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Clinical and Epidemiological MetabonomicsView this Special Issue
Research Article | Open Access
Don Otter, Mingshu Cao, Hui-Ming Lin, Karl Fraser, Shelley Edmunds, Geoff Lane, Daryl Rowan, "Identification of Urinary Biomarkers of Colon Inflammation in IL10-/- Mice Using Short-Column LCMS Metabolomics", BioMed Research International, vol. 2011, Article ID 974701, 12 pages, 2011. https://doi.org/10.1155/2011/974701
Identification of Urinary Biomarkers of Colon Inflammation in IL10-/- Mice Using Short-Column LCMS Metabolomics
The interleukin-10-deficient (IL10-/-) mouse develops colon inflammation in response to normal intestinal microflora and has been used as a model of Crohn's disease. Short-Column LCMS metabolite profiling of urine from IL10-/- and wild-type (WT) mice was used, in two independent experiments, to identify mass spectral ions differing in intensity between these two genotypes. Three differential metabolites were identified as xanthurenic acid and as the glucuronides of xanthurenic acid and of α-CEHC (2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman). The significance of several differential metabolites as potential biomarkers of colon inflammation was evaluated in an experiment which compared metabolite concentrations in IL10-/- and WT mice housed, either under conventional conditions and dosed with intestinal microflora, or maintained under specific pathogen-free (SPF) conditions. Concentrations of xanthurenic acid, α-CEHC glucuronide, and an unidentified metabolite m/z 495-/497+ were associated with the degree of inflammation in IL10-/- mice and may prove useful as biomarkers of colon inflammation.
1. Introduction
Crohn's disease is a chronic relapsing inflammatory disorder of the bowel which is characterized by recurring episodes of inflammation especially in the small and large intestine accompanied by abdominal pain and persistent diarrhea [1]. Crohn's disease has been attributed to a dysregulation of the intestinal mucosal immune response towards normal intestinal microflora and results from a complex interaction of genetic [2, 3] and environmental factors [4]. The complex aetiology and absence of a cure means that the management of Crohn's disease requires the continuing assessment of the inflammation status of the patient so that the efficacy of treatments can be determined. Current methods of assessing bowel inflammation require the sampling of intestinal tissue for histological evaluation or blood for measuring plasma biomarkers of inflammation. Metabolomic characterization, largely by NMR, of urine [5], faeces [6–8], and colon mucosal tissues [9] from Crohn's disease patients has revealed a range of metabolites whose concentrations are perturbed in association with established inflammation. Urinary biomarkers predictive of inflammation status would be preferable to sampling of intestinal tissue or blood as the collection of urine samples is relatively noninvasive and multiple samples can more readily be obtained.
A number of metabolomic studies of mouse models of Crohn's disease have been used to study the biochemical changes associated with inflammation and to identify possible biomarkers. LCMS metabolite profiling of serum from mice treated with dextran sulphate sodium (DSS) to induce colitis showed that colon inflammation was increased by DSS-inhibition of stearoyl-CoA desaturase 1 (SCD1)-mediated oleic acid biogenesis [10]. Examination by NMR of colonic mucosal samples from Sprague Dawley rats treated with 2% carrageenan for 1 or 2 weeks to induce inflammation showed increases in concentrations of creatinine, phosphatidylcholine and unsaturated lipids [11]. The interleukin-10-deficient (IL10-/-) mouse shows physiological, and biochemical similarities to Crohn's disease [3] including the development of intestinal inflammation in response to the presence of intestinal microflora and has been widely used as a model of Crohn's disease [12]. Interleukin-10 (IL10) is an immunosuppressive cytokine that down-regulates cell-mediated immune responses and has an important role in maintaining intestinal mucosal immunity [13]. Metabolic profiling of plasma of IL10-/- mice by NMR indicated the inflamed state was characterised by impaired metabolism of glyco- and lipoproteins and loss of energy homeostasis with higher levels of fatty acid oxidation and glycolysis and interconversion of amino acids to produce energy [14]. NMR metabolite profiling of 24-hour pooled urine [15] and GCMS analysis of spot urine samples [16] from IL10-/- mice identified changes in urinary metabolites associated with colon inflammation providing insights into biochemical changes associated with disease progression. In particular, fucose and xanthurenic acid were identified as early biomarkers of inflammation in the urine of IL10-/- mice [17].
The use of multiple metabolomic platforms extends the range of metabolites and hence the possible perturbations in biochemical pathways that may be observed. Recently, in a cross-platform comparison of metabolomics methods, LCMS was recommended as providing the best combination of versatility and robustness on a single platform [18]. Direct infusion (DI) MS/MS [19, 20] is a recently developed metabolomic analysis tool developed for the rapid screening of complex biological samples. DI-MS/MS combines the analytical power of MS/MS for metabolite characterisation with the advantages of minimal sample handling and high sample throughput.
For nontargeted urinary metabolite profiling, we have modified the DI-MS/MS procedure using a short 20-mm reversed-phase column (Short-Column LCMS) to separate and discard to waste highly ionic material and urea which would otherwise dominate the MS data [21]. In this study, we demonstrate the use of this methodology to rapidly screen sample sets and to identify metabolite differences between the urine from IL10-/- and wild-type mice that were not detected with other techniques. Structural identification of three of these differential metabolites using MS/MS, high resolution MS, and chemical synthesis has provided further insight into the metabolic changes associated with the IL10-/- deficiency. Finally the usefulness of these metabolites as biomarkers of colon inflammation was tested in an experiment where IL10-/- mice were either dosed with intestinal microflora to initiate inflammation or maintained under specific pathogen-free (SPF) conditions to limit the development of inflammation.
2. Materials and Methods
2.1. Animal Experiments
All animal studies were reviewed and approved by the Crown Research Institute Animal Ethics Committee in Hamilton, New Zealand, according to the New Zealand Animal Welfare Act (1999). Male IL10-/- mice of C57BL/6 background strain (B6.129P2-Il10tm1Cgn) and wild-type C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Maine, USA) of average age upon arrival of 4, 5.3, and 4.4 weeks old were used for experiments 1, 2 [16], and 3 [17], respectively. For experiments 1 and 2 (Figure 1), each mouse was housed individually in a shoebox-style cage under conventional conditions and fed powdered AIN76A diet with food intake adjusted to equal the mean amount of food consumed by IL10-/- mice the previous week. Four days after arrival, mice were orally dosed with a mixture of Enterococcus faecalis, E. Faecium, and complex intestinal flora to ensure the same initial microbial exposure and the development of consistent intestinal inflammation [22]. In experiment 1, yellow-fleshed kiwifruit (Actinidia chinensis) and in experiment 2, green-fleshed kiwifruit (A. delicoisa "Hayward"), fruit extracts were added to the diets of some mice [16] to test their possible anti-inflammatory effects [23]. Transcriptomic and proteomic results of diet treatments will be reported elsewhere. In experiment 3, all mice were fed powdered AIN76A diet only. Approximately half the IL10-/- and wild-type mice were maintained under conventional conditions as above and the other half were maintained under SPF conditions and not dosed with intestinal microflora to maintain a lower level of colon inflammation in the IL10-/- mice throughout the experiment [17]. Spot urine samples were collected from each experiment on four collection days when the average age of mice was 5.5, 7, 8.5, and 10.5 weeks old (experiment 1), 7, 9, 11.5 and 12 weeks old (experiment 2) [16], and 6.3, 8.0, 9.4 and 10.4 weeks old (experiment 3) [17]. Final urine samples for experiments 1 and 2 were collected immediately before mice were euthanized and after a cycle of feeding, fasting, and feeding designed to standardize the timing of the final food intake. Mice from each treatment of experiment 3 were euthanized after three and six weeks for histology and measurement of serum amyloid-A (SAA) protein concentrations [17].
2.2. Sample Preparation of Urine for LCMS
Urine (4 μL) was diluted with 0.1% formic acid (200 μL) in an Eppendorf tube, centrifuged at 12100 g for 2 minutes to precipitate particulates and an aliquot transferred to a vial before injection into the LCMS. Randomised batches of samples for each time point were analyzed sequentially.
2.3. Short-Column LCMS for Biomarker Identification
Samples were analyzed by a rapid LC-MS procedure [21] using a Thermo Surveyor pump and auto-sampler connected to a Thermo LTQ linear ion-trap mass spectrometer (Thermo Electron Corporation, San Jose, CA) using negative electrospray ionization. The capillary temperature was 275°C and source ionization voltage was −4000 V. A 20 μL aliquot of diluted urine was loaded onto a Strata-X on-line extraction cartridge (20 × 2.0 mm, Phenomenex, Torrence, CA) with water (300 μL min-1). For the first 0.5 min. the flow from the cartridge was diverted away from the mass spectrometer to waste, after which all flow entered the electrospray source. Samples were eluted with a gradient comprising water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The cartridge was eluted with solvent A for 1.5 min, then with a linear gradient to 100% B over 3 min and held at 100% B for 3 min. The initial solvent (100% A) was restored over a 0.5-minute linear gradient and eluted for 1.5 min at 600 μL min-1 and for 0.5 min at 300 μL min-1. The ion trap was programmed to collect an MS1 spectrum from 100 to 1000 m/z followed by MS2 and MS3 spectra from collision-induced dissociation of the most intense ions in the MS1 spectrum, using a 12-second exclusion list to cycle through coeluting ions.
2.4. LCMS/MS for Metabolite Characterization
Samples selected for further metabolite characterisation were analyzed by LCMS/MS in negative and positive electrospray ionization mode using a Thermo LTQ ion-trap mass spectrometer with temperature and voltage settings as above. The HPLC system consisted of two binary Jasco X-LC 3185PU pumps (Jasco Corporation, Tokyo, Japan) connected to a HTS-PAL auto-sampler (CTC Analytics, Zwingen, Switzerland). A 15 μL aliquot of diluted urine was loaded onto a Luna C18 column (150 × 2.0 mm, 5 μm particle size, Phenomenex, Torrence, CA) with a mobile phase flow rate of 200 μL min-1. For the first 3 min, the flow from the column was diverted away from the mass spectrometer to waste, after which all flow entered the electrospray source. Samples were eluted with a gradient comprising water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient was 95% solvent A for the first 3 min, then a linear gradient to 98% B over 27 min. This was held at 98% B for 3 min and then the initial solvent (95% A) was restored over a 1 min linear gradient and re-equilibrated for 6 min. The ion trap was programmed to collect a MS1 spectrum from 150 to 850 m/z followed by MS2, MS3, and MS4 spectra from collision- induced dissociation of the most intense target ion from a parent ion list comprising candidate negative ions as in Table 1 or the appropriate product ion spectrum. MS data was analysed with XCalibur software (Thermo Electron Corporation, San Jose, CA). Ion trees were compiled from extracted product ion spectra.
