping prone declined from 29.9% in the cohort prior to publication of the cohort findings (1 May 1988-30 April 1991) to 5.4% in the post publication cohort (1 May 1991-30 April 1992), RR = 0.18 (0.15, 0.22). Teenage motherhood was associated with nonawareness (RR = 2.39 (1.41, 3.24)) of an association between prone position and SIDS. After adjusting for maternal age, nonawareness remained positively associated with maternal smoking, maternal education (
Background Self–reported physical activity has been inversely associated with mortality but the effect of objectively measured step activity on mortality has never been evaluated. The objective is to determine the prospective association of daily step activity on mortality among free-living adults. Methods and Findings Cohort study of free-living adults residing in Tasmania, Australia between 2000 and 2005 who participated in one of three cohort studies (n = 2 576 total participants). Daily step activity by pedometer at baseline at a mean of 58.8 years of age, and for a subset, repeated monitoring was available 3.7 (SD 1.3) years later (n = 1 679). All-cause mortality (n = 219 deaths) was ascertained by record-linkage to the Australian National Death Index; 90% of participants were followed-up over ten years, until June 2011. Higher daily step count at baseline was linearly associated with lower all-cause mortality (adjusted hazard ratio AHR, 0.94; 95% CI, 0.90 to 0.98 per 1 000 steps; P = 0.004). Risk was altered little by removing deaths occurring in the first two years. Increasing baseline daily steps from sedentary to 10 000 steps a day was associated with a 46% (95% CI, 18% to 65%; P = 0.004) lower risk of mortality in the decade of follow-up. In addition, those who increased their daily steps over the monitoring period had a substantial reduction in mortality risk, after adjusting for baseline daily step count (AHR, 0.39; 95% CI, 0.22 to 0.72; P = 0.002), or other factors (AHR, 0.38; 95% CI, 0.21–0.70; P = 0.002). Conclusions Higher daily step count was linearly associated with subsequent long term mortality among free living adults. These data are the first to quantify mortality reductions using an objective measure of physical activity in a free living population. They strongly underscore the importance of physical inactivity as a major public health problem.
Purpose: To investigate the association between maternal smoking in pregnancy, early-life environment and childhood vision. Methods: Twin and triplet children enrolled in the Twins Eye Study in Tasmania underwent a comprehensive ophthalmic examination and their parents/guardians retrospectively answered a questionnaire regarding crawling, walking and other measures. A subset of these twins was also in the Tasmanian Infant Health Survey, which prospectively collected data on antenatal smoking, gestation, birth weight and other factors. Results: The mean age of the 346 individuals (172 multiple birth sets) at the time of examination was 9.25 ± 2.4 years. Mean unaided visual acuity was 0.0 (6/6). The mean spherical equivalent was +0.87D, and decreased with increasing child age (p < 0.01). A prospective analysis, accounting for birth set clustering and relevant confounders, showed increasing levels of maternal smoking in the third trimester was associated with poor stereoacuity on the Titmus test (worse (>) than 100″, p = 0.05) and Lang test (p = 0.001) and also with the presence of esotropia (p = 0.02). These associations persisted after adjustment for infant postnatal smoke exposure at one month of age. Poor stereoacuity on Titmus stereo test circles was associated with late age of first crawling (RR = 1.23 (1.06, 1.42) p = 0.005 per month) and late age of first walking (RR 1.18 (1.05, 1.22) p = 0.001 per month). Conclusions: Antenatal smoking was independently associated with poor stereovision and the presence of esotropia. Poor stereoacuity may be associated with delayed age at first crawling or walking.
Recent studies reported an association between smaller birth size and narrower retinal vascular caliber, but it remains unclear whether this association is attributed to confounding by shared environment or genetic factors. At a mean age of 9.3 years, 266 twins (49 monozygotic and 84 dizygotic pairs) in the Twins Eye Study in Tasmania underwent an ophthalmic examination including retinal photography. Retinal vascular caliber was measured using a validated protocol. The majority of these twins were also in the Tasmanian Infant Health Study, which prospectively collected data on birth parameters and antenatal maternal factors. We conducted the main analysis using linear mixed models, accounting for birth set clustering. Both the within-pair (-9.73; 95% CI: -14.68 to -4.77 microm per 5-cm decrease in birth length) and between-pair associations (-7.15; 95% CI: -11.54 to -3.01) with retinal arteriolar caliber were significant and of similar magnitude (difference in effect, P=0.61), after adjusting for age, sex, maternal smoking, mean arterial blood pressure, and other confounders. These associations remained within dizygotic and monozygotic pairs. Analyses of head circumference and retinal arteriolar caliber were similar to those of birth length (within-pair regression coefficient: -2.41; 95% CI: -5.09 to 0.28; between-pair regression coefficient: -2.60; 95% CI: -5.00 to -0.19). For birth weight, only a between-pair association was evident (-7.28; 95% CI: -13.07 to -1.48). This study demonstrates a consistent association between smaller birth size and narrower retinal arterioles in twins. The independent effect of shorter birth length on retinal arteriolar caliber supports a role for twin-specific supply line factors affecting fetal growth on vascular structure.
Studies on the role of non‐milk fluids in the development of child atopic disease are scarce. We had a unique opportunity to investigate prospective association between the introduction of fruit syrup, orange juice, sterilized water, vitamins and honey at 1 month and the development of child atopic disease. The exposure of interest was measured by parental report of non‐milk fluids introduction to infants aged 1 month at the Tasmanian Infant Health Survey, 1988–89, Tasmania. Data on the outcomes of interest (atopic sensitization, asthma, eczema and hay fever) were collected during the 1997 Childhood Allergy and Respiratory Health Study when children were 8 yr old. Relative risks were derived from generalized linear model with a log link function and binomial error structure. None of the non‐milk fluids appeared to be a significant predictor of atopic sensitization. Only sterilized water was a significant risk factor for asthma (adjusted relative risk = 1.59; 95% confidence intervals: 1.14–2.22), which may be partly because of associated overall better hygienic conditions and decreased exposure to early infections in the household. In summary, we were unable to find evidence for an association between introduction of non‐milk fluids in infancy and childhood atopic disease.
Home gas appliance use has been associated with child respiratory illness but prospective data on the relationship between infant exposure and the development of child allergic disease has not been readily available.(a) To determine if home gas appliance use is associated with increased risk of house dust mite (HDM) sensitization. (b) To examine whether any association between current home gas use and airway obstruction is influenced by HDM sensitization.an 8-year follow-up birth cohort study of children born during 1988 and 1989.a population-based sample (n = 498) of children who participated in the Tasmanian Infant Health Survey (TIHS) and resided in Northern Tasmania in 1997 (84% of eligible children).(a) Skin prick test reaction to nine allergens, including Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1). (b) Spirometric lung function indices, including forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC).The relative risk for home gas appliance use at 1 month of age and HDM sensitization was 1.98 (1.04, 3.79) in a cohort analysis with confounder matching. Current home gas use was also associated with HDM sensitization (ARR 1.73 (1.43, 2.76)). Current home gas use was related to a stronger (P = 0.006) reduction in the FEV(1) : FVC ratio among HDM-sensitive children (adjusted difference - 6.2% (- 10.0 to - 2.4)) than non-HDM-sensitive children (adjusted difference - 0.3% (- 2.5 to 1.8)).Indoor pollutants from gas combustion may increase the likelihood of initial sensitization to HDM and play a role in the development of atopic asthma. HDM-sensitized children may be more vulnerable to indoor pollutant-induced airway obstruction. The ability of this study to detect such effects may partly reflect unflued gas appliance use among this sample.
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