Exposure to air pollution has adverse effects on cardiopulmonary health of adults. Exposure to air pollution in pregnancy may affect foetal development. However, the evidence of such effect remains inconsistent. We investigated the effects of exposure to air pollution during pregnancy on birth outcomes.2 exposure and birth outcomes.This study, based within the Norwegian Mother and Child Cohort Study (MoBa), includes 17,533 participants living in the two largest cities in Norway: Oslo and Bergen, and their two surrounding counties: Akershus and Hordaland. Air pollution levels at residential addresses were estimated using land use regression models and back-extrapolated to the period of each pregnancy using continuous monitoring station data. Birth outcomes were birth weight, low birth weight, gestational age, and preterm delivery obtained from the Medical Birth Registry of Norway. Information on lifestyle factors was collected from MoBa questionnaires completed by mothers during pregnancy. Linear and logistic regression models were used to analyse the associations between pregnancy NO2 and birth weight −43.6 (95%CI −55.8 to −31.5) g per 10 μg/m3 NO2. However, after adjusting for either area or the combination of parity and maternal weight, no substantive effects of air pollution exposure were evident.We found a statistically significant negative association between pregnancy exposure to NOExposure to air pollution during pregnancy was associated with decrease in birth weight, but area-related and lifestyle factors attenuated this association. We found no statistically significant associations of air pollution exposure with gestational age, low birth weight or preterm delivery.The online version of this article (doi:10.1186/s13690-016-0138-8) contains supplementary material, which is available to authorized users. The ass2 for the specific areas of Norway that we studied: two city areas (Oslo and Bergen) and their two surrounding counties . The selected areas represent the most populated areas in Norway and also include the territories with highest levels of ambient air pollution. Each of these four areas has different land use practices, road network density, industrial activities and landscape specifics. To our knowledge, previously the associations of modelled air pollution exposure with pregnancy outcomes were studied in Norway only in Oslo [2.5 with an increased risk of low birthweight at term but no significant decrease in birth weight [With the development of new methods, epidemiologic studies have been able to provide more individualized exposure assessment by using dispersion or land use regression (LUR) models for estimating air pollution exposure at residential addresses. In this paper we built LUR models for traffic-related air pollutant NO in Oslo , 7. In t in Oslo . Oslo daWe investigated the effects of exposure to traffic-related air pollution during pregnancy in selected urban and county areas of Norway on birth outcomes, including birth weight and the length of gestation.N = 22149). We excluded consequently participants with missing pregnancy NO2 exposure information (N = 3876), multiple births (N = 664) and non-live births (N = 76). Only data on singleton live births were used in the analysis. Total number of participants from the studied areas with non-missing air pollution exposure data was 17533 (born from 2001 to 2009), of which 4669 from Oslo, 7554 from Akershus, 3869 from Bergen and 1441 from Hordaland (Table th April 2011.The Norwegian Mother and Child Cohort Study (MoBa) is a prospective population-based pregnancy cohort study conducted by the Norwegian Institute of Public Health , 9. PartN = 718, 9.5 %), we used data from ultrasound measurements to determine gestational age. We also studied binary outcomes: low birth weight (<2500 g) and preterm delivery (<37 completed weeks of gestation).For assessing birth outcomes we used continuous MBRN variables birth weight (grams) and gestational age (weeks). Air pollution can potentially affect the size of developing foetus. Therefore estimating gestational age was based on the last menstrual periods as estimates based on ultrasound assessments are dependent on early foetal growth . If infoThe following characteristics were also extracted from the MBRN: sex of the child , parity , mother’s age at birth (years), marital status . Questionnaire information was used to determine: maternal education ), maternal smoking during pregnancy , maternal weight at the beginning of pregnancy (kg), maternal height (cm). Exposure to air pollution was estimated at the registered address at delivery. Women who did not change their address during pregnancy were used in a sensitivity analysis. Area variable was defined by the location of the address at delivery: Oslo, Akershus, Bergen, Hordaland.2 levels were built for the studied areas in order to account for regional specifics. In the models we used air pollution measurements conducted in 2010 for Oslo and Akershus, and in 2011 for Bergen and Hordaland. Measurement campaigns included three rounds of approximately two weeks duration with NO2 measurements  within a one year period. Measurement sites were selected to represent the range of residential exposure for each study area. For the analysis we included sites with no missing data, and no geocoding mismatches.Estimating air pollution exposure during pregnancy was based on the methodology developed for the ESCAPE project , 11. LUR2 level that was used for LUR modelling. LUR models were built separately for Oslo and Akershus. One model was built for Bergen and Hordaland together due to a low number of valid measurement sites for Hordaland. Predictors for building the LUR model were obtained from a GIS analysis of the N50 and VBASE maps (obtained in February 2013) providing information on land use, residential density, types of landscape and road network information. Predictor variables are listed in Additional file The mean exposures over the three measurement periods were averaged to obtain a yearly mean NO2 measurements were obtained from the Norwegian Institute for Air Research database “Luftkvalitet.info” for the period 2000–2012 in Oslo (used for Oslo and Akershus), and for the period 2003–2012 in Bergen . The daily estimates of exposure were calculated as the LUR-modelled yearly estimate multiplied by the ratio between daily NO2 routine monitoring station measurement and an annual average for the year when LUR measurement campaign took place. Daily NO2 exposure estimates were averaged separately for 1st, 2nd, and 3rd trimester, and also over the whole pregnancy. Exposures by trimester and the whole pregnancy exposure were highly correlated and therefore we decided to use one exposure estimate: the average NO2 exposure during the whole pregnancy.Yearly means of air pollution levels at residential address at birth were estimated using the resulting LUR models. Variables in models were truncated in accordance to the range of corresponding variables used for LUR model building. Negative modelled values were replaced with 0.01. In order to account for temporal variability, we used the ratio method of back-extrapolation to the period of each pregnancy using continuous routine monitoring station data . Daily N2 exposure and birth weight and gestational age. Logistic regression models were used to evaluate associations between pregnancy NO2 exposure and low birth weight and preterm delivery. The results are presented for both crude and adjusted models for a 10 μg/m3 increase in NO2. Adjustment variables were selected based on the literature analysis. Model 1 is adjusted for: maternal education, birth season, sex of child, maternal age, maternal marital status, maternal smoking during pregnancy, and maternal height. Model 2 contains Model 1 adjustment plus the area variable. Model 3 contains Model 1 adjustment plus parity and maternal weight. We performed stratified analysis by area , smoking status of the mother during pregnancy, parity, sex of child, maternal education level, and season of birth in order to explore potential effect modifiers. Sensitivity analysis of the results was performed in a subgroup of women who did not change address during pregnancy and in a subgroup of women for whom gestational age was determined from the last menstrual period – as a preferable method for assessment of potential effects of air pollution on the size of the developing foetus [We used linear regression models to analyse associations between pregnancy NOOverall birth weight was lower in cities (Oslo and Bergen) and higher in the two county areas , however, these differences were less prominent when comparing birth weight within each parity strata Table . Having 2 yearly means are presented in Table 2 model were area of water in the buffer of 500 m, distance to major road, and agricultural areas within the buffer of 1000 m. The model for levels of NO2 in Bergen and Hordaland included the following: lengths of major roads within the buffers 50 m, 50 to 100 m, 100 to 1000 m, inverse squared distance to port, high and low density residential land within the buffer of 1000 m, altitude, agricultural area within the buffer of 1000 m, and distance to the sea. The adjusted R2 of the models ranged from 59 to 87 % , and lowest in Hordaland county (6.3 μg/m3)  compared to those exposed to below 20 μg/m3 NO2, with estimated 60 g difference in means (p< 0.0001) between these groups   g per 10 μg/m3 NO2 (Table N = 15829) did not change substantially from the results in the main analyses.Comparing the overall birth weight distributions, we found a shift towards lower birth weight in children with pregnancy exposure to NOups Fig. . The sam47) Fig. . In the 2 and birth weight were seen only in children born in spring , Lee et al. 2003 (exposure between months 3 and 5) [Birth weight is associated with a variety of different factors: genetic and ethnic background, parity, maternal weight, socio-economic factors, medical conditions before or during pregnancy, and maternal lifestyle variables, most notably smoking during pregnancy . The rol3 and 5) , 14. How3 and 5) , 16 even2 and birthweight was attenuated and no longer statistically significant when adjusting for study area. Our area variable may reflect the spatial distribution of air pollution, representing by itself the proxy of exposure, or it may represent unmeasured factors in each study area which also influence birth weight and pregnancy length. The area means for modelled NO2 range from highest in Oslo to lowest in Hordaland (Table 2 levels on local scale. Areas may differ by mean levels of air pollution exposure, as well as by socioeconomic and lifestyle characteristics. For instance, social classes may be differently distributed within polluted areas depending on a region [In our study, the association between pregnancy exposure to NOnd Table , thus ada region . In the a region .Fig. 2SiAmong the potential area-specific factors that attenuate the association of birth weight with air pollution exposure, our study singled out the combination of parity and maternal weight. These factors may be related to lifestyle, socioeconomic situation, and health state of mothers, and can vary by study area. Specifically in Norway, families with more than one child tend to move out from large cities, which may explain the difference in distribution of parity by study area. Stronger associations of air pollution exposure with adverse birth outcomes have been reported in less economically advantaged classes , 20. In 2 exposure during pregnancy with gestational age and preterm delivery. This is in line with other studies also reporting no association of NO2 exposure with preterm delivery [Our study found no association of NOdelivery , 22.The important strengths of our study include standardized exposure assessment, detailed information on potential confounders, and a large study population. We highlighted the importance of area-specific factors that may confound the association between air pollution exposure and pregnancy outcomes. Our results suggest that these factors should be given a proper consideration in large multicenter studies. Further research is needed for identifying potential critical windows of exposure. In our data, exposures by trimester were highly correlated with whole pregnancy exposure. We assessed exposure at residence, but for more detailed individual exposure it is advisable to consider estimating exposures during travel and frequency of visits to city center. Most of our study participants did not move during pregnancy, but we did note a difference in distribution of multiparous women in our study areas, with higher proportion of them living in counties, and not in the cities. Besides the identification of the important confounders, such as parity and maternal weight, we might still have the residual confounding of unmeasured lifestyle factors in our data.2 during pregnancy was associated with a decrease in birth weight in some models. However, when adjusting for area-related and lifestyle factors this association was attenuated. We found no statistically significant associations of air pollution exposure with gestational age, low birth weight and preterm delivery.In this large pregnancy cohort with detailed information on lifestyle and pregnancy factors, land use regression modelled exposure to NO


DNA methylation plays an essential role in the regulation of gene expression. While its presence near the transcription start site of a gene has been associated with reduced expression, the variation in methylation levels across individuals, its environmental or genetic causes, and its association with gene expression remain poorly understood.cis to sequence variation, usually within 5 kb. Via eQTL analysis with microarray data combined with mapping of allelic expression regions, we obtained a set of 2,770 regions mappable in cis to sequence variation. In 9.5% of these expressed regions, an associated SNP was also a methylation QTL. Methylation and gene expression are often correlated without direct discernible involvement of sequence variation, but not always in the expected direction of negative for promoter CpGs and positive for gene-body CpGs. Population-level correlation between methylation and expression is strongest in a subset of developmentally significant genes, including all four HOX clusters. The presence and sign of this correlation are best predicted using specific chromatin marks rather than position of the CpG site with respect to the gene.We report the joint analysis of sequence variants, gene expression and DNA methylation in primary fibroblast samples derived from a set of 62 unrelated individuals. Approximately 2% of the most variable CpG sites are mappable in Our results indicate a wide variety of relationships between gene expression, DNA methylation and sequence variation in untransformed adult human fibroblasts, with considerable involvement of chromatin features and some discernible involvement of sequence variation. Perhaps the best studied of epigenetic phenomena, the methylation of CpG dinucleotides, has been known for many years to play a key role in X-chromosome inactivation , transcrMethylation has been shown to be highly variable across cell types with variable sites falling in two broad categories: those with inverse correlation between DNA methylation and chromatin accessibility, and those with variable chromatin accessibility and constitutive DNA hypomethylation . As reviet al. [et al. [et al.[et al.[et al.[Methylation also varies between healthy individuals in a population. Relationships between DNA methylation, gene expression and various other genetic and epigenetic biomarkers have been examined previously. Recent studies have identified SNPs whose genotype correlates with DNA methylation  in various human populations and cell types. Bell et al.  utilized [et al.  used the [et al. . Zhang e. [et al. performe. [et al. has examl.[et al. report 1l.[et al. report pcis do not, in general, show strong overlap with annotated genes or promoter regions - rather, CpG sites where this correlation is negative are most commonly seen in sites associated with active promoter marker H3K4me3 and DHS regions, while those with positive correlation are most commonly seen in the presence of the repressive chromatin marker H3K27me3 (histone 3 lysine 27 tri-methylation).To further understand the relationship between genetics, gene expression, DNA methylation, and other epigenetic marks, we present analyses of DNA methylation, gene expression  and DNA sequence polymorphisms from a set of 62 fibroblast cell lines derived from healthy human individuals, augmented with publicly available histone mark and DNase I hypersensitivity (DHS) data. We show that: widespread relationships exist between DNA polymorphisms and DNA methylation (mQTLs); widespread relationships exist between DNA methylation and gene expression, especially in developmentally significant genes, including all four HOX clusters; supplementing expression quantitative trait locus (eQTL) data with mapping of allelic expression to adjacent SNPs, a large set of regions and genes mapped to a QTL that also functions as an mQTL, comprising 242 genes and 23 regions not overlapping with an annotated gene; and CpG sites where methylation correlates with gene expression in We report on the joint analysis of inter-individual variation in the levels of DNA methylation, total and allelic expression, and DNA sequence of 62 healthy parents of 31 parent-child trios of European descent. Here, we start by introducing each data set individually before discussing the relationships among them.DNA methylation was assayed in forearm skin fibroblast samples using the Illumina 450 K assay . For each sample, methylation was measured at approximately 485,000 CpG sites, but we only considered the approximately 392,000 sites uniquely mapped in autosomes and containing no known SNPs. Methylation levels are measured in populations of diploid cells using beta values , which rThe Illumina 450 K assay includes both type I probes utilizing two query probes per CpG locus (largely concentrated around genes’ TSSs), and type II probes utilizing a single probe per locus . The distributions of methylation beta values differ for type I and type II probes due to their localization biases but both are bimodal, with modes corresponding to CpG sites that are unmethylated in most cells of the sample (hypomethylated), and those that are methylated in most cells of the sample (hypermethylated)  are usually located far away from the TSS (either in intergenic regions or in the gene body), or are located near the TSS of genes with low expression in fibroblasts and generally lack regulatory or evolutionary marks of function. The majority of these CpG sites show a unimodal distribution  were considered for further analyses.To complement total expression data, allelic expression (AE) was assayed at a set of approximately 900,000 SNP locations dispersed in annotated genes and intergenic regions of all autosomes using hybridization to genotyping arrays, as previously described  ; Materials and methods), and found 74,624 aeSNPs within annotated gene regions (corresponding to 15.8% of genic/intronic SNPs), and 25,467 outside (corresponding to 5.4% of intergenic SNPs). aeSNPs were clustered into 3,327 aeRegions (consisting of two or more consecutive aeSNPs), of which more than 80% had full or partial overlap with an annotated gene ). A set of 27,486 pairs . We then mapped CpG beta values to the imputed genotype at polymorphic sites within 250 kb  of mappable CpG probes have a significant mQTL within 5 kb but in 6% of cases the closest significant mQTL lies more than 100 kb away . Such eQTLs were found for 420 (4.4%) genes and involved 9,674 SNPs . A total of 95,949 aeQTL-aeRegion pairs were obtained  within 250 kb . This resulted in the identification of 587 genes with correlation to at least one of 1,793 CpG probes  and 145 (24%) involved in multicellular organismal development   of them being known transcription factors , expression to sequence (eQTLs and aeQTLs) and methylation to expression. To quantify the degree of overlap between the various relationships studied, we used genes, rather than CpG probes or SNPs, as the primary unit of interest. As seen in Figure C21orf56  but positively correlated probes in the body of the transcript (but near the TSS of PAX8). Indeed, RNA-seq data obtained from three individuals with differing genotypes in the cis-associated emQTLs suggest that the expression of PAX8 and its antisense transcript are positively correlated, ruling out an interference between the two but instead hinting at a possible chromatin-linked role of DKFZP686E10196 activation in regulating PAX8 transcription. , presenion, see ).GSTM and GSTT also show multiple genes being mappable to similar sets of CpG probes and SNPs . For each gene, the five SNPs (within 250 kb) jointly explaining the largest portion of the expression variation on the training data were sequentially identified and regressed out. Independently of this we regressed out the five CpG sites explaining the largest portion of the expression variation. We found a total of 25.5% of gene expression variation to be explained by sequence variation, whereas methylation explained only 8.9% of expression variation. We applied a third model in which the top five SNPs were regressed out and then the top five CpGs were regressed from the residuals, finding in this case the variation explained by methylation dropped to 5.9%. This suggests that 5.9/8.9 = 66% of methylation-facilitated gene expression variation was independent of sequence variation. These figures are considerably higher than the 1.2% and 3.3% variation of expression explained, respectively, by DNA sequence and DNA methylation found by Li t tumors , indicatWe have analyzed the inter-individual variability of and relationships between one of the most comprehensive set of biomarkers in untransformed adult cells to date, including a more expansive assay for DNA methylation containing a large and diverse set of CpG dinucleotide probes; gene expression data; SNP data and allelic expression data, augmented with publicly available histone mark and DHS data from other cell cultures.We chose primary skin fibroblasts as a model system. These comparatively easy to isolate and cultivate cells are a readily accessible source of patient material, and are in use as a model system for complex disease etiological studies . EpigenAlthough most CpG sites with variable methylation seem unrelated to variation in gene expression, a non-negligible portion show significant correlations. Remarkably, the properties of these relationships appear quite complex, and the location of CpG probes with respect to the gene provides relatively little information about the sign of the correlation. Instead, chromatin states, particularly those that are representative of active chromatin and transcribed regions (DHS and H3K4me3) were more strongly indicative of negative correlation. Using the publicly available ENCODE data, we found in general that negatively correlated probes most strongly overlapped with regions of constitutive DHS but variable H3K4me3 among the five fibroblast cell lines considered, whereas positively correlated probes most strongly overlapped with an indicator of inactive transcription, H3K27me3. Work published in the ENCODE paper on DHS  indicateWhether the associations between gene expression and methylation truly reflect variation in tissues or other differences acquired after sample collection is an important and challenging question. One possible source of post-sample collection variation is differences in cell proliferation rates. However, we have found that cell proliferation variation explained only 8% of the variance in methylation levels of expression-correlated CpG sites, and 13% of the variance in expression levels of methylation-correlated genes. Among mappable CpG sites and genes, the proportion of variance explained was negligible .C21orf56, a gene for which we find many emQTLs in fibroblasts, also exhibits the same property in whole blood [GSTM3, NAPRT1, SPG7 and WBSCR27) were also identified in our assay, indicating that genetic variation leading to both methylation and expression variation at the same locus is a relatively rare but reproducible phenomenon, the mechanism and implication of which merit further investigation. We report a total of 260 annotated genes or aeRegions that, to our knowledge, have not been previously reported as having emQTLs, including 23 aeRegions having no overlap to annotated genes. We attribute these discoveries to the usage of AE assays as well as a gene expression microarray experiments, together with use of the relatively recently developed Illumina Infinium HumanMethylation450 platform, interrogating methylation at a larger and more diverse set of CpG sites compared to most previous studies. As the effect of methylation on gene expression can in some cases involve cell-specific trans-acting factors [Relatively high overlap was seen with results from previous studies in terms of the rare genes where both expression and methylation could be linked to genetic variation (emQTLs). In particular, le blood  and LCLsle blood . Severalle blood   and the McGill Cellbank . Cells were grown in alpha MEM Medium  supplemented with 2 mmol/l L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (SigmaAldrich) at 37°C with 5% COGenomic DNA (gDNA) for SNP genotyping and DNA methylation analysis was extracted from cell lysates using the GenElute DNA Miniprep Kit (SigmaAldrich) and DNeasy Blood and Tissue Kit , respectively, according to manufacturer’s protocol. DNA concentrations were determined using the Quant-iT PicoGreen kit . Total RNA was extracted from cell lysates using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol, and treated with 6 U DNase I. RNA quality was confirmed to be high for all samples on the Agilent 2100 Bio-Analyzer , with an RNA integrity number (RIN) range of 8.1 to 10, and concentrations were determined using the Nanodrop ND-1000 .gDNA (500 ng) was used for bisulfite conversion employing the EZ DNA Methylation Kit , according to the manufacturer’s protocols. The modified gDNA was processed as described in the Infinium Assay Methylation Protocol Guide Rev. C (November 2010), and analyzed on Infinium HumanMethylation450 BeadChips  correlates with DNA concentration. The first principal component of expression levels for the set of these 42 genes was obtained and used as a vector estimating the level of cell proliferation effects in the full set of individuals. For each methylation probe correlated either to gene expression or sequence variation, we carried out linear regression with the probe’s beta values and the cell proliferation vector. The variance of the residuals was compared with the variance of the original methylation probe, and done so cumulatively across probes to obtain the total variation in methylation of correlated probes explained by cell proliferation effects (with separate categories for CpG sites correlated to DNA sequence and gene expression). The process was repeated with expression probes found to be correlated with eQTLs and/or methylation of adjacent CpG sites to obtain an estimation of the total variation in expression of correlated genes explained by cell proliferation effects.et al.