New Transcriptomic Biomarkers for Detection of the Recombinant Human Erythropoietin (rHuEPO) MirCERA in Horses.

Detection and monitoring of biomarkers related to doping is particularly suitable for the development of analytical strategies dedicated to indirect detection of banned substances. Previous studies in horses have already allowed the investigation of transcriptomic biomarkers in equine blood associated with reGH and rHuEPO administrations. Our most recent developments continue to focus on the discovery and monitoring of transcriptomic biomarkers for the control of ESAs, and a collaborative study with WADA-accredited doping control laboratories has recently been initiated to conduct a pilot study. In humans, three mRNAs (ALAS2, CA1, and SLC4A1) were previously observed to be differentially expressed after blood doping and were associated with immature red blood cells, the so-called circulating reticulocytes. In horses, circulating reticulocytes are rarely observed even after rHuEPO administration. With the improved primers that detect the equine orthologues of the human mRNAs from the ALAS2, CA1, and SLC4A1 genes, we can now report the first evidence of the detection of the three biomarkers in equine blood. In addition, an upregulation of the mRNA levels of the three genes was observed after analysis of blood samples collected from MirCERA-treated animals, with kinetics similar to those previously documented in humans. Our data suggest that ALAS2 and CA1 are promising indirect biomarkers for the detection of recombinant EPO abuse in horses, as observed in humans.

A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity.

Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

miRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker.

Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agent (MDA) abuses. The development of a quantitative polymerase chain reaction (qPCR) method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, lithium-heparin [LiHep]). Although analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in LiHep tubes is challenging, as heparin is a reverse transcription (RT) and a PCR inhibitor. Different strategies were considered, and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca-miR-144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post-administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.

Development of a Standardized Microflow LC Gradient to Enable Sensitive and Long-Term Detection of Synthetic Anabolic-Androgenic Steroids for High-Throughput Doping Controls.

Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected to mass spectrometry. Applying the newly designed elution procedures described in this paper to the analyses of stanozolol and its metabolites in complex matrixes revealed improved sensitivity compared to previously described high-throughput methods. Indeed, we report the consistent and reliable detection of 16ß-hydroxy-stanozolol down to 10 pg/mL in equine urine and being detectable up-to 3 months after a microdosing administration. Furthermore, a five months long elimination of stanozolol and its metabolites could be monitored on horse mane sections after a single dose administration. Our work highlights novel solutions to detect AAS with improved sensitivity. The application of such developments constitutes new landmarks for doping control laboratories and could be extended to other targeted compounds in residue analysis, toxicology, and metabolomics. Based on this work, the developed chromatographic method is now freely available within the Evosep Plus program.

Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis.

According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.

Innate promiscuity of the CYP706 family of P450 enzymes provides a suitable context for the evolution of dinitroaniline resistance in weed.

Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.

MetIDfyR: An Open-Source R Package to Decipher Small-Molecule Drug Metabolism through High-Resolution Mass Spectrometry.

With recent advances in analytical chemistry, liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) has become an essential tool for metabolite discovery and detection. Even if most of the common drug transformations have already been extensively described, manual search of drug metabolites in LC-HRMS/MS datasets is still a common practice in toxicology laboratories, complicating metabolite discovery. Furthermore, the availability of free open-source software for metabolite discovery is still limited. In this article, we present MetIDfyR, an open-source and cross-platform R package for in silico drug phase I/II biotransformation prediction and mass-spectrometric data mining. MetIDfyR has proven its efficacy for advanced metabolite identification in semi-complex and complex mixtures in in vitro or in vivo drug studies and is freely available at github.com/agnesblch/MetIDfyR.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

A Promiscuous CYP706A3 Reduces Terpene Volatile Emission from Arabidopsis Flowers, Affecting Florivores and the Floral Microbiome.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1.

Four Major Channels Detected in the Cytochrome P450 3A4: A Step toward Understanding Its Multispecificity.

We computed the network of channels of the 3A4 isoform of the cytochrome P450 (CYP) on the basis of 16 crystal structures extracted from the Protein Data Bank (PDB). The calculations were performed with version 2 of the CCCPP software that we developed for this research project. We identified the minimal cost paths (MCPs) output by CCCPP as probable ways to access to the buried active site. The algorithm of calculation of the MCPs is presented in this paper, with its original method of visualization of the channels. We found that these MCPs constitute four major channels in CYP3A4. Among the many channels proposed by Cojocaru et al. in 2007, we found that only four of them open in 3A4. We provide a refined description of these channels together with associated quantitative data.

ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D3) and 25-hydroxycholecalciferol [25(OH)D3] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco-2 cells and increased by ABCB1 overexpression in Griptites or Madin-Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes ( Abcb1-/-) displayed an accumulation of 25(OH)D3 in plasma, intestine, brain, liver, and kidneys, together with an increased D3 postprandial response after gavage compared with controls. 25(OH)D3 efflux through Abcb1-/- intestinal explants was markedly decreased compared with controls. This reduction of 25(OH)D3 transfer from plasma to lumen was further confirmed in vivo in intestine-perfused mice. Docking experiments established that both D3 and 25(OH)D3 could bind with high affinity to Caenorhabditis elegans P-glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single-nucleotide polymorphism (SNP) in ABCB1 (rs17064) was significantly associated with the fasting plasma 25(OH)D3 concentration. Thus, we showed here for the first time that ABCB1 is involved in neo-absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.-Margier, M., Collet, X., le May, C., Desmarchelier, C., André, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Role of Arginine 117 in Substrate Recognition by Human Cytochrome P450 2J2.

