Genetic and epigenetic regulation of Catechol-O-methyltransferase in relation to inflammation in chronic fatigue syndrome and Fibromyalgia.

BACKGROUND: Catechol-O-methyltransferase (COMT) has been shown to influence clinical pain, descending modulation, and exercise-induced symptom worsening. COMT regulates nociceptive processing and inflammation, key pathophysiological features of Chronic Fatigue Syndrome and Fibromyalgia (CFS/FM). We aimed to determine the interactions between genetic and epigenetic mechanisms regulating COMT and its influence on inflammatory markers and symptoms in patients with CFS/FM. METHODS: A case-control study with repeated-measures design was used to reduce the chance of false positive and increase the power of our findings. Fifty-four participants (28 patients with CFS/FM and 26 controls) were assessed twice within 4 days. The assessment included clinical questionnaires, neurophysiological assessment (pain thresholds, temporal summation, and conditioned pain modulation), and blood withdrawal in order to assess rs4818, rs4633, and rs4680 COMT polymorphisms and perform haplotype estimation, DNA methylation in the COMT gene (both MB-COMT and S-COMT promoters), and cytokine expression (TNF-α, IFN-γ, IL-6, and TGF-ß). RESULTS: COMT haplotypes were associated with DNA methylation in the S-COMT promoter, TGF-ß expression, and symptoms. However, this was not specific for one condition. Significant between-group differences were found for increased DNA methylation in the MB-COMT promoter and decreased IFN-γ expression in patients. DISCUSSION: Our results are consistent with basic and clinical research, providing interesting insights into genetic-epigenetic regulatory mechanisms. MB-COMT DNA methylation might be an independent factor contributing to the pathophysiology of CFS/FM. Further research on DNA methylation in complex conditions such as CFS/FM is warranted. We recommend future research to employ a repeated-measure design to control for biomarkers variability and within-subject changes.

Forensic Epigenetic Age Estimation and Beyond: Ethical and Legal Considerations.

Forensic geneticists are in a race to develop methods based on DNA methylation for various forensic applications, including age estimation. We argue that using epigenetic biomarkers could reveal a broad range of health and life-style related information, therefore it is necessary to develop adequate safeguards to protect the privacy of the individuals under scrutiny.

Carbon Nanotube- and Asbestos-Induced DNA and RNA Methylation Changes in Bronchial Epithelial Cells.

Carbon nanotubes (CNTs) are nanoscale tube-shaped carbon materials used in many industrial areas. Their fiber shape has caused concerns about their toxicity given their structural similarity with asbestos. The aim here was to elucidate the effect of CNTs and asbestos exposure on global DNA and RNA methylation and the methylation of genes associated with cell cycle, inflammation, and DNA damage processes in human lung cells. Human bronchial epithelial cells (16HBE14o-) were exposed for 24 h to 25 and 100 µg/mL CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) and 2.5 µg/mL asbestos (chrysotile, amosite, and crocidolite). Global DNA and RNA (hydroxy)methylation to cytosines was measured by a validated liquid chromatography tandem-mass spectrometry method. Global RNA methylation to adenines was measured by a colorimetric ELISA-like assay. Gene-specific DNA methylation status at certain cytosine-phosphate-guanine (CpG) sites of cyclin-dependent kinase inhibitor 1A ( CDKN1A), serine/threonine kinase ( ATM), and TNF receptor-associated factor 2 ( TRAF2) were analyzed by using bisulfite pyrosequencing technology. Only MWCNT-exposed cells showed significant global DNA hypomethylation of cytosine and global RNA hypomethylation of adenosine. SWCNT, MWCNT, and amosite exposure decreased DNA methylation of CDKN1A. ATM methylation was affected by chrysotile, SWCNT, and MWCNT. However, SWCNT exposure led to DNA hypermethylation of TRAF2. These findings contribute to further understanding of the effect of CNTs on different carcinogenic pathways.

Age determination through DNA methylation patterns in fingernails and toenails.

Over the past decade, age prediction based on DNA methylation has become a vastly investigated topic; many age prediction models have been developed based on different DNAm markers and using various tissues. However, the potential of using nails to this end has not yet been explored. Their inherent resistance to decay and ease of sampling would offer an advantage in cases where post-mortem degradation poses challenges concerning sample collection and DNA-extraction. In the current study, clippings from both fingernails and toenails were collected from 108 living test subjects (age range: 0-96 years). The methylation status of 15 CpGs located in 4 previously established age-related markers (ASPA, EDARADD, PDE4C, ELOVL2) was investigated through pyrosequencing of bisulphite converted DNA. Significant dissimilarities in methylation levels were observed between all four limbs, hence both limb-specific age prediction models and prediction models combining multiple sampling locations were developed. When applied to their respective test sets, these models yielded a mean absolute deviation between predicted and chronological age ranging from 5.48 to 9.36 years when using ordinary least squares regression. In addition, the assay was tested on methylation data derived from 5 nail samples collected from deceased individuals, demonstrating its feasibility for application in post-mortem cases. In conclusion, this study provides the first proof that chronological age can be assessed through DNA methylation patterns in nails.

Improving the age estimation model for toenails.

In a previous study, we have provided the first proof that chronological age can be estimated through DNA methylation (DNAm) patterns in fingernails and toenails. DNAm data of 15 CpGs located in 4 genetic markers (ASPA, EDARADD, ELOVL2 and PDE4C) were evaluated, of which variable selection yielded age prediction models with a mean absolute deviation (MAD) ranging from 7.68 to 9.36 years, depending on the sampling location. Three additional age-associated markers (KLF14, MIR29B2CHG and TRIM59) were assessed in the current study with the goal of increasing the prediction accuracy of the model initially constructed for toenails. This new and improved age estimation assay yielded an MAD of 4.82 and 5.61 years for the training and test set, respectively. The feasibility of the application for post-mortem cases was also demonstrated through testing a limited set of samples collected from deceased individuals.

Mild daily stress, in interaction with NR3C1 DNA methylation levels, is linked to alterations in the HPA axis and ANS response to acute stress in early adolescents.

BACKGROUND: Daily Hassles (DH) or daily stress - is a mild type of stressor with unique contributions to psychological distress. Yet, most prior studies that investigate the effects of stressful life experiences focus on childhood trauma or on early life stress and little is known about the effects of DH on epigenetic changes in stress system related genes and on the physiological response to social stressors. METHODS: In the present study, conducted among 101 early adolescents (mean age = 11.61; SD = 0.64), we investigated whether Autonomic Nervous System (ANS) (namely heart rate and heart rate variability) and Hypothalamic-Pituitary-Adrenal (HPA) axis functioning (measured as cortisol stress reactivity and recovery) are associated with DNA methylation (DNAm) in the glucocorticoid receptor gene (NR3C1), the level of DH and their interaction. To assess the stress system functioning the TSST protocol was used. RESULTS: Our findings show that higher NR3C1 DNAm in interaction with higher levels of daily hassles, is associated with blunted HPA axis reactivity to psychosocial stress. In addition, higher levels of DH are associated with extended HPA axis stress recovery. In addition, participants with higher NR3C1 DNAm had lower ANS adaptability to stress, specifically lower parasympathetic withdrawal; for heart rate variability this effect was strongest for participants with higher level of DH. CONCLUSIONS: The observation that interaction effects between NR3C1 DNAm levels and daily stress on the functioning of the stress-systems, are already detectable in young adolescents, highlights the importance of early interventions, not only in the case of trauma, but also daily stress. This might help to prevent stress-induced mental and physical disorders later in life.

Evaluation of infrared photography for latent bloodstain visualization and the influence of time.

The localization of latent blood traces at crime scenes is generally performed using fluorescent stains although infrared light has previously been recognized as an effective localization test for bloodstains as it is a non-destructive and non-contact technique. The goal of this study was to evaluate infrared photography for the detection of latent bloodstains on a large number of objects with different compositions frequently encountered at crime scenes. In this study we show that infrared light photography was able to detect bloodstains deposited on 71.7% of materials while bloodstains on 29.2% of materials could only be detected using infrared photography and not through visual photography. Bloodstain could be detected on most fabrics composed of 100% polyester, 100% cotton and 100% wool or a combination of these fibers with other types of fiber such as nylon or viscose. For other materials such as leather, tiles, wood, bricks, parquet, infrared did not improve the visibility of the bloodstains. Finally, the influence of the time of bloodstain deposition was tested over the period of 1 week and 1 month and did not reveal major differences compared to stains after 24 h drying time suggesting that time has little influence on the ability of infrared light and visual light to detect latent bloodstains.

Time since contact influences DNA profiling success of cartridges and fired cartridge casings.

Forensic DNA analysis of cartridges and fired cartridge casings remains challenging, possibly due to the heat and pressure generated during firing of the weapon as well as metal ions from the casings that have been suggested to initiate DNA degradation and inhibit PCR during the DNA profiling process. Even though recently developed DNA recovery protocols have shown to significantly improve DNA yields and DNA profile success rates no information is available on whether the time interval between contact and the DNA recovery process has an influence on these outcomes. In the current study 40 cartridges and 40 fired cartridge casings were left untreated for 24 h or 1 week after which the rinse-and-swab technique was used to collect DNA. Higher DNA yields and higher DNA profile success rates were obtained from cartridges compared to fired cartridge casings. The same general observation was made when cartridges and fired cartridge casings were processed after 24 h compared to after 1 week. In addition, DNA profiles suitable for comparison could still be generated from samples when real-time PCR quantification indicated DNA concentrations < 0.001 ng/µl, suggesting that quantification results may not be reliable when assessing the presence of DNA on such items. In conclusion, the results indicate that cartridges and fired cartridge casings should be processed for DNA profiling as soon as possible and that DNA quantification results should be interpreted with caution as DNA profiles suitable for comparison could be missed.

Influence of ink and smoke ATM security systems on dactyloscopy and subsequent DNA analysis after detonation.

Automated Teller Machine bombings are an increasing societal problem that are often committed using Improvised Explosive Devices. The evolution in IEDs and the negative consequences for society require new security measures to prevent these crimes. Ink staining and security smoke devices are added to cash cassettes, in order to protect ATMs and prevent ATM bombings. Traces found at crime scenes, such as fingerprints and DNA, can contribute to the identification of perpetrators. However, the effect of ink staining and security smoke devices on dactyloscopy and DNA profiling is still unknown. In the current study, we demonstrate that procedures using Citrus Cleaner or sulfosalicylic acid were successful in removing ink and security smoke deposited on plastic plates but did result in the massive loss of fingerprint information as only a low number (4%) of good quality fingerprints were recovered after smoke contamination. Secondly, security ink Sun Blue ES2, but not SICPA Green and Sun Blue ES1, had a significant impact on DNA profiling success. DNA concentrations obtained from blood spiked swabs decreased with increasing ink concentration resulting in a complete loss of genotype information with the addition of ≥10 µl Sun Blue ES2. No noticeable PCR inhibition or DNA degradation was detected during quantification, but a decreased efficiency of DNA extraction could not be excluded. Security smoke, on the other hand, does not seem to have a significant influence on DNA analysis. Precautions must therefore be taken in order to avoid contaminating DNA swabs with ink during sampling. Thirdly, only a single negative impression of a glove in ink and a single glove print were able to be visualized with white fingerprint powder on detonated cash cassettes. In conclusion, the detection of glove prints and fingerprints is limited and security ink, contrary to smoke, after detonation can have a negative influence on downstream DNA analysis procedures.

Role of NR3C1 and SLC6A4 methylation in the HPA axis regulation in burnout.

BACKGROUND: Work-related stress and burnout have become major occupational health concerns. Dysregulation of HPA axis is considered one of the central mechanisms and is potentially moderated through epigenetics. In the present study, we aim to investigate epigenetic regulation of the HPA axis in burnout, by focusing on salivary cortisol and cortisone and DNA methylation of the glucocorticoid receptor gene (NR3C1) and the serotonin transporter gene (SLC6A4). METHODS: We conducted a cross-sectional study with 59 subjects with burnout and 70 healthy controls recruited from the general population. All participants underwent a clinical interview and psychological assessment. Saliva samples were collected at 0, 30 and 60 min after awakening and were used to quantify cortisol and cortisone. Pyrosequencing was performed on whole blood-derived DNA to assess DNA methylation. RESULTS: There were no between-group differences in cortisol levels, whereas burnout participants had higher levels of cortisone. Job stress was associated with increased cortisol and cortisone. We observed both increased and decreased NR3C1 and SLC6A4 methylation in the burnout group compared to the control group. Some of these methylation changes correlated with burnout symptoms dimensionally. Increased methylation in a specific CpG in the SLC6A4 promoter region moderated the association between job stress and burnout. DNA methylation in this CpG was also associated with increased cortisol. In addition, average methylation of NR3C1 was negatively associated with cortisone levels. LIMITATIONS: This is a cross-sectional study and therefore no conclusions on causality could be made. CONCLUSIONS: We provide first evidence of changes in DNA methylation of NR3C1 and SLC6A4 in burnout, which were further associated with cortisol and cortisone. Further, increased cortisol and cortisone seemed to reflect job stress rather than burnout itself.

Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death.

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

Survival of forensic trace evidence on improvised explosive devices: perspectives on individualisation.

Improvised Explosive Devices (IEDs) are weapons of modern times, used by terrorist groups and thereby causing substantial damage to communities. There is a widespread misconception that destructive conditions like heat, water or pressure destroy all forensic evidence. Moreover, the plausibility to detect identifiable fingermarks and DNA on components of IEDs is insufficiently known. Therefore, this study investigated the effects of neutralisation and explosion on latent fingerprints and touch DNA. In a majority of the cases, comparative fingermark- and DNA testing resulted in individualisation. In some cases, despite extremely low amounts of contact DNA detected after deployment of render-safe tools or detonation, full STR profiles could be constituted, even after applying fingerprint development techniques. This research shows that latent fingerprints and touch DNA on improvised explosives can be successfully detected after destructive conditions and possibly be linked to the perpetrators of such crimes. This individualising power offers perspectives to enhance forensic investigations of terrorism-related crimes.

Epigenetic perspective on the role of brain-derived neurotrophic factor in burnout.

Brain-derived neurotrophic factor (BDNF) plays a potential role in the neurobiology of burnout, but there are no studies investigating the underlying genetic and epigenetic mechanisms. Our aim is to further explore the role of BDNF in burnout, by focusing on the Val66Met polymorphism and methylation patterns of the BDNF gene and serum BDNF (sBDNF) protein expression. We conducted a cross-sectional study by recruiting 129 individuals (59 with burnout and 70 healthy controls). Participants underwent a clinical interview, psychological assessment and blood sample collection. Polymorphism and DNA methylation were measured on DNA from whole blood, using pyrosequencing and sBDNF levels were measured using ELISA. We found significantly increased methylation of promoter I and IV in the burnout group, which also correlated with burnout symptoms. In addition, DNA methylation of promoter I had a significant negative effect on sBDNF. For DNA methylation of exon IX, we did not find a significant difference between the groups, nor associations with sBDNF. The Val66Met polymorphism neither differed between groups, nor was it associated with sBDNF levels. Finally, we did not observe differences in sBDNF level between the groups. Interestingly, we observed a significant negative association between depressive symptoms and sBDNF levels. The current study is the first to show that BDNF DNA methylation changes might play an important role in downregulation of the BDNF protein levels in burnout. The presence of depressive symptoms might have an additional impact on these changes.

Increased methylation of NR3C1 and SLC6A4 is associated with blunted cortisol reactivity to stress in major depression.

BACKGROUND: Epigenetic changes are considered the main mechanisms behind the interplay of environment and genetic susceptibility in major depressive disorder (MDD). However, studies focusing on epigenetic dysregulation of the HPA axis stress response in MDD are lacking. Our objective was to simultaneously asses DNA methylation of the glucocorticoid receptor gene (NR3C1) and serotonin transporter gene (SLC6A4) and HPA axis response to stress in MDD. METHODS: We recruited 80 depressed inpatients and 58 gender and age matched healthy controls. All participants underwent the Trier Social Stress Test (TSST) and salivary cortisol was repeatedly measured to assess HPA axis reactivity. DNA methylation of the NR3C1 (exon 1 F) and SLC6A4 CpG islands was quantified from whole blood DNA. In the MDD group, clinical assessment was repeated at 8-week follow-up to test the predictive potential of DNA methylation for symptom improvement. RESULTS: Depressed patients had blunted cortisol reactivity to TSST compared to healthy controls (p = 0.01). In addition, they presented with increased average SLC6A4 (p = 0.003) and NR3C1 methylation (p = 0.03), as well as methylation of two individual NR3C1 CpG loci overlapping with the NGFI-A-binding sites (CpG12 and CpG20). Methylation of one of these two loci (CpG20) predicted lower symptom improvement at the follow-up (p = 0.007). Both, average NR3C1 and SLC6A4 methylation were associated with lower cortisol reactivity in the MDD group and explained about 16% of variability in cortisol response to TSST. CONCLUSIONS: We provide evidence of the role of NR3C1 and SLC6A4 DNA methylation in HPA axis dysregulation in MDD, which needs to be further explored.

DNA Methylation and Brain-Derived Neurotrophic Factor Expression Account for Symptoms and Widespread Hyperalgesia in Patients With Chronic Fatigue Syndrome and Comorbid Fibromyalgia.

OBJECTIVE: The epigenetics of neurotrophic factors holds the potential to unravel the mechanisms underlying the pathophysiology of complex conditions such as chronic fatigue syndrome (CFS). This study was undertaken to explore the role of brain-derived neurotrophic factor (BDNF) genetics, epigenetics, and protein expression in patients with both CFS and comorbid fibromyalgia (CFS/FM). METHODS: A repeated-measures study was conducted in 54 participants (28 patients with CFS/FM and 26 matched healthy controls). Participants underwent a comprehensive assessment, including questionnaires, sensory testing, and blood withdrawal. Serum BDNF (sBDNF) protein levels were measured using enzyme-linked immunosorbent assay, while polymorphism and DNA methylation were measured in blood using pyrosequencing technology. To assess the temporal stability of the measures, participants underwent the same assessment twice within 4 days. RESULTS: Repeated-measures mixed linear models were used for between-group analysis, with mean differences and 95% confidence intervals (95% CIs) shown. Compared to controls, serum BNDF was higher in patients with CFS/FM (F = 15.703; mean difference 3.31 ng/ml [95% CI 1.65, 4.96]; P = 0.001), whereas BDNF DNA methylation in exon 9 was lower (F = 7.543; mean difference -2.16% [95% CI -3.93, -0.83]; P = 0.007). BDNF DNA methylation was mediated by the Val66Met (rs6265) polymorphism. Lower methylation in the same region predicted higher sBDNF levels (F = 7.137, ß = -0.408 [95% CI -0.711, -0.105]; P = 0.009), which in turn predicted participants' symptoms (F = 14.410, ß = 3.747 [95% CI 1.79, 5.71]; P = 0.001) and widespread hyperalgesia (F = 4.147, ß = 0.04 [95% CI 0.01, 0.08]; P = 0.044). CONCLUSION: Our findings indicate that sBDNF levels are elevated in patients with CFS/FM and that BDNF methylation in exon 9 accounts for the regulation of protein expression. Altered BDNF levels might represent a key mechanism explaining CFS/FM pathophysiology.

New aspects of dental implants and DNA technology in human identification.

Missing, ineligible or delayed reference data to establish conventional dental or DNA identification are common scenarios in forensic practice. Therefore, it is worthwhile to explore new avenues that facilitate human identification. Due to the recent remarkable evolution in the prosthetic dental restorations based on dental implants and the emergence of novel DNA technologies utilized to infer the biological profile, the identification process has become easier than ever before. We report on a characteristic case, which highlights the particular importance of dental implants and DNA approaches in the prospective investigations for human identification. The aim of this publication is to focus on the possibility of identifying the batch numbers, even if they were not engraved in dental implants, making antemortem dental records of dental implants more easily accessible to establish a comparative dental identification. In addition, the reported case presents the supplementary data yielded through estimating the epigenetic age using DNA methylation as well as the biogeographical origin using Y-Haplotype and mitochondrial DNA analyses. Our results demonstrate that expanded oral implant investigations that also include implants extraction and comprehensive microscopic measurements can lead to identifying their batch numbers despite the numerous number of implants systems manufactured and distributed worldwide. Data saved by dental implant manufacturers can be very supportive and represent additional reference data for dental identification, when antemortem dental records are still missing. Furthermore, DNA methylation and mitochondrial DNA analyses can support the progress of investigation.

The Influence of the Duration of Breastfeeding on the Infant's Metabolic Epigenome.

Nutrition in the postnatal period is associated with metabolic programming. One of the presumed underlying mechanisms involves epigenetic modifications (e.g., DNA methylation). Breastfeeding has an unknown impact on DNA methylation at a young age. Within the Maternal Nutrition and Offspring's Epigenome (MANOE) study, we assessed the effect of breastfeeding duration on infant growth and buccal methylation in obesity-related genes (n = 101). A significant difference was found between infant growth and buccal RXRA and LEP methylation at 12 months of breastfeeding. For RXRA CpG2 methylation, a positive association was found with duration of breastfeeding (slope = 0.217; 95% confidence interval (CI) 1.03, 0.330; p < 0.001). For RXRA CpG3 and CpG, mean methylation levels were significantly lower when children were breastfed for 4-6 months compared to non-breastfed children (only CpG3), and those breastfed for 7-9 months, 10-12 months, or 1-3 months. On the other hand, higher LEP CpG3 methylation was observed when mothers breastfed 7-9 months (6.1%) as compared to breastfeeding for 1-3 months (4.3%; p = 0.007) and 10-12 months (4.6%; p = 0.04). In addition, we observed that infant weight was significantly lower when children were breastfed for 10-12 months. Breastfeeding duration was associated with epigenetic variations in RXRA and LEP at 12 months and with infant biometry/growth. Our results support the hypothesis that breastfeeding could induce epigenetic changes in infants.

