Use of novel DNA methylation signatures to distinguish between human airway structural cell types

INTRODUCTION: Chronic inflammatory and fibrotic lung diseases like asthma, COPD and pulmonary fibrosis are characterised by modified phenotype of the airway structural cells. Airway walls are comprised of a robust epithelial layer that lines the lumen followed by the basement membrane, submucosa predominantly composed of fibroblasts and finally enveloped by a bulk of smooth muscle cells that determine the relaxation and constriction of the airways. The phenotype of airway structural cells is determined by epigenetic alterations such as DNA methylation, which alters the activation status of a range of important inflammatory and remodelling genes. Here we determined if airway structural cells (Epithelial cells, fibroblasts and smooth muscle cells) have different DNA methylome signatures that can be used to distinguish between them. This will offer a reference standard for identifying cell type specific DNA methylation changes induced by various inflammatory stimuli. EXPERIMENTAL METHODS: Illumina Human Methylation 450K Beadchip (HM450K) was used to perform genome-wide methylome screening on 17 bronchial fibroblast (BrF), 23 lung parenchymal fibroblast (LgF), 17 airway epithelial cell (Ep) and 6 airway smooth muscle cell (ASM) samples isolated from healthy individuals. The data was normalised using funtoonorm, a specialised algorithm in R developed for multiple tissue types. R packages minfi, limma and DMRcate was used for CpG site exclusion and identification of significant differentially methylated regions (DMR) specific to each of the four cell types. RESULTS AND DISCUSSION: Epithelial cells distinctly separated from other lung cells (791 DMR). LgF, BrF and ASM had 13, 10 and 1 signature DMR respectively. Despite close anatomical proximity, ASM and BrF displayed 2 DMR when compared to each other. Interestingly, fibroblasts obtained from airway showed 6 DMR in comparison to those obtained from lung parenchyma, suggesting that the same cell type obtained from different parts of the lung can have significantly different methylation patterns that might lead to phenotypic differences. CONCLUSION: We have identified cell and tissue specific methylation signatures which can be used to differentiate between different types of airway structural cells. The airway epithelial cells showed the greatest separation from other airway structural cells. The Bronchial fibroblasts varied minimally from airway smooth muscle cells despite its significant separation from airway epithelial cells and parenchymal fibroblasts

The biological embedding of early-life socioeconomic status and family adversity in children's genome-wide DNA methylation.

AimTo examine variation in child DNA methylation to assess its potential as a pathway for effects of childhood social adversity on health across the life course.Materials & methodsIn a diverse, prospective community sample of 178 kindergarten children, associations between three types of social experience and DNA methylation within buccal epithelial cells later in childhood were examined.ResultsFamily income, parental education and family psychosocial adversity each associated with increased or decreased DNA methylation (488, 354 and 102 sites, respectively) within a unique set of genomic CpG sites. Gene ontology analyses pointed to genes serving immune and developmental regulation functions.ConclusionFindings provided support for DNA methylation as a biomarker linking early-life social experiences with later life health in humans

Natural Variation in Maternal Care and Cross-Tissue Patterns of Oxytocin Receptor Gene Methylation in Rats

Since the first report of maternal care effects on DNA methylation in rats, epigenetic modifications of the genome in response to life experience have become the subject of intense focus across many disciplines. Oxytocin receptor expression varies in response to early experience, and both oxytocin signaling and methylation status of the oxytocin receptor gene (Oxtr) in blood have been related to disordered social behavior. It is unknown whether Oxtr methylation varies in response to early life experience, and whether currently employed peripheral measures of Oxtr methylation reflect variation in the brain. We examined the effects of early life rearing experience via natural variation in maternal licking and grooming during the first week of life on behavior, physiology, gene expression, and epigenetic regulation of Oxtr across blood and brain tissues (mononucleocytes, hippocampus, striatum, and hypothalamus). Rats reared by “high” licking-grooming (HL) and “low” licking-grooming (LL) rat dams exhibited differences across study outcomes: LL offspring were more active in behavioral arenas, exhibited lower body mass in adulthood, and showed reduced corticosterone responsivity to a stressor. Oxtr methylation was significantly lower at multiple CpGs in the blood of LL versus HL rats, but no differences were found in the brain. Across groups, Oxtr transcript levels in the hypothalamus were associated with reduced corticosterone secretion in response to stress, congruent with the role of oxytocin signaling in this region. Methylation of specific CpGs at a high or low level was consistent across tissues, especially within the brain. However, individual variation in methylation relative to these global patterns was not consistent across tissues. These results suggest that blood Oxtr methylation may reflect early experience of maternal care, and that Oxtr methylation across tissues is highly concordant for specific CpGs, but that inferences across tissues are not supported for individual variation in Oxtr methylation

Investigating genome wide DNA methylation in bronchial and lung fibroblasts from healthy individuals and individuals with COPD

Rationale: Lung fibroblasts are implicated in respiratory disease pathology including chronic obstructive pulmonary disease (COPD). Phenotypic differences between fibroblasts isolated from the bronchi versus the lung parenchyma have been described but no studies have compared the cell types on a genome wide scale. DNA methylation is a reversible modification of the DNA structure with the ability to affect cell function via the alteration of gene expression. Here we compared genome wide DNA methylation profiles from bronchial and lung fibroblasts and assessed modification to these profiles in cells isolated from individuals with COPD. Methods: DNA was isolated from lung (LgF) and bronchial fibroblasts (BrF) at passage 4 and bisulphite treated. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Linear modelling and DMRcate functions identified differentially methylated sites and regions respectively between BrF and LgF and from cells isolated from healthy individuals versus those with COPD. Results: 3980 CpG (methylation) sites significantly differed, following Bonferroni correction, between BrF and LgF isolated from healthy individuals. These sites had a broad distribution of effect size, with 240 CpG sites displaying a difference in methylation of >50%. 78 of these sites were validated in a second cohort of matched BrF and LgF isolated from the same individuals. There was genomic proximity to these sites and DMRcate was used to refine the individual CpG sites to 5 regions of interest associated with 5 genes; HLX, TWIST1, CREB5, SKAP2 and PRDM16. Differences in methylation were less pronounced when comparing cells isolated from healthy individuals to those with COPD. In BrF 47 DMRcate regions were identified with a maximum difference in methylation of at least 20%. In LgF 3 DMRcate regions were identified with a maximum difference in methylation of at least 20%. Conclusions: DNA methylation profiles are significantly different between BrF and LgF but only small modifications are associated with COPD. Future work will focus on validating a methylation based marker of lung versus bronchial fibroblasts to differentiate cell types by validating our differential DNA methylation observations with gene/protein expression

Inhalation of diesel exhaust and allergen alters human bronchial epithelium DNA methylation

Background Allergic disease affects 30% to 40% of the world's population, and its development is determined by the interplay between environmental and inherited factors. Air pollution, primarily consisting of diesel exhaust emissions, has increased at a similar rate to allergic disease. Exposure to diesel exhaust may play a role in the development and progression of allergic disease, in particular allergic respiratory disease. One potential mechanism underlying the connection between air pollution and increased allergic disease incidence is DNA methylation, an epigenetic process with the capacity to integrate gene-environment interactions. Objective We sought to investigate the effect of allergen and diesel exhaust exposure on bronchial epithelial DNA methylation. Methods We performed a randomized crossover-controlled exposure study to allergen and diesel exhaust in humans, and measured single-site (CpG) resolution global DNA methylation in bronchial epithelial cells. Results Exposure to allergen alone, diesel exhaust alone, or allergen and diesel exhaust together (coexposure) led to significant changes in 7 CpG sites at 48 hours. However, when the same lung was exposed to allergen and diesel exhaust but separated by approximately 4 weeks, significant changes in more than 500 sites were observed. Furthermore, sites of differential methylation differed depending on which exposure was experienced first. Functional analysis of differentially methylated CpG sites found genes involved in transcription factor activity, protein metabolism, cell adhesion, and vascular development, among others. Conclusions These findings suggest that specific exposures can prime the lung for changes in DNA methylation induced by a subsequent insult

Prenatal Alcohol Exposure: Profiling Developmental DNA Methylation Patterns in Central and Peripheral Tissues