Samples were analyzed using a Jasco X-LC UHPLC and HTS-PAL autosampler connected to the LTQ with instrument settings as described above. A 20 μL aliquot of diluted urine was loaded onto a Zorbax SB-C18 UPHLC column (50 × 3.0 mm, 1.8 μm particle size, Agilent Technologies, Santa Clara, CA, USA) with a mobile phase flow rate of 900 μL min-1. For the first 0.7 min, the flow from the column was diverted away from the mass spectrometer to waste, after which all flow entered the electrospray source. Samples were eluted with a gradient comprising water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient was 95% solvent A for the first 0.5 min, then with a linear gradient to 100% B over 4.5 min and then restored to the initial solvent (95% A) over a 0.5 min linear gradient and re-equilibrated for 2 min. The ion trap was programmed to collect an MS1 spectrum from 100–1000 m/z followed by MS2 and MS3 spectra from collision-induced dissociation of the most intense ions in the MS1 spectrum, using a 4-second exclusion list to cycle through coeluting ions.
2.6. High Resolution (HR) Mass Spectrometry
Salts and urea were removed from a pooled IL10-/- urine sample by elution of the sample and an aqueous wash through a C-18 SPE cartridge, and the 80% methanol eluate was collected for MS analysis. The eluate was infused into an LTQ-FTMS (Thermo Electron Corporation, San Jose, CA, USA) for MS/MS and high resolution MS analysis under both positive and negative electrospray ionisation conditions. HR ions observed for xanthurenic acid were negative ions, MS2 product ion (m/z 204 → 160) m/z 160.0405 ([M–H–CO2]-, C9H5O2N- requires 160.0398) and positive ions MS1m/z 206.0450 ([M+H]+, C10H7O4N+ requires 206.0448), and MS2 product ion (m/z 206 → 188) m/z 188.0344 (C10H5O3N+ requires 188.0347). HR ions observed for α-CEHC glucuronide were MS1m/z 453.1773 ([M–H]-, requires 453.1761) and m/z 435.1664 ([M–H–H2O]-, source fragment), requires 435.1655). HR LCMS ions observed for xanthurenic acid glucuronide were MS1m/z 382.0762 ([M+H+], C16H16NO16 requires 382.0774) and MS2 product ion m/z 206.0450 (C10H8NO4 requires 206.0453).
2.7. Bioinformatic Analysis of Short-Column LCMS/MS Data
Raw data (positive and negative ionization) were converted to mzXML format. Nominal mass binning (integer m/z ± 0.5) was carried out for the full m/z range of 100 to 1000 resulting in 901 nominal bins for each individual sample. Signal intensity at nominal mass resolution was retrieved using continuous wavelet transform (CWT) algorithms [24] for peaks within each bin with S/N > 3 (noise defined as 95% quantile of absolute CWT coefficients of scale one). The median peak intensity of the identified peaks in each bin was used to represent the intensity of that bin. Peak intensity was then normalised by a linear regression of log ion intensity against run sequence. Empirical Bayes moderated statistics [25] (R Limma package) were applied to identify MS1 ions that were significantly different (FDR adjusted ) between samples from IL10-/- and wild-type mice in each experiment. Linear modelling of the full factorial design with three factors (genotype, diet (experiments 1 and 2), and day of collection of urine samples) was also carried out for each experiment. No significant diet effects were found for the metabolites discussed below.
2.8. Metabolite Identification
Possible metabolite structures were inferred from MS/MS ion trees and high resolution FTMS data using web-based metabolite databases [26] and by comparison with authentic compounds and published mass spectral fragmentation pathways where available. Xanthurenic acid was obtained from Aldrich Chemical Co. Xanthurenic acid sulphate dipotassium salt was prepared by chemical synthesis [27]. α-CEHC glucuronide was synthesised following the method of Pope et al. [28].
3. Results
3.1. Short-Column LCMS Data Analysis
To identify metabolic differences between IL10-/- and wild-type mice, urine samples were collected at four time points from each of two independent experiments (experiments 1 and 2) [16] (Figure 1). Short-Column LCMS using a short (20 mm) solid phase extraction column enabled extremely rapid sample analysis (8 minutes per sample) and avoided blocking the source with salt build-up but provided only minimal chromatographic separation of analytes. The LCMS data for each sample consisted of broad peaks of varying signal intensity on each of the unit mass channels of the mass spectrometer (Figure 2). LCMS data files were processed based on methods developed for the direct-infusion LCMS data analysis [29] but with notable modifications. With the short solid phase extraction column used in these experiments, the signal distributions within mass bins varied with time with variable and non-Gaussian chromatographic peaks. Robust statistics were needed to summarise the signal intensities within each mass bin. Wavelet-based signal identification was used to identify any m/z peaks within each bin with S/N > 3 [24] with the median average of the identified peaks in each bin being used to represent the intensity of that bin.
A progressive decline in ion intensity, as observed by Koulman et al. [20], was observed during the infusion of the large number of urine samples. Several normalization approaches [30] including linear regression, local regression loess and quantile were tested to correct for this decline. The loss of ion intensity was best explained by a linear relationship between the log of ion intensity and the position of samples in the run sequence. Normalization based on this model was carried out for all MS ions and the normalized data were subjected to statistical analysis.
3.2. Selection of Biomarker Candidate Ions from Short-Column LCMS Data
MS1 ions that were significantly different (FDR adjusted -value ) between samples from IL10-/- and wild-type mice in each experiment were identified using empirical Bayes moderated -statistics [25] (R Limma package) (Supplementary Materials available online at doi: 10.1155/2011/974701, Table S1). More differential metabolites were found by negative mode Short-Column LCMS, however, there was also a pleasing correspondence between the top candidate ions identified in both experiments (1 and 2) and to a lesser extent between candidate ions identified in both positive and negative ionisation modes. From this initial list, biomarker candidates were selected for further investigation based on additional criteria: the detection and level of significant of ions in both experiments, detection early in the experiment, detection as both positive and negative ions, and the presence of sufficient peak intensity to allow detailed MS/MS analysis. A more complex analysis using linear mixed modelling to incorporate effects of diet and the metabolic variation of individual mice was used to evaluate changes in the metabolic responses over time but did not produce additional candidate ions discriminating the IL10-/- mice. LC-MS/MS analysis of selected samples was then carried out to identify which candidate ions (and ion trees) identified from Short-Column LCMS analysis corresponded to single or to multiple metabolites, to eliminate source fragment ions and adducts, and to obtain expanded MS/MS ion trees of chromatographically resolved metabolites of interest. The mass spectral ions selected by these criteria are listed in Table 1.
3.3. Structural Characterization of Biomarker Candidates
3.3.1. Xanthurenic Acid (m/z 160, 204 [M–H]- and m/z 206 [M+H]+)
Three major metabolites with m/z 204- were detected in mouse urine by LCMS of which only the major, and earliest eluting metabolite, was elevated in the urine from IL10-/- mice. This metabolite co-eluted with the only significant m/z 160- ion in the samples (also elevated in IL10-/- mice), a likely source fragment which was shown to arise from the m/z 204- ion, (loss of 44 amu) by MS/MS. High resolution FTMS and LCMS comparison with authentic material identified this metabolite as xanthurenic acid.
3.3.2. Xanthurenic Acid Glucuronide (m/z 380 [M–H] - and m/z 382 [M+H]+)
LCMS identified the negative ions at m/z 380 and the positive ion at m/z 382 as resulting from the same metabolite that occurred at lower concentrations in the urine from IL10-/- mice (Table 1). LC-MS/MS showed loss of a glucuronide-derived fragment (176 amu) in both negative and positive ionization modes to form a product ion (m/z 204 or 206 resp.) which underwent further fragmentations as observed for the corresponding xanthurenic acid ions (Figure 3). High resolution MS data is consistent with xanthurenic acid glucuronide, although this remains to be confirmed by chemical synthesis. Xanthurenic acid glucuronide was identified as a differentiating metabolite (FDR adjusted ) in both positive and negative ion LCMS and in both experiments 1 and 2, but with concentrations changing in the opposite direction to that of its aglycone (Table 1).
3.3.3. α-CEHC Glucuronide (2,5,7,8-Tetramethyl-2-(2′-Carboxyethyl)-6-Hydroxychroman Glucuronide, m/z 453 [M–H]-)
The ion (m/z 453-) occurred at lower concentrations in urine from IL10-/- mice. A metabolite (M+ 454) not containing nitrogen was assumed. LC-MS/MS analysis identified this metabolite as a second glucuronide (MS2 loss of 176 amu to m/z 277) with a subsequent loss of 44 amu, implying a glucuronide acid conjugate. The molecular formula therefore requires a minimum of nine oxygen atoms. FTMS analysis indicated that the major m/z 453- ion in the sample showed the required ion tree and had an accurate mass of 453.1773. Constraining any molecular formula to a minimum of 9 oxygen atoms gave as the most likely formula for this ion (theoretical value 453.1761). Similarly, the major fragment ion at m/z 435.1664 (loss of 18 amu) is assigned a formula of C22H27O9 (theoretical value 435.1655). The molecular formula is then C22H30O10 with C16H22O4 being the formula of the aglycone (a carboxylic acid). A search for such aglycones in the Human Metabolome Database [31] suggested that this metabolite was the glucuronide of α-CEHC. The MS/MS fragmentation appeared consistent with this structure (Figure 4). α-CEHC glucuronide was prepared by synthesis [28] and the MS/MS and LCMS matched that of the urine metabolite. α-CEHC is a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted in the urine [28].
3.3.4. Unidentified Metabolites
The m/z 284- ion was detected amongst the most significant differentiating species between IL10-/- and wild-type mice (Table S1), and xanthurenic acid sulphate, a known urinary metabolite of xanthurenic acid [27], seemed a likely candidate. UHPLC-MS analysis, however, showed that this ion was an isotopologue of m/z 283-, also a highly significant differentiating species (Table S1), and eluted at a later retention time than authentic xanthurenic acid sulphate [27]. Xanthurenic acid sulphate (m/z 284 [M–H]-) was not detected by UHPLC-MS (Experiment 3) using an authentic standard.
Two additional ions (m/z 308- and m/z 497+/495-) were significantly reduced in urine from IL10-/- mice (Table 1). The first metabolite (m/z 308) was found only as a negative ion, and LC-MS/MS analysis (MS2 m/z 124, MS3 m/z 107, 80) suggested a taurine conjugate. The second pair of ions (m/z 497+/495- ) would seem to be derived from the same metabolite as they appear at the same retention time on LCMS/MS with the same losses of 147 and 75 (glycine) amu from the corresponding pseudomolecular ions. The structures of these metabolites remain unknown.
3.4. Evaluation of Candidates as Biomarkers of Colon Inflammation
The biological significance of these metabolic differences as biomarkers of colon inflammation was evaluated in experiment 3 that compared IL10-/- and wild-type mice housed either under conventional conditions and dosed with intestinal microflora or maintained under SPF conditions without dosing with intestinal microflora [17]. The rationale for this experiment was that biochemical differences arising from colon inflammation in IL10-/- mice should be reduced when these mice were maintained under SPF conditions [3]. Biochemical differences arising from other genetic differences between the mouse strains [32] or as a consequence of microbial dosing should not be similarly affected. IL10-/- mice housed under these SPF conditions showed decreased colon inflammation as measured by histology scores and SAA levels in serum [17]. Urine samples were analyzed using UHPLC-MS in both positive and negative ionisation modes with both targeted analysis of biomarker candidates and untargeted metabolic profiling of all metabolites. The top scoring metabolites identified as differing between IL10-/- and wild-type mice by untargeted analysis included xanthurenic acid, xanthurenic acid glucuronide, and α-CEHC glucuronide among the top 20 candidates (Supplementary Table S2: ion labels below refer to this Table). Amongst the positive ions m/z 206 (M206T94), with isotopologues m/z 207 and 208, was the top ranking candidate showing an average 2.4-fold increase () in urine from IL IL10-/- mice. However, the corresponding negative ion (M204T91) was only the 21st ranking candidate despite the high ranking for this ion in previous experiments. Targeted analysis of xanthurenic acid monitoring the m/z 204- MS1 ion in negative mode showed interference from a closely eluting metabolite.
Xanthurenic acid concentrations were significantly higher in dosed mice () with this increase being more apparent between the dosed and SPF IL10-/- animals (). In addition, SPF IL10-/- mice showed an increase in xanthurenic acid concentrations from the earliest timepoint () supporting the hypothesis that xanthurenic acid is an early biomarker of inflammation and consistent with the increasing histology scores and SAA concentrations measured for these mice [17].
UHPLC-MS analysis confirmed that xanthurenic acid glucuronide (M382T85 and M380T82) occurred at significantly higher concentrations in urine from WT mice () (Figure 5) and showed that, while concentrations of this metabolite increased throughout the experiment in both types of mice (), concentrations in IL10-/- mice were not affected by housing conditions (SPF or conventional) (). We therefore conclude that the difference in concentrations of this metabolite between IL10-/- and WT mice represents a metabolic difference between the two mouse strains unrelated to the state of inflammation [17, 32]. Concentrations of this metabolite increased with time in IL10-/- mice under SPF conditions. However, similar increases also occurred in WT mice and would seem to be related to the age of the mice rather than to inflammation.
UHPLC-MS analysis showed that α-CEHC glucuronide (M453T145) occurred at significantly higher concentrations in urine from WT mice (Figure 6) and at higher concentrations when either IL10-/- or WT mice were housed under SPF conditions (). The concentration of this metabolite in urine from WT mice was unaltered over the course of the experiment () but decreased () in the IL10-/- mice under both SPF and conventional conditions. Overall these results suggest that reduced concentrations of α-CEHC glucuronide may serve as a marker of inflammation status in the IL10-/- mice
The metabolite with ions M495T117/M497T116 appeared as the 29th and 41st ranking ions that were significantly different between IL10-/- and wild-type samples. Both ions occurred at significantly lower concentrations () in urine from IL10-/- mice (Figure 7) confirming results obtained by Short-Column LCMS; however, this suppression was partially removed by SPF treatment (Treatment effect for m/z 495 and for the less intense m/z 497 ion). Metabolite concentrations in urine from IL10-/- mice were significantly reduced as a result of microbial dosing (). Concentrations of the more intense m/z 495- ion were also significantly reduced with time ( overall; for IL10-/- mice). These results suggest that reduced concentrations of this metabolite were related to the extent of colon inflammation in the IL10-/- mouse.
UHPLC-MS analysis confirmed that the metabolite with m/z 308 (M308T85) occurred at slightly higher concentrations in urine from IL10-/- mice () and showed that concentrations were reduced in urine from IL10-/- mice under SPF conditions () (Figure 8). However, the downward trend in urinary concentrations of this metabolite with time for all treatments () suggested that its elevated concentrations in IL10-/- mice was not associated with colon inflammation.
4. Discussion
Xanthurenic acid was previously identified by GCMS as an early biomarker of inflammation in urine from IL10-/- mice [16, 17] and is a product of tryptophan catabolism through the kynurenine pathway. This pathway is activated by proinflammatory stimuli such as bacterial lipopolysaccharides and interferon-γ cytokine [33]; however, metabolites of this pathway are also associated with the induction of immune tolerance [33–35]. Thus the elevated levels of xanthurenic acid in urine from IL10-/- mice may result from the absence of negative feedback control by IL10 on the production of kynurenine metabolites.