[Whole genome bisulfite sequencing was carried out for cell lines GM02316, GM02317, GM02456, and GM02555 as described  with theet al..AE measurement was carried out as described previously . In shorImputation of HapMap genotypes and phasing of Infinium HumanOmni1 and HumanOmni2.5-derived genotyping data were done using Beagle . The SNPGene expression levels for 58 of the 62 individuals were determined using the Illumina HumanRef-8 Expression BeadChip according to the manufacturer’s protocol, giving expression levels for 21,916 probes mapping to a total of 16,952 genes.P-values, and a P-value of 1.4 × 10-5 was obtained as the cutoff yielding a 5% FDR.These expression values were quantile normalized, the genes filtered such that only those in the top 50% variance of expression were retained, and expression values of these genes correlated using Spearman’s correlation coefficient to all SNPs within the gene boundaries or up to 250 kb upstream of the TSS or downstream of the TES. Expression values of the top 50% variable genes were permuted and the correlation analysis repeated to obtain a null distribution of P-values. A P-value threshold of 0.0029 was set based upon an FDR of 5%.All HapMap (release 28) SNPs at a distance of ±250 kb flanking each aeRegion and having a minor allele frequency >10% were correlated using Spearman’s correlation coefficient to their respective aeRegion. For each aeRegion, allelic expression values were permuted amongst the samples and the regression repeated to obtain an overall null distribution used in determining the FDR of P-value of 6 × 10-6 for an FDR cutoff of 5%.Only probes having variance across samples in the top 25% were kept for correlation analysis with SNPs. Spearman’s rho was calculated between the highest 25% variance probes and HapMap SNPs at a distance of ±250 kb flanking each CpG probe and having minor allele frequency >10%. For each variable CpG probe, the analysis was repeated with methylation values permuted across individuals, in order to obtain a P-value cutoff of 5.132 × 10-5 for a 5% FDR.The same set of top 25% variable methylation probes and top 50% variable genes in the Illumina HumanRef-8 Expression BeadChip were used, obtaining the Spearman correlation coefficient between any methylation probe located within the body of an annotated gene or up to 250 kb on either side. Expression levels for each gene were permuted across the samples and the same set of Spearman correlation coefficients obtained, in order to set the A CpG probe was labeled as being in the 'TSS' a gene if it was ±1,500 bp from its TSS. It was labeled as 'body' if it was not located within 1,500 bp of any TSS but was within an annotated transcript. Finally, it was labeled as 'intergenic' if it was neither 'TSS' nor 'body'.2 between the expression values and the predicted expression values using the models was obtained as the percentage of expression variation explained by sequence variation. The same procedure was repeated with the residuals of the gene expression values from the sequence-expression model as response variables and methylation beta values of CpG probes in the neighborhood as explanatory variables, in order to obtain the percentage of expression variation explained by methylation variation.The percentage contribution of methylation and sequence variation to expression variation was assessed using five-fold cross-validation and step-wise feature selection. For the training subset , a linear model with expression of a particular gene as the response variable and genotypes of SNPs in the neighborhood of that gene as explanatory variables was selected using the stepAIC function in R ; the modSignificantly overrepresented GO categories were obtained for variable CpG probes and genes correlated to DNA methylation using Fisher’s exact test via GOStat , using dP-values were calculated by the GOStat web server, whereas fold enrichment was determined by dividing the proportion of genes in the test set with a given GO term by the proportion of genes in the background set with the same GO term.In the case of enrichment for highly variable CpG sites, genes with at least one top 25% variable CpG site at a TSS ± 1,500 bp were used as the test set; the set of all autosomal genes overlapping with at least one Illumina 450 K CpG probe was used as the background set. In the case of methylation-expression correlation, the set of all genes whose expression correlated significantly at 5% FDR with methylation of at least one CpG site were used in the test set; the set of all genes containing at least one CpG site within 250 kb was used as the background set. Data were downloaded from the ENCODE Data Consortium Center at UCSC at  on 15 OcThe set of all SNPs that are categorized as correlated both to gene expression (aeQTL having relationship to an aeRegion and/or an eQTL correlated to a gene in the Ref8 array) and to DNA methylation (mQTL) at an FDR threshold of 5% in both of the respective analyses described above are categorized as methylation-regulatory SNPs (emQTLs).Methylation, gene expression, and SNP genotyping data for this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO SuperSeries accession number GSE53261.AE: allelic expression; aeQTL: allelic expression quantitative trait locus; aeSNP: allelic expression single nucleotide polymorphism; bp: base pair; DHS: DNase I hypersensitivity; emQTL: expression and methylation quantitative trait loci; eQTL: expression quantitative trait locus; FDR: false discovery rate; gDNA: genomic DNA; GO: Gene Ontology; H3K27me3: histone 3 lysine 27 tri-methylation; H3K4me3: histone 3 lysine 4 tri-methylation; LCL: lymphoblastoid cell line; LTOR-HMM: left-to-right hidden Markov model; mQTL: methylation quantitative trait locus; SNP: single-nucleotide polymorphism; TES: transcription end site; TSS: transcription start site.The authors declare that they have no competing interests.TP, MB, SB and JW conceived of the study. JW, MB, TP and SB wrote the article. TP, SB, BG and TK performed DNA methylation, gene expression, allelic expression and SNP genotyping experiments. JW, MB, and SB performed the computational analyses. All authors have read and approved the manuscript for publication.Replicability of beta values in samples GM02456.Click here for fileDistribution of methylation beta values in type I probes across the genome, partitioned by position relative to (A) CpG islands and (B) annotated genes.Click here for fileProportion of type I CpG probes falling in various types of genomics regions identified by ENCODE, partitioned by (A) CpG probe mean beta value and (B) percentile of beta value standard deviation. All data types, except for 28-way conservation, are derived from broad peaks in BJ human foreskin fibroblast cells.Click here for fileMean (A) and standard deviation (B) of type I CpG probes with respect to their position relative to transcription start sites (TSSs) of annotated genes. Each green dot corresponds to a CpG probe, and the four lines show the running median for probes based on the quartile of the expression level  of the gene they are associated with.Click here for fileProportion of CpG sites determined to have various numbers of modes in the set of individuals in the present study. Independently for each CpG site, we applied the kernel smoothing algorithm in R [thm in R , obtainiClick here for fileEnrichment/depletion of Gene Ontology terms, obtained using GoStat [, for highly variable CpG probes (worksheet 1) and genes with expression correlated to DNA methylation.g GoStat , for higClick here for fileOverlap of aeRegions with annotated genes.Click here for fileSet of aeRegions.Click here for fileSignificant mQTL-CpG probe pairs.Click here for fileWhole genome bisulfite sequencing (WGBS) statistics.Click here for fileSignificant eQTL-Ref8 gene pairs.Click here for fileSignificant aeQTL-aeRegion gene pairs.Click here for fileSignficant CpG probe-Ref8 gene methylation-expression correlation pairs.Click here for file


The Institute of Medicine recommends that lactating women ingest 290 μg iodide/d and a nursing infant, less than two years of age, 110 μg/d. The World Health Organization, United Nations Children’s Fund, and International Council for the Control of Iodine Deficiency Disorders recommend population maternal and infant urinary iodide concentrations ≥ 100 μg/L to ensure iodide sufficiency. For breast milk, researchers have proposed an iodide concentration range of 150–180 μg/L indicates iodide sufficiency for the mother and infant, however no national or international guidelines exist for breast milk iodine concentration. For the first time, a lactating woman and nursing infant biologically based model, from delivery to 90 days postpartum, was constructed to predict maternal and infant urinary iodide concentration, breast milk iodide concentration, the amount of iodide transferred in breast milk to the nursing infant each day and maternal and infant serum thyroid hormone kinetics. The maternal and infant models each consisted of three sub-models, iodide, thyroxine (T4), and triiodothyronine (T3). Using our model to simulate a maternal intake of 290 μg iodide/d, the average daily amount of iodide ingested by the nursing infant, after 4 days of life, gradually increased from 50 to 101 μg/day over 90 days postpartum. The predicted average lactating mother and infant urinary iodide concentrations were both in excess of 100 μg/L and the predicted average breast milk iodide concentration, 157 μg/L. The predicted serum thyroid hormones , and T3) in both the nursing infant and lactating mother were indicative of euthyroidism. The model was calibrated using serum thyroid hormone concentrations for lactating women from the United States and was successful in predicting serum T4 and fT4 levels (within a factor of two) for lactating women in other countries. T3 levels were adequately predicted. Infant serum thyroid hormone levels were adequately predicted for most data. For moderate iodide deficient conditions, where dietary iodide intake may range from 50 to 150 μg/d for the lactating mother, the model satisfactorily described the iodide measurements, although with some variation, in urine and breast milk. Predictions of serum thyroid hormones in moderately iodide deficient lactating women (50 μg/d) and nursing infants did not closely agree with mean reported serum thyroid hormone levels, however, predictions were usually within a factor of two. Excellent agreement between prediction and observation was obtained for a recent moderate iodide deficiency study in lactating women. Measurements included iodide levels in urine of infant and mother, iodide in breast milk, and serum thyroid hormone levels in infant and mother. A maternal iodide intake of 50 μg/d resulted in a predicted 29–32% reduction in serum T4 and fT4 in nursing infants, however the reduced serum levels of T4 and fT4 were within most of the published reference intervals for infant. This biologically based model is an important first step at integrating the rapid changes that occur in the thyroid system of the nursing newborn in order to predict adverse