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Asthma and allergy are complex disorders influenced by both inheritance and environment, a relationship that might be further clarified by epigenetics. Neuropeptide S Receptor 1 (NPSR1) has been associated with asthma and allergy and a study suggested modulation of the genetic risk by environmental factors. We aimed to study DNA methylation in the promoter region of NPSR1 in relation to asthma and environmental exposures. Electrophoretic Mobility Shift Assay (EMSA) was used to investigate potential functional roles of both genotypes and methylation status in the NPSR1 promoter. DNA methylation was analysed using EpiTYPER in blood samples from two well-characterized cohorts; the BIOAIR study of severe asthma in adults and the Swedish birth cohort BAMSE. We observed that DNA methylation and genetic variants in the promoter influenced the binding of nuclear proteins to DNA, suggesting functional relevance. Significant, although small, differences in methylation were related to both adult severe asthma (p = 0.0001) and childhood allergic asthma (p = 0.01). Furthermore, DNA methylation was associated with exposures such as current smoking in adults for two CpG sites (p = 0.005 and 0.04), parental smoking during infancy in the children (p = 0.02) and in which month the sample was taken (p = 0.01). In summary, DNA methylation levels in the promoter of NPSR1 showed small but significant associations with asthma, both in adults and in children, and to related traits such as allergy and certain environmental exposures. Both genetic variation and the methylated state of CpG sites seem to have an effect on the binding of nuclear proteins in the regulatory region of NPSR1 suggesting complex regulation of this gene in asthma and allergy.
Citation: Reinius LE, Gref A, Sääf A, Acevedo N, Joerink M, Kupczyk M, et al. (2013) DNA Methylation in the Neuropeptide S Receptor 1 (NPSR1) Promoter in Relation to Asthma and Environmental Factors. PLoS ONE 8(1): e53877. https://doi.org/10.1371/journal.pone.0053877
Editor: Susanne Krauss-Etschmann, Ludwig-Maximilians-University Munich, Germany
Received: May 21, 2012; Accepted: December 4, 2012; Published: January 23, 2013
Copyright: © 2013 Reinius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the Swedish Foundation for Strategic Research (the Epigene study) and the Swedish Research Council. LER and MK were supported by the Osher Initiative for Research on Severe Asthma at Karolinska Institutet, AG and NA were supported by a PhD grant from Karolinska Institutet, and MJ was supported by a Marie Curie Intra-European Fellowship. The BAMSE birth cohort was supported by grants from the Swedish Foundation for Strategic Research, the Swedish Research Council, Swedish Heart-Lung Foundation, the Stockholm County Council and the Swedish Foundation for Health Care Sciences and Allergy Research, and the European Union. The BIOAIR study was supported by the Fifth and Sixth Framework Programs of the European Union, contract numbers: QLG1-CT-2000-01185 (BIOAIR) and FOOD-CT-2004-506378 (GA2LEN), and several national funding bodies (Sweden: Heart-Lung Foundation and Asthma and Allergy Foundation; Greece: unrestricted competitive research grant "The Herakleitos project 2002", from the Hellenic Ministry of Education). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Asthma and allergy are complex disorders influenced by both inheritance and environment. In recent decades there has been a marked increase in their prevalence that today has reached a plateau, a development that cannot be explained by genetics alone. Epigenetics might help to explain this development of asthma and allergy, but studies on this topic are so far few. DNA methylation is an epigenetic modification that occurs mostly on cytosines followed by guanines, so called CpG sites. There are approximately 28 million such CpG sites in the human genome and of these, 70 to 80% are methylated [1]. Methylation of DNA has previously been shown to regulate gene expression [2] and it is believed that the occurrence of these marks/modifications can be affected by the environment and that this could partly explain the increase in the prevalence of asthma and allergy [3]. For example, exposure to smoking in utero may influence DNA methylation in the offspring [4], and has been associated with an increased risk of childhood asthma [5], [6]. This effect was also observed in grandchildren of grandmothers that smoked during the mother's fetal period which indicates a transgenerational effect that might be mediated by a change in DNA methylation patterns.
The Neuropeptide S Receptor 1 (NPSR1) gene on chromosome 7p14.3 encodes a G-protein coupled receptor that has been associated to many complex disorders such as asthma, irritable bowel syndrome, rheumatoid arthritis and psychiatric disorders [7]–[12]. The receptor was first identified as an asthma candidate gene by positional cloning and has since then been replicated several times in different ethnic populations [10], [13]–[20]. Recent data suggest that the genetic background of NPSR1 can modify the effect of environmental factors such as farm exposure on the development of allergic symptoms [21]. The underlying mechanism of this effect in asthma is still unknown.
Since NPSR1 is a strong candidate gene for asthma and one study suggested that its genetic risk is modulated by the environment, we aimed to study DNA methylation in the regulatory region of NPSR1 in relation to asthma and environmental exposures. We used Electrophoretic Mobility Shift Assay (EMSA) to investigate a potential functional role of both genotypes and methylation status in the regulatory region of NPSR1. We used two well characterized cohorts that offer unique opportunities to investigate genetics and epigenetics in relation to asthma phenotypes and environmental exposures; the BIOAIR study of severe asthma in adults [22] and the Swedish birth cohort BAMSE [23].
Transcription factor binding sites, DNA methylation, and genetic variants
The NPSR1 promoter contains twelve CpG sites (Figure 1) [24]. To evaluate potential functionality of these CpG sites, we performed EMSA to test for possible differential protein binding based on both genotype and methylation status. Four suggestive transcription factor binding sites in the promoter were selected based on the presence of a CpG site within the predicted binding site. The sites studied included CpG site 2 coinciding with rs2168890 in a predicted Hmx2 binding site, CpG site 3 in a predicted STAT1 binding site, CpG site 8 coinciding with rs2530547 in a predicted binding site for Myb, and CpG site 9 coinciding with rs887020 in a predicted binding site for AP1 (Figure 1). EMSA using nuclear extracts from both Colo205 and HEK293 cells showed clear differences in binding of nuclear proteins based both on genotype and methylation status of the C allele in the CpG site 2/rs2168890 (Figure 2). CpG site 3 showed differences in binding of nuclear proteins between unmethylated and methylated cassettes with loss of bands for the methylated cassette. The results for CpG site 8 showed no or very weak binding for either allele of the SNP rs2530547 while the methylated C allele bound nuclear proteins. CpG site 9 showed loss of protein binding for the C allele, both methylated and unmethylated, as compared to the T allele (upper band, Figure 2). Taken together, the EMSA results suggest that DNA methylation, in the promoter of NPSR1, could functionally be relevant based on the differential binding of transcription factors due to not only genetic variation but also the methylated state of the allele creating a CpG site.
Polymorphisms are marked by triangles, transcription factor binding sites are underlined and CpG sites are shaded in gray. Green color of the transcription factor indicates neurologically associated factors, blue indicates immunologically associated factors, and black indicates general factors.
The experiment was performed three separate times using nuclear protein extracts from two different cell types (Colo205 and HEK293). Data presented here is a representative gel using nuclear extract from Colo205. Data was similar for nuclear cell extracts from both cell lines. The sites studied included CpG site 2 coinciding with rs2168890 in a predicted HMX2 binding site, CpG site 3 in a predicted STAT1 binding site, CpG site 8 coinciding with rs2530547 in a predicted binding site for MYB, and CpG site 9 coinciding with rs887020 in a predicted binding site for AP1. Arrows indicates sites showing differential binding.
Variation of DNA methylation in main blood cell types
To assess whether it would be meaningful to measure DNA methylation levels in peripheral blood samples, we compared the degree of methylation in the NPSR1 region in a set of samples of whole blood and fractionated white blood cells from six healthy adult male donors [25]. Three of the probes included in the Illumina Infinium 450K array for DNA methylation were overlapping with CpG sites in the NPSR1 promoter region analyzed here, i.e. CpG sites 3, 7 and 10, described below. The results show that across NPSR1, the degree of methylation in different types of white blood cells is relatively constant, suggesting that methylation measurements are reliable for NPSR1 in whole blood irrespective the differential count of white blood cells (Figure 3). Furthermore, to get a more complete picture of the cell specific DNA methylation pattern in the NPSR1 promoter region, we use the EpiTYPER technology to determine DNA methylation levels of CpG site 1–12 in the same samples used for the array based method (Figure S5). Again, we confirm that the degree of methylation is similar across different cell types in whole blood for the promoter region studied in NPSR1.
Peripheral blood donations were obtained from six healthy adult male donors. Data were obtained using the Illumina Infinium 450K bead array and the M-value represents methylation degree [25]. Dark grey bars represent lymphoid cells, light grey bars myeloid cells and black bar whole blood. TSS – Transcription start site.
DNA methylation and genetic variation in the NPSR1 promoter
