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10 résultats trouvés.
  • Advancing non-invasive detection of doping via in vivo genome editing

    Advancing non-invasive detection of doping via in vivo genome editing

    By

    Investigateur principal
    K. Cromer

    Regents of the University of California

    États-Unis   ―   2024   ―   En vigueur

    Sommaire

    Code: 242E03KC

    Gene doping, which is banned by WADA, is the alteration of an athlete’s DNA in order to obtain athletic advantage. Rapid advances in genome editing technologies pose a significant future threat to fair competition and athlete safety. This project aims to address this challenge by focusing on detecting performance-enhancing genome modifications.

    This research will begin with animal models to study how effectively genome editing can be achieved in tissues relevant for doping and then be detected non-invasively. From there, the research will work towards developing accurate, affordable, and scalable methods to detect genome editing in humans in the context of performance enhancement.

    The project consists of three main objectives:

    1. Animal studies. Using state-of-the-art genomic technologies, we will perform genome editing experiments in mice. This will help us assess the efficiency of genome editing across different tissues and identify non-invasive detection methods.
    2. Human genome editing detection assay. We will create a targeted deep sequencing tool to detect edits in 20-30 human genes linked to doping. We will develop software to differentiate genuine edits from experimental errors and naturally occurring variations.
    3. Validation. We will validate the assay by applying it to lab-edited primary human hematopoietic stem cells and employing a blinded computational analysis to identify genome editing events.

    This pioneering research will provide crucial insights into how new genomic technologies could be used in doping and on the factors that affect editing detectability. Our goal is to create novel, cost-effective methods for detecting genome editing in humans, supporting anti-doping efforts worldwide.

    Voir le projet à propos de Advancing non-invasive detection of doping via in vivo genome editing
  • Effect of exercise induced proteinuria and hypoxia on the analysis of rEPO

    Effect of exercise induced proteinuria and hypoxia on the analysis of rEPO

    By

    Investigateur principal
    S. Voss

    IDAS Dresden

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 242B05SV

    ERA analysis has been repeatedly legally challenged. One of the criticised points is potential protein overload related to exercise induced proteinuria. Although exercise induced proteinuria has been investigated for IEF and SDS-PAGE and samples are immunopurified, no standardized data are available for the currently used SAR-PAGE method.

    Although both methods apply the same measurement principle, which is basically separation by migration behaviour based on molecular weight and posttranslational modifications such as glycosylation or pegylation of the molecule, the detergent is different. In addition, the analysis method evolved with the introduction of the biotinylated version of the AE7A5 Anti-EPO antibody being now used for the analysis, and no data exist which can exclude the possibility that contents of exercise urines might cross-react with, for example, the Streptavidin-HRP complex which is used for the visualization by chemiluminescence.

    Further, it has not been demonstrated whether high intensity exercise in a hypoxic environment could further affect the ERA analysis, for example by a higher excretion of erythropoietin. Therefore, it is planned to investigate urine samples from 30 athletes who are subjected to an exercise protocol on a treadmill or cycling ergometer known to induce proteinuria. In addition, urine samples from 10 athletes following an acute hypoxic exercise protocol will be collected and examined.

    Voir le projet à propos de Effect of exercise induced proteinuria and hypoxia on the analysis of rEPO
  • Development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification and CRISPR/Cas12a sensing system

    Development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification and CRISPR/Cas12a sensing system

    By

    Investigateur principal
    C. Sung

    Korea Institute of Science and Technology

    Corée du Sud   ―   2024   ―   En vigueur

    Sommaire

    Code: 241E07CS

    The detection of gene and cell doping in athletes is crucial to maintain fair competition and prevent the use of performance-enhancing substances that can be harmful to an athlete's health. In this project, we propose the development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a system with on-chip target gene amplification and detection functions. This chip will facilitate the sequential execution of multiple isothermal nucleic acid amplification and CRISPR/Cas12a sensing reactions directly on small blood samples, eliminating the need for separate pre-processing steps. Our proposed all in one gene doping chip paper features three key components: a sample pad, four conjugation pads, and a test membrane. The sample pad, preloaded with dried reaction buffer, allows for simple heat treatment of whole blood, releasing transgene DNA directly onto the chip without pre-preparation step. Each conjugation pad, linked to a specific target gene, combines on-chip RPA with a Cas12a reaction. In just 15 minutes at 37°C, RPA amplifies the target gene using specific primers. Next, Cas12a cleaves a reporter molecule (biotin-FAM) in the presence of the target exogenous DNA, but leaves it intact in its absence. Finally, the test membrane, pre-coated with streptavidin and anti-anti-FAM lines, captures the reporter depending on the target presence.

    Voir le projet à propos de Development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification and CRISPR/Cas12a sensing system
  • Quantitative criteria for the evaluation of gel imagesfor SAR-PAGE analysis of rEPO

    Quantitative criteria for the evaluation of gel imagesfor SAR-PAGE analysis of rEPO

    By

    Investigateur principal
    C. Reichel

    Austrian Research Centers GmbH- ARC

    Autriche   ―   2024   ―   En vigueur

    Sommaire

    Code: 24T04CR

    Unfortunately, the mass difference between rEPO and uEPO/bEPO is not large enough to result in a clear separation on SAR- and SDS-PAGE. Instead, a “mixed” band containing both compounds is frequently observed. The lower the rEPO concentration becomes, the fainter the area above the endogenous band becomes (“diffuse or faint area” according to TD2024EPO, p. 17). In case of rEPO-doping, this faint area must extend beyond the cut-off line defined by the band apex of Epoetin-δ (Dynepo) or Epoetin-α.

    The weaker the area becomes (e.g. due to intravenous rEPO microdosing), the more difficult the band profile is to interpret. So far, the interpretation relies solely on the experience of the analyst and second opinion provider(s) and thus has been challenged by the athletes’ attorneys in the past. The aim of this project is to define a criterion that enables an objective assessment of the diffuse area. Since all WADA labs use GASepo platform for image analysis, the criterion will be developed using this software. Two approaches will be evaluated: (1) the ratio of relative pixel volumes above the rEPO migration cut-off line (“Dynepo-line”), and (2) the differentiation of the band side plot.

    The evaluation will be first done with urine samples after SAR-PAGE analysis. If successful, studies on serum/plasma and possibly DBS will be performed immediately. As part of this project, a revision of GASepo will be implemented to enable an automated cutting of bands along the “Dynepo” line. The project consists of three phases, namely (1) the intralaboratory phase phase (including a GASepo revision) performed by our lab leading to the proposal of threshold criteria for "AAF", "ATF" and "negative findings", (2) the iterlaboratory validation phase performed by 4 WADA accredited laboratories, and (3) the blinded test samples phase

    Voir le projet à propos de Quantitative criteria for the evaluation of gel imagesfor SAR-PAGE analysis of rEPO
  • A randomised controlled trial to assess whether tapentadol and dihydrocodeine have the potential to enhance performance and represent an actual or potential health risk to athletes.

    A randomised controlled trial to assess whether tapentadol and dihydrocodeine have the potential to enhance performance and represent an actual or potential health risk to athletes.

    By

    Investigateur principal
    A. Mauger

    University of Kent

    Royaume-Uni   ―   2024   ―   En vigueur

    Sommaire

    Code: 241C18AM

    Tapentadol and dihydrocodeine are potent narcotic analgesics. They are currently on WADA's Monitoring Programme as there is concern that they may be used as replacement drugs for the recently prohibited tramadol, to confer a performance advantage by reducing exertional pain and allowing an athlete to work even harder. The drugs' centrally mediated side effects may also present a risk to athletes by reducing motor control which may increase risk of injury to the athlete and their competitors.

    This project will employ an experimental design that identifies whether these narcotics allow

    athletes to work harder by reducing pain, and thus allow for a better performance. It will also assess whether they impair a rider's stability and control of their bicycle. Therefore, the main outcome of this project will be to provide robust experimental evidence to inform whether the in-competition use of tapentadol and dihydrocodeine should be regulated due to potential ergogenicity and athlete safety.

    Twenty-one highly trained cyclists will complete a randomised, controlled, double-blinded crossover study design. Participants will ingest tapentadol, dihydrocodeine, or a placebo and complete an assessment of their balance/stability during intense cycling followed by a laboratory cycling task that replicates the time/intensity demands of professional road cycling. Together, the cycling tasks will involve fixed-intensity and self-paced time trial cycling, amounting to approximately 1.5 hours of hard cycling - a duration and intensity that is representative of the context in which narcotic analgesics are purportedly taken to enhance performance. Exercise performance (completion time), perceptual responses (perceived pain and effort) and participant's control of their bike (lateral movement) will be compared between conditions, with results used to inform consultation with WADA regarding the S7 Narcotics category of the Prohibited List.

    Voir le projet à propos de A randomised controlled trial to assess whether tapentadol and dihydrocodeine have the potential to enhance performance and represent an actual or potential health risk to athletes.
  • Determination of the metabolic pathways of different Rycals in in vitro samples for doping control purposes

    Determination of the metabolic pathways of different Rycals in in vitro samples for doping control purposes

    By

    Investigateur principal
    M. Thevis

    German Sport University Cologne

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 241C07MT

    With the ongoing development of new drugs, the addition of newly developed substances to the World Anti-Doping Agency's Prohibited List has been and continues to be a critical part of anti-doping research. Also, the knowledge of the metabolism of such new compounds is an important tool to identify suspicious samples in routine doping controls. In recent years, ryanodine channel complex stabilizers (Rycals) have received growing attention in the anti doping community due to their potential performance-enhancing effects, by stabilizing the calcium channels which are located in the skeletal muscle. To date, the compounds S107, JTV-519, ARM036 and ARM210 have been investigated for such properties and hence are potential doping agents. While the metabolic behavior of S107 has been investigated, little is known on the metabolism of JTV 519, ARM036 and ARM210. Additionally, the ARM 036 and ARM 210 are either of limited availability or not commercially available.

    In this project, we describe the synthesis of ARM 036 and ARM 210 using a multi step approach. After synthesis, the Rycals S107, JTV 519, ARM 036 and ARM 210 are metabolized in vitro using human liver preparations. The samples are analyzed by means of liquid chromatography (LC) coupled with to a high resolution mass spectrometer (HRMS) to identify possible transformation products. These possible metabolites are then analyzed by tandem mass spectrometry (MS/MS) to gain further information on their possible structures. Suitable metabolites are subjected to chemical synthesis in order to confirm their proposed structures. Precursor compounds as well as identified metabolites are potential targets for doping control analysis and are therefore of particular interest for anti doping research

    Voir le projet à propos de Determination of the metabolic pathways of different Rycals in in vitro samples for doping control purposes
  • Analysis of GLP-1 receptor agonists (Semaglutide, Liraglutide etc.) from blood and dried blood spots by means of LC-MS

    Analysis of GLP-1 receptor agonists (Semaglutide, Liraglutide etc.) from blood and dried blood spots by means of LC-MS

    By

    Investigateur principal
    M. Thevis

    German Sport University Cologne

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 241A15MT

    In 2024, the Glucagon-like-peptide-1 (GLP-1) analog Semaglutide has been included in the WADA monitoring program. While the underlying idea for the development of this peptide-based drug was the improved therapy of diabetes mellitus type II, the resulting anti-obesity properties cause an alarming run in the non-diabetic community (incl. athletes) for Semaglutide-containing drugs like Ozempic or Wegovy, presumably used for weight management purposes. The drug is administered once a week by subcutaneous injection of 0.25 to 2.5 mg and owns a half-life of approx. 8 days. In contrast to other peptide-based drugs, circulating time and levels are comparably high, thus, the analysis of blood and DBS samples by means of LC-MS appears sensible and advantageous. Due to the extensive metabolism and low renal clearance, neglectable levels of intact Semaglutide are excreted, and relatively non-characteristic metabolites appear in urine only.

    Besides Semaglutide, also other GLP-1 receptor agonists are available (e.g. Liraglutide, Lixisenatid and Tirzepatide) as approved medications with comparable biological effects. Noteworthy, the most important analogs Semaglutide, Liraglutide and Tirzepatide share an incorporation of artificially modified lysine residues, which enables the aimed action profile after application. This modification facilitates the analysis by LC-MS considerably due to the explicitly unnatural property of these structures. In this study, it is planned to include several of these GLP-1 analogs into one testing procedure for blood and DBS samples. After successful development, also the analysis of post administration samples with paired blood/DBS and urine-samples from volunteers treated with different GLP-1 receptor agonists is planned in order to enable the comparison of plasma/whole blood and urine levels.

    Voir le projet à propos de Analysis of GLP-1 receptor agonists (Semaglutide, Liraglutide etc.) from blood and dried blood spots by means of LC-MS
  • Investigations into the metabolism and analysis of Kisspeptin and analogs for doping controls by means of LC-MS

    Investigations into the metabolism and analysis of Kisspeptin and analogs for doping controls by means of LC-MS

    By

    Investigateur principal
    M. Thevis

    German Sport University Cologne

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 241A14MT

    Kisspeptins play an important role in human reproductive health by regulating the pituitary secretion of gonadotropin-releasing hormone (GnRH). This renders them therapeutically interesting for the treatment of hypogonadism or anovulation, as well as hormone-dependent cancers. However, the stimulation of GnRH release (and thus indirect stimulation of gonadal testosterone synthesis) also harbors the potential for misuse for doping purposes, which is why Kisspeptins have been introduced to the WADA Prohibited List in 2024.

    Within this research project, we aim to develop a liquid chromatography-mass spectrometry (LC-MS) based method to detect the potentially endogenously occurring peptide hormone Kisspeptin-54, its fragments Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, as well as the synthetic Kisspeptin receptor 1 (KISSR1) agonist TAK-448 in human urine and serum.

    Following method optimization and validation, a suitable number of urine and serum samples will be analyzed as a reference population, to investigate tentative endogenous Kisspeptin levels and to assess a possible need for threshold levels in doping control analysis. Furthermore, metabolism studies will be included, employing suitable in vitro approaches. The resulting data will be evaluated with regard to specific peptide metabolites, that can help to improve the method’s detection capacity for authentic samples following Kisspeptin administration

    Voir le projet à propos de Investigations into the metabolism and analysis of Kisspeptin and analogs for doping controls by means of LC-MS
  • Synthesis, clean-up and mass spectrometric characterization of both YK-11 metabolites 5β‐19‐Nor‐pregnane‐3α,17β,20‐triol and 5β‐19‐Nor‐pregnane‐3α,17β‐diol‐20‐one

    Synthesis, clean-up and mass spectrometric characterization of both YK-11 metabolites 5β‐19‐Nor‐pregnane‐3α,17β,20‐triol and 5β‐19‐Nor‐pregnane‐3α,17β‐diol‐20‐one

    By

    Investigateur principal
    M. Thevis

    German Sport University Cologne

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 241A04MT

    Already in 2006 the synthesis of (17α,20E)‐17,20‐[(1‐methoxyethylidene) bis (oxy)]‐3‐oxo‐19‐norpregna‐4,20‐diene‐21‐carboxylic acid methyl ester (YK-11) was published and investigations into its pharmacological properties demonstrated the potential of YK-11 as a selective androgen receptor modulator (SARM). Albeit YK-11 has not been approved for humans until now, it was first detected in a black-market product in 2017 and several times thereafter. According to the Dietary Supplement Label Database issued by the National Institutes of Health, at least 6 different preparations are currently available in the United States alone claiming to contain YK-11.

    In 2018 the human metabolism of YK-11 was investigated and two main urinary metabolites were detected suitable to be implemented into routine doping control procedures. YK-11 itself was not detected in urine, presumably due to the fact that it undergoes fast metabolism in humans. As the parent compound YK-11 will not be found in urine after administration, doping controls can only rely on the identified urinary metabolites. Both metabolites were synthesized in order to elucidate their chemical structure, but no reference material at a larger scale was synthesized at that time suitable to be provided to all doping control laboratories worldwide.

    In order to enable all laboratories to implement the urinary metabolites of YK-11 into their screening procedures, the production of a suitable reference material of both YK-11 metabolites will be inevitable. Therefore, the aim of this research project is to reinvestigate the different potential routes of chemical synthesis, optimize these and produce reference material of both YK-11 metabolites in order to distribute this to all accredited doping control laboratories.

    Voir le projet à propos de Synthesis, clean-up and mass spectrometric characterization of both YK-11 metabolites 5β‐19‐Nor‐pregnane‐3α,17β,20‐triol and 5β‐19‐Nor‐pregnane‐3α,17β‐diol‐20‐one
  • Accelerating the sample preparation for isotope ratio mass spectrometry-based determinations employing supercritical fluid chromatography

    Accelerating the sample preparation for isotope ratio mass spectrometry-based determinations employing supercritical fluid chromatography

    By

    Investigateur principal
    M. Thevis

    German Sport University Cologne

    Allemagne   ―   2024   ―   En vigueur

    Sommaire

    Code: 241A02MT

    In sports drug testing, isotope ratio mass spectrometry is employed to enable the differentiation between endogenous and exogenous sources of urinary steroids, i.e. steroids produced inside the body or administered. Testosterone or testosterone-prohormones may be used by athlete for performance enhancement. As these steroids are endogenous, their presence alone in human urine is not suspicious. Only if urinary concentrations may exceed individual thresholds, a confirmation of the exogenous source becomes necessary and carbon isotope ratios proved to be the method of choice here.

    Sample preparation protocols for isotope ratio mass spectrometry-based method are complicated and time-consuming as the purity of analytes is crucial and a prerequisite for valid isotope ratio determinations. Currently applied sample preparations encompass solid and liquid-liquid extraction steps to pre-purify and concentrate urine samples prior to the final clean-up step employing high performance liquid chromatography and fraction collection. This powerful tool for sample clean-up comes along with relatively long run-times per sample of more than 45 min and it may be necessary to use even a two-fold separation here.

    This research project aims for improving this crucial step in sample preparation and shorten the run-times per sample by employing supercritical fluid chromatography coupled to a novel fraction collection tool. In supercritical fluid chromatography, pressurized carbon dioxide is used as solvent offering unique features for chromatographic separation not directly comparable to the commonly applied mixtures of water and organic solvent. Therefore, a novel sample preparation method suitable for isotope ratio mass spectrometry will be developed and validated within this project investigating the benefits of supercritical fluid chromatography in sports drug testing. To evaluate the new method and facilitate its implementation into routine doping controls, reference population-based values will be determined and compared to existing routine applications.

    Voir le projet à propos de Accelerating the sample preparation for isotope ratio mass spectrometry-based determinations employing supercritical fluid chromatography
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