Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. METHODS: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. RESULTS: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. CONCLUSIONS: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.

Genes possibly related to symbiosis in early life stages of Acropora tenuis inoculated with Symbiodinium microadriaticum.

Due to the ecological importance of mutualism between reef-building corals and symbiotic algae (Family Symbiodiniaceae), various transcriptomic studies on coral-algal symbiosis have been performed; however, molecular mechanisms, especially genes essential to initiate and maintain these symbioses remain unknown. We investigated transcriptomic responses of Acropora tenuis to inoculation with the native algal symbiont, Symbiodinium microadriaticum, during early life stages, and identified possible symbiosis-related genes. Genes involved in immune regulation, protection against oxidative stress, and metabolic interactions between partners are particularly important for symbiosis during Acropora early life stages. In addition, molecular phylogenetic analysis revealed that some possible symbiosis-related genes originated by gene duplication in the Acropora lineage, suggesting that gene duplication may have been the driving force to establish stable mutualism in Acropora, and that symbiotic molecular mechanisms may vary among coral lineages.

Genomic and transcriptomic analyses illuminate the molecular basis of the unique lifestyle of a tubeworm, Lamellibrachia satsuma.

Vestimentiferan tubeworms are representative members of deep-sea chemosynthetic ecosystems. In this study, we developed a draft genome and gene models and performed genomic and transcriptomic analyses of Lamellibrachia satsuma, the only vestimentiferan reported from the euphotic zone. The quality of the genome assembly and gene models is comparable to or higher than those of previously reported vestimentiferan tubeworms. Tissue-specific transcriptome sequencing revealed that Toll-like receptor genes and lineage-specific expanded bacteriolytic enzyme genes are highly expressed in the obturacular and vestimental regions, respectively, suggesting the importance of these tissues in defense against pathogens. On the other hand, globin subunit genes are expressed almost exclusively in the trunk region, supporting the hypothesis that the trophosome is the site of haemoglobin biosynthesis. Vestimentiferan-specific expanded gene families included chitinases, ion channels, and C-type lectins, suggesting the importance of these functions for vestimentiferans. C-type lectins in the trunk region, in particular, may be involved in recognition of pathogens, or in interactions between tubeworms and symbiotic bacteria. Our genomic and transcriptomic analyses enhance understanding of molecular mechanisms underlying the unique lifestyle of vestimentiferan tubeworms, particularly their obligate mutualism with chemosynthetic bacteria.

Establishment of a post-race biomarkers database and application of pathway analysis to identify potential biomarkers in post-race equine plasma.

In the context of doping control, conventional direct chemical testing detects only a limited scope of target substances in equine biological samples. To expand the ability to detect doping agents and their detection windows, metabolomics has recently become a common approach for monitoring alteration of biomarkers caused by doping agents in relevant metabolic pathways. In horse racing, remarkable changes in metabolic profiles between the rest state and racing are likely to affect the identification of doping biomarkers. Previously, we reported a limited number of significantly upregulated metabolites after racing, based on a non-targeted metabolomics approach using out-of-competition and post-race equine plasma samples. In this study, we performed a more thorough analysis of the data set, using pathway analysis to establish a post-race biomarkers database (PBD) that includes upregulated and downregulated metabolites, their fold changes, and relevant pathways, with the main objective of improving our understanding of changes in physiological status related to horse racing. Statistical analysis of the PBD revealed that two peak combinations of pentadecanoyl carnitine/galactosylglycerol (P/G) and heptadecanoyl carnitine/galactosylglycerol (H/G) could be used as potential biomarkers for the discrimination of the rest and post-race groups. To demonstrate the applicability of the PBD, we validated the post-race biomarkers P/G and H/G (highly involved in lipid metabolism) by a single-blind test. This strategy, which combines establishment of a biomarker database with pathway analysis, represents a powerful tool for discovering potential doping biomarkers in the future.