UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

Benzoxazinoid Metabolites Regulate Innate Immunity against Aphids and Fungi in Maize

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR) is a major pest of maize that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR, but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using carbon-11 with positron emission tomography, root autoradiography and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth which are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of glutamine. Finally, the regrowth zones of WCR attacked roots show an increase in glutamine turnover which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin flux network as a promising marker for induced WCR tolerance.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.