Perikles Simon

Randomized Response Estimates for the 12‐Month Prevalence of Cognitive‐Enhancing Drug Use in University Students

To estimate the 12‐month prevalence of cognitive‐enhancing drug use.

Randomized response estimates for doping and illicit drug use in elite athletes.

BACKGROUND To date, there are estimates for the percentage of unknown cases of doping and illicit drug use in fitness sports, but not for elite sports. This can be attributed to the problem of implementing questionnaires and surveys to get reliable epidemiological estimates of deviant or illicit behaviour. METHODS All athletes questioned were subject to doping controls as members or junior members of the national teams. In order to estimate the prevalence of doping and illicit drug abuse, the athletes were either issued an anonymous standardized questionnaire (SQ; n=1394) or were interviewed using randomized response technique (RRT; n=480). We used a two-sided z-test to compare the SQ and RRT results with the respective official German NADA data on the prevalence of doping. RESULTS Official doping tests only reveal 0.81% (n=25,437; 95% CI: 0.70-0.92%) of positive test results, while according to RRT 6.8% (n=480; 95% CI: 2.7-10.9%) of our athletes confessed to having practiced doping (z=2.91, p=0.004). SQ and RRT both revealed a prevalence of about 7% for illicit drug use, but SQ failed to indicate a realistic prevalence of doping (0.20%; 95% CI: 0.02-0.74%). CONCLUSIONS We demonstrate for the first time that data from official doping tests underestimate the true prevalence of doping in elite sports by more than a factor of eight. Our results indicate that implementing RRT before and after anti-doping measures could be a promising method for evaluating the effectiveness of anti-doping programs.

Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer.In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15–20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis or apoptosis of cells in acute exercise settings. Recently, rapid DNA release mechanisms of activated immune-competent cells like NETosis (pathogen-induced cell death including the release of neutrophil extracellular traps [NETs]) have been discovered. cfDNA accumulations might comprise a similar kind of cell death including trap formation or an active release of cfDNA. Just like chronic diseases, chronic high-intensity resistance training protocols induced persistent increases of cfDNA levels. Chronic, strenuous exercise protocols, either long-duration endurance exercise or regular high-intensity workouts, induce chronic inflammation that might lead to a slow, constant release of DNA. This could be due to mechanisms of cell death like apoptosis or necrosis. Yet, it has neither been implicated nor proven sufficiently whether cfDNA can serve as a marker for overtraining. The relevance of cfDNA with regard to overtraining status, performance level, and the degree of physical exhaustion still remains unclear. Longitudinal studies are required that take into account standardized and controlled exercise, serial blood sampling, and large and homogeneous cohorts of different athletic achievement. Furthermore, it is important to establish standardized laboratory procedures for the measurement of genomic cfDNA concentrations by quantitative real-time polymerase chain reaction (PCR). We introduce a new hypothesis based on acute exercise and chronic exposure to stress, and rapid active and passive chronic release of cfDNA fragments into the circulation.

Use of illicit and prescription drugs for cognitive or mood enhancement among surgeons.

BackgroundSurgeons are usually exposed to high workloads leading to fatigue and stress. This not only increases the likelihood of mistakes during surgery but also puts pressure on surgeons to use drugs to counteract fatigue, distress, concentration deficits, burnout or symptoms of depression. The prevalence of surgeons taking pharmacological cognitive enhancement (CE) or mood enhancement (ME) drugs has not been systematically assessed so far.MethodsSurgeons who attended five international conferences in 2011 were surveyed with an anonymous self-report questionnaire (AQ) regarding the use of prescription or illicit drugs for CE and ME and factors associated with their use. The Randomized Response Technique (RRT) was used in addition. The RRT guarantees a high degree of anonymity and confidentiality when a person is asked about stigmatizing issues, such as drug abuse.ResultsA total of 3,306 questionnaires were distributed and 1,145 entered statistical analysis (response rate: 36.4%). According to the AQ, 8.9% of all surveyed surgeons confessed to having used a prescription or illicit drug exclusively for CE at least once during lifetime. As one would expect, the prevalence rate assessed by RRT was approximately 2.5-fold higher than that of the AQ (19.9%; 95% confidence interval (CI), 15.9% to 23.9%, N = 1,105). An even larger discrepancy between the RRT and AQ was observed for the use of antidepressants with a 6-fold higher prevalence (15.1%; 95% CI, 11.3% to 19.0%, N = 1,099) as compared to 2.4% with the AQ. Finally, logistic regression analysis revealed that pressure to perform at work (odds ratio (OR): 1.290; 95% CI, 1.000 to 1.666; P = 0.05) or in private life (OR: 1.266; 95% CI, 1.038 to 1.543; P = 0.02), and gross income (OR: 1.337; 95% CI, 1.091 to 1.640; P = 0.005), were positively associated with the use of drugs for CE or ME.ConclusionsThe use of illicit and prescription drugs for CE or ME is an underestimated phenomenon among surgeons which is generally attributable to high workload, perceived workload, and private stress. Such intake of drugs is associated with attempts to counteract fatigue and loss of concentration. However, drug use for CE may lead to addiction and to overestimation of one’s own capabilities, which can put patients at risk. Coping strategies should be taught during medical education.