outcomes from exposure to thyroid acting chemicals, drugs, radioactive materials or iodine deficiency. Thyroid hormones are involved in many cellular activities essential to normal body function, including maturation and growth. Elemental iodine is vital for thyroid hormone synthesis within the thyroid gland. Severe iodine deficiency has several adverse health outcomes on growth and development. Pregnant women and fetuses are susceptible to iodine deficiency. An infant with severe iodine deficiency in utero may experience impaired intellectual development, growth retardation, and hypothyroidism . Recent In the healthy euthyroid nursing or bottle-fed full term infant, studies demonstrating hypothyroxinemia and central nervous system effects were not located in the published literature. However, for infants born preterm, transient hypothyroxinemia  has been reported –13. The For exclusively breast-fed infants, mammary gland milk is the only source of elemental iodine. The mammary gland is capable of concentrating iodide in breast milk via the sodium iodide symporter (NIS), a membrane bound transport protein, that is under the control of prolactin and other hormones . Semba aThe Food and Nutrition Board of the Institute of Medicine (IoM) has a Recommended Dietary Allowance (RDA) for dietary iodine intake of 290 μg/d for lactating women . For infRecently our in silico laboratory has developed computational models to describe the effects of iodine insufficiency on serum thyroid hormone concentrations in the lactating rat  and pregIn this paper, we describe a biologically based model for the lactating woman and nursing infant, employing equations used for the lactating rat and nursing pup and the pregnant woman , 19. TheIn the remainder of the paper, the term iodide, if used, replaces the terms, elemental iodine, iodine, and other forms of iodine such as salts of iodide.The infant portion of the biological model for the lactating mother and nursing infant predicts serum thyroid hormones from near 5–7 days of age to 90 days of age. At birth, a mechanistic understanding of the HPT axis is lacking. Surges in production of thyroid stimulating hormone (TSH) and T4 result in both high serum levels of TSH and T4 after birth. Serum TSH and T4 concentrations decline in the newborn over the first week after birth . SufficiExtreme iodide deficiency, accompanied by maternal and infant goiter and hypothyroidism, has been reported. For example, in Germany, an area well known for iodide deficiency, Heidemann and Stubbe  describerd trimester mother at birth [The model compartments for the mother consist of plasma, slowly perfused with blood , richly perfused with blood , thyroid gland, thyroid plasma, mammary gland plasma, mammary gland fat and mammary gland milk . Some moat birth , were usat birth   into fat stores  of the mammary tissue (very minor pathway) or was transported unidirectional by the NIS protein into breast milk (major pathway) . The PAmThe infant iodide model (delivery to 90 days postpartum) was a physiologically based pharmacokinetic model  with theInfant body weight growth  was expressed using an algebraic equation to less than 11 months of age . Plasma Urinary excretion of iodide  increases from birth to 90 days of age   was targeted to be less than 8% of the T3 formed . Fisher The sub-model model structures for T4 and T3 are one compartment , the samT4 secretion (production) from the thyroid gland  for the first few days of life in the infant is 0.535 nmol/hr/kg, then decreases over the first month of life to 0.375 nmol/hr/kg, and then remains stable at 0.322 nmol/hr/kg for up to 11 months of age . These dThe volume of distribution of T4  was set to 0.31 L/kg based on an analysis of 7 children up to 12 months of age, who were injected with radiolabeled thyroxine . DeiodinAn age dependent factor was incorporated to describe deiodinase III conversion of T4 to rT3. At birth, deiodinase III is present at relatively high levels and decreases with increasing age , 55. ChoApproximately 8% of T4 was assumed to be secreted into feces in the infant based on adult experimental findings  of 10–15The volume of distribution for T3 in the infant was set equal to the fetus . The proUsing the model parameter values found in The lactating mother and infant studies discussed below represent moderately low intake of iodide over a long duration. Simulations for model evaluation of moderate iodide deficiency were carried out using the model calibrated for the euthyroid lactating mother and infant and simply decreasing the daily intake of iodide. The ability of the model to predict urinary concentrations of iodide in both the lactating mother and infant and breast milk concentrations of iodide were used as a guide to estimate the daily intake of iodide. The model compartments (iodide and thyroid hormones) for the calibrated euthyroid lactating mother and nursing infant were set to starting values at birth (mass for each compartment) determined from simulations in the pregnant mother and term birth fetus , assuminCosteira et al.  reportedAndersen et al.  conducteEltom et al.  conducteHannan et al.  reportedBouhouch et al.  conducteThe most data rich, moderate iodine deficient study, for early postpartum maternal-infant pairs was from New Zealand . A randoi is the model output resulting from the 1% increase in the parameter value, P is the original parameter value, and Pi is the parameter value increased by 1%. Parameters with maximum absolute NSC values over 90 days exceeding 0.1 were considered to be sensitive. Manual time dependent  local sensitivity analyses were performed for model parameters described with Table functions.A time-dependent 90 d local sensitivity analysis (SA) was carried out using acslX to assess the impact of a 1% increase in model parameter values in predicting serum fT4 for the lactating mother and for the nursing infant. The local SA was conducted assuming a dietary iodide intake 250 μ/d (iodide sufficiency) or 50 μ/d (iodide deficiency). The normalized sensitivity coefficient (NSC) was calculated using the following equation :NSC=. The iodide ingestion rates of 250 and 400 μg/d were divided equally among 3 meals over a 12 hr period each day.−5 L/hr/kg (0.0002 L/hr). The maximal rate of uptake of iodide from mammary plasma (across the mammary duct tissue) into milk (VmaxmamC) was visually fit to milk iodide concentrations [Some model parameters were set as described in trations  using a The lactating mother urinary iodide from Leung et al.  was incrPredictions of urinary iodide in the infant  were conAssuming a maternal iodide intake of 250 or 400 μg/d, the model predicted the central tendency of the 15 maternal spot urinary iodide concentrations , which rThe calibration of the iodide sub-models for mother and infant required thyroid hormones. An iterative process was employed to calibrate the lactating mother and nursing infant sub-models for T4 and T3 and iodide.−6 /hr/kg), and deiodination or metabolism of T4 . The serum fT4 fraction  was also fit to NHANES 2007–2012 data [Using the maternal iodide model parameters in 012 data . Model p−3 /hr/kg) values were fit to NHANES 2007–2012 data  and metabolism of T3  and 3 months postpartum . Mean and reference intervals were reported for T4 for lactating women from Belgium (range only = 50–150 nmol/L), Hungry , Nigeria , and Sweden .The model predicted euthyroid serum T3 concentration in the lactating mother was 1.4 nmol/L at 90 days postpartum, which is similar to the reported values from other countries. Serum total T3 concentrations were reported for 90 days postpartum in lactating women from Sudan , Hungry , Nigeria , and Sweden  .With the thyroid hormone model for the nursing infant calibrated using the data set of Lem et al.  and mateFor lactating women-infant pairs from Portugal , 64 simuIn the nursing infant good agreement was obtained between observation and prediction  urinary iodide . As statOther moderately iodide deficient data sets were simulated by lowering the maternal intake of iodide from 250 to 50 μg/d . GeneralFor the lactating mother-infant pairs from New Zealand, a maternal iodide intake of 50 μg/d for iodide provided partial agreement between model predictions and observations (mean ±SD) for maternal urinary . For breAs expected, the most sensitive parameters (SA >0.5 and <1.1) for the euthyroid and iodide-deficient mother and infant included parameters directly related to fT4 in serum  in many cases by extracting the data from the literature to describe the infant HPT axis. Scaling of these age dependent model parameters based simply on body weight during this rapid growth period was not adequate.The data sets for iodide in breast milk and urine of the lactating mother and infant were variable, perhaps reflecting several factors such as time since last meal, variable dietary intake of iodide and for urine, and fluid hydration. Model predictions, generally speaking, were adequate for many data sets. Recommendations for sufficient iodide (median concentrations on a population basis) during lactation have been reported for urinary iodide in infants less than two years of age (≥ 100 μg/L ), and ioThe American Thyroid Association and the Endocrine Society have recommended that lactating women take vitamins containing 150 μg of iodide daily to supplement their dietary intake of iodide. This recommendation stems from NHANES reports of low individual maternal urinary iodide concentrations in women of childbearing age and pregnant women –75. The Model predictions of thyroid hormones in the lactating mother, calibrated using a modest data set reported from NHANES, and compared to other data sets from several countries, was moderately successful, within a factor of two for T4 and fT4, and in agreement with serum T3 levels. These results raise questions about the reproducibility of analytical methods for measuring thyroid hormones in serum  and poteThe infant model parameters were primarily derived from data in infants, both for iodide and thyroid hormones, including reference intervals for serum thyroid hormones. This was important for the successful description of thyroid hormones in the infant because of the rapid changes that occur in early life. Nevertheless, the calibrated infant model did under predict serum total T4 levels for a few data sets, while acceptable model predictions occurred for infant serum fT4 and T3 concentrations. This was considered an excellent outcome. The variation in measured serum levels of thyroid hormones and iodide in breast milk and urine of mother and the infant demonstrate that a computational description of the HPT axis is a multifaceted problem.Using the calibrated model to predict iodide concentrations for lactating women who were moderately iodide deficient was successful for an intake of 150 μg/d of iodide  and for Although model parameters were not adjusted, to obtain better agreement between observation and prediction, iodide deficiency may enable non-TSH mechanisms to maintain serum thyroid hormones levels. Vermiglio et al. , who werPrevious investigators have speculated about the importance of lactational transfer of thyroid hormones from mother to infant , 81, 82.The construction of a pharmacodynamic model for thyroid hormones and a kinetic model for iodide require the integration of many published human datasets. While the assimilation of data from diverse areas is a strength of this methodology, it also represents a challenge. There was substantial variability in the reported datasets for iodide concentrations in biological samples. To reduce the uncertainty in model predictions, Monte Carlo methods would help address population differences in both iodide intake and resulting serum, breast milk, and urine iodide concentrations and provide statistical inferences on serum thyroid hormone concentrations as a function of iodide intake. Even with its limitations, this deterministic model provides a quantitative tool to help understand complex interactions of an endocrine system during early development of an infant. The most sensitive model parameter for the euthyroid and iodide deficient infant was thyroidal iodide stores, which was only measured for near birth. More ontogeny information is required to improve the model predictions, perhaps using imaging technology.The model code will be provided upon request, written to be executed using acslX simulation language (Version 3.0.2.1) coupled m files containing data sets and other programming information . All differential equations have been previously described , 19–21, S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 TableModel predictions of serum thyroid hormones were in agreement, less than (↓), or greater than (↑) statistically derived upper and lower serum thyroid concentrations.(DOC)Click here for additional data file.