Xanthurenic acid glucuronide, identified on the basis of its high resolution LC-MS/MS fragmentation pattern, was also a differential metabolite. Concentrations of the glucuronide were lower in urine from IL10-/- mice suggesting a shift to higher free xanthurenic concentrations associated with colon inflammation. However, the low urinary concentration of the glucuronide in IL10-/- mice kept under SPF conditions suggested its concentration was not related to inflammation status. Similar observations have been made for short chain dicarboxylic acids that occurred at reduced concentrations in the urine of IL10-/- mice regardless of inflammation status [17]. The origin of such metabolic differences was attributed to residual 129P2 embryonic stem cell-derived genetic material flanking the IL10 gene in the IL10-/- C57BL/6 mouse [17, 32]. The UDP glucuronosyltransferase (UGT1) gene family (http://www.ensembl.org/) also lies within this region of embryonic stem cell-derived genetic material and may contribute to this metabolic difference between mouse strains. Xanthurenic acid 8-O-sulphate and 8-O-glucoside have also been identified in human urine as natriuretic hormones that regulate sodium excretion by the kidney [27]. Xanthurenic acid sulphate was not detected in the present study while xanthurenic acid glucoside (m/z 366 [M-H]-) was only tentatively detected by UHPLC as the differential metabolite (M366T84) (Table S2); higher in the urine from IL10-/- mice.
α-CEHC is a degradation product of vitamin E normally present in urine [28] where its concentration may reflect that of α-tocopherol and of α-CEHC in the liver [36]. Vitamin E is reduced in serum from Crohn's disease patients possibly as a result of reduced concentrations of serum lipids and of reduced absorption of lipids from the diet [37]. The reduced concentrations of α-CEHC glucuronide observed in urine of IL10-/- mice, and their recovery under SPF conditions, is consistent with reduced availability, degradation, and excretion of vitamin E in mice with impaired intestinal function. Reduced concentrations of α-CEHC glucuronide, and of the related metabolite γ-CEHC β-D-glucoside, have also been identified in urine of mice treated with a pregnane X receptor (PXR) activator [38]. It was suggested that these conjugates in urine may be useful urinary biomarkers of PXR activation. PXR is highly expressed in the intestines where it functions as a xenosensor to regulate the expression of metabolic enzymes and the excretion of xenobiotics as well as of toxic endogenous metabolites such as bile acids [39]. PXR activation was shown to be protective in the DSS-induced colitis mouse model of inflammatory bowel disease [40]. In IL10-/- mice, increased intestinal permeability may likewise result in an increased ingress of xenobiotics and toxic endogenous metabolites such as bile acids resulting in activation of PXR and reduced urinary vitamin E metabolites. Urinary α-CEHC glucuronide may thus serve as a good biomarker of intestinal inflammation.
The discovery of α-CEHC glucuronide and other unknown metabolites as potential inflammatory biomarkers illustrates both the power and the limitations of current analytical techniques in metabolomic analysis. Short-Column LCMS analysis enables extremely rapid metabolic profiling of multiple samples with simultaneous collection of characterising MS/MS data. However, the limited separation of individual metabolites hinders the structural and biochemical characterisation of candidate ions and their biological validation. UHPLC-MS allows rapid measurement of chromatographically individual metabolites but with reduced mass spectral data, so that additional MS experimentation is required to characterise metabolites of interest. The challenge of metabolite identification remains a major constraint but is essential for biological validation of biomarker candidates. Comparison of our results with those obtained from other metabolomics studies of urine from Crohn's disease patients or from animal models indicates how the different analytical tools each brings their own selectivity [18] so that the application of new analytical technology, as here, will reveal new metabolic biomarkers. NMR profiling of urine from Crohn's and ulcerative colitis patients has identified a number of metabolites affected by gut microbes, such as formate, hippurate, and p-cresol sulphate, which were able to discriminate between the patient cohorts [5]. Methylhistidine, glycine, guanidoacetate, and citrate were further metabolites important in distinguishing between cohorts. Methylhistamine has previously been reported as a urinary marker of disease activity in Inflammatory Bowel Disease [41]. However, hippurate, p-cresol sulphate, methylhistidine, and methylhistamine were not detected as differentiating metabolites in the present study. More recently, Lin et al. using GCMS [16, 17] have proposed xanthurenic acid and fucose as early discriminating metabolites of colon inflammation in the urine of the IL10-/- mouse. This paper extends those observations raising wider questions about the role of tryptophan catabolism and xanthurenic acid conjugates in the regulation of urinary salt balance [27] and inflammation.
The authors thank M. Barnett, N. Roy, and R. Broadhurst for their help with mouse experiments and D. Greenwood for FTMS. and They also acknowledge funding from the Foundation for Research, Science and Technology (Contract C06X0702) and Nutrigenomics New Zealand (a collaboration between AgResearch Limited, Plant & Food Research, and The University of Auckland).
Supplementary Materials
Tables of mass spectral ions (Table S1) and mass time identifiers (Table S2) that were significantly different between IL10-/- and WT mice.
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Copyright © 2011 Don Otter et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Old English (Ænglisc, Anglisc, Englisc), or Anglo-Saxon, is the earliest historical form of the English language, spoken in England and southern and eastern Scotland in the early Middle Ages. It was brought to Great Britain by Anglo-Saxon settlers probably in the mid-5th century, and the first Old English literary works date from the mid-7th century. After the Norman conquest of 1066, English was replaced, for a time, as the language of the upper classes by Anglo-Norman, a relative of French. This is regarded as marking the end of the Old English era, as during this period the English language was heavily influenced by Anglo-Norman, developing into a phase known now as Middle English.
