Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

X-ray mosaic nanotomography of large microorganisms.

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.

Morphometric Vertebral Assessments via the Use of Dual X-ray Absorptiometry for the Evaluation of Radiographic Damage in Ankylosing Spondylitis: A Pilot Study.

We performed a pilot study to compare vertebral fracture assessments (VFA) and lateral X-rays in terms of inter- and intraobserver reliability and degree of correlation for the detection of syndesmophytes in ankylosing spondylitis (AS). We recruited 19 patients with AS and recent lumbar or cervical lateral X-rays with at least one syndesmophyte. Each patient underwent dual-energy X-ray absorptiometry with measurement of bone mineral density and dorso-lumbar VFA. Intra- and interreader reliability for VFA and X-rays were measured using 2 independent, blinded observers and Cohen's kappa values. An adapted modified Stoke Ankylosing Spondylitis Spinal Score (amSASSS) was generated with each method, and these 2 values correlated. For X-rays, intraobserver and interobserver agreement were 94.3% (κ = 0.83) and 98.6% (κ = 0.96), respectively; for VFA, corresponding values were 92.8% (κ = 0.79) and 93.8% (κ = 0.82). Overall agreement between the 2 techniques was 88.6% (κ = 0.72). The Pearson correlation coefficient for the 2 methods was 0.95 for the modified Stoke Ankylosing Spondylitis Spinal Score . Per dual-energy X-ray absorptiometry-generated bone mineral density, >50% of patients were osteopenic and 10% osteoporotic. In terms of reproducibility and correlation with X-rays, performing a VFA appears to be a candidate for assessing radiographic damage in AS, thought further research is necessary to justify this indication.

Phase Contrast X-Ray Tomographic Microscopy for Biological and Materials Science Applications

The application of two approaches for high-throughput, high-resolution X-ray phase contrast tomographic imaging being used at the tomographic microscopy and coherent radiology experiments (TOMCAT) beamline of the SLS is discussed and illustrated. Differential phase contrast (DPC) imaging, using a grating interferometer and a phase-stepping technique, is integrated into the beamline environment at TOMCAT in terms of the fast acquisition and reconstruction of data and the availability to scan samples within an aqueous environment. A second phase contrast method is a modified transfer of intensity approach that can yield the 3D distribution of the decrement of the refractive index of a weakly absorbing object from a single tomographic dataset. The two methods are complementary to one another: the DPC method is characterised by a higher sensitivity and by moderate resolution with larger samples; the modified transfer of intensity approach is particularly suited for small specimens when high resolution (around 1 mu m) is required. Both are being applied to investigations in the biological and materials science fields.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

X-ray microtomography of Larger Foraminifera

X-ray microtomography has become a new tool in earth sciences to obtain non-destructive 3D-image data from geological objects in which variations in mineralogy, chemical composition and/or porosity create sufficient x-ray density contrasts.We present here first, preliminary results of an application to the external and internal morphology of Permian to Recent Larger Foraminifera. We use a SkyScan-1072 high-resolution desk-top micro-CT system. The system has a conical x-ray source with a spot size of about 5µm that runs at 20-100kV, 0-250µA, resulting in a maximal resolution of 5µm. X-ray transmission images are captured by a scintillator coupled via fibre optics to a 1024x1024 pixel 12-bit CCD. The object is placed between the x-ray source and the scintillator on a stub that rotates 360°around its vertical axis in steps as small as 0.24 degrees. Sample size is limited to 2 cm due to the absorption of geologic material for x-rays. The transmission images are back projected using a Feldkamp algorithm into a vertical stack of up to 1000 1Kx1K images that represent horizontal cuts of the object. This calculation takes 2 to several hours on a Double-Processor 2.4GHz PC. The stack of images (.bmp) can be visualized with any 3D-imaging software, used to produce cuts of Larger Foraminifera. Among other applications, the 3D-imaging software furnished by SkyScan can produce 3D-models by defining a threshold density value to distinguish "solid" from "void. Several models with variable threshold values and colors can be imbricated, rotated and cut together. The best results were obtained with microfossils devoid of chamber-filling cements (Permian, Eocene, Recent). However, even slight differences in cement mineralogy/composition can result in surprisingly good x-ray density contrasts.X-ray microtomography may develop into a powerful tool for larger microfossils with a complex internal structure, because it is non-destructive, requires no preparation of the specimens, and produces a true 3D-image data set. We will use these data sets in the future to produce cuts in any direction to compare them with arbitrary cuts of complex microfossils in thin sections. Many groups of benthic and planktonic foraminifera may become more easily determinable in thin section by this way.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Marked hemiatrophy in carriers of Duchenne muscular dystrophy.

