Biological aging markers in blood and brain tissue indicate age acceleration in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is associated with increased mortality and morbidity risk. A reason for this could be accelerated biological aging, which is strongly influenced by disease processes such as inflammation. As recent studies of AUD show changes in DNA methylation and gene expression in neuroinflammation-related pathways in the brain, biological aging represents a potentially important construct for understanding the adverse effects of substance use disorders. Epigenetic clocks have shown accelerated aging in blood samples from individuals with AUD. However, no systematic evaluation of biological age measures in AUD across different tissues and brain regions has been undertaken. METHODS: As markers of biological aging (BioAge markers), we assessed Levine's and Horvath's epigenetic clocks, DNA methylation telomere length (DNAmTL), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in postmortem brain samples from Brodmann Area 9 (BA9), caudate nucleus, and ventral striatum (N = 63-94), and in whole blood samples (N = 179) of individuals with and without AUD. To evaluate the association between AUD status and BioAge markers, we performed linear regression analyses while adjusting for covariates. RESULTS: The majority of BioAge markers were significantly associated with chronological age in all samples. Levine's epigenetic clock and DNAmTL were indicative of accelerated biological aging in AUD in BA9 and whole blood samples, while Horvath's showed the opposite effect in BA9. No significant association of AUD with TL and mtDNAcn was detected. Measured TL and DNAmTL showed only small correlations in blood and none in brain. CONCLUSIONS: The present study is the first to simultaneously investigate epigenetic clocks, telomere length, and mtDNAcn in postmortem brain and whole blood samples in individuals with AUD. We found evidence for accelerated biological aging in AUD in blood and brain, as measured by Levine's epigenetic clock, and DNAmTL. Additional studies of different tissues from the same individuals are needed to draw valid conclusions about the congruence of biological aging in blood and brain.

MicroRNA-Related Polymorphism and Their Association with Fibromyalgia.

MicroRNAs are tissue-specific expressed short RNAs that serve post-transcriptional gene regulation. A specific microRNA can bind to mRNAs of different genes and thereby suppress their protein production. In the context of the complex phenotype of fibromyalgia, we used the Axiom miRNA Target Site Genotyping Array to search genome-wide for DNA variations in microRNA genes, their regulatory regions, and in the 3'UTR of protein-coding genes. To identify disease-relevant DNA polymorphisms, a cohort of 176 female fibromyalgia patients was studied in comparison to a cohort of 162 healthy women. The association between 48,329 markers and fibromyalgia was investigated using logistic regression adjusted for population stratification. Results show that 29 markers had p-values < 1 × 10-3, and the strongest association was observed for rs758459 (p-value of 0.0001), located in the Neurogenin 1 gene which is targeted by hsa-miR-130a-3p. Furthermore, variant rs2295963 is predicted to affect binding of hsa-miR-1-3p. Both microRNAs were previously reported to be differentially expressed in fibromyalgia patients. Despite its limited statistical power, this study reports two microRNA-related polymorphisms which may play a functional role in the pathogenesis of fibromyalgia. For a better understanding of the disease pattern, further functional analyses on the biological significance of microRNAs and microRNA-related polymorphisms are required.

Treatment-associated mRNA co-expression changes in monocytes of patients with posttraumatic stress disorder.

PTSD is a prevalent mental disorder that results from exposure to extreme and stressful life events and comes at high costs for both the individual and society. Therapeutic treatment presents the best way to deal with PTSD-the mechanisms underlying change after treatment, however, remain poorly understood. While stress and immune associated gene expression changes have been associated with PTSD development, studies investigating treatment effects at the molecular level so far tended to focus on DNA methylation. Here we use gene-network analysis on whole-transcriptome RNA-Seq data isolated from CD14+ monocytes of female PTSD patients (N = 51) to study pre-treatment signatures of therapy response and therapy-related changes at the level of gene expression. Patients who exhibited significant symptom improvement after therapy showed higher baseline expression in two modules involved in inflammatory processes (including notable examples IL1R2 and FKBP5) and blood coagulation. After therapy, expression of an inflammatory module was increased, and expression of a wound healing module was decreased. This supports findings reporting an association between PTSD and dysregulations of the inflammatory and the hemostatic system and mark both as potentially treatment sensitive.

Interactive teaching enhances students' physiological arousal during online learning.

The pure transfer of face-to-face teaching to a digital learning environment can be accompanied by a significant reduction in the physiological arousal of students, which in turn can be associated with passivity during the learning process, often linked to insufficient levels of concentration and engagement in the course work. Therefore, the aim of this study was to investigate whether students' psychobiological stress responses can be enhanced in the context of anatomical online learning and how increased physiological parameters correlate with characteristics of learning experiences in a digital learning environment. Healthy first-year medical students (n = 104) experienced a regular practical course in Microscopic Anatomy either in face-to-face learning, in passive online learning or in an interaction-enhanced version of online learning. Compared to passive online learning, students engaged in the interaction-enhanced version of online learning displayed a significantly reduced Heart Rate Variability (P 0.001, partial η2 = 0.381) along with a strong increase in salivary cortisol (P 0.001, partial η2 = 0.179) and salivary alpha-amylase activity (P 0.001, partial η2 = 0.195). These results demonstrated that the physiological arousal of students engaged in online learning can be enhanced via interactive teaching methods and pointed towards clear correlations between higher physiological responses and elementary criteria of learning experience such as engagement and attention.

Transgene detection by digital droplet PCR.

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

Epigenetics in sports.

The heritability of specific phenotypical traits relevant for physical performance has been extensively investigated and discussed by experts from various research fields. By deciphering the complete human DNA sequence, the human genome project has provided impressive insights into the genomic landscape. The hope that this information would reveal the origin of phenotypical traits relevant for physical performance or disease risks has proven overly optimistic, and it is still premature to refer to a 'post-genomic' era of biological science. Linking genomic regions with functions, phenotypical traits and variation in disease risk is now a major experimental bottleneck. The recent deluge of genome-wide association studies (GWAS) generates extensive lists of sequence variants and genes potentially linked to phenotypical traits, but functional insight is at best sparse. The focus of this review is on the complex mechanisms that modulate gene expression. A large fraction of these mechanisms is integrated into the field of epigenetics, mainly DNA methylation and histone modifications, which lead to persistent effects on the availability of DNA for transcription. With the exceptions of genomic imprinting and very rare cases of epigenetic inheritance, epigenetic modifications are not inherited transgenerationally. Along with their susceptibility to external influences, epigenetic patterns are highly specific to the individual and may represent pivotal control centers predisposing towards higher or lower physical performance capacities. In that context, we specifically review how epigenetics combined with classical genetics could broaden our knowledge of genotype-phenotype interactions. We discuss some of the shortcomings of GWAS and explain how epigenetic influences can mask the outcome of quantitative genetic studies. We consider epigenetic influences, such as genomic imprinting and epigenetic inheritance, as well as the life-long variability of epigenetic modification patterns and their potential impact on phenotype with special emphasis on traits related to physical performance. We suggest that epigenetic effects may also play a considerable role in the determination of athletic potential and these effects will need to be studied using more sophisticated quantitative genetic models. In the future, epigenetic status and its potential influence on athletic performance will have to be considered, explored and validated using well controlled model systems before we can begin to extrapolate new findings to complex and heterogeneous human populations. A combination of the fields of genomics, epigenomics and transcriptomics along with improved bioinformatics tools and precise phenotyping, as well as a precise classification of the test populations is required for future research to better understand the inter-relations of exercise physiology, performance traits and also susceptibility towards diseases. Only this combined input can provide the overall outlook necessary to decode the molecular foundation of physical performance.

Detection of EPO gene doping in blood.

Gene doping--or the abuse of gene therapy--will continue to threaten the sports world. History has shown that progress in medical research is likely to be abused in order to enhance human performance. In this review, we critically discuss the progress and the risks associated with the field of erythropoietin (EPO) gene therapy and its applicability to EPO gene doping. We present typical vector systems that are employed in ex vivo and in vivo gene therapy trials. Due to associated risks, gene doping is not a feasible alternative to conventional EPO or blood doping at this time. Nevertheless, it is well described that about half of the elite athlete population is in principle willing to risk its health to gain a competitive advantage. This includes the use of technologies that lack safety approval. Sophisticated detection approaches are a prerequisite for prevention of unapproved and uncontrolled use of gene therapy technology. In this review, we present current detection approaches for EPO gene doping, with a focus on blood-based direct and indirect approaches. Gene doping is detectable in principle, and recent DNA-based detection strategies enable long-term detection of transgenic DNA (tDNA) following in vivo gene transfer.

A quick one-tube nested PCR-protocol for EPO transgene detection.

