Persistent Oxytetracycline Exposure Induces an Inflammatory Process That Improves Regenerative Capacity in Zebrafish Larvae

BACKGROUND: The excessive use of antibiotics in aquaculture can adversely affect not only the environment, but also fish themselves. In this regard, there is evidence that some antibiotics can activate the immune system and reduce their effectiveness. None of those studies consider in detail the adverse inflammatory effect that the antibiotic remaining in the water may cause to the fish. In this work, we use the zebrafish to analyze quantitatively the effects of persistent exposure to oxytetracycline, the most common antibiotic used in fish farming. METHODOLOGY: We developed a quantitative assay in which we exposed zebrafish larvae to oxytetracycline for a period of 24 to 96 hrs. In order to determinate if the exposure causes any inflammation reaction, we evaluated neutrophils infiltration and quantified their total number analyzing the Tg(mpx:GFP)(i114) transgenic line by fluorescence stereoscope, microscope and flow cytometry respectively. On the other hand, we characterized the process at a molecular level by analyzing several immune markers (il-1β, il-10, lysC, mpx, cyp1a) at different time points by qPCR. Finally, we evaluated the influence of the inflammation triggered by oxytetracycline on the regeneration capacity in the lateral line. CONCLUSIONS: Our results suggest that after 48 hours of exposure, the oxytetracycline triggered a widespread inflammation process that persisted until 96 hours of exposure. Interestingly, larvae that developed an inflammation process showed an improved regeneration capacity in the mechanosensory system lateral line

Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

BACKGROUND: Metabolic phenotyping has become an important 'bird's-eye-view' technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of 'top-down' chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems. METHODOLOGY/PRINCIPAL FINDINGS: The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 (1)H and (13)C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each (13)C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient (13)C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts. CONCLUSIONS/SIGNIFICANCE: Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of (13)C atoms of given metabolites on development-dependent changes in the 56 identified (13)C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism

Incomplete Inhibition of Sphingosine 1-Phosphate Lyase Modulates Immune System Function yet Prevents Early Lethality and Non-Lymphoid Lesions

BACKGROUND: S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments. METHODOLOGY: In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models. PRINCIPAL FINDINGS: Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation. SIGNIFICANCE: These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs