A novel transcriptomic based approach to the detection of recombinant human erythropoietin doping

Administration of recombinant human erythropoietin (rHuEpo) improves endurance performance. Hence rHuEpo is, allegedly, frequently subject to abuse by athletes, although rHuEpo is prohibited by the World Anti-Doping Agency. A transcriptomic-based longitudinal screening approach has the potential to improve further the performance of current detection methods. AIM: To assess the effects of rHuEpo on blood gene expression profiles in order to identify a “molecular signature” of rHuEpo doping. METHODS: 19 Caucasian trained males at sea-level (Scotland – SCO) and 20 Kenyan endurance runners at moderate altitude (~2,150 m, Kenya – KEN) received rHuEpo injections of 50 IU∙kg-1 body mass every two days for 4 weeks. Blood was obtained 2 weeks before, during and 4 weeks after administration. 3,000 m time trial performance was measured pre, post administration and at the end of the study. RNA was extracted from blood stabilized in Tempus RNA tubes, amplified, labelled and hybridized to Illumina HumanHT-12v4 Expression BeadChips. Expression data was analyzed using Rank Products with a 5% false discovery rate and an additional 1.5 fold-change threshold. A subset of target and housekeeping genes was further validated using QuantiGene Plex assay. RESULTS: Despite markedly different baseline values between SCO and KEN, as exemplified by the haematocrit (41.9 ± 1.8% vs. 45.3 ± 2.6, p 0.188). These results confirmed that the perturbation involving rHuEpo worked effectively. Relative to baseline, the expression of hundreds of genes were found to be altered by rHuEpo. In particular, 30 transcripts were already differentially expressed two days after the first injection while 15 transcripts were profoundly up-regulated during and subsequently down-regulated up to 4 weeks post administration in both groups. Importantly, the same pattern was observed in all subjects. The functions of the discovered genes were mainly related to either the functional or structural properties of the erythrocyte or to the cell cycle and its regulation. CONCLUSION: This research successfully identified the blood “molecular signature” of rHuEpo administration and provides the strongest evidence to date that gene biomarkers have the potential to substantially improve the performance of current anti-doping methods such as the Athlete Biological Passport for rHuEpo detection

Laminated Metal Composites by Infiltration

We show that steel-magnesium alloy laminated metal composites (LMCs) can be produced by gas pressure infiltration of a liquid magnesium alloy between layers of stacked dimpled steel sheets. Resulting LMCs are amenable to subsequent warm rolling. The LMCs are free of pores or brittle intermetallics and feature, in the as-cast condition, metal layers of uniform thickness and spacing. The ultimate tensile strength of the as-cast LMCs, of 260MPa, obeys the "rule-of-mixtures” (ROM). The uniform tensile elongation, of around 20pct, makes the infiltrated LMC nearly as ductile as the bulk steel it contains, implying that the magnesium alloy in the as-cast LMCs has a substantially increased tensile ductility in comparison to its metallurgically equivalent bulk state. Rolling reduces the metal layer thicknesses, causes waviness in the interface, and makes the LMCs stronger but less ductile, by factors in the vicinity of 2 for both properties; the main cause for this is work hardening in the steel layer

Laminated Metal Composites by Infiltration

We show that steel-magnesium alloy laminated metal composites (LMCs) can be produced by gas pressure infiltration of a liquid magnesium alloy between layers of stacked dimpled steel sheets. Resulting LMCs are amenable to subsequent warm rolling. The LMCs are free of pores or brittle intermetallics and feature, in the as-cast condition, metal layers of uniform thickness and spacing. The ultimate tensile strength of the as-cast LMCs, of 260 MPa, obeys the "rule-of-mixtures" (ROM). The uniform tensile elongation, of around 20 pct, makes the infiltrated LMC nearly as ductile as the bulk steel it contains, implying that the magnesium alloy in the as-cast LMCs has a substantially increased tensile ductility in comparison to its metallurgically equivalent bulk state. Rolling reduces the metal layer thicknesses, causes waviness in the interface, and makes the LMCs stronger but less ductile, by factors in the vicinity of 2 for both properties; the main cause for this is work hardening in the steel layers