Background: Prenatal alcohol exposure (PAE) can alter the development of neurobiological systems, leading to lasting neuroendocrine, neuroimmune, and neurobehavioral deficits. Although the etiology of this reprogramming remains unknown, emerging evidence suggests DNA methylation as a potential mediator and biomarker for the effects of PAE due to its responsiveness to environmental cues and relative stability over time. Here, we utilized a rat model of PAE to examine the DNA methylation profiles of rat hypothalami and leukocytes at four time points during early development to assess the genome-wide impact of PAE on the epigenome and identify potential biomarkers of PAE. Our model of PAE resulted in blood alcohol levels of ~80–150 mg/dl throughout the equivalent of the first two trimesters of human pregnancy. Hypothalami were analyzed on postnatal days (P) 1, 8, 15, 22 and leukocytes at P22 to compare central and peripheral markers. Genome-wide DNA methylation analysis was performed by methylated DNA immunoprecipitation followed by next-generation sequencing.Results: PAE resulted in lasting changes to DNA methylation profiles across all four ages, with 118 differentially methylated regions (DMRs) displaying persistent alterations across the developmental period at a false-discovery rate (FDR) < 0.05. In addition, 299 DMRs showed the same direction of change in the hypothalamus and leukocytes of P22 pups at an FDR < 0.05, with some genes overlapping with the developmental profile findings. The majority of these DMRs were located in intergenic regions, which contained several computationally-predicted transcription factor binding sites. Differentially methylated genes were generally involved in immune function, epigenetic remodeling, metabolism, and hormonal signaling, as determined by gene ontology analyses.Conclusions: Persistent DNA methylation changes in the hypothalamus may be associated with the long-term physiological and neurobehavioral alterations in observed in PAE. Furthermore, correlations between epigenetic alterations in peripheral tissues and those in the brain will provide a foundation for the development of biomarkers of fetal alcohol spectrum disorder (FASD). Finally, findings from studies of PAE provide important insight into the etiology of neurodevelopmental and mental health disorders, as they share numerous phenotypes and comorbidities

Sex differences in epigenetic age in Mediterranean high longevity regions

Sex differences in aging manifest in disparities in disease prevalence, physical health, and lifespan, where women tend to have greater longevity relative to men. However, in the Mediterranean Blue Zones of Sardinia (Italy) and Ikaria (Greece) are regions of centenarian abundance, male-female centenarian ratios are approximately one, diverging from the typical trend and making these useful regions in which to study sex differences of the oldest old. Additionally, these regions can be investigated as examples of healthy aging relative to other populations. DNA methylation (DNAm)-based predictors have been developed to assess various health biomarkers, including biological age, Pace of Aging, serum interleukin-6 (IL-6), and telomere length. Epigenetic clocks are biological age predictors whose deviation from chronological age has been indicative of relative health differences between individuals, making these useful tools for interrogating these differences in aging. We assessed sex differences between the Horvath, Hannum, GrimAge, PhenoAge, Skin and Blood, and Pace of Aging predictors from individuals in two Mediterranean Blue Zones and found that men displayed positive epigenetic age acceleration (EAA) compared to women according to all clocks, with significantly greater rates according to GrimAge (β = 3.55; p = 1.22 × 10-12), Horvath (β = 1.07; p = 0.00378) and the Pace of Aging (β = 0.0344; p = 1.77 × 10-08). Other DNAm-based biomarkers findings indicated that men had lower DNAm-predicted serum IL-6 scores (β = -0.00301, p = 2.84 × 10-12), while women displayed higher DNAm-predicted proportions of regulatory T cells than men from the Blue Zone (p = 0.0150, 95% Confidence Interval [0.00131, 0.0117], Cohen's d = 0.517). All clocks showed better correlations with chronological age in women from the Blue Zones than men, but all clocks showed large mean absolute errors (MAE >30 years) in both sexes, except for PhenoAge (MAE <5 years). Thus, despite their equal survival to older ages in these Mediterranean Blue Zones, men in these regions remain biologically older by most measured DNAm-derived metrics than women, with the exception of the IL-6 score and proportion of regulatory T cells

YARSI UNIVERSITY PROGRAM TO MEET THE DEMAND OF STUDENT’S ENGLISH FLUENCY WITH TOEIC AS THE ASSESSMENT TOOL

Abstract: A test is only a measurement tool of a learning process. The important part is the learning process itself; how the process can help learners acquire English as a foreign language that enables them to compete in the working environment. To measure the process, TOEIC with all its parts was meant to measure learners’ ability to communicate in English. Teachers should not be focusing on the test but more on the approaches that allow the students to have adequate and sophisticated listening, reading, and writing skills to exchange information and to negotiate meaning in real life. Many university level English teachers are trapped within the rules that students should achieve a 550 or 605 TOEIC score to graduate. Instead of helping the students to acquire the language as a communication tool, they tend to focus more on getting the students to master the test. This is what teachers should deal with, not only facilitate students to learn the language but at the same time help them to do the test well. Despite the challenge of facing students who lack motivation and have very basic English skills, Yarsi University Language Lab is setting up several programs and approaches that allow students to acquire the language and enable them to communicate in the target language which is eventually measured by an instrument called TOEIC. Keywords: Language Acquisition, direct and indirect test, discrete and intregativ

Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(βNon-exposed = 0.06, βSRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05) was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight