Assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility.

Endometriosis is the cause of infertility. The eutopic endometrium of women with endometriosis showed an aberrant expression pattern of multitude genes. The role of TET1 protein in the pathogenesis of endometriosis and related infertility is not sufficiently known. Further, knowledge on TET1 transcriptional control still remains incomplete. The aim of the study was assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility. The study included 44 infertile patients with endometriosis (IWE) and 77 infertile (IW) and fertile (FW) patients without endometriosis. The research material was eutopic endometrium. The TET1 mRNA level was analyzed by qPCR. Western blot was used to evaluate the level of TET1 protein. The level of DNA methylation and H3K27me3 level of TET1 gene's promoter region were assessed using HRM and ChIP qPCR, respectively. The level of TET1 expression (TET1 mRNA; TET1 protein level) was lower in IWE during the implantation window (p < 0.001; p = 0.0329). The level of TET1 DNA methylation was higher in the secretory endometrium in mild and advanced IWE (p < 0.004; p < 0.008). H3K27me3 level did not differ between the study groups. The diminished expression of TET1 gene during the secretory phase, may account for the aberrant process of embryonic implantation in infertile endometriosis patients. DNA hypermethylation of TET1 gene is a potential relevant regulator of its expression. H3K27me3 occupancy does not affect the expression of TET1 gene in our study group.

Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Prognostic value of test(s) for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide.

BACKGROUND: Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide. OBJECTIVES: To determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide. SEARCH METHODS: We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014. SELECTION CRITERIA: Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis. DATA COLLECTION AND ANALYSIS: Two review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals. MAIN RESULTS: We included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status. AUTHORS' CONCLUSIONS: PSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma.

BACKGROUND: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the MGMT methylation status of 28 isocitrate dehydrogenase-wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values. RESULTS: We designed three primers for separate regions (regions 1-3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan-Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

Assessment of tumor suppressor promoter methylation in healthy individuals.

BACKGROUND: The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. RESULTS: In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. CONCLUSIONS: Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage.

DNA methylation analysis is increasingly used in stress research. Available methods are expensive, laborious and often limited by either the analysis of short CpG stretches or low assay sensitivity. Here, we present a cost-efficient next generation sequencing-based strategy for the simultaneous investigation of multiple candidate genes in large cohorts. To illustrate the method, we present analysis of four candidate genes commonly assessed in psychoneuroendocrine research: Glucocorticoid receptor (NR3C1), Serotonin transporter (SLC6A4), FKBP Prolyl isomerase 5 (FKBP5), and the Oxytocin receptor (OXTR). DNA methylation standards (100 %; 75 %; 50 %; 25 % and 0 %) and DNA of a female and male donor were bisulfite treated in three independent trials and were used to generate sequencing libraries for 42 CpGs from the NR3C1 1 F promoter region, 84 CpGs of the SLC6A4 5' regulatory region, 5 CpGs located in FKBP5 intron 7, and additional 12 CpGs located in a potential enhancer element in intron 3 of the OXTR. In addition, DNA of 45 patients with borderline personality disorder (BPD) and 45 healthy controls was assayed. Multiplex libraries of all samples were sequenced on a MiSeq system and analyzed for mean methylation values of all CpG sites using amplikyzer2 software. Results indicated excellent accuracy of the assays when investigating replicates generated from the same bisulfite converted DNA, and very high linearity (R2 > 0.9) of the assays shown by the analysis of differentially methylated DNA standards. Comparing DNA methylation between BPD and healthy controls revealed no biologically relevant differences. The technical approach as described here facilitates targeted DNA methylation analysis and represents a highly sensitive, cost-efficient and high throughput tool to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes.

The value of endothelin receptor type B promoter methylation as a biomarker for the risk assessment and diagnosis of prostate cancer: A meta-analysis.