We next performed a pilot study to evaluate if differences in DNA methylation status can be identified in whole blood from patients with asthma and other obstructive airway disease, including adults with mild asthma, severe asthma or chronic obstructive pulmonary disease (COPD) from the BIOAIR study (n = 171, Table 1) and a subset of 260 randomly selected children with or without asthma from the BAMSE birth cohort. DNA methylation levels for twelve CpG sites in the promoter region of NPSR1, approximately 600 base pairs 5′ of the transcription start site (Figure 1) were analysed using EpiTYPER [26]. There was a difference in both age and gender distribution for the adults in the BIOAIR study. Patients with COPD were older and consisted of more males compared to both mild and severe asthmatics (Table 1). The age and gender distributions among healthy and asthmatic children were similar between those 260 randomly chosen from the BAMSE birth cohort (healthy children 8.3±0.4 years and 52% boys, asthmatic children 8.4±0.4 years and 61% boys). The difference between minimum and maximum measured levels in DNA methylation for all the CpG sites in the promoter region (Δmax-min) varied from lowest 15% to highest 47% in the total adult sample set and the total childhood sample set varied from lowest 10% to highest 52%. Most CpG sites in the promoter showed very high levels of average methylation (>75%) with the exception of CpG site 8 showing lower methylation levels (<60%). There were indications of difference in DNA methylation based on adult asthma and other obstructive airway disease for CpG site 2 (homozygous C allele carriers only) and CpG site 5 in the promoter region (Table S1). None of the CpG sites analysed for the smaller selection of BAMSE samples (n = 260) were differentially methylated between the groups but they showed overall similar degree of methylation as compared to the adult BIOAIR samples (Table S1).
Next, we investigated whether SNPs located in the predicted NPSR1 promoter region were associated with asthma and other obstructive airway disease in the 171 adult samples from BIOAIR and the 260 BAMSE children. Four SNPs were genotyped, rs2168891, rs2168890, rs2530547 and rs887020, which coincide with CpG sites 1, 2, 8 and 9, respectively (Figure 1). The genotype distribution for the four SNPs were similar between the groups (mild asthma vs. severe asthma vs. COPD or healthy vs. asthmatic children) showing no significant association with asthma in neither the BIOAIR study nor the BAMSE randomised children (data not shown). Importantly, we found that the level of DNA methylation was affected by genotypes in the predicted NPSR1 promoter region. For example, at CpG site 2 (rs2168890) and CpG site 8 (rs2530547), a step-wise decrease in DNA methylation was observed depending the CC, CT or TT genotype, respectively. However, there were no effects on DNA methylation neither for the rs2168891 at CpG site 1 nor for the rs887020 at CpG site 9. Only homozygous carriers of the allele creating a CpG site were considered for further analysis both in the BIOAIR and the BAMSE analysis.
NPSR1 promoter methylation status, asthma and other obstructive airway disease, lifestyle and exposure in adults
The BIOAIR study is an extensive collection of data including a one year follow up of the patients [22] and was therefore sought to be investigated in more detail with the respect to DNA methylation, asthma and environmental exposures. The effect of phenotypes related to asthma and COPD and lifestyle factors (including BMI and smoking) on DNA methylation of the predicted NPSR1 promoter region (CpG site 1–12), are shown in Table 2. For analyses regarding the CpG site 1, 2, 8 and 9, only homozygous individuals for the allele creating the CpG site were included due to confounding effects of the SNP on the methylation analysis (n = 147, 116, 62 and 40, respectively). An adjusted linear regression model was used to identify associations between DNA methylation and asthma and other obstructive airway disease. There was significantly decreased methylation levels for CpG site 5 in severe asthma patients compared to mild asthma patients when adjusting for age, gender and country of origin (p = 0.0001, Table 2). Current smoking showed largest effects on DNA methylation levels. CpG site 1 showed significantly increased levels of methylation in relation to current smoking (p = 0.005) but not to former smoking (p = 0.13). For CpG site 8, the effect of current smoking resulted in 8% change in DNA methylation (p = 0.042) while former smoking resulted in a 6% change (p = 0.013, Table 2). Small but significant changes in the level of DNA methylation were also linked to BMI for CpG site 2, 3 and 5 and to age for CpG site 4 (p = 0.045, Table 2). Given the known relationship between asthma and obesity/BMI, we also adjusted the analyses between methylation status at CpG site 5 and severe asthma for BMI and found the results unchanged (p = 0.002). However, the association between CpG site 5 and BMI became non-significant (p = 0.12) after adjustment for asthma status, which indicates that this effect at CpG site 5 is mediated by asthma status rather than BMI per se. Results were unchanged between BMI and CpG sites 2 and 3 after adjustment for affection status. There were no relationships found between lung function tests (FEV1 and FVC) or blood eosinophil counts and DNA methylation.
NPSR1 promoter methylation status and asthma in children
Based on the results from EMSA as well as the suggestive associations between asthma and life style factors, and DNA methylation in the 5′ end of the predicted NPSR1 promoter for the BIOAIR study, we analysed CpG sites 1 to 5 more in depth in a larger number of children from the BAMSE study (n = 546). The children were of the same age but gender distribution differed among healthy and asthmatic children and asthmatic children were more frequently exposed to parental smoking in infancy (Table 3). To answer the question if methylation levels were associated with childhood asthma, adjusted linear regression was performed between methylation status and different asthma outcomes. For analyses regarding the CpG site 1 and 2, only homozygous individuals for the allele creating the CpG site were included due to confounding effects of the SNP on the methylation analysis (n = 470 and 397, respectively). Four models were considered to test for differences in methylation status between healthy controls and cases diagnosed with: 1) asthma ever up to 8 years, 2) allergic asthma up to 8 years, 3) non-allergic asthma up to 8 years, and 4) current asthma at 8 years. The results showed a small, but significantly lower NPSR1 promoter methylation level in children diagnosed with asthma ever up to age 8 as compared to healthy children at CpG site 4 (p = 0.04, Table 4). Lower DNA methylation levels of the promoter were also seen in children with allergic asthma at CpG site 1 (p = 0.01) and at CpG site 4 (p = 0.04) as compared to healthy children, whereas no differences were seen between non-allergic asthmatics and healthy children. The level of significance remained after adjustment for gender, age at blood sampling and plate used for sodium bisulfite treatment. The results showed no significant difference in NPSR1 promoter methylation levels between children with current asthma as compared to healthy children. CpG site 5 was not associated with any asthma outcome in children.
NPSR1 promoter methylation status, lifestyle and environmental factors in children
Extended analyses of the 5′end of the predicted NPSR1 promoter was also performed to test if the methylation status was affected by lifestyle and environmental factors in children up to 8 years of age (Table 5). Parental smoking in infancy was associated with a small, but significantly lower level of DNA methylation in CpG site 1 (p = 0.02, Table 5). However, there was no significant association to current parental smoking. High current dietary folate intake was associated with decreases in DNA methylation levels at CpG site 1 (p = 0.01) and CpG site 4 (p = 0.04). No association was observed in NPSR1 promoter methylation in relation to exposure to traffic-NOx during infancy or currently, or to current BMI (Table 5). Interestingly, a seasonal effect was found on the DNA methylation status of the predicted NPSR1 promoter region. A significant increase in DNA methylation of CpG site 5 was observed for samples obtained during July to September (p = 0.01), and October to December (p = 0.02), as compared to January to March, which was used as reference category. After additional adjustment for birth month, the significance remained at CpG site 5 July to September (p = 0.003). Further analyses showed that sensitization to any inhalant allergen (positive Phadiatop) was associated with decreased methylation in CpG site 1 (p = 0.01), similar to the results for allergic asthma (Table 4). Specifically, sensitization to birch and dog were associated with methylation changes (p = 0.05 and 0.04, respectively).
This study presents an extensive analysis of DNA methylation in the promoter of NPSR1 in relation to asthma, lifestyle and exposures in both adults and children. We found a functional relevance of DNA methylation at certain CpG sites in the promoter based on differential binding of nuclear proteins, an effect also shown for genetic variants. Furthermore, there were small but significant differences in DNA methylation in relation to adult severe asthma (but not COPD) as well as to allergic asthma in children. Furthermore, we observed associations between DNA methylation changes in the promoter region and current smoking in adults and passive smoking in children. The previous genetic associations seen to IgE levels and allergy in relation to asthma for NPSR1 polymorphisms [10], [14], [20], [27], in combination with our results in this study, highlight the importance this gene might have in the corresponding biology.
Many of the genetic associations concerning NPSR1 and asthma are related to allergic asthma [10], [14], [20], [27]. Up-regulation of Npsr1 expression in the lung has been observed in one mouse model of allergen-induced airway inflammation [10] but not in other models [28], [29]. Low levels of methylation are generally associated with higher levels of gene expression [2]. We found that DNA methylation in the predicted NPSR1 promoter was slightly lower in peripheral blood from children that have allergic asthma as compared to healthy children. Although it remains to compare methylation patterns in blood and lung tissue specimens from subjects with asthma, recent data suggest that the pattern in blood inflammatory cells reflect those in the lung tissue [30]. It should be noted that parental report of doctor's diagnosis of asthma was used as a basis for all asthma phenotypes in BAMSE, and that the cohort data did not allow for analyses of e.g. severe asthma in children as evaluated by a paediatrician. Further studies on other asthma phenotypes such as severe asthma are warranted.
We identified methylation sites in the promoter for NPSR1 that showed differential methylation in relation to exposures of smoking, either current smoking in adults or passive smoking in children. Several studies have shown effects of smoking on DNA methylation, both on candidate gene level and globally [31]–[34]. Interestingly, a previous report from the BAMSE birth cohort found a dose-response relation between exposure to parental smoking in infancy and IgE sensitisation [35], supporting a link between variations in NPSR1 methylation and allergic asthma.