Medium Chain Acylcarnitines Dominate the Metabolite Pattern in Humans under Moderate Intensity Exercise and Support Lipid Oxidation

Background Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise. Methodology and Principal Findings First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% VIAT) or a 120 min run (at 70% VIAT) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial β-oxidation was verified in skeletal muscle cell culture experiments by probing 13C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine. Conclusions Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.

Detection, Validation, and Downstream Analysis of Allelic Variation in Gene Expression

Common sequence variants within a gene often generate important differences in expression of corresponding mRNAs. This high level of local (allelic) control—or cis modulation—rivals that produced by gene targeting, but expression is titrated finely over a range of levels. We are interested in exploiting this allelic variation to study gene function and downstream consequences of differences in expression dosage. We have used several bioinformatics and molecular approaches to estimate error rates in the discovery of cis modulation and to analyze some of the biological and technical confounds that contribute to the variation in gene expression profiling. Our analysis of SNPs and alternative transcripts, combined with eQTL maps and selective gene resequencing, revealed that between 17 and 25% of apparent cis modulation is caused by SNPs that overlap probes rather than by genuine quantitative differences in mRNA levels. This estimate climbs to 40–50% when qualitative differences between isoform variants are included. We have developed an analytical approach to filter differences in expression and improve the yield of genuine cis-modulated transcripts to ∼80%. This improvement is important because the resulting variation can be successfully used to study downstream consequences of altered expression on higher-order phenotypes. Using a systems genetics approach we show that two validated cis-modulated genes, Stk25 and Rasd2, are likely to control expression of downstream targets and affect disease susceptibility.

Differential regulation of serum- and glucocorticoid-inducible kinase 1 (SGK1) splice variants based on alternative initiation of transcription.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a key-regulator of transport, cell volume and cell survival. SGK1 transcription is under genomic control of a wide variety of hormones and cell stressors. Little is known, however, about sequence variation in SGK1 transcripts. Thus, we took an in silico approach to determine sequence variations in the N-terminal region of SGK1, which is considered particularly important for subcellular SGK1 localization. Expressed Sequence Tag analysis revealed two novel phylogenetically highly conserved SGK1 mRNAs with different promoter sites based on alternative initiation of transcription at -2981, -850 upstream of the transcription initiation site (+1) of the reference mRNA. RT-PCR in various human cell lines and tissues confirmed the expression of the 3 alternative splice variants, which differed exclusively in their first exons. The two novel variants were devoid of the localization and degradation signal with otherwise unchanged and intact open reading frames. Spatial distribution of transcription factor binding sites among the three promoter sites indicated common responsiveness to glucocorticoids but different responsiveness to hypoxia and cellular differentiation. Differential expression under those conditions was confirmed for all variants in cultured myoblasts and myotubes. p53 and ETF-1 binding sites were overrepresented at the promoter site of the reference sequence variant SGK1(+1). Transcript levels were 4.1-fold [SGK1(+1)] and 3.1-fold [SGK1(-850)] higher in renal clear cell carcinoma than in remote tissue. The transcript levels were 42-fold [SGK1(+1)], 26-fold [SGK1(-850)] and 17-fold [SGK1(-2981)] higher in highly malignant human glioma cells than in non-neoplastic brain tissue. SGK1 transcript levels were differentially increased by differentiation or hypoxia (treatment with CoCl2). In conclusion, the present observations disclose the transcription of three distinct SGK1 splice variants, which are all markedly upregulated in tumor tissue but differentially upregulated following differentiation or hypoxia.

Direct and long-term detection of gene doping in conventional blood samples

The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as ‘gene doping’. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.

Reactive astrocytes and activated microglial cells express EAAT1, but not EAAT2, reflecting a neuroprotective potential following ischaemia

Aims:  Glutamate receptor antagonists have failed clinical stroke trials and it has been proposed that the action of N‐methyl d‐aspartate receptors is necessary for neuronal survival. Thus, excitatory amino acid transporters (EAATs) might be a promising therapeutic target. The aim of this study was to investigate glial expression of EAATs following ischaemia.

Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

BACKGROUND Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.