Previous researches have demonstrated that the methylation status of the EDNRB promoter was associated with the prostate cancer (PCa), but these conclusions remained controversial. Thus, the aim of this meta-analysis was to evaluate the association between EDNRB promoter methylation and the PCa. According to the PRISMA statement, the Web of Science, PubMed, EMBASE, and Cochrane Library databases were retrieved. The ORs and 95 % CIs were analyzed to evaluate the associations between EDNRB promoter methylation and the risk and clinical features of PCa. Heterogeneity among the included studies was estimated by I2 statistic and Q test. Publication bias and sensitivity analysis were utilized to test the robustness of our outcomes. In addition, the pooled sensitivity and specificity were calculated to assess the diagnostic value of EDNRB methylation for PCa. Ultimately, 11 eligible studies were included. Under the random-effects model, the pooled OR shown that the frequency of EDNRB methylation was substantially higher in cases compared with controls (OR = 5.42, 95 % CI = 1.98-14.88, P = 0.001). The similar results were also found by the data from TCGA database. Subgroup analysis according to the methylation detection method showed that the heterogeneity in quantitative methylation-specific polymerase chain reaction (qMSP) group was insignificant (I2 = 0.0 %, P = 0.669). Moreover, the pooled sensitivity for all-inclusive studies was 0.55 (95 % CI: 0.26-0.81), and the pooled specificity was 0.93 (95 % CI: 0.55-0.99). The methylation of EDNRB promoter might increase the risk of PCa. Meanwhile, EDNRB promoter methylation test combined with PSA testing and/or other biomarkers could be promising diagnostic biomarkers for more accurate detection of PCa.

Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

DNMHMM: An approach to identify the differential nucleosome regions in multiple cell types based on a Hidden Markov Model.

Nucleosome occupancy changes across cell types and environmental conditions and such changes often have profound influence in transcription. It's of importance to identify the differential nucleosome regions (DNRs) where the nucleosome occupancy level differs across cell types. Here we developed DNMHMM, a Hidden Markov Model (HMM) based algorithm, to detect the DNRs with nucleosomal DNA sequenced dataset. The performance evaluation indicates that DNMHMM is advisable for multi-cell type comparison. Upon testing this model in yeast mutants, where the modifiable histone residues were mutated into alanine, we found that DNA sequences of the dynamic nucleosomes lack 10-11 bp periodicities and harbor binding motifs of the nucleosome remodelling complex. Moreover, the highly expressed genes have more dynamic nucleosomes at promoters. We further compared nucleosome occupancy between resting and activated human CD4+ T cells with this model. It was revealed that during the activation of CD4+ T cells, dynamic nucleosomes are enriched at regulatory sites, hence, up to some extent can affect the gene expression level. Taken together, DNMHMM offers the possibility to access precise nucleosome dynamics among multiple cell types and also can describe the closer association between nucleosome and transcription.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line.

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

Assessment of Interleukin-18 gene polymorphism and serum levels in oral lichen planus in an Indian population.

BACKGROUND: Oral lichen planus (OLP) is a chronic, inflammatory disease with uncertain etiology. The aim of this study was to assess Interleukin-18 (IL-18) gene polymorphism and serum levels in OLP cases of Indian origin and to compare them with a control population of similar background. METHODS: The assessment of single-nucleotide polymorphisms (SNPs) of IL-18 gene at promoter regions -137(G/C) and -607(C/A) was done in 70 OLP cases and 70 healthy controls using sequence-specific primer-polymerase chain reaction (SSP-PCR). In a subset of this cohort, comprising of 41 OLP cases and 41 controls, serum IL-18 levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: Mean serum levels of IL-18 among OLP cases were significantly higher when compared to controls. Genotypic and allelic frequencies of IL-18 at position -137(G/C) showed that GG genotype and allele G was significantly higher in OLP cases, whereas, GC genotype and C allele was high in the control group. Polymorphism of IL-18 at position -607(C/A) showed no significant differences. CONCLUSIONS: Gene polymorphism at -137GG genotype and allele G seems to be associated with genetic susceptibility to OLP whereas -137GC and allele C may have a protective role against its development. However, our study lacks clear statistical correlation, the differences observed could be caused by sampling problems and the results could not be fully representative of Indian patients with OLP. Further studies are warranted to explore the role of IL-18 genetic polymorphisms in OLP development.

Prognostic Value of TERT Alterations, Mutational and Copy Number Alterations Burden in Urothelial Carcinoma.