The influence of Arginine 117 of human cytochrome P450 2J2 in the recognition of ebastine and a series of terfenadone derivatives was studied by site-directed mutagenesis. R117K, R117E, and R117L mutants were produced, and the behavior of these mutants in the hydroxylation of ebastine and terfenadone derivatives was compared to that of wild-type CYP2J2. The data clearly showed the importance of the formation of a hydrogen bond between R117 and the keto group of these substrates. The data were interpreted on the basis of 3D homology models of the mutants and of dynamic docking of the substrates in their active site. These modeling studies also suggested the existence of a R117-E222 salt bridge between helices B' and F that would be important for maintaining the overall folding of CYP2J2.

Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13.

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.

A multidrug ABC transporter with a taste for GTP.

During the evolution of cellular bioenergetics, many protein families have been fashioned to match the availability and replenishment in energy supply. Molecular motors and primary transporters essentially need ATP to function while proteins involved in cell signaling or translation consume GTP. ATP-Binding Cassette (ABC) transporters are one of the largest families of membrane proteins gathering several medically relevant members that are typically powered by ATP hydrolysis. Here, a Streptococcus pneumoniae ABC transporter responsible for fluoroquinolones resistance in clinical settings, PatA/PatB, is shown to challenge this concept. It clearly favors GTP as the energy supply to expel drugs. This preference is correlated to its ability to hydrolyze GTP more efficiently than ATP, as found with PatA/PatB reconstituted in proteoliposomes or nanodiscs. Importantly, the ATP and GTP concentrations are similar in S. pneumoniae supporting the physiological relevance of GTP as the energy source of this bacterial transporter.

Visualization Within the Ventricles of the Brain: A Micro-Focus X-Ray Study.

Conceptualization of the ventricular system of the brain by macroscopic studies is complicated by the lack of physical structure of these interconnected cavities. Dissection procedures designed to display the structures in the walls of the ventricles are destructive and not conducive for the appreciation of the ventricular system in its entirety. The application of Micro-focus X-ray tomography affords the possibility to appreciate hidden structures in a nondestructive manner. The aim of this study was to explore the possibility of using micro-focus X-ray tomography in the three-dimensional (3D) visualization of the ventricular system as well as the various neuroanatomical structures within its walls for educational purposes. Randomly selected embalmed human cadaver brains were scanned at Necsa (South African Nuclear Energy Corporation) housing the MIXRAD laboratory consisting of a Nikon XTH 225 ST micro-focus X-ray tomography facility. A 3D flythrough video of the ventricular system was reconstructed from these scans using software to view the inner surface of the ventricles. Micro-focus X-ray tomography provides feasible means of delivering high-resolution images in a nondestructive way to design a representation of the ventricular system. In addition, structures in the walls of the ventricular system could be appreciated in a novel way. It is envisaged that this 3D-fly-through video of the ventricular system will be valuable when integrated with standard prosections and atlas pictures in the educational setting. Further studies evaluating the use of this integrative visualization of the ventricular system of the brain for its applicability in the educational setting should be performed. Anat Rec, 301:1138-1147, 2018. © 2017 Wiley Periodicals, Inc.

The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme.

The exponential increase of genomes' sequencing has revealed the presence of NO-Synthases (NOS) throughout the tree of life, uncovering an extraordinary diversity of genetic structure and biological functions. Although NO has been shown to be a crucial mediator in plant physiology, NOS sequences seem present solely in green algae genomes, with a first identification in the picoplankton species Ostreococcus tauri. There is no rationale so far to account for the presence of NOS in this early-diverging branch of the green lineage and its absence in land plants. To address the biological function of algae NOS, we cloned, expressed and characterized the NOS oxygenase domain from Ostreococcus tauri (OtNOSoxy). We launched a phylogenetic and structural analysis of algae NOS, and achieved a 3D model of OtNOSoxy by homology modeling. We used a combination of various spectroscopies to characterize the structural and electronic fingerprints of some OtNOSoxy reaction intermediates. The analysis of OtNOSoxy catalytic activity and kinetic efficiency was achieved by stoichiometric stopped-flow. Our results highlight the conserved and particular features of OtNOSoxy structure that might explain its ultrafast NO-producing capacity. This integrative Structure-Catalysis-Function approach could be extended to the whole NOS superfamily and used for predicting potential biological activity for any new NOS.

Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking.

BACKGROUND: Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. METHODS: We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. RESULTS: The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. MAJOR CONCLUSION AND GENERAL SIGNIFICANCE: This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation.

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme.

We computed the channels of the 3A4 isoform of the cytochrome P450 3A4 (CYP) on the basis of 24 crystal structures extracted from the Protein Data Bank (PDB). We identified three major conformations (denoted C, O1 and O2) using an enhanced version of the CCCPP software that we developed for the present work, while only two conformations (C and O2 ) are considered in the literature. We established the flowchart of definition of these three conformations in function of the structural and physicochemical parameters of the ligand. The channels are characterized with qualitative and quantitative parameters, and not only with their surrounding secondary structures as it is usually done in the literature.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.