Effects of EPO on blood parameters and running performance in Kenyan athletes

Introduction: Recombinant human erythropoietin (rHuEpo) administration enhances oxygen carrying capacity and performance at sea level. It remains unknown whether similar effects would be observed in chronic altitude-adapted endurance runners. The aim of this study was to assess the effects of rHuEpo on hematological and performance parameters in chronic altitude-adapted endurance runners as compared to sea level athletes. Methods: Twenty well-trained Kenyan endurance runners (KEN) living and training at approximately 2150 m received rHuEpo injections of 50 IU·kg−1 body mass every 2 d for 4 wk and responses compared with another cohort (SCO) that underwent an identical protocol at sea level. Blood samples were obtained at baseline, during rHuEpo administration and 4 wk after the final injection. A maximal oxygen uptake (V˙O2max) test and 3000-m time trial was performed before, immediately after and 4 wk after the final rHuEpo injection. Results: Hematocrit (HCT) and hemoglobin concentration (HGB) were higher in KEN compared to SCO before rHuEpo but similar at the end of administration. Before rHuEpo administration, KEN had higher V˙O2max and faster time trial performance compared to SCO. After rHuEpo administration, there was a similar increase in V˙O2max and time trial performance in both cohorts; most effects of rHuEpo were maintained 4 wk after the final rHuEpo injection in both cohorts. Conclusions: Four weeks of rHuEpo increased the HGB and HCT of Kenyan endurance runners to a lesser extent than in SCO (~17% vs ~10%, respectively) and these alterations were associated with similar improvements in running performance immediately after the rHuEpo administration (~5%) and 4 wk after rHuEpo (~3%)

Haemoglobin mass and running time trial performance after recombinant human erythropoietin administration in trained men

<p>Recombinant human erythropoietin (rHuEpo) increases haemoglobin mass (Hbmass) and maximal oxygen uptake (v˙ O2 max).</p> <p>Purpose: This study defined the time course of changes in Hbmass, v˙ O2 max as well as running time trial performance following 4 weeks of rHuEpo administration to determine whether the laboratory observations would translate into actual improvements in running performance in the field.</p> <p>Methods: 19 trained men received rHuEpo injections of 50 IUNkg21 body mass every two days for 4 weeks. Hbmass was determined weekly using the optimized carbon monoxide rebreathing method until 4 weeks after administration. v˙ O2 max and 3,000 m time trial performance were measured pre, post administration and at the end of the study.</p> <p>Results: Relative to baseline, running performance significantly improved by ,6% after administration (10:3061:07 min:sec vs. 11:0861:15 min:sec, p,0.001) and remained significantly enhanced by ,3% 4 weeks after administration (10:4661:13 min:sec, p,0.001), while v˙ O2 max was also significantly increased post administration (60.765.8 mLNmin21Nkg21 vs. 56.066.2 mLNmin21Nkg21, p,0.001) and remained significantly increased 4 weeks after rHuEpo (58.065.6 mLNmin21Nkg21, p = 0.021). Hbmass was significantly increased at the end of administration compared to baseline (15.261.5 gNkg21 vs. 12.761.2 gNkg21, p,0.001). The rate of decrease in Hbmass toward baseline values post rHuEpo was similar to that of the increase during administration (20.53 gNkg21Nwk21, 95% confidence interval (CI) (20.68, 20.38) vs. 0.54 gNkg21Nwk21, CI (0.46, 0.63)) but Hbmass was still significantly elevated 4 weeks after administration compared to baseline (13.761.1 gNkg21, p<0.001).</p> <p>Conclusion: Running performance was improved following 4 weeks of rHuEpo and remained elevated 4 weeks after administration compared to baseline. These field performance effects coincided with rHuEpo-induced elevated v˙ O2 max and Hbmass.</p&gt

An integrative 'Omics' solution to the detection of recombinant human erythropoietin and blood doping

Administration of recombinant human erythropoietin (rHumanEPO) improves sporting performance and hence is frequently subject to abuse by athletes, although rHumanEPO is prohibited by the WADA. Approaches to detect rHumanEPO doping have improved significantly in recent years but remain imperfect. A new transcriptomic-based longitudinal screening approach is being developed that has the potential to improve the analytical performance of current detection methods. In particular, studies are being funded by WADA to identify a ‘molecular signature’ of rHumanEPO doping and preliminary results are promising. In the first systematic study to be conducted, the expression of hundreds of genes were found to be altered by rHumanEPO with numerous gene transcripts being differentially expressed after the first injection and further transcripts profoundly upregulated during and subsequently downregulated up to 4 weeks postadministration of the drug; with the same transcriptomic pattern observed in all participants. The identification of a blood ‘molecular signature’ of rHumanEPO administration is the strongest evidence to date that gene biomarkers have the potential to substantially improve the analytical performance of current antidoping methods such as the Athlete Biological Passport for rHumanEPO detection. Given the early promise of transcriptomics, research using an ‘omics’-based approach involving genomics, transcriptomics, proteomics and metabolomics should be intensified in order to achieve improved detection of rHumanEPO and other doping substances and methods difficult to detect such a recombinant human growth hormone and blood transfusions

Letter to the editor

Santos-Concejero and Tucker argued in their letter to the editor that in our study the running economy in achieving superior performance was overlooked due to some methodological aspects. Having given the remarks of Santos-Concejero and Tucker appropriate consideration, in this letter to the editor, we argue that our paper does not downplay the influence of running economy on the determinants of the East African running phenomenon, but rather adds novel insights into the interaction between running economy, maximal oxygen uptake and performance

Auto detecting deliveries in elite cricket fast bowlers using microsensors and machine learning

Cricket fast bowlers are at a high risk of injury occurrence, which has previously been shown to be correlated to bowling workloads. This study aimed to develop and test an algorithm that can automatically, reliably and accurately detect bowling deliveries. Inertial sensor data from a Catapult OptimEye S5 wearable device was collected from both national and international level fast bowlers (n = 35) in both training and matches, at various intensities. A machine-learning based approach was used to develop the algorithm. Outputs were compared with over 20,000 manually recorded events. A high Matthews correlation coefficient (r = 0.945) showed very good agreement between the automatically detected bowling deliveries and manually recorded ones. The algorithm was found to be both sensitive and specific in training (96.3%, 98.3%) and matches (99.6%, 96.9%), respectively. Rare falsely classified events were typically warm-up deliveries or throws preceded by a run. Inertial sensors data processed by a machine-learning based algorithm provide a valid tool to automatically detect bowling events, whilst also providing the opportunity to look at performance metrics associated with fast bowling. This offers the possibility to better monitor bowling workloads across a range of intensities to mitigate injury risk potential and maximise performance

Dissociation between running economy and running performance in elite Kenyan distance runners

The purpose of this study was to investigate the relationship between running economy (RE) and performance in a homogenous group of competitive Kenyan distance runners. Maximal aerobic capacity (VO2max) (68.8 ± 3.8 ml∙kg−1∙min−1) was determined on a motorised treadmill in 32 Kenyan (25.3 ± 5.0 years; IAAF performance score: 993 ± 77 p) distance runners. Leg anthropometry was assessed and moment arm of the Achilles tendon determined. While Achilles moment arm was associated with better RE (r2 = 0.30, P = 0.003) and upper leg length, total leg length and total leg length to body height ratio were correlated with running performance (r = 0.42, P = 0.025; r = 0.40, P = 0.030 and r = 0.38, P = 0.043, respectively), RE and maximal time on treadmill (tmax) were not associated with running performance (r = −0.01, P = 0.965; r = 0.27; P = 0.189, respectively) in competitive Kenyan distance runners. The dissociation between RE and running performance in this homogenous group of runners would suggest that RE can be compensated by other factors to maintain high performance levels and is in line with the idea that RE is only one of many factors explaining elite running performance

Precision of the optimized carbon monoxide rebreathing method to determine total haemoglobin mass and blood volume

The aim of this study was to evaluate the influence of different apparatuses, procedures and calculations on the precision of the optimized carbon monoxide method. Total haemoglobin mass was determined twice on consecutive days in 11 subjects using both venous and capillary blood samples. To estimate loss of carbon monoxide due to exhalation, carbon monoxide concentration was measured by two portable carbon monoxide analysers (Fluke CO-220, Fluke, Norwich, UK and Pac 7000 Carbon Monoxide, Draeger Safety, Northumberland, UK) and alveolar ventilation was specified using an automated metabolic gas analysis system (Cosmed Quark b2, Cosmed, Rome, Italy). Blood volume was derived from total haemoglobin mass using haemoglobin concentration and haematocrit obtained in both the supine and the seated position. Two different formulae to calculate blood volume were also compared. Precision was good for both total haemoglobin mass and blood volume measurements performed on consecutive days (typical error < 2%). Using Fluke CO-220 analyser, an estimated alveolar ventilation and capillary blood, total haemoglobin mass (917±136 g) was similar when compared to the Pac 7000 Draeger CO-analyser (904±137 g; mean bias –13 g with 95% limits of agreement –26 to + 1 g, P=0.76), specified alveolar ventilation (911±132 g, mean bias –6 g with 95% limits of agreement –18 g to + 6 g, P =0.87) and venous blood (917±134 g, mean bias 0 g with 95% limits of agreement –38 to + 38 g, P=0.99), respectively. Blood volume determination was also not significantly affected by the supine vs. seated position (6.7±0.8 l vs. 6.6±0.8 l, P=0.56) but can deviate by ~0.6 l (P=0.01) depending on the formula applied. Thus, the good precision of the assessment of total haemoglobin mass and blood volume using the optimized carbon monoxide rebreathing method is not significantly influenced by the make of CO analyser, method of obtaining alveolar ventilation, blood sampling method and subject position, but should for longitudinal monitoring purposes use the same formula