Point mutations in the TERT gene promoter occur at high frequency in multiple cancers, including urothelial carcinoma (UC). However, the relationship between TERT promoter mutations and UC patient outcomes is unclear due to conflicting reports in the literature. In this study, we examined the association of TERT alterations, tumor mutational burden per megabase (Mb), and copy number alteration (CNA) burden with clinical parameters and their prognostic value in a cohort of 398 urothelial tumors. The majority of TERT mutations were located at two promoter region hotspots (chromosome 5, 1 295 228 C>T and 1 295 250 C>T). TERT alterations were more frequently present in bladder tumors than in upper tract tumors (73% vs 53%; p=0.001). ARID1A, PIK3CA, RB1, ERCC2, ERBB2, TSC1, CDKN1A, CDKN2A, CDKN2B, and PTPRD alterations showed significant co-occurrence with TERT alterations (all p<0.0025). TERT alterations and the mutational burden/Mb were independently associated with overall survival (hazard ratio[HR] 2.31, 95% confidence interval [CI] 1.46-3.65; p<0.001; and HR 0.96, 95% CI 0.93-0.99; p=0.002), disease-specific survival (HR 2.23, 95% CI 1.41-3.53; p<0.001; and HR 0.96, 95% CI 0.93-0.99; p=0.002), and metastasis-free survival (HR 1.63, 95% CI 1.05-2.53; p=0.029; and HR 0.98, 95% CI 0.96-1.00; p=0.063) in multivariate models. PATIENT SUMMARY: The majority of TERT gene mutations that we detected in urothelial carcinoma are located at two promoter hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden had more favorable outcome compared to tumors with low mutational burden.

Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of cytochrome P450 3A4 and pregnane X receptors.

Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids, FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays, respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity, and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXRCYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with other drugs.

Transcriptional processes: Models and inference.

Many biochemical events involve multistep reactions. One of the most important biological processes that involve multistep reaction is the transcriptional process. Models for multistep reaction necessarily need multiple states and it is a challenge to compute model parameters that best agree with experimental data. Therefore, the aim of this work is to design a multistep promoter model which accurately characterizes transcriptional bursting and is consistent with observed data. To address this issue, we develop a model for promoters with several OFF states and a single ON state using Erlang distribution. To explore the combined effects of model and data, we combine Monte Carlo extension of Expectation Maximization (MCEM) and delay Stochastic Simulation Algorithm (DSSA) and call the resultant algorithm as delay Bursty MCEM. We apply this algorithm to time-series data of endogenous mouse glutaminase promoter to validate the model assumptions and infer the kinetic parameters. Our results show that with multiple OFF states, we are able to infer and produce a model which is more consistent with experimental data. Our results also show that delay Bursty MCEM inference is more efficient.

Overcoming the Cost of Positive Autoregulation by Accelerating the Response with a Coupled Negative Feedback.

A fundamental trade-off between rapid response and optimal expression of genes below cytotoxic levels exists for many signaling circuits, particularly for positively autoregulated systems with an inherent response delay. Here, we describe a regulatory scheme in the E. coli PhoB-PhoR two-component system, which overcomes the cost of positive feedback and achieves both fast and optimal steady-state response for maximal fitness across different environments. Quantitation of the cellular activities enables accurate modeling of the response dynamics to describe how requirements for optimal protein concentrations place limits on response speed. An observed fast response that exceeds the limit led to the prediction and discovery of a coupled negative autoregulation, which allows fast gene expression without increasing steady-state levels. We demonstrate the fitness advantages for the coupled feedbacks in both dynamic and stable environments. Such regulatory schemes offer great flexibility for accurate control of gene expression levels and dynamics upon environmental changes.

Rapid Assessment of Site Specific DNA Methylation through Resistive Pulse Sensing.

Many diseases are defined by patterns of DNA methylation which result in aberrant gene expression. We present a rapid assay based upon resistive pulse sensing, RPS, to characterize sequence specific DNA methylation sites in genomic DNA. We modify the surface of superparamagnetic beads, SPBs, with DNA (capture probe). The particles are added to solution where they bind to and extract sequence specific DNA (target DNA). The target loaded SPBs are then incubated with antibodies which bind to the methylation sites, and the velocity of the SPBs through the nanopore reveals the number and location of the epigenetic markers within the target. The approach is capable of distinguishing between different methylation sites within a DNA promoter region. Crucially the approach is not dependent on accurate sequencing of assayed DNA, with genomic regions targeted through complementary probes. As such the number of stages and reagents costs are low and the assay is complete in under 60 min which includes the incubation and run times. The format also allows simultaneous quantification of number of copies of methylated DNA, and we illustrate this with a dose response curve.

Risky driving and the persistent effect of a randomized intervention focusing on impulsivity: The role of the serotonin transporter promoter polymorphism.