To further support an effect between DNA methylation in NPSR1 and allergic asthma, we saw an increase in DNA methylation related to blood sampling during the months July to December compared to January to March in both asthmatic and healthy children. Seasonal variation has been shown to influence DNA methylation at certain genomic regions when considering prenatal nutritional variation during periods of famine [36], [37]. Our data may indicate an environmental effect of, for example, allergens such as airborne pollen exposure during spring and/or a greater exposure to certain viruses and other infections during winter time, both relevant factors for the exacerbation of asthma. Further studies are however needed to clarify the role of seasonal effects on methylation patterns.
A methyl-rich diet can affect methylation status and enhance allergic airway disease in mice offspring [38], and periconceptional maternal folic acid use is associated with increased methylation in the growth promoting gene IGF2 [39]. Here we report a decreased methylation level in the predicted NPSR1 promoter in association with high dietary folate intake in children. The conflicting results may partly be explained by the fact that we consider a daily intake of folate from the diet whereas others have looked at intake from supplements. Further, compared to others we have investigated the child's current intake which might not affect methylation to the same degree as for example intrauterine exposure. Nevertheless, our results should be interpreted with caution until further replicated.
Traffic related air pollution exposure has been linked to incident airway disease in a previous study in the BAMSE birth cohort [40] and this effect appeard to be modified by genetic polymorphisms [41]. Traffic related air pollution has also been reported to affect DNA methylation [42], [43]. We found no effect between NOx from traffic and methylation in the promoter of NPSR1. The NOx levels in Stockholm are comparatively low in an international comparison, which may contribute to the absence of association.
In this study, we have focused on asthma and used DNA derived from peripheral blood as target for analysis. Importantly, there are several tissues expressing NPSR1 mRNA and protein such as immune cells, gut, skin and lung epithelium as well as the brain [9], [10], [44]–[47]. Furthermore, NPSR1 has not only been associated to asthma and related phenotypes but also to other diseases and target tissues. While the expression of NPSR1 is rather low in immune cells, in accordance with our finding of high levels of DNA methylation, other tissues might show larger variations in both expression and DNA methylation and therefore further, more detailed studies in various tissues are required in the future. However, even though peripheral blood is a mixture of many cell types in different developmental stages we could here show that the cell specific patterns for DNA methylation regarding NPSR1 in blood show little variation indicating that using whole blood could still be relevant. Also, although our data suggest that the region we have studied is a functional promoter [24], other alternative promoters may exist that could show cell specificity.
Age is a factor that long has been associated with changes in DNA methylation [48], [49]. It has been shown that genome-wide DNA methylation generally decreases with age [48], [50] but certain sites also show increased methylation [48]. Furthermore, the rate of methylation changes in blood may be higher in children compared to adult populations [48]. In our study we saw some effects by age on the levels of DNA methylation for certain CpG sites, specifically for CpG site 3. Here, younger study participants showed lower levels of methylation when comparing all adults to all children irrespective of disease (Figure S1).
In summary, DNA methylation levels in the promoter of NPSR1 showed significant associations with asthma and related traits such as allergy, as well as with certain exposures, when measured in DNA from whole blood, although absolute differences were generally small. Importantly, EMSA performed in the same region suggest that both genetic variation and the methylated state of the CpG sites have an effect on the binding of transcription factors in the regulatory region of NPSR1. These results, however, need to be further evaluated in other target tissues and/or specific cell types in an independent sample material.
Materials and Methods
Ethical statement
This study was performed in accordance with the principles expressed in the Declaration of Helsinki. The study was approved by the local ethics committee at Karolinska Institutet and the BIOAIR study (clinicaltrials.gov.NCT00555607; EU Contract QLG1-CT-2000-01185) was approved by the ethics review boards and drug regulatory authorities in the twelve participating centers (Stockholm, Athens, Heraklion, Gent, Montpellier, London, Southampton, Krakow, Palermo, Ferrara, Hamburg and Leiden). Written informed consent for genetic testing was obtained either by the adult study participant or from the parents of the participating children.
Study design
This study was conducted in four steps: 1) a functional study to evaluate the relevance of DNA methylation in the predicted NPSR1 promoter, 2) testing of DNA methylation at CpG sites in the promoter region of NPSR1 in a smaller set of human DNA samples (both adults and children) obtained from peripheral blood, and 3) general analysis of all CpG sites in the promoter in relation to respiratory phenotypes, life style factors and exposures in the BIOAIR study [22], 4) in depth analysis of CpG sites shown in previous steps to be of interest for further analysis in a larger data set from the BAMSE birth cohort [23] which would allow for studying the influence of environmental exposures.
The aim of BIOAIR (the BIOmarkers of severe chronic AIRways disease) study is to clarify the natural course of severe asthma, elucidate mechanisms, risk factors, markers of severity, exacerbations and poor outcome [22]. Adult patients with mild controlled asthma, severe uncontrolled asthma and chronic obstructive pulmonary disease (COPD) were followed for one year. Data were collected at baseline and at several visits during one year of follow up and included: spirometry (FIV1, FEV1, FVC, FEV1/FVC), clinical characteristics, medical history, induced sputum, urine, medication use and questionnaires (respiratory quality of life and asthma control). Blood samples from 56 mild asthmatics, 67 severe asthmatics and 48 COPD patients were collected at baseline and DNA was extracted for investigation of DNA methylation status and genetic variation in NPSR1 (Table 1).
The BAMSE (Swedish abbreviation for Children Allergy Environment Stockholm Epidemiology) study is a Swedish prospective birth cohort [23] with 4,089 infants recruited between 1994 and 1996. Data related to environmental exposures and symptoms of asthma and allergic diseases in the children have been obtained by questionnaires sent to the parents at different occasions. Peripheral blood samples drawn at 8 years of age were used for analysing serum IgE antibodies to inhalant allergens using Phadiatop (Phadia AB, Uppsala, Sweden) as well as for the current study on DNA methylation.
All children in BAMSE with a doctor's diagnosis of asthma (ever) up to 8 years of age as reported by the parents, with DNA available for analyses were selected as cases (n = 273) and children with no history of asthma or other allergic diseases with DNA available for analyses were selected as controls (n = 273) [51]. Cases and controls were obtained after exclusion of individuals with non-successful DNA methylation analyses (n = 19) resulting in 273 cases and 273 controls (Table 3). The pilot study testing the assays in human peripheral blood derived DNA was based on a random selection of all the 273 children in BAMSE with a doctor's diagnosis of asthma (ever) up to 8 years of age (n = 136) and a subsequent random selection of the 273 healthy children (n = 124). For the in depth investigation of DNA methylation status, a selection of CpG sites (number 1 to 5) were studied in relation to asthma phenotypes and the effect of exposure to environmental factors.
Four asthma phenotypes were investigated in relation to DNA methylation status; 1) asthma up to 8 years of age (doctor's diagnosed reported by the parents), 2) allergic asthma (defined as doctor's diagnosis of asthma up to 8 years in combination with an IgE-value for Phadiatop ≥0.35 kU/l; a mixture of cat, dog, horse, birch, timothy, mugwort, Dermatophagoides pteronyssinus, and Cladosporium allergens), 3) non-allergic asthma (defined as doctor's diagnosis of asthma up to 8 years in combination with an IgE-value for Phadiatop ≤0.35 kU/l), and 4) current asthma at 8 years (defined as doctor's diagnosis of asthma up to 8 years in combination with at least one episode of wheeze in the last 12 months). In addition, association between sensitization (IgE-value for Phadiatop ≥0.35 kU/l) and methylation levels were also analyzed.
Exposure to environmental tobacco smoke was classified as parental tobacco smoke exposure in infancy (daily parental smoking at birth of the child, yes/no) and current parental tobacco smoke exposure (daily parental tobacco smoke exposure at child age 8 years, yes/no). Information on air pollution exposure from road traffic (NOx) at residential, day care, and school addresses was estimated using dispersion modelling, and has been described elsewhere [52]. The mean level of traffic-NOx to which the child was exposed during infancy was 19.5 µg/m3 (median: 15.0 µg/m3 and 5th–95th percentile: 3.2-48.9 µg/m3) and current mean exposure level at 8 years of age was 9.6 µg/m3 (median: 7.3 µg/m3 and 5th–95th percentile: 1.7–27.7 µg/m3) in the study population (cases and controls combined). Associations were investigated for the 5th–95th percentile difference in NOx exposure levels corresponding to 45.7 µg/m3 during first year of life (here defined as NOx from traffic during infancy), and 26.0 µg/m3 between 7 and 8 years of age (here defined as current NOx from traffic). Current dietary folate intake (energy adjusted intake at 8 years of age, in µg per day) was divided into quartiles (Q1 = 142.6 to <192.7 µg per day, Q2 = 192.7 to <212.6, Q3 = 212.6 to <235.3 µg per day, Q4 = 235.3 to 401.6 µg per day) before investigation. Current BMI (Body mass index at 8 years of age) was measured in kg/m2. Sampling season is the time of the year when blood samples were collected at 8 years of age, and were investigated in three-month seasons using January to March as reference category.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts from Colo205 (colon adenocarcinoma, ATCC CCL-222) and HEK293 (human embryonic kidney cells, ATCC CRL-1573) cells were prepared according to standard protocols [53]. Duplex probes encompassing the four sites CpG site 2 coinciding with rs2168890, CpG site 3, CpG site 8 coinciding with rs2530547, and CpG site 9 coinciding with rs887020 in the predicted NPSR1 promoter (Figure 1) were produced by annealing oligonucleotide pairs specific for each allele at the polymorphic site (2.5 µmol/l, probe sequences available upon request). The four sites were chosen according to the central location of the CpG site in relation to binding of the transcription factor and the value of the transcription factor itself in relation to NPSR1 and/or asthma. Binding reactions were performed in 25 mM HEPES pH 7.9, 150 mM KCl, 10% glycerol and 5 mM dithiothreitol, with 3 µg nuclear extract, approximately 0.5 ng [α-32P] Klenow-labeled probe and 3 µg poly dI-dC in a total volume of 20 µL. Polyacrylamide (6%) gels were run in 0.5× tris-borate-EDTA at 4°C for 3 h at 300 V, dried, and autoradiographed to visualize DNA protein complexes. Experiments were repeated three times for each nuclear cell extract (Colo205 and HEK293). Both cell extracts gave similar results and data shown is a representative gel from the Colo205 analyses.