The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real-time PCR-based approach and one uses a primer-internal, intron-spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate. Based on spiPCR, here we present a novel one-tube nested PCR approach that minimizes the chances for cross-contamination and shows increased sensitivity compared to non-nested PCR techniques. To further reduce the occurrence of false-positives based on cross-contamination, a multi-functional 19bp extended erythropoietin standard (EPO) was cloned which can be easily differentiated from transgenic EPO DNA (tEPO) and can be used as an internal or external positive control in PCR-based applications. We found that one-tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR, and shows similar sensitivity compared to real-time based PCR assays. Although it did not reach sensitivity of spiPCR, the one-tube nested PCR technique described here is less laborious, less expensive and much faster than spiPCR. This technique might therefore be useful as a pre-screening tool for gene doping in the future.

Principle considerations for the use of transcriptomics in doping research.

Over the course of the past decade, technical progress has enabled scientists to investigate genome-wide RNA expression using microarray platforms. This transcriptomic approach represents a promising tool for the discovery of basic gene expression patterns and for identification of cellular signalling pathways under various conditions. Since doping substances have been shown to influence mRNA expression, it has been suggested that these changes can be detected by screening the blood transcriptome. In this review, we critically discuss the potential but also the pitfalls of this application as a tool in doping research. Transcriptomic approaches were considered to potentially provide researchers with a unique gene expression signature or with a specific biomarker for various physiological and pathophysiological conditions. Since transcriptomic approaches are considerably prone to biological and technical confounding factors that act on study subjects or samples, very strict guidelines for the use of transcriptomics in human study subjects have been developed. Typical field conditions associated with doping controls limit the feasibility of following these strict guidelines as there are too many variables counteracting a standardized procedure. After almost a decade of research using transcriptomic tools, it still remains a matter of future technological progress to identify the ultimate biomarker using technologies and/or methodologies that are sufficiently robust against typical biological and technical bias and that are valid in a court of law.

Interaction of serotonin transporter gene-linked polymorphic region and stressful life events predicts cortisol stress response.

There has been significant controversy whether stressful life events (SLEs) experienced over the lifespan may elevate the risk of depression in individuals who are homozygous for the short (S) allele of the repeat length polymorphism (5-HTTLPR) in the regulatory region of the serotonin transporter gene (SLC6A4), compared with individuals homozygous for the long (L) allele. On the basis of the hypothesis that age may be a critical variable, by which such a gene-by-environment interaction may be present in younger adults, but not in older adults and in children, aim of this study was to investigate the role of 5-HTTLPR and SLEs on the endocrine stress response in multiple age cohorts. A total of 115 children (8-12 years), 106 younger adults (18-31 years), and 99 older adults (54-68 years) were subjected to the Trier Social Stress Test (TSST) and structured interviews on SLEs. The TSST induced significant endocrine stress responses in all groups. There was a main effect of genotype in younger and older adults with individuals homozygous for the more active L allele showing a significantly larger cortisol response to the TSST than individuals carrying at least one of the low-expressing S alleles. As predicted, there was a significant interaction of 5-HTTLPR genotype and SLEs, but this interaction was only significant in younger adults and only when the measured SLEs had occurred during the first 5 years of life, suggesting that both age and the specific type of SLE has a role in whether a significant gene-environment interaction is observed.

Stathmin, a gene regulating neural plasticity, affects fear and anxiety processing in humans.

The identification of biological mechanisms underlying emotional behavior is crucial for our understanding of the pathogenesis of mental disorders. Besides genes modulating neural transmission and influencing amygdala reactivity and anxiety-related temperamental traits a different plasticity regulating genes affect interindividual differences in emotional regulation. Recently it has been demonstrated that stathmin, a regulator of microtubule formation which affects long-term potentiation (LTP), controls learned and innate fear responses in rodents, but its role in human emotion regulation is unknown. We hypothesized that in humans the gene coding for stathmin (STMN1), which is highly expressed in the lateral nucleus of the amygdala and associated thalamic and cortical structures, influences behavioral responses to fear and anxiety stimuli by way of two common single nucleotide polymorphisms (rs182455, SNP1; rs213641, SNP2). These polymorphisms are located within or close to the putative transcriptional control region. We used the acoustic startle paradigm and a standardized laboratory protocol for the induction of fear and psychosocial stress in 106 healthy volunteers to investigate the impact of stathmin gene variation on two fear- and anxiety-controlling effector-systems of the amygdala. We found that STMN1 genotype interacting with individuals' gender significantly impacts fear and anxiety responses as measured with the startle and cortisol stress response. We therefore conclude that STMN1 genotype has functional relevance for the acquisition and expression of basic fear and anxiety responses also in humans.