Randomized controlled trial of the effect of selenium supplementation on thyroid function in the elderly in the United Kingdom.

BACKGROUND: Thyroid function depends on the essential trace mineral selenium, which is at the active center of the iodothyronine deiodinase enzymes that catalyze the conversion of the prohormone thyroxine (T(4)) to the active form of thyroid hormone, triiodothyronine (T(3)). OBJECTIVE: Because selenium intake in the United Kingdom has fallen during the past 25 y, we wanted to determine whether current selenium status might be limiting conversion of T(4) to T(3) in the elderly, in whom marginal hypothyroidism is relatively common. DESIGN: We investigated the effect of selenium supplementation in a double-blind, placebo-controlled trial in 501 elderly UK volunteers. Similar numbers of men and women from each of 3 age groups, 60-64 y, 65-69 y, and 70-74 y, were randomly allocated to receive 100, 200, or 300 microg Se/d as high-selenium yeast or placebo yeast for 6 mo. As part of the study, plasma selenium, thyroid-stimulating hormone, and total and free T(3) and T(4) were measured. Data from 368 euthyroid volunteers who provided blood samples at baseline and 6 mo were analyzed. RESULTS: Although selenium status at baseline correlated weakly with free T(4) (r = -0.19, P < 0.001) and with the ratio of free T(3) to free T(4) (r = 0.12, P = 0.02), we found no evidence of any effect of selenium supplementation on thyroid function, despite significant increases in plasma selenium. However, baseline plasma selenium in our study (x: 91 microg/L) was somewhat higher than in previous supplementation studies in which apparently beneficial effects were seen. CONCLUSION: We found no indication for increasing selenium intake to benefit T(4) to T(3) conversion in the elderly UK population.

Evaluation of three statistical prediction models for forensic age prediction based on DNA methylation.

DNA methylation is a promising biomarker for forensic age prediction. A challenge that has emerged in recent studies is the fact that prediction errors become larger with increasing age due to interindividual differences in epigenetic ageing rates. This phenomenon of non-constant variance or heteroscedasticity violates an assumption of the often used method of ordinary least squares (OLS) regression. The aim of this study was to evaluate alternative statistical methods that do take heteroscedasticity into account in order to provide more accurate, age-dependent prediction intervals. A weighted least squares (WLS) regression is proposed as well as a quantile regression model. Their performances were compared against an OLS regression model based on the same dataset. Both models provided age-dependent prediction intervals which account for the increasing variance with age, but WLS regression performed better in terms of success rate in the current dataset. However, quantile regression might be a preferred method when dealing with a variance that is not only non-constant, but also not normally distributed. Ultimately the choice of which model to use should depend on the observed characteristics of the data.

Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells.

Recent studies have identified carbon nanotube (CNT)-induced epigenetic changes as one of the key players in patho-physiological response. In the present study, we investigated whether CNT exposure is associated with epigenetic changes in human bronchial epithelial cells (16 HBE), in vitro. We focused on global DNA methylation, methylation of LINE-1 elements and promoter sequence of twelve functionally important genes (SKI, DNMT1, HDAC4, NPAT, ATM, BCL2L11, MAP3K10, PIK3R2, MYO1C, TCF3, FGFR 1 and AGRN). Additionally, we studied the influence of CNT exposure on miRNA expression. Using a LC-MS/MS method and pyrosequencing for LINE-1, we observed no significant changes in global DNA methylation (%) between the concentrations of multi-walled and single-walled CNT (MWCNT and SWCNT, respectively). Significant changes in sequence-specific methylation was observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). While changes in DNA methylation of the genes were relatively small, these changes were associated with changes in RNA expression, especially for MWCNT. However, the study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Based on our results, mainly MWCNT influence DNA methylation and expression of the studied genes and could have significant impact on several critical cellular processes.