Isolated cell types from blood
Peripheral blood, 450 ml, was collected from six healthy adult male blood donors, age 38±13.6 years as described elsewhere [25]. Briefly, whole blood, peripheral blood mononuclear cells (PBMC), CD4+ T cells, CD8+ T-cells, CD56+ NK cells, CD19+ B cells, CD14+ monocytes, granulocytes, neutrophils and eosinophils were isolated. PBMC and granulocytes were isolated using Ficoll-Paque Plus™ (GE Healthcare, Sweden). Magnetic-activated cell sorting (MACS, Miltenyi Biotech, Germany) was used to obtain T cells, B cells, monocytes, and NK cells from the PBMC:s and neutrophils and eosinophils from the granulocyte containing pellet. Cell purities were controlled by fluorescence activated cell sorting (FACS) and ranged from lowest 81.8±18.8% for eosinophils to highest 97.8±1.7% for neutrophils. DNA was extracted from cells using QIAmp DNA Micro Kit (QIAGEN, Germany). 500 ng DNA was bisulfite treated using EZ-96 DNA Methylation Kit (Zymo Research Corporation, USA) according to the manufacturer's instructions, and analyzed on the Illumina Infinium 450K Bead array.
SNP genotyping
Primers for multiplex PCR and extension reactions were designed by the SpectroDesigner software (Sequenom GmbH, San Diego, CA, USA). All primer sequences are available on request. PCR and extension reactions were performed according to the manufacturer's standard protocols. The SNP analysis in the BAMSE samples was performed by MALDI-TOF mass spectrometry (matrix-assisted laser desorption/ionisation-time of flight; Sequenom GmbH). Each assay was validated using a set of 14 trios families, in total 42 individuals. Genotype data from these individuals are available through the HapMap consortium (http://hapmap.ncbi.nlm.nih.gov/). Concordance analyses with the HapMap data as well as analysis of the parent-offspring-compatibility with the produced genotypes were performed. No significant deviation from Hardy-Weinberg equilibrium (P>0.05 using χ2 test) was seen for any of the SNPs and the average genotyping success rate was >95%. BIOAIR samples were genotyped using TaqMan allelic discrimination on the ABI Prism 7500 detection system according to the manufacturer's protocol (Assays used: C__16245540_10, C__16245541_10, C___2959938_10, and C__2959937_10, Applied Biosystems, CA, USA). Reruns of a small set of samples for all four genotypes showed 100% success rate.
DNA methylation
The promoter region [24] used for primer design contained 12 CpG sites and four of these were affected by polymorphisms (rs2168891 affecting CpG site 1, rs2168890 affecting CpG site 2, rs2530547 affecting CpG site 8 and rs887020 affecting CpG site 9, Figure 1). As a quality check for the methylation pattern in the promoter of NPSR1, a second region with a predicted CTCF binding site which is of interest for DNA methylation was chosen for analysis in the intron 4 region. This region constituted 1053 bp (NCBI36, chr7:34829940–34830992) and contained 11 CpG sites (Figure S2). Two sites, CpG site 2 and 5, were affected by polymorphism (rs17199818 and rs17199853, respectively). Primers for the regions were designed using EpiDesigner (www.epidesigner.com, Sequenom, CA, USA, Table S2). One µg of DNA obtained from peripheral blood cells was modified by sodium bisulfite using the EZ DNA Methylation™ Kit (Zymo Research, CA, USA). Sodium bisulfite DNA was analysed using EpiTYPER according to manufacturer's recommendations & protocol [26]. All EpiTYPER designs were quality tested by analysing standard curves with methylated and unmethylated DNA mixed in ratios resulting in 0%, 25%, 50%, 75%, and 100% methylated DNA. This information resulted in the exclusion of CpG site 7 in the promoter region and CpG site 5 in the CTCF binding region from further analyses due to poor estimation of DNA methylation (Figure S3). All samples were distributed randomly on all plates based on disease phenotype, samples were analysed in duplicates and samples with a standard deviation above 0.3 were removed from further analysis. Two CpG sites (CpG site 5 and 8 in the promoter) were included in two separate designs and showed similar methylation levels for both assays (Table S1). A quick analysis of the intron 4 region revealed a similar pattern of DNA methylation compared to the promoter region. No differences in DNA methylation based on respiratory phenotype were found and therefore, this region was not studied further.
Statistical analyses
None of the continuous variables deviated from normal distribution including DNA methylation levels (Figure S4). For basic comparisons of clinical characteristics, DNA methylation in relation to asthma and other obstructive airway disease and genotype effects on DNA methylation, Analysis of variance (ANOVA) when considering three groups or Student's T-test when considering two groups were used. Distribution regarding genotype and gender was compared using chi square test.
For the investigation of promoter methylation status and asthma and other obstructive airway disease in the larger selection of samples from the BIOAIR study (n = 171) and the BAMSE cohort (n = 546), crude and adjusted linear regression model was used (methylation levels as a continuous outcome). In the BIOAIR analyses, the linear regression models were adjusted for the potential confounders gender, age and country of origin for all variables analysed. Linear regression analyses performed for the BAMSE data were adjusted for the potential confounders gender, age at blood sampling and plate used for sodium-bisulfite treatment. For the investigation of DNA methylation status and the effect of exposure to environmental factors linear regression models were used adjusting for gender, age at blood sampling and plate used for sodium-bisulfite treatment. For analyses of air pollution effects, adjustment for municipality was also done. In addition, adjustment for all the other exposure variables analyzed in Table 5 did not change the results; neither did adjustment for doctor's diagnosis of asthma (ever) up to 8 years of age. STATA (StataCorp LP, TX, USA) was used for all statistical analyses.
Supporting Information
Figure S1.
The levels of DNA methylation (%) for each CpG site studied in relation to age in years (dot blots, left column of graphs) or material (box plots, right column of graphs). Dots with green color in the box plot defines mild outliers (values that are under three interquartile range (IQR) from the 25th and 75th percentiles) and red dots defines severe outliers (values that are outside of three IQR:s from the 25th and 75th percentiles). BAMSE - Swedish abbreviation for Children Allergy Environment Stockholm Epidemiology, BIOAIR - the BIOmarkers of severe chronic AIRways disease.
Figure S2.
The genetic sequence for the regions defined to bind CTCF in intron 4 of Neuropeptide S Receptor 1 (NPSR1) gene. Yellow regions indicate the CpG sites and red nucleotides indicate genetic variants.
Figure S3.
Standard curves obtained from the EpiTYPER analysis of bisulfite treated samples. We designed three assays in order to cover the Neuropeptide S Receptor 1 (NPSR1) gene promoter region. CpG site 5 and CpG site 8 are covered by two designs. CpG site 7 failed the validation and was excluded from analysis.
Figure S4.
The CpG sites analyzed showed normal distribution. Data is displayed via histogram plots in the left column of graphs and via probability plots in the right column of graphs.
Figure S5.
DNA methylation levels (%) in the NPSR1 promoter region in blood cells from six healthy adult male blood donors (age 38±13.6 years). DNA methylation was analyzed using EpiTYPER and the designs provided (Table S2).
Table S1.
DNA methylation (%) in the Neuropeptide S Receptor 1 (NPSR1) gene analyzed using EpiTYPER in two two well characterized cohorts that offer unique opportunities to investigate genetics and epigenetics in relation to respiratory disease phenotypes and environmental exposures; the BIOAIR study of severe asthma in adults and the Swedish birth cohort BAMSE.
Table S2.
EpiTYPER primer sequences for the Neuropeptide S receptor 1 (NPSR1) gene.
We thank Francesca Anedda for kindly helping out with the EMSA, the Mutation Analysis Core Facility (MAF) at Karolinska Institutet for the EpiTYPER analyses of DNA methylation and genotyping, and the Bioinformatic and Expression Analysis (BEA) Core facility for the laboratory work on Illumina methylation arrays.
Additional contributors to the BIOAIR study (Longitudinal Assessment of Clinical Course and BIOmarkers in Severe Chronic AIRway Disease): Barbro Dahlén1, Elisabeth H. D. Bel2, Pascal Chanez3, Leo M. Fabbri4, Mina Gaga5, Mark Gjomarkaj6, Pieter S. Hiemstra7, Peter H. Howarth8, Sebastian L. Johnston9, Guy F. Joos10, Frank Kanniess11, Ewa Nizankowska-Mogilnicka12, Alberto Papi13, Eleni Tzortzaki14, Isabell Vachier15. Affiliations: 1Karolinska Institutet, Stockholm, Sweden, 2University of Amsterdam, The Netherlands, 3University of Marseille, France, 4University of Modena, Italy, 5University of Athens, Greece, 6Italian National Research Council, Palermo, Italy, 7University of Leiden, The Netherlands, 8University of Southampton, UK, 9Imperial College, London, UK, 10Ghent University Hospital, Ghent, Belgium, 11 Pulmonary Research Institute, Grosshansdorf, Germany, 12 The Jagiellonian University, Krakow, Poland, 13University of Ferrara, Italy, 14University of Crete, Greece, 15University of Montpellier, France.
Author Contributions
Conceived and designed the experiments: LER EM GP SED CS JK. Performed the experiments: LER NA MJ. Analyzed the data: LER AG AS EM CS. Contributed reagents/materials/analysis tools: MK MD AB AS SED GP CS. Wrote the paper: LER AG.
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text:
Axel Trucks
Axel loves trucks of all kinds. Construction trucks, monster trucks, race cars, emergency vehicles, off-road vehicles, and more! Come join Axel and his Dad as they learn to use their imagination by playing hard core with all kinds of toy vehicles. Subscribe to the channel, and don't miss a single epic kids truck adventure! Our goal is to encourage kids to use their imagination while playing with their trucks... and play hard! www.axelbooks.com for a construction book made by Axel and his Parents.