Old English developed from a set of Anglo-Frisian or Ingvaeonic dialects originally spoken by Germanic tribes traditionally known as the Angles, Saxons and Jutes. As the Anglo-Saxons became dominant in England, their language replaced the languages of Roman Britain: Common Brittonic, a Celtic language, and Latin, brought to Britain by Roman invasion. Old English had four main dialects, associated with particular Anglo-Saxon kingdoms: Mercian, Northumbrian, Kentish and West Saxon. It was West Saxon that formed the basis for the literary standard of the later Old English period, although the dominant forms of Middle and Modern English would develop mainly from Mercian. The speech of eastern and northern parts of England was subject to strong Old Norse influence due to Scandinavian rule and settlement beginning in the 9th century.
Old English is one of the West Germanic languages, and its closest relatives are Old Frisian and Old Saxon. Like other old Germanic languages, it is very different from Modern English and difficult for Modern English speakers to understand without study. Old English grammar is quite similar to that of modern German: nouns, adjectives, pronouns and verbs have many inflectional endings and forms, and word order is much freer. The oldest Old English inscriptions were written using a runic system, but from about the 9th century this was replaced by a version of the Latin alphabet.
View More On Wikipedia.org
The year is 866 and the last remaining independent Anglo-Saxon kingdom, Wessex, whose forces are commanded by King Aethelred and his younger...
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subset: commoncrawl


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The use of this website is subject to the following terms of use:
Al Khoory Inn, Bur Dubai
17th Street, Bur Dubai, P.O.Box: 6237, Dubai, UAE.
Tel.: +971 4 310 7999, Fax: +971 3 107979 Email.: email@example.com
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Lattitude: 25.2584491
Longitude: 55.3007539
There is construction happening around the hotel. Please note that there may be sound disruptions.


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The state of mind - Liu Wa @ Sabsay
Right now and until June 30. you can experience Liu Wa's first solo show and her debut in Europe. The show Glimpse: A Passing Look comprises an immersive installation of color-coded paintings, experienced in a way triggered by the viewer wearing an electroencephalogram (EEG) headband, controlling the color of light in the room. The paintings, installed all over 3 walls, shows some kind of ecological catastrophe done in what looks like traditional Chinese style.
The real appearance of the paintings remains unknown, alluding to one's perceptions of the external world, both subjective and fluid, because of the changings of the color of the light. Indeed a very sophisticated fusing of technology and art.
"The development of contemporary technology has marked a new era of completely different media within the arts. It is interesting to see how young artists so fervently immerse into the technological opportunities. Liu Wa's exhibition at Sabsay is a bright example of graceful overlaying of the traditional and the cutting edge", says Masha Faurschou, founder of Sabsay - and we agree.
We where lucky enough to meet Liu Wa at Sabsay and have her talk further about her work.
Liu Wa (Beijing 1994), have a B.A. in Anthropology & B.A. in Art from Yale University, USA. She now lives and work in New York and Beijing.
Recent exhibitions include: Art Nova 100, Today Art Museum, Beijing, China, 2017; Heart of the Tin Man, M Woods Museum, Beijing, China, 2017; All happens after sunset, Museum of Contemporary Art Shanghai, Shanghai, China, 2017; Art Utopia, Ullens Center for Contemporary Art, Beijing, China, 2016, and Arts First Festival, Harvard University, Cambridge, USA, 2015.
OBS: May 24. there will be a talk in Sabsay. Director of The National Gallery SMK Mikkel Bogh and brain researcher Martin Skov will talk about neuro science and art. Sabsay 17.30-18.30
Glimpse: A Passing Look
Liu Wa
04.05.2018 - 30.06.2018
The technology is called EEG, ElectroEncephaloGram, and it basically measures the different kinds of brainwaves that will occur during brain activities. The way it works is that it senses the fluctuations of voltage from the bioelectrical activity in the brain and how it is reflected on the skin. It measures just the skin, but the minor fluctuations of activity is already enough to measure the attention level of the wearer of the device and when the wearer has concentration in a higher level it will affect the colour of the light. So in different states of mind people will see different colors and different parts of the painting will emerge from the darkness and some parts will fade away.
I have been exploring how this kind of technology can be combined with a more traditional medium like painting. It was my last project. Because the last project incorporated some light as well, but this time I just wanted to use the color of the light. So I was interested in the color and then I just remembered that red, green and blue are the primary colors of light. Then I would try to play around with it and did a lot of experiments just to see the effects and I thought it was really amazing. And then I just had the idea of creating a painting, composed of just red, green and blue.
The colors are not connected to the mood, this device only measures the intensity of the attention level, so it's like a spectrum of attention levels, of concentration. I don't want there to be an answer or a goal that the audience want to head to. It's more like you are trying to explore and feel your own self throughout the process, and try to figure out what you are thinking, like being really self aware in a way that is similar to meditation or mindfulness.
It really depends on the person and also the mental state of the person at that point. That's also the part I find really interesting; the subjectivity of the viewer. The painting can be read as a projection of one's subjective feelings. So you're projecting your own filter onto the paintings and then you will see different impressions in different states of mind. Just like people can see different colours in different states of mind there is also this kind of momentary glimpses of the painting. The perceptions of the external world it can be really subjective and it can be really fluent and people can have completely different views facing different kinds of situations and this can be irreconcilable and give rise to conflicts between different groups, like religious groups or social classes. It describes how people see the world differently through their own lenses.
My next project will probably still deal with this red, green and blue colour, but it will extend. Right now the painting is two-dimensional but then I'm trying to do something that extends from the two dimensional to the space, so some of the objects of the painting will extend, become real objects in the space. I haven't finalised my idea yet but i really do want to play with the space more.