Perennial ryegrass endophyte toxicosis in sheep, caused by ingestion of alkaloids, is characterized by staggers and heat stress resulting in poor production or, in some cases, death. This study investigated effects of alkaloid consumption in sheep housed under thermoneutral versus hot ambient temperature on some production and physiological responses. Alkaloid ingestion and extreme heat produced a similar heat stress response and, when combined, this response was exacerbated. Productivity and health and welfare may be compromised when sheep are consuming alkaloids, and this effect is most severe when alkaloid consumption occurs under hot environmental conditions.p = 0.008), and tended to reduce liveweight (p = 0.07). Rectal temperature and respiration rate were increased by both alkaloid and heat . Respiration rate increased to severe levels when alkaloid and heat were combined, indicating the short term effects which may be occurring in perennial ryegrass toxicosis (PRGT) areas during severe weather conditions, a novel finding. When alkaloid ingestion and heat were administered separately, similar physiological responses occurred, indicating alkaloid ingestion causes a similar heat stress response to 38 °C heat.A controlled feeding study was undertaken to determine the physiological and production effects of consuming perennial ryegrass alkaloids (fed via seed) under extreme heat in sheep. Twenty-four Merino ewe weaners  were selected and the treatment period lasted 21 days following a 14 day acclimatisation period. Two levels of two factors were used. The first factor was alkaloid, fed at a nil (NilAlk) or moderate level . The second factor was ambient temperature applied at two levels; thermoneutral  or heat , resulting in four treatments, NilAlk TN, NilAlk Heat, Alk TN and Alk Heat. Alkaloid consumption reduced dry matter intake ( Lolium perenne) is the primary forage used in the cool temperate, winter-spring rainfall region of Australia because of its persistence and productivity [Epichloe festucaevar Lolii.), an internal living fungi which exists in a symbiotic relationship with the plant. The endophyte produces alkaloids during stressful times of the year (mainly summer and autumn) for the plant to reduce insect attack and overgrazing and increase plant persistence and productivity [Perennial ryegrass (PRG) , is a significant animal disease in Australia. During a severe outbreak in 2002, there were 29,000 confirmed sheep deaths, however, it is thought this number only represented a third of farms affected . Body tevs. hot ambient temperature, as experienced in summer and autumn in Australia, on some production and physiological responses. A previous study undertaken by the authors  investigThe experimental protocol was reviewed and approved by the University of Melbourne, Science, Optometry and Vision Sciences and Land and Environment Animal ethics Committee (704727.1). Twenty four Merino ewe weaners aged 6 months (30.8 ± 1.0 kg) were sourced from The University of Melbourne , Dookie campus. Sheep were housed in group pens for seven days , followed by a seven day acclimatisation period to the metabolism crates (steel crates with a wire mesh floor measuring 1.5 m long and 0.6 m wide and raised 0.5 m from the ground) and controlled temperature room before a 21 day treatment period. Sheep were fed a high fibre pellet (9.9 MJ/kg ME and 11.6% CP) with the addition of whole barley (12.8 MJ/kg ME and 12.0% CP), throughout all stages of the experiment, twice daily, ad libitum. Ad libitum feed intake was maintained by increasing feed offered by 10% to sheep which consumed 90% of feed the prior day.Sheep were PRG naïve at the beginning of the experiment. Alkaloid was administered via whole wild-type endophyte infected PRG seed and this was mixed with whole barley to improve palatability of the seed. Most sheep consumed the mix of seed and barley presented, however, there was some individual sheep variability, with some sheep sorting through the feed, avoiding the seed. The use of this simple mixing method proved to be the most effective way of monitoring and ensuring sheep consumed the seed presented. A previous study using goats found only a small percentage (1.6%) of whole perennial ryegrass seed passes through to the faeces  so seed Alkaloid dosing levels were based on ergovaline and lolitrem B concentrations used in previous studies ,16, whenSheep were fed seed/barley at 8:00 AM while pellets were fed twice daily (8:00 AM and 4:00 PM) with the seed/barley mix provided first to ensure consumption. Feed and water intake were measured daily. Alkaloid intake was estimated by measuring the amount of seed in the feed refusal each day. Liveweight was measured on day 0 and 21, prior to feeding. Urine and faecal separators were placed on the metabolism crates on days 5 and 11–17, and 24 h 10% volume/weight samples of faeces were collected and volume of urine was recorded. Faecal samples were weighed and dried at 100 °C for 24 h or until samples reached a stable weight and the dry matter (DM) content was determined. Rectal temperature, respiration rate and skin temperature (measured on the back and leg) were measured at three hourly intervals commencing at 8:00 AM and finishing at 5:00 PM on days 2, 5, 9, and 12 of the treatment period. Rectal temperature was measured using a hand held digital thermometer . Respiration rate was measured by counting the number of breaths taken, via flank movements, in 30 s intervals. Skin temperature was measured on the back and hind leg by parting the wool and holding a hand held digital thermometer  to the skin for 30 s.Ambient temperature and relative humidity were measured using a temperature and humidity probe with a data logger attached . vs. Alk), temperature (TN vs. Heat) and time , and the random effect was sheep ID. For DMI, faecal water, urine output, and physiological parameters the data was analysed for weekly responses. To analyse changes over a 9 h period on day 12 for physiological parameters the model included fixed effects of alkaloid, temperature and time (hour), and the random effect was sheep ID. Covariates were also included in the analyses using baseline data or day 0 data where appropriate.Statistical analysis was undertaken using the Genstat statistical package . For all parameters significance was analysed using general analysis of variance. The model included fixed effects of alkaloid .Sheep consumed 88% of the infected seed offered in the Alk TN treatment and 68% of infected seed in the Alk Heat treatment . There was a significant alkaloid × week interaction , while temperature had no effect . There were no significant interactions for liveweight or ADG. Average daily gain over the experimental period tended to be less for the Alk treatment . There was an interaction between alkaloid and week, such that water intake increased over the treatment period in the Alk treatment (p = 0.008). Urine output was variable in the Alk treatment over the experimental period, increasing largely over the first week  and in the Heat treatment . There was an alkaloid x week interaction  and Heat treatment . There was a tendency for a temperature × week (p = 0.07) interaction such that respiration rate increased over time in the Heat treatment but not in the TN treatment. Data for rectal temperature and respiration rate are presented in p = 0.004) and Heat treatment . Similarly, leg skin temperature was higher in the Alk  and Heat treatments . There was a significant alkaloid × week interaction for leg skin temperature  interaction such that the Heat treatment was variable over time compared to the TN treatment.Over the nine hour sampling period on day 12, rectal temperature increased in the Alk and Heat treatments , while the Alk TN and NilAlk Heat groups were similar at most time points (apart from 8:00 AM and 3:00 PM).Respiration rate increased in the Alk and Heat treatments  such that the Heat treatment increased skin temperature over time, while the TN treatment remained lower.Back and leg skin temperature increased in the Alk and Heat treatments  and in response to Heat (+15%) with these effects being additive. A previous study measured water intake when sheep were exposed to wild-type and AR37 perennial ryegrass pasture. This study found water intake to increase by 25% when sheep consumed wild-type pasture and when ambient temperature stayed above 20 °C  which isAlkaloid ingestion and heat resulted in increased rectal temperature and respiration rate. This is probably due to the relatively high heat load imposed by the heating regimen. Presumably, the magnitude of this was such that elevation of respiration rate was insufficient for heat to be dissipated adequately during the TN period as indicated by the 9 h physiological profiles presented in Leg and back skin temperature increased due to alkaloid consumption and heat. This may have been due to vasoconstriction occurring in the extremities  of the sThis study demonstrated that feeding alkaloids under extreme heat conditions can compromise sheep productivity in the form of decreased feed intake and may reduce sheep liveweight over a longer period of time. Alkaloid intake, when fed via seed, combined with 38 °C heat resulted in respiration rate increasing to severe levels. This is the first study to elucidate the severity of PRGT heat stress under ambient temperature conditions similar to those experienced during a severe southern Australian summer, when toxin intake was known. Interestingly, the findings also indicate that alkaloid ingestion and 38 °C heat produce a similar heat stress response. Overall, the findings suggest that producer profitability along with sheep health and welfare may be compromised when sheep are consuming alkaloids or during severe weather events, such as extreme heat, and this effect is most severe when alkaloid consumption occurs under hot conditions. Further study investigating longer term effects of alkaloids under natural southern Australian summer conditions, when toxin intake is known, is warranted to further elucidate the effects of alkaloid consumption and heat on animal production and heat stress, reflecting that experienced in the field.