subset: commoncrawl


text:
A trio of strangers spent a single weekend this October innovating and stepping outside of their comfort zones. Now, entrepreneurs Grace Pesch, Jay Franson, and James Perreault are seeking ways to further develop their board game to teach empathy and understanding.
Their product, called 'What Were You Thinking?', breaks down the nine Enneagram personality types to better inform players' professional and personal relationships. The Enneagram is, an arguably, complicated model of the human psyche conveying basic fears, desires, and motivations.
At the surface, the 'What Were You Thinking?' game is simple. It's composed of character cards and scenario cards. Each character card describes a person with one of the nine Enneagram personality types. The scenario cards describe a real-life situation, the funnier the better, plus reactions to that scenario. Each participant plays a character card they think best describes the reaction to the scenario. Whoever convinces the judge that their character card matches the scenario behavior the best, wins.
On one level it's just a matching game. But on a deeper note, the game involves rationalizing why your character, with the indicated personality type and tendencies, would react to a situation in a certain manner.
"It causes the person that drew the card to really think outside of themselves and relate more to the card in hand, to the persona that's on that card," Franson explained. "The goal is to bring more awareness to the Enneagram as well as give language to different people's personality types."
The team believes this card game could be a unique way to teach empathy, improve relationships, and enhance team building.
Pesch, Franson, and Perreault developed a prototype for the game during Techstars' Startup Weekend Rochester. This fifty-four-hour event, held over Halloween weekend, helped participants explore their entrepreneurial tendencies, ideate, perform customer validation, and develop simple prototypes all in a single weekend.
"I like to do things I've never done before. I like to try and expand my horizons and push myself," Pesch explained. "I knew I was kind of scared and uncomfortable with the idea of doing Startup Weekend and I said, well I have to do it!"
While Franson entered the weekend with the original idea for the game, Pesch and Perreault quickly joined on to further develop and test the concept.
By the end of Startup Weekend, the team developed a prototype with a minimal set of cards, built a website, and had pre-orders of the product. They also identified a potential game creation company to construct the product, set a price point, and researched drop shipping for product distribution.
All complete strangers at the beginning of the weekend, the team believes their flexibility, open mindedness, and unique skill sets helped them to succeed.
"I think that personality is more important than actual knowledge. You don't need an expert as much as you need someone that you can communicate with," Franson said.
This was the first time that both Franson and Pesch ever participated in a Startup Weekend. Pesch, a person who prefers pacing herself and working well in advance on large projects, appreciated being forced to change her workstyle to deliver in such a limited time span.
"I think I was most inspired by the lack of instruction [at Startup Weekend] because that's real life. Really for the most part you go and figure it out. You can find a mentor or take a class, but it's still up to you," Franson said. "I thought that was immensely valuable because you learned so much more by doing it yourself."
Now, the team is looking for help from the community to write more cards to complete their game. You can learn more and follow their progress by visiting their website https://wwyt.squarespace.com


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While the speakers at an annual Alaska Native storytelling event on Saturday stuck with traditional forms of oration, the added category of "new media" offered glimpses of change in their cultural expression.
"I think every village, city and town has a few elders … If you don't find too many elders, learn what you can, listen deeply and become the elder that your community needs," said keynote speaker Ishmael Angaluuk Hope.
Hope, whose heritage includes Tlingit and Inupiaq, is known as a Native writer, poet and playwright. More recently, he worked on the video game "Never Alone" as a writer and helped ensure the accuracy of its source material.
The theme of Saturday's event was "Let Your Spirit Speak: Finding Our Voices." The University of Alaska Anchorage's Alaska Native studies director, Maria Shaa Tlaa Williams, said the theme focused on allowing the state's indigenous peoples to discuss their identities, or "spirits."
The event featured orators from UAA, Kenai Peninsula College and the Lower Kuskokwim School District. The participants offered humorous stories about fishing with elders and lessons from elders told in Yup'ik.
Williams said she was particularly excited about the additional category because technology offers young people a new way to express their ideas and share their culture.
Williams said nearly 40 percent of the Alaska Native population is younger than 19.
"This is happening right now," she said. "My view is that indigenous people are very good with tools, and that's why we've survived for so long. And technology is just a new tool. It's not culture-specific. You make it culture-specific."
UAA freshman Ivana Ash had planned to present her work on a Sugstun language application but was traveling to Kodiak on Saturday. The Alutiiq people of southern coastal Alaska speak Sugstun.
"The app gives contextual information about the language, so it goes a little deeper than a dictionary," Williams said.
And discussion has moved online as UAA graduate student Maria Crouch is leading a research study of attitudes about Natives. Crouch's handout about the study included a smartphone QR code to direct participants to the 30-minute questionnaire.
The survey will increase awareness of Native experiences, Crouch said.
Student Crystalynn Lemiux was the first speaker to share her experience with the small crowd gathered in a lecture hall at the university's Fine Arts Building. Her speech was titled "When I became aware."
Hailing from the southeastern village of Angoon, Lemiux now identifies herself as a proud member of the Deisheetaan clan. That wasn't always the case, she said.
"I didn't feel comfortable with being Native," she said.
When Lemiux arrived at UAA, she viewed the Alaska Native courses through a "Western lens," she said. She luckily had less shy students encourage her to visit the university's Alaska Native Language Center.
As the encouragement continued, she said she realized the importance of asking difficult questions about her culture. She thanked her mentors and fellow students.
"I wouldn't be here," Lemiux said. "I wouldn't finally be fine with being Native."


text:
After years of customer requests, GT Imports opened an affiliated business, GT Import Auto Sales in 2006. We specialize in quality pre-owned Hondas and Acuras. Every one of the cars on our lot has been subjected to our rigorous 120 point inspections to make sure that it meets our tough standards. And we offer a 3 year / 36,000 mile warranty and $500 voucher for first year service. Also, all of the prep work we have done to bring the vehicle we sell to the GT Certified Standard is under a 2 year/24,000 mile warranty (whichever interval comes last)!
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ENGINE SPECIFICATIONS Engine 3.5L Gas V6 Horsepower 255 @ 5750 RPM Torque 250 @ 4500 RPM DRIVETRAIN Drivetrain Front Wheel Drive Transmission 5 speed Automatic MPG (MILES PER GALLON) Fuel Tank Capacity, Approx 21.0 gal Engine 3.5L Gas V6 Transmission 5 speed Automatic EPA Fuel Economy Est (MPG) City: 20 / Hwy: 28 SAFETY Air Bag-Frontal-Driver Air Bag-Frontal-Passenger Air Bag-Side Body-Front Air Bag-Side Head-Front Air Bag-Side Head-Rear Brakes-ABS Brakes-Type-4 Wheel DISC Child Safety Rear Door Locks Engine Immobilizer Headlights-Automatic Security System Traction Control COMFORT & CONVENIENCE Air Conditioning-Auto Climate Control Air Conditioning-Front Air Conditioning-Multi-Zone Air Conditioning-Rear Cruise Control Keyless Entry Max Seating Capacity: 8 Mirror(s)-Power Mirrors-Vanity-Driver Mirrors-Vanity-Driver Illumination Mirrors-Vanity-Passenger Mirrors-Vanity-Passenger Illumination Power Locks Reading Lamps-Front Reading Lamps-Rear Seat Trim-Leather Seat(s)-Heated Front Seat-Adjustable Lumbar-Driver Seat-Power Driver Seat-Rear Pass-Through Seats-Front Bucket Seats-Rear Bucket Steering Wheel-Adjustable Steering Wheel-Audio Controls Steering-Power Universal Garage Door Opener Windows-Power MUSIC & ENTERTAINMENT Audio-AM/FM Stereo Audio-CD Changer Audio-CD Player INTERIOR Floor Mats Power Outlet Seat-3rd Row EXTERIOR Doors: 4 Door-Sliding-Rear Driver Side Door-Sliding-Rear Passenger Side Mirror(s)-Heated Rear Spoiler Rear Window Defogger Roof-Generic-Sun/Moon Roof-Sun/Moon Windows-Deep Tinted Wipers-Intermittent Wipers-Variable Speed Intermittent TIRES Front Tire Size: P235/65R16 Rear Tire Size: P235/65R16 Spare Tire Size: Compact WHEELS Front Wheel Size (in): 16 x 7 Rear Wheel Size (in): 16 x 7 Front Wheel Material: Alloy Rear Wheel Material: Alloy WEIGHT INFORMATION Base Curb Weight 4,537 lbs INTERIOR SPECIFICATIONS Passenger Capacity 8 Front Head Room (in) 39.2 Front Leg Room (in) 40.8 Front Shoulder Room (in) 63.5 Front Hip Room (in) 57.0 Second Head Room (in) 39.6 Second Leg Room ... [read more] (in) 40.0 Second Shoulder Room (in) 63.1 Second Hip Room (in) 64.4 Third Head Room (in) 38.4 Third Leg Room (in) 41.1 Third Shoulder Room (in) 61.2 Third Hip Room (in) 48.5 EXTERIOR SPECIFICATIONS Turning Diameter - Curb to Curb (ft) 36.7 Turning Diameter - Wall to Wall (ft) - TBD - Wheelbase (in) 118.1 Length, Overall (in) 201.0 Width, Max w/o mirrors (in) 77.1 Height, Overall (in) 70.0 Track Width, Front (in) 66.7 Track Width, Rear (in) 66.8 Min Ground Clearance (in) - TBD -


text:
This is a new project for one of our customers -radio shuttle racking system.
Knowing customer's requests, our technician designed a drawing for him.
After our communication for about one month,we won customer's order finally.
Usually, we need to know below information to make the drawing:
1. Warehouse house size or warehouse racking system size,including the length/width/height(mm).
2. Load capacity of each layer of the warehouse racking system.
3. Pallet size and pallet load capacity.
4. The max lifting height of the forklift.
5. Forklift driving direction-FIFO (first in first out) or FILO (first in last out).


text:
Service Desk Engineer
Job Title: Service Desk Engineer Location: Headquarters (Clayton, MO)
Job Category: 100% Technical Reports To: Vice President of Service
Department/Group: Service Level/Salary Range: Level 2
Required Skills:
· Exceptional analytic and problem solving skills
· Understanding of the scientific method
· Assertiveness
· Ability to coach and train teammates
· Experience with Linux
· Experience with iOS
· Advanced Experience with Powershell and command line
· Advanced Experience with HTML / FTP / Websites / Hosting
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· Expert with Office 365
· Expert with mail flow / Outlook / Exchange / Smart Filters
· Expert with SAN / NAS and virtual environments
· Expert ability to diagnose and fix hardware problems
· Expert with Group Policy
· Expert with Active Directory
· Expert LAN / WAN concepts and troubleshooting
· Expert Windows 7/8/10
· Expert Microsoft Office Products
· Expert Windows Server 7/8/10
· Expert Windows Windows Server 2008, 2012, 2016
· Expert Backup Systems
· Expert in user account management
· Expert in troubleshooting printers and copiers
· Expert in installing and troubleshooting Windows Operating Systems and 3rd party Applications
· Expert in Virus removal
· Understand and execute excellent customer service
· Strong belief in customer service and business ethics
· Sense of urgency and ability to change focus quickly
· Strong desire to learn new technologies and coachability
· Patience, listening, empathy and clear communication
· Assertiveness
· Ability to extract technical details
Job Duties
· Perform excellent customer service for each and every client
· Improve customer service, perception and satisfaction
· Accurately relay and retrieve customer information from and to technicians through ticketing system, instant messaging and phone
· Windows user account management
· Manage Active requests in ticketing system
· Schedule Field technicians for onsite visits
· Fast turnaround of customer requests
· Participate in oncall rotation
· Solve customer problems and communicate expected times
· Manage documentation
· Analyze customer issues for trends
· Vendor Management
· Accurately document issues in ticketing system and create clear plans for troubleshooting and remediation
· Identify customer needs and opportunities
· Coach technicians and administrators
Positional Goals
· Customer Service: Provide exceptional customer service at all times; Conducting yourself in a professional and personable manner.
· Utilization: Maintain a minimum of 75% billable time per month while continually increasing value to the clients
· Time Management: Ensure all client issues are taken care of in the scheduled time allotted and are recorded in ticketing system
· Performance: Work must be high quality and presentable to clients and staff.
· Microsoft Certification within 90 days of hire
· 5-10 years of customer support experience
· Previous technical training, school, on the job, self-study is all accepted
Send resume to: firstname.lastname@example.com