OBJECTIVE: To describe the clinical and molecular genetic findings in 2 carriers of Duchenne muscular dystrophy (DMD) who exhibited marked hemiatrophy. Duchenne muscular dystrophy is an X-linked disorder in which affected male patients harbor mutations in the dystrophin gene. Female patients with heterozygous mutations may be manifesting carriers. DESIGN: Case study. SETTING: Neurology clinic. PATIENTS: Two manifesting carriers of DMD. INTERVENTIONS: Clinical and radiologic examinations along with histologic and molecular investigations. RESULTS: Both patients had marked right-sided hemiatrophy on examination with radiologic evidence of muscle atrophy and fatty replacement on the affected side. In each case, histologic analysis revealed a reduction in dystrophin staining on the right side. Genetic analysis of the dystrophin gene revealed a tandem exonic duplication in patient 1 and a multiexonic deletion in patient 2 with no further point mutations identified on the other chromosome. CONCLUSIONS: Marked hemiatrophy can occur in DMD manifesting carriers. This is likely to result from a combination of skewed X-inactivation and somatic mosaicism.

In vivo study comparing an X-shaped polymethylmethacrylate and a cylindrical collagen implant for deep sclerectomy.

Background:  Study in vivo characteristics of a polymethylmethacrylate (PMMA) implant compared to the standard cylindrical collagen implant for deep sclerectomy (DS). Design:  Six-month comparative study. Samples:  Twenty eyes of ten rabbits. Methods:  Eyes were randomized to have DS with PMMA implant in one eye and collagen implant in the opposite eye. The growth of the new subconjunctival drainage vessels was assessed by combined fluorescein and indocyanin green anterior segment angiography; intrascleral and subconjunctival blebs were imaged by ultrasound biomicroscopy (UBM). At six months, outflow facility (C) was measured by anterior chamber perfusion and portions of one side of the DS were compared to portions on the 180° opposite side and native sclera on histology. Results:  The mean IOP preoperatively and at one, four, twelve, and twenty-four weeks was comparable in both groups (P > 0.1). UBM showed a statistically insignificant quicker regression of the subconjunctival bleb as well as a durable intrascleral lake in the PMMA group (P > 0.05). New drainage vessels were initially observed one month after surgery; they were more numerous in the PMMA group on angiographic and histological findings at 6 months (P < 0.05). The mean C increased significantly after surgery compared to preoperative values (P < 0.05) and no difference was observed between the implants (0.24 ± 0.06 µl/min/mmHg [PMMA] and 0.23 ± 0.07 µl/min/mmHg [collagen implant]) (P = 0.39). Conclusions:  Deep sclerectomy performed with PMMA or collagen implants showed similar IOP lowering effects, outflow facility increase, and degree of inflammatory reaction.

Analyse UBM du site de filtration après sclérectomie profonde postérieure modifiée utillisant un tube Ex-PRESS? X-50