Conventional, intravenous, patient-controlled analgesia, which is only administered by demand bolus without basal continuous infusion, is closely associated with inappropriate analgesia. Pharmacokinetic model-based dosing schemes can quantitatively describe the time course of drug effects and achieve optimal drug therapy. We compared the efficacy and safety of a conventional dosing regimen for intravenous patient-controlled analgesia that was administered by demand bolus without basal continuous infusion (group A) versus a pharmacokinetic model-based dosing scheme performed by decreasing the dosage of basal continuous infusion according to the model-based simulation used to achieve a targeted concentration (group B) following robot-assisted laparoscopic prostatectomy.In total, 70 patients were analyzed: 34 patients in group A and 36 patients in group B. The postoperative opioid requirements, pain scores assessed by the visual analog scale, and adverse events  were compared on admission to the postanesthesia care unit and at 0.5, 1, 4, 24, and 48 h after surgery between the 2 groups. All patients were kept for close observation in the postanesthesia care unit for 1 h, and then transferred to the general ward.P < 0.05). There were no differences in the adverse events between the 2 groups.The fentanyl requirements in the postanesthesia care unit for groups A and B were 110.0 ± 46.4 μg and 77.5 ± 35.3 μg, respectively. The pain scores assessed by visual analog scale at 0.5, 1, 4, and 24 h after surgery in group B were significantly lower than in group A (all We found that the pharmacokinetic model-based dosing scheme resulted in lower opioid requirements, lower pain scores, and no significant adverse events in the postanesthesia care unit following robot-assisted laparoscopic prostatectomy in comparison with conventional dosing regimen. Therefore, conventional dosing regimens for intravenous PCA,2 which are performed by intermittent bolus administration without the basal continuous infusion of opioids, may exhibit inadequate analgesic effects during the immediate postoperative period in patients in the postanesthesia care unit who have undergone robot-assisted laparoscopic prostatectomy (RALP).Laparoscopic surgery may induce more severe pain in comparison with open laparotomy due to inappropriate pain management during the immediate postoperative period.3 Together with these properties, its tolerability and wide therapeutic range make fentanyl a suitable opioid for PCA.2 In terms of the respiratory effects of fentanyl, the basal continuous infusion of 0.12 to 0.67 μg/kg/h fentanyl does not result in significant respiratory depression.5 However, the basal continuous infusion of intravenous PCA is not usually recommended for postoperative pain management due to the risk of opioid accumulation and concerns about respiratory depression.6 As respiratory depression is closely related to high plasma concentrations of fentanyl, which may be caused by not only continuous infusion but also by high bolus doses,8 target-concentration strategies may be safer and more effective than conventional dosing regimens. However, little is known about the efficacy and safety of pharmacokinetic model-based fentanyl dosing schemes, which can quantitatively describe the time course of drug effects and achieve optimal drug therapy,9 for adequate pain management following RALP.The pharmacokinetic characteristics of fentanyl include its short duration of action, high lipid solubility, rapid distribution to fat and skeletal muscle, and a high extraction ratio drug.In our present study, we compared the opioid requirements in the postanesthesia care unit, visual analog scale (VAS) scores for pain, and adverse events during the postoperative period between a conventional dosing regimen and pharmacokinetic model-based dosing scheme in patients who underwent RALP.2. Patients who had taken opioids or analgesics before surgery were also excluded. Before surgery, all patients were instructed how to use the PCA pump and VAS for pain scoring by our acute pain service team. No patients were premedicated before surgery.The protocol was approved by the institutional review board of Asan Medical Center, Seoul, Republic of Korea , and written informed consent was obtained from patients with prostate cancer who underwent RALP between April 2015 and September 2015. This study was registered with the ClinicalTrials. gov database (NCT02402621). Exclusion criteria included allergies to opioids, history of chronic pain, history of alcohol or drug abuse, history of sleep apnea or respiratory complications, American Society of Anesthesiologists physical status ≥ 3, age < 20 years or ≥ 70 years, and body mass index > 30 kg/mAfter applying a routine monitoring system , general anesthesia was induced using a bolus intravenous injection of 5 mg/kg thiopental sodium and 0.6 mg/kg rocuronium and maintained using 5 to 6 vol% desflurane plus an effect-site target-controlled infusion 2 to 5 ng/mL remifentanil with 50% oxygen in medical air. Patients were mechanically ventilated at a constant tidal volume of 6 to 8 mL/kg, and the respiratory rate was adjusted to maintain the end-tidal carbon dioxide partial pressure between 35 and 40 mm Hg during the operation.10Patients were randomly allocated to receive the conventional dosing regimen performed by intermittent bolus administration without basal continuous infusion (group A) or the pharmacokinetic model-based dosing scheme performed by decreasing the dose of basal continuous infusion according to the model-based simulation in order to achieve a targeted concentration (group B) Figure . Figure 12 which indicates severe pain, the physician intravenously administered a bolus of 25 μg fentanyl. The opioid requirements in the postanesthesia care unit were measured. All patients were kept for close observation in the postanesthesia care unit for 1 h and then transferred to the general ward. VAS scores and adverse events  were measured on admission to the postanesthesia care unit and at 0.5, 1, 4, 24, and 48 h after surgery by an investigator who was blind to the treatment groups. The definitions of respiratory depression, desaturation, and sedation were a respiratory rate < 12 breaths/min, oxygen saturation < 90%, and level of sedation so that the patient has a normal response to verbal stimuli, respectively. The total fentanyl dose administered during the postoperative period was calculated by combining the infused fentanyl doses and any other opioids or analgesics that were administered during the postoperative period. All opioids and analgesics were recorded and converted to fentanyl-equivalent doses using morphine sulfate equivalents.15 The total duration of surgery (from the start of surgery to the end of surgery in minutes) and time to extubation from the end of surgery (from the end of surgery to extubation in minutes) were also recorded.In the postanesthesia care unit, if the VAS score  of the patient was ≥ 6,16 and below the concentration that can result in unwanted side effects4—was determined according to the pharmacokinetic parameters of fentanyl as follows:10Vcentral (L): 26.6, k10 (min–1): 0.0332, k12 (min–1): 0.172, k13 (min–1): 0.131, k21 (min–1): 0.1, k31 (min–1): 0.0177, and Ke0 (min–1): 0.147. In this present study, we determined the targeted concentration, which is slightly higher than the minimal effective concentration throughout the postoperative period, and then simulated the cumulative drug amount in order to maintain these concentrations using NONMEM® VII level 2 . After simulating the time-concentration curve using various dosing schemes, we selected the optimal dosing regimen that could maintain the appropriate concentrations and entered the basal rate into the intravenous PCA infusion pump . Deterministic simulations using point estimates of the fixed-effect parameters were performed to provide an illustration of the predicted time courses of the plasma and effect-site concentrations after fentanyl administration. Variances in the random-effect parameters  were set to 0. Figure The optimal dosing scheme for model-based PCA infusion—that is, maintaining the target concentration above the minimal effective concentration of 0.23 ng/mL17 The total estimated number of patients was 73 after considering a 10% dropout rate. Data such as demographics, surgical duration, time to extubation from the end of surgery, opioid requirements, and VAS score were compared with using Student's t test or Mann–Whitney U test, as appropriate. The American Society of Anesthesiologists physical status and incidences of adverse events were measured by using the χ2 test or Fisher's exact test, as appropriate. Statistical analysis and randomization were conducted using R , SigmaStat 3.5 for Windows , and SPSS 22 for Windows . Data are expressed as the mean ± SD, or number (percentage) as appropriate. In this study, P < 0.05 was considered statistically significant.The sample size was estimated based on the power calculation, which showed that ≥33 patients per group were necessary to achieve 80% power and detect a 20% difference between computer-controlled alfentanil infusion and conventional PCA morphine infusion in order to reduce opioid requirements.This study included 73 patients who underwent RALP. After randomization, however, 3 patients were not included in the analysis due to the development of intraoperative paroxysmal atrial fibrillation with a rapid ventricular rate, refusal to participate in the trial just before induction, or an unanticipated change to laparotomy due to severe peritoneal adhesion, respectively Figure .The patient characteristics and outcomes for groups A and B are compared in Table Among our patients who underwent RALP, the pharmacokinetic model-based dosing scheme for fentanyl PCA demonstrated significant reductions in opioid requirements in the postanesthesia care unit and pain scores assessed by VAS during the postoperative period without developing any significant opioid-related complications in comparison with the conventional dosing regimen. Our data suggest that both the plasma and effect-site concentrations did not decrease below the minimal effective concentration of fentanyl in the model-based PCA group. However, in the conventional PCA group, we believe the fentanyl concentration decreased to below the minimal effective concentration around admission to postanesthesia care unit, which may have resulted in moderate to severe pain.20 Although fentanyl has a wide therapeutic window, large on-demand doses or high basal infusion rates are associated with the risk of respiratory depression.22 In addition, the long context-sensitive half-time of fentanyl after a long period of infusion results in a dramatic increase in its concentration, which may be associated with postoperative complications such as respiratory depression, nausea, and vomiting.24 For these reasons, basal continuous infusion is not routinely recommended as a postoperative analgesia.Fentanyl is a highly lipophilic drug, which facilitates its distribution into the brain, and is well suited for intravenous PCA. Furthermore, using fentanyl for intravenous PCA demonstrates considerable analgesic efficacy with good tolerability.1 Furthermore, in our present study, the administration of only the demand dose of fentanyl—without basal continuous infusion—demonstrated inadequate pain relief in the postanesthesia care unit in patients who underwent RALP. Similar to the previous simulation results, the fentanyl concentration progressively decreased to the minimal effective concentration at ∼30 min after the first fentanyl bolus injection. This time course of the concentration was inversely related to the trends of the pain scores, which increased abruptly after admission to the postanesthesia care unit and peaked at 30 min after admission. In addition, the simulated fentanyl concentration of the demand dose plus an additional fentanyl bolus in the postanesthesia care unit showed not only an abrupt increase in the fentanyl effect-site concentration to > 2 ng/mL—which may produce fentanyl-related complications—but also that the fentanyl concentration could have decreased to below the minimal effective concentration of 0.23 ng/mL and thereby resulted in inappropriate pain management. Considering the appropriate dosing scheme for managing postoperative analgesia, there are problematic concerns regarding the lack of basal continuous infusion with a demand bolus dose, which may have led to an insufficient fentanyl concentration below the minimal effective concentration.However, pain after laparoscopic surgery is known to be more intense during the immediate postoperative period in comparison with after open laparotomy.9 The model-based dosing scheme is very popular in drug development area, especially in first-in-human studies and trials on optimal dosing regimens.26 In addition, these approaches helped to achieve successful registration of the drug and were identified as a more efficient trial design that reduced the required number of subjects and thereby saved time. Therefore, model-based PCA, which can restrict concentrations within the therapeutic range, can provide tailored postoperative analgesic management approaches.Taken together, we adapted pharmacokinetic model-based PCA as an advanced method for reducing the period during which the fentanyl concentration is below the minimal effective concentration during the postoperative period. We found that the model-based dosing scheme reduced analgesic requirements in the postanesthesia care unit and pain scores during the postoperative period among patients who underwent RALP. Pharmacokinetic model-based dosing schemes, which can provide a quantitative description of the time course of drug effects and offer great potential for achieving optimal drug therapy, are increasingly used within most therapeutic areas.27 except children and overweight patients.29 Therefore, our results are promising for the postoperative pain management of patients who undergo RALP.In terms of study limitations, we did not examine the predictive performance of the simulated PCA regimen, which should take blood samples at predetermined time points. In addition, our simulation did not include interindividual variations in the pharmacokinetic parameters of fentanyl. However, the pharmacokinetic model of fentanyl is largely accepted to be valid for all patients,In conclusion, pharmacokinetic model-based PCA, which can provide optimal basal infusion in order to achieve a concentration above the minimal effective concentration, significantly decreases the opioid requirements and pain scores without inducing serious adverse events immediately following RALP. These results provide a better understanding of effective pain management following RALP.


