Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA.

BACKGROUND: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. METHODS: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). RESULTS: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. CONCLUSIONS: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

The Salzburg 10/7 HIIT shock cycle study: the effects of a 7-day high-intensity interval training shock microcycle with or without additional low-intensity training on endurance performance, well-being, stress and recovery in endurance trained athletes-study protocol of a randomized controlled trial.

BACKGROUND: Performing multiple high-intensity interval training (HIIT) sessions in a compressed period of time (approximately 7-14 days) is called a HIIT shock microcycle (SM) and promises a rapid increase in endurance performance. However, the efficacy of HIIT-SM, as well as knowledge about optimal training volumes during a SM in the endurance-trained population have not been adequately investigated. This study aims to examine the effects of two different types of HIIT-SM (with or without additional low-intensity training (LIT)) compared to a control group (CG) on key endurance performance variables. Moreover, participants are closely monitored for stress, fatigue, recovery, and sleep before, during and after the intervention using innovative biomarkers, questionnaires, and wearable devices. METHODS: This is a study protocol of a randomized controlled trial that includes the results of a pilot participant. Thirty-six endurance trained athletes will be recruited and randomly assigned to either a HIIT-SM (HSM) group, HIIT-SM with additional LIT (HSM + LIT) group or a CG. All participants will be monitored before (9 days), during (7 days), and after (14 days) a 7-day intervention, for a total of 30 days. Participants in both intervention groups will complete 10 HIIT sessions over 7 consecutive days, with an additional 30 min of LIT in the HSM + LIT group. HIIT sessions consist of aerobic HIIT, i.e., 5 × 4 min at 90-95% of maximal heart rate interspersed by recovery periods of 2.5 min. To determine the effects of the intervention, physiological exercise testing, and a 5 km time trial will be conducted before and after the intervention. RESULTS: The feasibility study indicates good adherence and performance improvement of the pilot participant. Load monitoring tools, i.e., biomarkers and questionnaires showed increased values during the intervention period, indicating sensitive variables. CONCLUSION: This study will be the first to examine the effects of different total training volumes of HIIT-SM, especially the combination of LIT and HIIT in the HSM + LIT group. In addition, different assessments to monitor the athletes' load during such an exhaustive training period will allow the identification of load monitoring tools such as innovative biomarkers, questionnaires, and wearable technology. TRIAL REGISTRATION: clinicaltrials.gov, NCT05067426. Registered 05 October 2021-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05067426 . Protocol Version Issue date: 1 Dec 2021. Original protocol. Authors: TLS, NH.

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy.

Physical activity impacts immune homeostasis and leads to rapid and marked increase in cell-free DNA (cfDNA). However, the origin of cfDNA during exercise remains elusive and it is unknown if physical activity could improve or interfere with methylation based liquid biopsy. We analyzed the methylation levels of four validated CpGs representing cfDNA from granulocytes, lymphocytes, monocytes, and non-hematopoietic cells, in healthy individuals in response to exercise, and in patients with hematological malignancies under resting conditions. The analysis revealed that physical activity almost exclusively triggered DNA release from granulocytes, highlighting the relevance as a pre-analytical variable which could compromise diagnostic accuracy.

Association of Innate and Acquired Aerobic Capacity With Resilience in Healthy Adults: Protocol for a Randomized Controlled Trial of an 8-Week Web-Based Physical Exercise Intervention.

BACKGROUND: Physical activity alleviates chronic stress. The latest research suggests a relationship between resilience and physical fitness. Beneficial adaptations of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system, endocannabinoid system, and tryptophan pathway, which are induced by an active lifestyle, are considered to be conducive to resilience. However, detailed knowledge on the molecular link between the effects of acute and chronic physical exercise and improved resilience to stress in humans is missing. Moreover, the relationship between innate and acquired aerobic capacity and resilience is poorly understood. OBJECTIVE: The aim of this study is to implement a human exercise intervention trial addressing the following main hypotheses: a high innate aerobic capacity is associated with high resilience to stress, and web-based physical exercise training improves aerobic capacity of physically inactive adults, which is accompanied by improved resilience. In this setting, we will analyze the relationship between resilience parameters and innate and acquired aerobic capacity as well as circulating signaling molecules. METHODS: A total of 70 healthy, physically inactive (<150 minutes/week of physical activity) adults (aged 18-45 years) will be randomly assigned to an intervention or control group. Participants in the intervention group will receive weekly training using progressive endurance and interval running adapted individually to their remotely supervised home training performance via web-based coach support. A standardized incremental treadmill exercise test will be performed before and after the intervention period of 8 weeks to determine the innate and acquired aerobic capacity (peak oxygen uptake). Before and after the intervention, psychological tests and questionnaires that characterize parameters implicated in resilience will be applied. Blood and saliva will be sampled for the analysis of cortisol, lactate, endocannabinoids, catecholamines, kynurenic acid, and further circulating signal transducers. Statistical analysis will provide comprehensive knowledge on the relationship between aerobic capacity and resilience, as well as the capacity of peripheral factors to mediate the promoting effects of exercise on resilience. RESULTS: The study was registered in October 2019, and enrollment began in September 2019. Of the 161 participants who were initially screened via a telephone survey, 43 (26.7%) fulfilled the inclusion criteria and were included in the study. Among the 55% (17/31) of participants in the intervention group and 45% (14/31) of participants in the control group who completed the study, no serious adverse incidents were reported. Of 43 participants, 4 (9%) withdrew during the program (for individual reasons) and 8 (19%) have not yet participated in the program; moreover, further study recruitment was paused for an indeterminate amount of time because of the COVID-19 pandemic. CONCLUSIONS: Our study aims to further define the physiological characteristics of human resilience, and it may offer novel approaches for the prevention and therapy of mental disorders via an exercise prescription. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/29712.