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Here's a quick rundown of all of the happenings in the CAM industry in this week's CAM Mashup!
First and foremost, it's important to talk about the elephant in the room--the persisting government shutdown. We make an effort not to delve too deeply into politics, only commenting when it becomes relevant to the CAM industry. Right now, there's definitely some relevance, because tax season is in full effect, shutdown or no shutdown, and you will still be responsible for filing on time, as the IRS has issued a statement officially acknowledging that filing WILL start on time. For more information on the IRS's statement and what impact the the government shutdown may have, see AICPA's blog post about the ordeal here.
In that same vein, End of Year is also going strong, and it's important to remember that there's more than just taxes to be done. While you're gathering everything you need for taxes, it's also a good time to take a look at these annual financial reports that your HOA and condo association should be keeping, and how TOPS [ONE] can make those reports easy and stress-free.
It's no secret that some people (misguidedly) hate their HOAs. They see them as nothing more than corrupt entities filled with power-hungry greed monsters, and sometimes they're right. Just last week I was crawling around on reddit and stumbled across a story from the perspective of a homeowner who had taken down his corrupt HOA.
But the reality is that those kinds of HOAs are not the standard. Unfortunately, that doesn't mean your homeowners aren't waiting for you to fail. The best you can do is, well, your best! Ensuring that you're not under- or oversharing sensitive information with board members, making a point to be vigilant about your (actual or perceived) conflicts of interest when dealing with community funding and activities, and even something as simple as keeping HOA meetings productive and efficient, and making sure you take to task only the problems that really matter, can go a long way toward creating and maintaining trust within your HOA or condo association.
Do You Have Big News This Week?
If you've got big news that would be relevant to other CAM professionals, please share it with us in the comments, or email email@example.com and share a link to your news with us for inclusion in the next mash-up!
About the CAM Mash-up: As an educated member of the Community Association Management industry, you already know how crucial it is to stay informed and up-to-date on the latest news, trends and happenings within the industry. Each week, we bring together our favorite reads of the week and share them with you! Enjoy the CAM Mash Up and please share it too!
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Bitcoin (+7.9%) and US 10Y Treasuries (+1.3%) SURGE while US stocks (-2.0%) TUMBLE on President Trump's abrupt escalation of a trade war.
President Donald Trump abruptly escalated his trade war with China.
According to Trump's tweet, a 10% tariff will be imposed on $300 billion in Chinese goods on Sept 1, and the tariffs can be raised beyond 25%.
The 25% tariff already imposed on $250B in Chinese goods will remain in place.
How did the financial markets react?
Trump's escalation of a trade war wasn't on anyone's radar and hit the markets by surprise.
Now we see a complete re-pricing of expectations surrounding the trade war.
It's outright negative for stocks. Investors flee into safe-haven assets, which could be seen by market price changes following the news announcement:
• The S&P 500 declined by 2.03% due to investors selling US equities to buy 10-year Treasuries and gold futures .
• The prices of the US 10-year Treasury notes surged by 1.31% to 128.8 - the highest price (the lowest yield) since 2016.
• Gold futures on CME spiked by 2.32% to $1,436.0 - the highest price since 2013.
• Bitcoin IS SURGING +7.86% on the news. The chart shows that traders are increasingly using Bitcoin to hedge against macroeconomic and geopolitical risks.
Now is a perfect time to buy Bitcoin.
What effect will tariffs have on the US economy?
Tariffs are taxes that a government levies on imported goods.
Tariffs will reduce the demand for Chinese goods in the US by increasing their prices above the free trade price.
The economic impact is going to be harmful to both countries. However, the economic impact for China is going to be worse, as compared to the US.
When the 10% tariff is imposed, the Chinese businesses are more likely to decrease prices for their goods to retain their market shares. This reduction in prices for some products will effectively lead to a redistribution of income from China to the US. The prices for the end consumers in the US might not change, while the profit margins of Chinese business will decline, and the revenue from tariffs to the US government will rise.
Is Bitcoin a true safe-haven asset?
Looking at this chart gives me an increased conviction that Bitcoin is perceived by traders as an increasingly safe-haven asset to hedge against macroeconomic and geopolitical risks.
It is different from other safe-haven assets like the US Dollar , Gold , and the U.S. Treasury notes.
Investors perceive Bitcoin as a true hedge of Black Swan events, particularly events which lead to the broad market declines. This becomes reasonable if we think about the design philosophy of Bitcoin created to function without any centralized institutions or trust.
When investors foresee a risk of a failure of major financial institutions or political institutions and policymakers, Bitcoin surges in price.
What's the upside potential for Bitcoin?
We expect the equity markets to continue declining, which will be a strong catalyst for Bitcoin price appreciation over the near-term. Bitcoin has broken its short-term significant resistance at $10,000 and the trend is gaining momentum upwards.
Technically, based on Bitcoin trend analysis and the analysis of chart patterns, Bitcoin price may reach at least $12,000 over the next week, representing a 15% upside potential.
Now is a perfect time to buy Bitcoin.
If you agree with this analysis, then Follow Us at Trading View!
This report is for information purposes only and should not be considered a solicitation to buy or sell any cryptocurrency or cryptocurrency product. Monfex accepts no responsibility for any consequences resulting from the use of this material. Any person acting on this trade idea does so entirely at their own risk.


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Image Details
Choose export citation format:
Detection of Continuum Submillimeter Emission Associated with Candidate Protoplanets
2019 The Astrophysical Journal Letters 879
Caption: Figure 2.
Measured astrometry for PDS 70 b and c with 3σ error bars (see Table 2). The labels "smm cont" and "smm CO" refer to the position of the ALMA continuum sources relative to the center of the continuum emission and the center of the disk rotation as measured from the CO line emission, respectively.
© 2019. The American Astronomical Society. All rights reserved.
Additional terms of reuse


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Sweet Shoppe Saturday 3/9
Hey Hey Sweet Shoppers! Another week has come and gone, and we hope it's been a memorable one for you and yours. Our Designers and Babes have been busy behind the scenes this week creating lots of new products, tutorials and inspiration around the site! So read on for all of the details on this week's new additions to the sweetest spot in town…
I have to tell you guys that I have been loving putting the newsletter together each week this year! It gives me such a great opportunity to sit down and take in what our staffers have been putting out there for you on a regular basis. I am so inspired by our tutorials, and I love seeing the products that excite our blog team in each month's theme! There is so much going on here at SSD, so I hope you guys are enjoying the highlights in our weekly newsletters as much as I am.
On that note, I'll be back in your inbox next weekend with lots more sweet new stuff. Until then, keep making memories and scrap MORE!


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News | 12 July 2019
The Visual Arts 'Public Open Space – art program' has commissioned three new street art murals, and they are well underway to brightening our streets.
The first activation is an abstract work by 'Resio' at Phoenix Park Community Centre. This artwork incorporates a mixture of bright colours, artistic splatters and tonal blended sections. The artist has just confirmed completion of the project, which took 5 days to paint. Community consultation was conducted while finalising the design – the local response has been very positive, both in planning stages and during installation.
Secondly, Porter Street pocket park is getting some 'star' dogs on its walls. Artist 'Sugar' is well known for his nature inspired murals which incorporate local flora and fauna. Three dogs which frequent the pocket park were photographed to feature in the mural alongside local birdlife. The installation is underway and expected to take over 10 days to complete.
A third project was coordinated through Youth Services, which has resulted in a bright and colourful new look on The Hub roller doors and building. We have arranged anti-graffiti coatings to be applied to the murals, to ensure the longevity of our commissioned projects. Each activation has a desired presence for at least two years.
In conjunction with this program, the Visual Arts team provided two scissor lift training sessions for street artists at Holmesglen Tafe. The education aims to provide safe working practices and professional development opportunities for our creative community.
Last updated: 12 July 2019
The City of Stonnington acknowledges that we are on the traditional land of the Boonwurrung and Wurundjeri people and offer our respects to the elders past and present. We recognise and respect the cultural heritage of this land.


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|ian at airs dot com
|[Bug gold/17432] internal error in relocate_tls, at gold/x86_64.cc:3829
|Thu, 25 Sep 2014 00:51:04 +0000
https://sourceware.org/bugzilla/show_bug.cgi?id=17432 --- Comment #1 from Ian Lance Taylor <ian at airs dot com> --- Can you show how gold is invoked? Can you show the input files? -- You are receiving this mail because: You are on the CC list for the bug.
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"Close Tihange in 2015 as well? Electrabel is bluffing"
Key ministers decided yesterday to close the two oldest nuclear reactors in Doel (East Flanders) in 2015 as agreed, but also to keep the nuclear power plant in Tihange (Wallonia) open 10 years longer than originally planned. This is in order to exclude any risk of power cuts or so-called "blackouts" in energy supplies, as Belgian does not yet have enough alternatives like sustainable energy.
A Belgian compromise was worked out to close Doel but to give Tihange an extended lifespan. The government is asking Electrabel to sell part of the Tihange electricity to challengers. At the same time, Electrabel will have to make a big investment to keep the old infrastructure up to modern safety standards.
Electrabel is not amused with the outcome of the talks yesterday, and has threatened to close down Tihange in 3 years' time, together with Doel 1 and 2.
Mr Wathelet does not believe that Electrabel really means it. "If nuclear energy wouldn't be profitable, this would be big news. But we know that this is not the case." He thinks Electrabel is bluffing. The green opposition MP Meyrem Almaci calls Electrabel's move "blackmail."
However, the federal government is ready to take action if Electrabel should carry out its threat. Economy Minister Johan Vande Lanotte says that a bill will be passed that allows another company to take things over if Electrabel refuses to keep Tihange open longer. There has been a lot of debate about the high profits Electrabel is making at the moment with nuclear energy. The government has demanded that Electrabel share part of this profit with the government and the tax payer.
200 jobs on the line
In an official statement, Electrabel said that nobody working at Doel 1 and 2 will actually be fired. People who retire will not be replaced, while specific deals will be offered to older employees. A total of 200 jobs will be lost due to the closure of Doel 1 and 2, Electrabel has calculated.