The aim of this study was to evaluate the clincal value of multi-slice spiral computed tomography (MSCT) in the diagnosis of double aortic arch (DAA) and analyze the associated angiography and 3D reconstruction imaging to increase the accuracy of DAA diagnosis. Clinical and imaging data from 15 patients diagnosed with DAA by MSCT were summarized and compared with the corresponding surgical observations. The DAA diagnosis by MSCT for the 15 patients was confirmed by the surgical observations. A total of 13 cases were categorized as type I (double arches are open), including nine with a larger right arch, two with a larger left arch and two with balanced arches. Two cases were categorized as type II (one atretic arch), characterized by left atretic arch. Among the 15 patients, ultrasound diagnosis succeeded in nine cases and failed in the rest. A single malformation was identified in only two cases, whereas the rest had single or multiple combination(s) of intracardiac and extracardiac malformations, including seven with different levels of tracheobronchial stenosis. MSCT was demonstrated to precisely diagnose DAA complicated by malformation and tracheobronchial stenosis. MSCT is an essential therapeutic strategy and serves as a primary method in DAA diagnosis. Double aortic arch (DAA) is the most common form of vascular ring and accounts for 33–73% of vascular ring malformations . DAA entA total of 15 cases  diagnosed with DAA malformation through computed tomography (CT) and confirmed by surgery were recruited from the Wuhan Asia Heart Hospital  between May 2005 and March 2013. All patients underwent a chest X-ray and echocardiography concurrently. The pediatric patients exhibited different levels of wheezing, coughing, and shortness of breath or failure of the respiratory system, and nine of them had feeding difficulties. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of Wuhan Asia Heart Hospital. Written informed consent was obtained from the subjects or their legal guardians.A Brilliance CT 64-channel scanner  or Somatom Definition Flash dual-source CT scanner  was utilized for the scanning. Patients incapable of coordination fasted for 4–6 h and were then anesthetized (polyphenols: 2.0–2.5 mg/kg) and scanned. Patients capable of respiratory coordination were asked to hold their breath for 15–20 sec after inspiration during the scanning. Electrocardiogram (ECG)-gating and oxygen inhalation were employed, and ECG and saturation of blood oxygen were detected during the scanning. A non-ionic contrast agent  and a dual syringe injector  were used, and the injection duration was 15–22 sec. The injection dose and flow speed of the Ultravist were as follows: If ≤12 years old, 2.0–2.5 ml/kg and 2.0–2.5 ml/sec; and if >12 years old, 60–80 ml and 3.0–3.8 ml/sec; followed with 4–15 ml saline injection at 0.8–3.0 ml/sec. If ≤12 years old, delayed scanning was employed after the injections, with a delay time of 15–19 sec; and if >12 years old, bolus tracking and its automatic triggering were employed to monitor the enhancement of the descending aorta, and the view of interest was set in the descending aorta on the aortopulmonary window level, with a threshold value of 100–140 HU. The scanning scope ranged from the upper edge of the sixth cervical vertebra to 1 cm below the lower edge of the apex. The Brilliance CT 64-channel scanning parameters were as follows: 80–120 kV, 100–300 mAsec, pitch of 0.2–0.3, X-ray tube rotation time of 0.4 sec/round, 180×180 to 300×300 mm field of view (FOV), matrix size of 512×512, slice thickness of 0.67 mm, reconstruction interval of 0.33 mm, reconstruction phase 40% and 75%. The Somatom Definition Flash dual-source CT scanning parameters were as follows: 80–120 kV, 100–300mAsec, pitch of 0.2–0.5, X-ray tube rotation time of 0.33 sec/round, 180×180 to 300×300 mm FOV, matrix size of 512×512, slice thickness of 0.75 mm, reconstruction interval of 0.40 mm, and the reconstruction phase was automatically determined by a computer to select the best systolic and diastolic periods. Following the scanning, the original data were uploaded to the Philips Extended Brilliance Workspace (Koninklijke Philips N.V.).All subjects were asked to wear lead eyeglasses, scarves and aprons below the abdomen to reduce the radiation damage to the thyroid, gonads and eyes.All diagnoses were provided by at least two experts in cardiovascular imaging following the independent analysis of the CT imaging results and clinical features according to the principle of DAA pathological anatomy. The tracheal stenosis and its location were also assessed. Agreements were obtained and in-depth discussions were held when disagreements occurred. The post analysis included multi-planar reconstruction (MPR), maximum intensity projection (MIP), minimum intensity projection (MinIP) and volume rendering (VR).All 15 patients with DDA were precisely diagnosed using MSCT and they were as follows: 13 cases of type I (double arches are open), including nine with a larger right arch , two witSeven patients exhibited complications caused by different levels of tracheobronchial stenosis. The MPR, VR and MIP analyses revealed the stenosis of the lower trachea or the bronchus that was involved due to compression  and 6.et al  The two aortic arches are open, generally with a larger right arch and a smaller left arch  and ii) MSCT is a non-invasive method characterized by a short scanning time, rapid imaging, high time and spatial resolution, and higher isotropic of Z-axis direction. The powerful post-processing function of MSCT allows for the observation of lesions from different angles. MSCT not only clearly reveals the shape and direction of an abnormal vascular ring but also accurately identifies the situation of the tracheal and esophageal compression, which is essential in the diagnosis of pediatric cardiovascular and respiratory diseases ,14. The Angiocardiography is a conventional method that is considered the gold standard in diagnosing DAA due to its high selectivity and excellent capability to indicate anatomy and blood flow in the main pulmonary artery and its branches. However, angiocardiography is invasive, difficult for patients (particularly pediatric patients) to tolerate, and unable to show the compression of the esophagus and trachea as a 2D image. Plain chest radiography and the barium swallow test have been used as auxiliary methods in DAA diagnosis; the two methods reveal indirect signs, including compression of the trachea and esophagus by the aorta and the pulmonary artery and its branches . EchocarIn summary, patients with DAA commonly suffer from complications caused by intracardiac and extracardiac malformations and different degrees of airway stenosis, whereas DAA with one-side arch atresia mostly involves left arch atresia. MSCT precisely diagnoses DAA complicated by anomalies and airway stenosis. MSCT is important in treating DAA and thus serves as the primary tool for DAA diagnosis.