Investigation of the Sympathetic Regulation in Delayed Onset Muscle Soreness: Results of an RCT.

Sports-related pain and injury is directly linked to tissue inflammation, thus involving the autonomic nervous system (ANS). In the present experimental study, we disable the sympathetic part of the ANS by applying a stellate ganglion block (SGB) in an experimental model of delayed onset muscle soreness (DOMS) of the biceps muscle. We included 45 healthy participants (female 11, male 34, age 24.16 ± 6.67 years [range 18-53], BMI 23.22 ± 2.09 kg/m2) who were equally randomized to receive either (i) an SGB prior to exercise-induced DOMS (preventive), (ii) sham intervention in addition to DOMS (control/sham), or (iii) SGB after the induction of DOMS (rehabilitative). The aim of the study was to determine whether and to what extent sympathetically maintained pain (SMP) is involved in DOMS processing. Focusing on the muscular area with the greatest eccentric load (biceps distal fifth), a significant time × group interaction on the pressure pain threshold was observed between preventive SGB and sham (p = 0.034). There was a significant effect on pain at motion (p = 0.048), with post hoc statistical difference at 48 h (preventive SGB Δ1.09 ± 0.82 cm VAS vs. sham Δ2.05 ± 1.51 cm VAS; p = 0.04). DOMS mediated an increase in venous cfDNA -as a potential molecular/inflammatory marker of DOMS- within the first 24 h after eccentric exercise (time effect p = 0.018), with a peak at 20 and 60 min. After 60 min, cfDNA levels were significantly decreased comparing preventive SGB to sham (unpaired t-test p = 0.008). At both times, 20 and 60 min, cfDNA significantly correlated with observed changes in PPT. The 20-min increase was more sensitive, as it tended toward significance at 48 h (r = 0.44; p = 0.1) and predicted the early decrease of PPT following preventive stellate blocks at 24 h (r = 0.53; p = 0.04). Our study reveals the broad impact of the ANS on DOMS and exercise-induced pain. For the first time, we have obtained insights into the sympathetic regulation of pain and inflammation following exercise overload. As this study is of a translational pilot character, further research is encouraged to confirm and specify our observations.

Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1-20.3), and intermediate precision ≤ 12.1% (95% CI 9.2-17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise.

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Considerations for the Analysis of Small Extracellular Vesicles in Physical Exercise.

Physical exercise induces acute physiological changes leading to enhanced tissue cross-talk and a liberation of extracellular vesicles (EVs) into the circulation. EVs are cell-derived membranous entities which carry bioactive material, such as proteins and RNA species, and are important mediators of cell-cell-communication. Different types of physical exercise interventions trigger the release of diverse EV subpopulations, which are hypothesized to be involved in physiological adaptation processes leading to health benefits and longevity. Large EVs ("microvesicles" and "microparticles") are studied frequently in the context of physical exercise using straight forward flow cytometry approaches. However, the analysis of small EVs (sEVs) including exosomes is hampered by the complex composition of blood, confounding the methodology of EV isolation and characterization. This mini review presents a concise overview of the current state of research on sEVs released upon physical exercise (ExerVs), highlighting the technical limits of ExerV analysis. The purity of EV preparations is highly influenced by the co-isolation of non-EV structures in the size range or density of EVs, such as lipoproteins and protein aggregates. Technical constraints associated with EV purification challenge the quantification of distinct ExerV populations, the identification of their cargo, and the investigation of their biological functions. Here, we offer recommendations for the isolation and characterization of ExerVs to minimize the effects of these drawbacks. Technological advances in the ExerV research field will improve understanding of the inter-cellular cross-talk induced by physical exercise leading to health benefits.

Children's Cortisol and Cell-Free DNA Trajectories in Relation to Sedentary Behavior and Physical Activity in School: A Pilot Study.

The worldwide prevalence of mental disorders in children and adolescents increased constantly. Additionally, the recommended amount of physical activity (PA) is not achieved by this age group. These circumstances are associated with negative impacts on their health status in later life and can lead to public health issues. The exposure to natural green environments (NGE) seems to be beneficial for human health. The compulsory school system offers great opportunities to reach every child with suitable health-related contents and interventions at an early stage. The concept of Education Outside the Classroom (EOtC) uses NGE and sets focus on PA. Therefore, EOtC might be a beneficial educational intervention to promote students health. The association between biological stress markers and sedentary behavior (SB) plus PA is insufficiently evaluated in school settings. This exploratory study aims to evaluate the association between students' cortisol, plus circulating cell-free deoxyribonucleic acid (cfDNA) levels, and their SB, light PA (LPA), and moderate-to-vigorous PA (MVPA). We assessed data from an EOtC program (intervention group [IG], n = 37; control group [CG], n = 11) in three seasons (fall/spring/summer) in outdoor lessons (IG) in a NGE and normal indoor lessons (CG). SB and PA were evaluated by accelerometry, and cortisol and cfDNA levels by saliva samples. Fitted Bayesian hierarchical linear models evaluated the association between cortisol and cfDNA, and compositional SB/LPA/MVPA. A steady decline of cortisol in the IG is associated with relatively high levels of LPA (posterior mean = -0.728; credible interval [CRI 95%]: -1.268; -0.190). SB and MVPA tended to exhibit a similar effect in the CG. A high amount of cfDNA is positively associated with a relatively high amount of SB in the IG (posterior mean, 1.285; CRI: 0.390; 2.191), the same association is likely for LPA and MVPA in both groups. To conclude, LPA seems to support a healthy cortisol decrease in children during outdoor lessons in NGEs. Associations between cfDNA and SB/PA need to be evaluated in further research. This study facilitates the formulation of straightforward and directed hypotheses for further research with a focus on the potential health promotion of EOtC.

Boost Me: Prevalence and Reasons for the Use of Stimulant Containing Pre Workout Supplements Among Fitness Studio Visitors in Mainz (Germany).

The rapidly increasing interest in fitness related sports over the past few years has been accompanied by a booming industry of nutritional supplements. Many of these substances have unproven benefits and are even potentially harmful to the user. The aim of this study was to determine the prevalence and reasons for nutritional supplement (NS) use among fitness studio visitors in Mainz (Germany), emphasizing new multi-ingredient based supplements such as pre workout boosters (PWBs). Some of the PWBs contain stimulants such as DMAA, N,α-DEPEA, DMAE and DMBA with so far unknown risks, harms and benefits. Four-Hundred and Ninety Two participants in 13 fitness studios completed a questionnaire on the use of nutritional supplements. Descriptive statistics and chi-square tests were used to examine differences in supplement use regarding training- and intake-reasons. About 57.0% of the participants reported the use of NS during the last 4 weeks. The all-time prevalence of creatine use was 28.7%, whereas 12.2% of the participants stated creatine use during the past 4 weeks. The all-time prevalence of PWB intake was 25.8%, whereas the last month prevalence was 11.8%. Among the group of PWB users, 20.5% stated to search specifically for substances such as DMAA, N,α-DEPEA, DMAE or DMBA. Logistic regression analysis showed positive relations between creatine use and the predictor variables gender, strength training and bodybuilding, as well as the stated exercisers' training reasons to increase physical- and sports-performance, and quality of life. PWB consumption was related to the variables gender, training frequency, and the reason for sports performance enhancement. Specific ingredient focus was related to the predictor variables competition participation and increase of mental performance. The results of the study show a high prevalence of PWB consumption among fitness studios visitors, which is comparable with creatine use. The predicting variables for consumption seem to be slightly different between the supplements, especially if the users are searching for stimulating agents. The current findings help to create preliminary consumption patterns and can help to identify potential endangered fitness studio visitors for prevention and risk communication, especially for PWBs.

Gene and Cell Doping: The New Frontier - Beyond Myth or Reality.

The advent of gene transfer technologies in clinical studies aroused concerns that these technologies will be misused for performance-enhancing purposes in sports. However, during the last 2 decades, the field of gene therapy has taken a long and winding road with just a few gene therapeutic drugs demonstrating clinical benefits in humans. The current state of gene therapy is that viral vector-mediated gene transfer shows the now long-awaited initial success for safe, and in some cases efficient, gene transfer in clinical trials. Additionally, the use of small interfering RNA promises an efficient therapy through gene silencing, even though a number of safety concerns remain. More recently, the development of the molecular biological CRISPR/Cas9 system opened new possibilities for efficient and highly targeted genome editing. This chapter aims to define and consequently demystify the term "gene doping" and discuss the current reality concerning gene- and cell-based physical enhancement strategies. The technological progress in the field of gene therapy will be illustrated, and the recent clinical progress as well as technological difficulties will be highlighted. Comparing the attractiveness of these technologies with conventional doping practices reveals that current gene therapy technologies remain unattractive for doping purposes and unlikely to outperform conventional doping. However, future technological advances may raise the attractiveness of gene doping, thus making it easier to develop detection strategies. Currently available detection strategies are introduced in this chapter showing that many forms of genetic manipulation can already be detected in principle.

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.

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.

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.