Purpose: To study the filtering site using ultrasound biomicroscopy. (UBM) after posterior deep sclerectomy with Ex-PRESS? X-50 implant in patients undergoing filtering surgery.¦Methods: Twenty-six patients that participated in this prospective, non comparative study underwent a posterior deep sclerectomy and an Ex- PRESS? X-50 tube implantation. Clinical outcome factors recorded include: intraocular pressure, number of antiglaucoma medications, best corrected visual acuity (BCVA), frequency and types of complications. Six months postoperatively, an ultrasound biomicroscopy examination was performed.¦Results: Mean follow up was 12.0±3.4 months. Mean IOP decreased from 21 ±5.7 mmHg to 12.4±3 mmHg. At last follow-up examination, 65% of eyes had a complete success and 30% a qualified success. The mean number of antiglaucoma medications decreased from 2.5±1.2 preoperatively to 0.7±1 at the last follow-up postoperatively. BCVA was not changed. 27 complications were observed. On the UBM images, the mean intrascleral space volume was 0.25±0.27 mm3 and no relationship was found between volume and intraocular pressure reduction. We noted in 5/26 (19%) eyes a suprachoroïdal hypoechoic. Low-reflective blebs (L-type) were the most common: 15/26 (58%). No correlation between UBM findings and surgical success was evident.¦Conclusions: Deep sclerectomy with Ex-PRESS? X-50 tube implantation seems an efficient glaucoma surgery. It allows satisfactory IOP reduction with a low number of post operative complications. The advantages of deep sclerectomy with collagen implant are maintained with this modified technique. In both, the same reflective types of filtering blebs are present (high, low, encapsulated and flat). The UBM underlines the three mechanisms of aqueous humor resorption previously identified but no correlation with surgical success can be proved.¦-¦Ce travail de thèse est une analyse par ultrasonographic biomicroscopique (UBM) du site de filtration après sclérectomie profonde postérieure modifiée avec implantation d'un tube Ex- PRESS? X-50.¦Vingt six patients atteints d'un glaucome à angle ouvert, ont participé à cette étude prospective et non-comparative. Le critère d'inclusion est un glaucome à angle ouvert non contrôlé malgré un traitement topique maximal.¦Différents types de chirurgie filtrante sont effectués dans la chirurgie du glaucome dont la trabéculectomie et la sclérectomie profonde.¦L'intervention chirurgicale pratiquée dans cette étude consiste en l'implantation d'un tube Ex-PRESS? X-50 de format défini (3 mm de longueur et 50 μπι de diamètre interne) dans la chambre antérieure,au niveau du trabeculum, sous un volet scléral, ce qui permet le drainage de l'humeur aqueuse vers les espaces sous-conjonctivaux, avec diminution de la pression intraoculaire.¦Cette technique implique uniquement une dissection d'un volet scléral superficiel , sans volet scléral profond comme d'une sclérectomie profonde classique.¦Les modes de fonctionnement de cette sclérectomie profonde modifiée sont explorés par UBM, qui donne des images à haute résolution, semblables à des coupes anatomiques. Le volume de l'espace intrascléral créé artificiellement peut en effet être mesuré et mis en corrélation avec la pression intraoculaire et donc avec le taux de succès. Les différents types d'échogénécité de la bulle de filtration sous-conjonctivale provoquée par la dérivation de l'humeur aqueuse sont également observés. La présence éventuelle d'une filtration supplémentaire au niveau choroïdien est aussi détectée.¦De février 2007 à juin 2008, nous avons suivi chez les vingt six yeux des vingt six patients le volume intrascléral, la filtration sous-conjonctivale et la filtration choroïdienne le cas échéant, de même que l'acuité visuelle, la pression intraoculaire, le nombre de traitement antihypertenseur topique et les complications.¦Les résultats démontrent une réduction de 41 % par rapport à la pression intraoculaire préopératoire, ce qui est statistiquement significatif (p<0.0005). En ce qui concerne l'acuité visuelle, les valeurs demeurent stables. Par ailleurs, le nombre de médicaments antiglaucomateux diminue de façon significative de 2.5 ± 1.2 en préopératoire à 0.7 ± 1.0 au dernier examen (p<0.0005). Le volume de l'espace intrascléral, apparaissant toujours en échographie d'aspect fusiforme, n'est pas corrélé de façon significative avec un meilleur succès chirurgical bien que l'on aperçoive une tendance à une corrélation entre un plus grand volume et une pression intraoculaire plus basse.¦La classification la bulle de filtration se fait selon les 4 catégories de bulle de filtration décrites dans la littérature. La répartition révèle une majorité de type L soit hypoéchogène: 15/26 (58%) et une proportion identique, soit, 4/26 (16%), de bulles hyperéchogènes (type H) et encapsulées (type E); les bulles de filtration plates et hyperéchogènes (type F) sont les moins nombreuses 3/26 (11 %).¦La ligne hyporéflective visible dans 19 % des cas entre la sclère et la choroïde représentant potentiellement un drainage suprachoroïdien, n'est pas associée statistiquement à une meilleure filtration et une pression intraoculaire plus basse mais demeure une troisième voie de filtration, en plus de la filtration sous-conjonctivale et intrasclérale.¦En conclusion, cette technique différente, offrant une plus grande sécurité et des résultats satisfaisants sur l'abaissement de la pression intraoculaire, peut être, dans certains cas, une alternative à la sclérectomie profonde classique ,dont elle partage les mécanismes de filtration objectivés par ultrasonographic biomicrioscopique.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Origène face au problème du désaccord (diaphonia) entre les évangiles (CIo X, 3-36)

(Résumé de l'ouvrage) Il "Commento a Giovanni" si presenta con caratteri di eccezionalità nell'ambito di una produzione origeniana, da collocare sotto il segno del genio.

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players.

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide in urine was developed. Sample preparation was performed by liquid-liquid extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10-10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10-5000 ng/mL for nicotine-N'-oxide and cotinine-N-oxide, with calculated coefficients of determination (R(2)) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ?9.4 and ?9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide and cotinine-N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.