Road traffic accidents are a serious public health issue, and real-life traffic offences are an excellent indicator of the behavioural tendencies of impulsivity and risk-taking. We have previously reported on short-term efficacy of a brief intervention in driving schools to reduce traffic risks (Paaver et al., Accid. Anal. Prev., 2013; 50, 430-437), and have now addressed the question of whether does the impact of the intervention last for a few years, and whether traffic behaviour and the intervention effect are associated with the serotonin transporter polymorphism (5-HTTLPR) genotype as the central serotonin system is strongly associated with impulse control. Participants of the study were 1866 novice car-drivers (mean age 23.0, SD = 7.2 years). Data on traffic violations were obtained four years after intervention from the police database and on traffic collisions from the national traffic insurance database. DNA samples were available for 767 participants and 5-HTTLPR genotypes were classified using the triallelic model. For the observation period after the intervention, speeding, drunk driving and involvement in traffic accidents were significantly lower in the intervention group. 5-HTTLPR genotype was associated with traffic behaviour: The S'-allele carriers had significantly lower odds for speeding offences and traffic accidents. The lower prevalence of S'-allele carriers among those who had committed speeding offences was statistically significant in females, while the lower prevalence of having been involved in a traffic accident was rather observed in males. Statistically significant intervention effects were observed only in the L'/L' homozygotes who had higher prevalence of traffic incidents. Conclusively, the brief intervention in traffic schools had a significant impact on traffic safety within subsequent four years, and traffic behaviour was associated with the serotonin transporter genotype. These findings suggest that subjects who are less likely to self-regulate their driving habits while gaining experience would benefit from training of impulsivity recognition.

MGMT assessment in pituitary adenomas: comparison of different immunohistochemistry fixation chemicals.

PURPOSE: Despite the established role of O6-methyl-guanine-DNA methyltransferase (MGMT) as a marker for temozolomide response, consensus of the most reliable method to assess MGMT expression in pituitary adenomas is still missing. Currently, immunohistochemistry (IHC) assessment of formaldehyde fixed tissue samples is most widely used in a semiquantitative description. As formaldehyde fails to completely preserve nucleic acids, RCL2, an alcohol-based formaldehyde-free fixative, has been proposed as a more reliable alternative in terms of cell stability. Furthermore, as the current method of IHC is semiquantitative and observer-dependent, pyrosequencing, an objective tool to evaluate the methylation status of the MGMT promoter, has emerged as a reliable and accurate alternative. The aim of this study was to validate the current IHC method for assessment of MGMT protein expression in pituitary adenomas. METHODS: The tissue samples of 8 macroadenomas with positive IHC MGMT expression (> 50%) were investigated: first, we compared the time dependent stability of MGMT protein expression after pituitary adenoma removal between formaldehyde vs. RCL2. Then, we compared positive IHC MGMT expression with methylated promoter status using pyrosequencing. RESULTS: In the first 12 h after adenoma removal, tissue samples remained MGMT positive in significantly more samples when fixated with formaldehyde than with RCL2, respectively (96 vs. 81%, p = 0.025). CONCLUSION: Our data confirm that the current method using formaldehyde tissue fixation and IHC reveals stable and reliable results of MGMT assessment in pituitary adenomas.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Assessment of Brain Derived Neurotrophic Factor in hair to study stress responses: A pilot investigation.

To study pathogenic stress-effects in health and disease, it is paramount to define easy access parameters for non-invasive analysis of biological change in response to stress. Hair samples successfully provide this access for the study of hypothalamus-pituitary-adrenal axis (HPA) changes. In this study, we assess the hair expression and corresponding epigenetic changes of a neurotrophin essential for autonomic nervous system function and mental health: brain derived neurotrophic factor (BDNF). In three independent studies in healthy academic volunteers (study I: German students, N=36; study II, German academic population sample, N=28; study III: Mexican students, N=115), BDNF protein expression or BDNF gene (BDNF) histone acetylation was determined. Simultaneously, mental distress and distress-associated somatic complaints were assessed by self-report. In study I, we found a negative correlation between hair-BDNF protein level and hair-cortisol as well as between hair-BDNF and somatic complaints, while hair-cortisol correlated positively with mental distress. In study II, we found a negative correlation between H4 histone acetylation at the BDNF gene P4-promoter and somatic complaints. Regression analysis confirmed confounder stability of associations in both studies. In study III, we confirmed study I and found lower hair-BDNF protein level in volunteers with high somatic complaints, who also reported higher mental distress during the end of term exams. The results indicate that BDNF protein levels can be detected in clipped hair and are associated with somatic complaints and stress in life. In addition, we concluded that plucked hair can provide material for the study of epigenetic changes in stress-affected tissues. These tools can prove valuable for future studies on distress, both under experimental and field conditions.