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Bellydance: the mother of all dance styles
I began bellydancing at 15 years of age under Sandra Hayes and Kelashi Bellydance, learning tribal style and improvisation techniques. I performed with Kelashi Bellydance at weddings, shows and events in Ireland as I continued training. I choreographed and coordinated bellydancers to perform at the annual St. Patrick's Day Parade in 2006 and 2007, the first time bellydancers had been part of the Cork parade. Soon after, I began my Egyptian style bellydance training under Adriana Echeveri, learning how to dance with the Wings of Isis and performing for the first time with them with Moondance Productions in the Firkin Crane (2006). It was at this show that I performed my Sword Ballet; a self choreographed fusion of pointe work and sword bellydance. I left bellydance for several years, coming back to it in 2014 to dance at the Zenith Cabaret, in Agadir, Morocco, with choreography by Mounia Dadi. I continue to freelance as a bellydance performer and teacher in London and recently competed in the London Bellydance Festival (July 2018).
Want to book a bellydance performance for your wedding, show, production or event?
(Please note, due to the commonly misunderstood nature of bellydance, restrictions apply).
Interested in a private bellydance class? Please see AK Dance Studio for prices and how to book.
Evening of Burlesque, solo bellydance veil performance, Theatre du Leman (Geneva), 11th December 2018.
Coco Cabaret, Southend on Sea, Bellydance solo performer, September and October 2018.
City Dance Academy, Bellydance teacher, London Contemporary Dance School, January- August 2018.
Innovations Dance, bellydance teacher, Innovations Ealing, February 2018 to date.
Weddings, parties, events across London, solo bellydance performancer (September 2016 to date)
Bellydolls, bellydancer soloist and group dancer for various weddings and events in the UK (September 2016)
Ishq Karenge, bellydancer and bellydance assistant choreographer for title song music video, April 2015
Zenith Cabaret, bellydancer and soloist, Agadir, Morocco, November 2014- February 2015.


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I have recently been given the task to evaluate applications. Note that I am not responsible for hiring, nor do I take any active role in the decision making. Instead I am only asked to provide feedback on my judgment of the candidate based on my technical experience related to the corresponding job position(s).
After having received these applications I am unsure of what the best way to proceed would be. Interestingly enough, I have seen other questions and answers around here, which simply state that a cover letter should not be included. I don't know about other countries, but at least in Germany I haven't ever seen or heard of an application without a cover letter. In these cases though, cover letters were present, but bad ones.
I am torn apart in this case, because on one side it is not strictly my job to judge the cover letter as such, but the candidate as a whole, in particular technical skills. On the other hand, I have been growing up with a certain importance being placed on cover letters, and hence, would not want to omit it from my evaluation.
The latter got the better of me and I did point out various deficiencies and what I inferred from them about the candidate to my superior - none of which was very positive of course. What surprised me though was that I got back opinions that I was being too elitist, so I would like to get a second opinion as a sort of reality check:
If a cover letter is included in an application, and it strikes you as being a badly written one, how would you evaluate the overall application based on that? Do you flat out ignore it and stick to the facts, or how much of an impact does/should it have ?
Minor details on what I considered bad in case it plays any role:
- Grammatical mistakes. Fortunately, spelling mistakes are a thing of the past, but Word just doesn't fix grammar mistakes yet.
- Logical fallacies - I may be prejudiced here due to having a strong background in predicate (and other) logics. Mostly, these are based on trigger words, which form logical implicitions (like therefore, hence, because of, etc.), but the two connected sentence have nothing to do with each other in terms of that relation. Other fallacies are maybe a risky try to impress with something the candidate doesn't have. For example, he first states that he worked on some jobs solo, emphasizing the corresponding soft-skills w.r.t. trust, responsibility, etc. and then following this up with what working in a team is like - although I have no evidence that he ever did work in one (which I don't expect from a fresh graduate for example, but it still leaves a red flag on that sentence).
- Unfounded assumptions - A candidate claims to know what's best for the company
- Inconsistencies - The candidate explains how he spends all his time to focus on improving in topic X, just before telling me the same thing two sentences later for topic Y, followed by topics A, B, and C.
- Generally weak wording - Again I may be prejudiced, as for me and everyone in my peer group, a cover letter is usually written at least 3-5 times over in order to make everything sound just right. It is easy to see that these cover letters were just simply written once without giving it much thought.


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Order Your Bridesmaid Dress
Ordering a bridesmaids dress through Bon Bon Belle Bridal Boutique is easy!
Simply take your measurements, refer to the designer size chart to determine your size, and complete the form below.
Then just "Submit" your information and you will be phoned by a Bon Bon Belle consultant to collect payment and you're all set!
What you will need: flexible measuring tape and a paper/pen to record your measurements.
Please note: Half down is required for dress orders to begin production with designers.


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This year's Annual Recognition Dinner will be held on Thursday, April 18th, 2019 at 6pm at Blackhawk Auto Museum in Danville, CA.
Las Aguilas de Diablo is offering five $1,000 college financial aid scholarships this year. These awards will be based on Scouting leadership, community participation, academic achievement and financial need. Details of the scholarships and their applications can be found here. Scholarship applications are due April 1st. We will announce the scholarship recipients at the Eagle Dinner on April 18th and if you have applied for a scholarship, we request you attend this event.
This year's speaker is Colin Braudrick. Colin has been a member of The Walt Disney Company for two years, where he serves as the Manager of Global Workplace Security. In his role, Colin mitigates the threat posed by workplace violence, active shooter incidents, and other global workplace security issues, such as vehicular attacks and kidnapping. Prior to joining Disney, Colin retired after a 20-year career with the Los Angeles Police Department. His last assignment with LAPD was as the gang unit supervisor in Watts. Prior to that spent many years as a homicide detective in South Los Angeles, being involved in the investigation of over 100 homicide cases. Early in his career, he was involved in LAPD's updated response to active shooter incidents.
Colin is the proud father of three kids, one of whom, Owen, is an Eagle Scout and is currently a Freshman at UCLA.
The youth attire for the evening will be full scout uniform. If that is not possible, a coat and tie are appropriate.
Parents who wish to attend must register separately from their Scout. The cost will be $50 per person. All Scouts are paired with a Las Aguilas Member or sponsor by career interest.
Each year Las Aguilas de Diablo honors young people who have achieved the rank of Eagle Scout, Quartermaster or Summit Award at a special banquet. Besides being an enjoyable evening for our honorees, the dinner is also an opportunity for them to spend some time with a successful member of the career field they are interested in joining. The success of our dinner depends on having our members and sponsors present as examples to the youth in our council.
2016 Dinner Speaker
2018 Dinner Gallery
Our annual banquet is hosted by local Scouting businesses, professional and civic leaders. Each honoree will have an alumni sponsor from an area of their career interest for the evening. Please be aware that this is not just a dinner to honor our Scouts and their accomplishments, but to also begin networking for future employers or mentors.
The cost to sponsor an honoree at the annual dinner is $125. If you are an Eagle Scout, Quartermaster or Summit Award recipient, please consider becoming a member of Las Aguilas de Diablo for $250. This includes the dinner, a contribution to the MDSC Endowment Fund, a contribution towards our scholarships, and a general operating fund for the group. You can find out more about our Membership options here.


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By Chris Seiter. Essentially you ignore your ex for a period of time and by doing so you get all kind of unconscious benefits. So, I went further and compiled a list of psychological research on ignoring an ex and what benefits that can bring you. Currently she is three years old which means that routines are very important for her. One of these routines is that she likes a hug and a kiss before she takes a nap or goes to sleep for the night.


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Custom Blinds WA supports local businesses by utilising their network of premium curtain and blind suppliers that create their blinds curtains and blinds with Australian fabrics. As a locally and family owned business Custom Blinds considers it extremely important to keep their business local however, there are more benefits to you, as a customer, when you buy local.
Opening Hours:
Monday - Thursday 8.00am - 4.00pm
Friday - 8.00am - 1.00pm
Saturday - By Appointment
Public Holidays: Closed
Unit 5 12-14
Baretta Road, Wangara
Phone: (08) 9409 1555