To summarize the relationship between type 2 diabetes mellitus (T2DM) and risk of colorectal adenomas (CRA), we performed a meta-analysis of observational studies.To find studies, we searched PubMed, Embase, the Cochrane Library, Web of Science and conference abstracts and related publications for American Society of Clinical Oncology and the European Society of Medical Oncology. Studies that reported relative risks (RRs) or odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between T2DM and risk of CRA were included. The meta-analysis assessed the relationships between T2DM and risk of CRA. Sensitivity analyses were performed in two ways: (1) by omitting each study iteratively and (2) by keeping high-quality studies only. Publication bias was detected by Egger’s and Begg’s tests and corrected using the trim and fill method.This meta-analysis included 17 studies with 28,999 participants and 6798 CRA cases. We found that T2DM was a risk factor for CRA , and also for the advanced adenoma . Patients with existing T2DM  or newly diagnosed T2DM  have a risk of CRA. Similar significant results were found in retrospective studies  and population based cross-sectional studies , but not in prospective studies .Our results suggested that T2DM plays a risk role in the risk of developing CRA. Consequently, medical workers should increase the rate of CRA screening for T2DM patients so that they can benefit from behavioural interventions that can help prevent the development of colorectal cancer. Additional, large prospective cohort studies are needed to make a more convincing case for these associations.The online version of this article (doi:10.1186/s12885-016-2685-3) contains supplementary material, which is available to authorized users. Diabetes mellitus (DM) is the fourth or fifth leading cause of death in developed countries and one of the biggest threats to human health worldwide . More thResearch on risk factors for CRA has focused on several epidemiological factors, including smoking , alcoholTwo investigators (FY and YG) independently conducted a systematic literature searches on January 10, 2016 in PubMed, Embase, the Cochrane Library and Web of Science without limiting the publication date range. The following search terms were used: (diabetes mellitus OR diabetes OR diabetic OR glucose) AND  AND (adenomas OR adenoma OR adenomatous OR polyp). No language restrictions and any other limitations were imposed. Conference abstracts on the websites of American Society of Clinical Oncology’s (ASCO) and the European Society for Medical Oncology’s (ESMO) annual meetings were also searched, along with the reference lists of the identified publications. Additional file The titles and abstracts of all of the studies from the searches were screened independently by three reviewers . To be included in this meta-analysis, studies had to be at least one of the following criteria: (1) retrospective or perspective observational study of the association between diabetes mellitus and CRA, or (2) a study reporting the relative risks (RRs) or odds ratios (ORs) for T2DM on CRA with 95 % confidence intervals (95 % CIs) adjusted for gender, age, or other factors. Studies reporting on the CRA recurrence were excluded.Data extraction was performed by three reviewers , and verified independently for accuracy by a forth reviewer (JH). The following information was collected for each study: title and author, publication year, population, location, sample size, proportion of males and covariates controlled for by matching or multivariate analysis. For studies that reported several multivariate adjusted ORs, the effect estimate that adjusted for the maximum potential confounders was selected. Two investigators (FY and ZJ) conducted a quality assessment using the 9-star Newcastle-Ottawa Scale (NOS) , which w2) statistic [p < 0.10 [We examined the relationship between T2DM and CRA risk on the basis of the adjusted RRs and ORs and corresponding 95 % CI published in each study. A fixed effects model was used to estimate the pooled RR and OR with 95 % CIs if there was no evidence of heterogeneity; otherwise, a random effect model was used , 24. Bectatistic . Statistp < 0.10 .Several methods were used to test and adjust for potential publication bias. Visual inspection of funnel plots was performed, and the Egger’s regression test  and Beggp < 0.05 Stata  was used for all analyses.All statistical tests were two-sided and regarded as statistically significant at Until January 10, 2016, 2522 records were retrieved by using our search strategy. After reviewing the titles and abstracts, 113 articles were further evaluated by reviewing the full texts. Of those remaining articles, we excluded studies that : (1) reported the data of adenoma recurrence were excluded , 32, (2)I2 = 65.6 %, P < 0.001). Figure The summary RR of diabetes on CRA was statistically significant . Evidence of the heterogeneity was identified  and Egger’s regression test (p = 0.003) results showed potential publication bias that is described in Fig. The Begg’s rank correlation test  might take effect in the adenoma–carcinoma process. High insulin levels could promote tumor growth , 45, 46.Some studies reported a difference in risk between males and females , 53–55; Our analysis revealed that with T2DM have about a 5 % higher risk of CRA than newly diagnosed diabetes patients, revealing the duration of T2DM as a risk factor for CRA. A possible explanation is that known T2DM patients’ bowels are exposed to hyperinsulinemia or a hyperglycemic environment for a longer time, and such hormonal or metabolic abnormalities (according to former study ) could aSeveral limitations of in this meta-analysis that should be taken into consideration. First, results for several subgroups, such as gender and adenoma sub-site subgroup, were based on a limited number of studies. Therefore, we cannot rule out the possibility that insufficient statistical power is present. Second, in the present analysis, some small studies with inverse associations between T2DM and risk of CRA risk seemed to be suppressed. The presence of possible publication bias could have led to an overestimation of the effect of T2DM on CRA risk. However, the adjusted result was comparable after trim and fill method corrections. Third, we could not account for all of the confounding factors in the meta-analysis, though most confounders were adjusted in the original RRs. Many factors might induce the adenomas, such as age, ethnicity, inactivity, regular aspirin use, obesity, and family history of CRA, and menopausal status. We could not control for these covariates because of lack of relevant data. Relevant studies with additional data on these other factors may be found by searching by searching beyond the sources used for this study. Furthermore, we could not determine whether using insulin as a therapy for T2DM is an important factor because CRA risk might be altered by hyperinsulinemia, thought to be an important promoter of carcinogenesis , 63. At In conclusion, the results of our meta-analysis indicated that patients with T2DM have higher risks for the development of CRA, which is an important inducement for colorectal cancer. Our study has important implications for clinical and public health. Because T2DM and CRA are prevalent in the developed and developing countries , medical


Moso bamboo can rapidly complete its growth in both height and diameter within only 35–40 days after shoot emergence. However, the underlying mechanism for this “explosive growth” remains poorly understood. We investigated the dynamics of non-structural carbohydrates (NSCs) in shoots and attached mature bamboos over a 20-month period. The results showed that Moso bamboos rapidly completed their height and diameter growth within 38 days. At the same time, attached mature bamboos transferred almost all the NSCs of their leaves, branches, and especially trunks and rhizomes to the “explosively growing” shoots via underground rhizomes for the structural growth and metabolism of shoots. Approximately 4 months after shoot emergence, this transfer stopped when the leaves of the young bamboos could independently provide enough photoassimilates to meet the carbon demands of the young bamboos. During this period, the NSC content of the leaves, branches, trunks and rhizomes of mature bamboos declined by 1.5, 23, 28 and 5 fold, respectively. The trunk contributed the most NSCs to the shoots. Our findings provide new insight and a possible rational mechanism explaining the “explosive growth” of Moso bamboo and shed new light on understanding the role of NSCs in the rapid growth of Moso bamboo. As the main product of plant photosynthesis, carbohydrates can largely be partitioned into structural carbohydrates (SCs) and non-structural carbohydrates (NSCs) according to their roles1256123910Previous investigations have usually focused on the responses of NSCs to drought stress1213516171917211617181617Phyllostachys pubescens Mazel ex H. de Lehaie) forests are currently the most important source of non-wood forest products in China, covering an area of 3.87 million ha, representing 70% of the country’s bamboo forest area and 80% of the global distribution of P. pubescens262829Moso bamboo  needed for the construction and metabolism of bamboo shoots during EGP are provided by attached mature bamboos via underground rhizomes.In approximately the first 15 days after Moso bamboo shoots emerge, the height growth and biomass accumulation of the shoots were relatively slow . ApproxiP < 0.05).In approximately the first 7 days after Moso bamboo shoots emerged, the NSC content of the bamboo shoots showed an increase to 8.25% . HoweverThe changes in the NSC content of the leaves, branches, trunks and rhizomes of mature bamboos attached to each sampled shoot are shown in et al.Moso bamboo shoots rapidly completed their height growth within 38 days after emerging and reached an average height of 13.26 m, which was consistent with the previous studies2929Pinus sylvestris L.) decreased during bud break16Populus tremuloides clones during leaf flushing. It was also reported that the bud break of broad-leaved trees depended on last season’s carbon reserveset al.After height growth was completed (approximately May 7), new leaves and branches began to grow out from the young bamboo, and this period continued until the middle-to-end of July35After August 2, the NSC pool in the leaves, branches and trunks of young bamboos gradually recovered . It has 38The leaves of young bamboo all fall in the following spring, and then new leaves with a lifespan of two years growWithin 38 days after emerging, Moso bamboo shoots completed their “explosive growth” and reached an average height of 13.26 m and biomass of 3.89 kg. During this period, almost all the NSC content of the leaves, branches, and especially trunks and rhizomes of attached mature bamboos was re-allocated and transferred to the “explosively growing” shoots via underground rhizomes for both the structural growth and the metabolism of the shoots. The transfer process of NSCs from attached mature bamboos to shoots (young bamboos) stopped when the leaves of the young bamboos could provide enough carbohydrates (photoassimilates) to meet the immediate carbon demands of the young bamboos. Afterward, the depleted NSC pool of the attached mature bamboos gradually recovered.The study site was located in Qingshan Town, Lin’an city , Zhejiang Province, China. This area has a monsoonal subtropical climate with four distinct seasons. The mean annual precipitation is 1,420 mm, and the mean annual temperature is 15.6 °C, with maximum and minimum temperatures of 41.7 °C and −13.3 °C, respectively. The area receives an average of approximately 1,847 hours of sunshine per year and an average of 230 frost-free days per year.2O5:K2O: 15:6:20, 450 kg ha−1) are distributed and followed by a deep plowing to 0.3 m. Twelve understory species, dominated by Viola prionantha, achieve a mean height of 0.1 m, a forest floor coverage of 7%, and a total herbal biomass of 16.3 kg ha−1.The Moso bamboo plantations were originally established in the late 1970 s from native evergreen broadleaf forest in sites of similar topography (southwest slope of approximately 6 degrees) and soil type. The soils are classified as Ferrisols derived from graniteThe growth of the shoots of Moso bamboo is strongly seasonal. Shoots are usually initiated from solitary lateral buds on below-ground rhizomes every summer (June–August), then begin to emerge aboveground at the end of the following March and attain their full size approximately two months after emergence. By the middle-to-end of May, the shoots reach the top of the canopy and develop to their full height and DBH, becoming young bamboos282935In November 2013, three plots of 30 × 30 m were established. The initial stand and soil characteristics are summarized in The oven-dried samples were ground with a grinder . The NSC concentration was determined using an improved colorimetric method43One-way analysis of variance (ANOVA) and least significant difference (LSD) tests were used to determine the statistical significance of the differences in the NSC content of leaves, branches, trunks and rhizomes among sampling events. The data were checked and satisfied the assumptions of homogeneity of variance. These analyses were conducted using SPSS  16.0 for Windows software .How to cite this article: Song, X. et al. Dynamic allocation and transfer of non-structural carbohydrates, a possible mechanism for the explosive growth of Moso bamboo (Phyllostachys heterocycla). Sci. Rep. 6, 25908; doi: 10.1038/srep25908 (2016).