The painting shows an ecological apocalypse and how people are trying to employ different kinds of means to survive. And when people are facing these kinds of situations, people in a safer spot will be more relaxed whereas people in the water will be struggling to survive. People act differently to the same situation depending on their own position. So it gets really dramatic when it is a really extreme and dangerous situation.
I guess I've always been interested in tragical and sad moments in life. In general I use these themes a lot. The ecological apocalypse is something not happening right now, but we've been discussing how global warming and flood and refugee crises can come into play, but I don't want to emphasise only one theme. In the project I wanted to create an open discussion. You can refer to the refugee crisis, because there are people struggling in the flood not necessarily. But you can also see it in another way.
Thank you


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Provence is everything the guidebooks say it is – picturesque villages, views over vineyards, olives, lavender and hilltops. And nestled into the middle of it are arguably 2 of the most lovely of them all. Less than 20 minutes drive from each other, they make for a perfect day in Provence.
Roussillon is noteworthy and unusual for the ochre quarries that surround it. While most Provencal villages are known for their grey stone buildings and cool blue and purple accents, Roussillon radiates warm oranges and reds, which surround you in the rock quarries themselves and the buildings which clearly drew from their environment for supplies and inspiration.


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The Patron Saint of Liars
Author: Ann Patchett
Format: Paperback
Pages: 352
Language: English
Publisher: HarperCollins Publishers
Release Date: April 01, 2003
Since her first publication in 1992, celebrated novelist Ann Patchett has crafted a number of elegant novels, garnering accolades and awards along the way. Now comes a reissue of the best-selling debut novel that launched her remarkable career. St. Elizabeth's, a home for unwed mothers in Habit, Kentucky, usually harbors its residents for only a little while. Not so Rose Clinton, a beautiful, mysterious woman who comes to the home pregnant but not unwed, and stays. She plans to give up her child, thinking she cannot be the mother it needs. But when Cecilia is born, Rose makes a place for herself and her daughter amid St. Elizabeth's extended family of nuns and an ever-changing collection of pregnant teenage girls. Rose's past won't be kept away, though, even by St. Elizabeth's; she cannot remain untouched by what she has left behind, even as she cannot change who she has become in the leaving.


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"No act of kindness, no matter how small, is ever wasted." By Aesop
Random Acts of Kindness Week, February 12-18, is a week-long celebration dedicated to encouraging people to do one thing: be kind. You can follow the thread at #RAKweek2017.
The Random Acts of Kindness (RAK) Foundation is a great site to help you integrate RAK in your classroom. RAK goal is to "ultimately striving to make kindness the standard in every aspect of life. Whether it's helping a stranger in need on your way to work, instilling the importance of kindness in students in a classroom, or a mutual demonstration of appreciation of those closest to us, our end-goal is to make kindness not an act at all, but a reflex. And to make the need for kindness obsolete by the overwhelming and undeniable presence of it everywhere."
RAK is apart of the hidden curriculum in schools but there are lots of ways to integrate it seamlessly so that it is apart of your classroom culture. Below are five ways you can integrate RAK:
- Talk about RAK during morning meetings and/or advisory time.
- Start a bucket filling program in your classroom or school
- Make it apart of your Interactive Read Aloud. Here are some book titles to get you started:
Have You Filled A Bucket Today? A Guide To Daily Happiness For Kids by Carol McCloud
Ordinary Mary's Extraordinary Deed by Emily Pearson, Fumi Kosaka
We All Sing With The Same Voice by J. Philip Miller and Sheppard M. Greene
Each Kindness by Jacqueline Woodson
A Sick Day For Amos McGee by Philip C. Stead and Erin E. Stead
Last Stop On Market Street by Matt de la Pena
Horton Hears A Who by Dr. Seuss
Enemy Pie by Derek Munson
The Giving Tree by Shel Silverstein
The Three Questions by Jon J Muth
- Show video's demonstrating RAK. You can use the RAK Video Library as a resource!
- Ripil App is a free "kindness tracker" where you can post daily RAK goals.
You can start integrating RAK by also becoming a RAKtivists = Random Acts of Kindness activist! You can apply to be a RAKtivist here.
More Articles and Information about RAK:
Random Acts of Kindness Week: Encouraging Empathy and Appreciation of Diversity in Your Classroom
102 Random Acts of Kindness – Ideas to Inspire Kindness
9 Ways to Introduce Students to Random Acts of Kindness


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Municipal Calendar
Saturday, November 30, 2019
Old Fashioned Christmas
Sat. 30 Nov, 2019 9:00 am
Make plans now to attend this years' Sherbrooke Village Old Fashioned Christmas Celebrations taking place Friday, Nov. 22nd to Sunday, Nov. 24th
Opening night features the Christmas tree lighting, Kidd Brothers Performance, visit from Santa, treats and fireworks!
The weekend will be filled with activities including a Christmas craft market & workshops, Victorian & Children's Tea, heritage building & house tours, children's activities, free skate, breakfast, lunches and Dinner theatre, visits to "Whoville", the artists co-op, and a Christmas Variety concert.
Festivities continue November 30th
Sherbrooke Village Old Fashioned Christmas is back for another great day of activities, on Saturday November 30th. Activities will include breakfast, lunches, Christmas craft market & workshops, decorated house tours, Amazing Santa Race, closing night parade and fireworks followed by a Victorian Yuletide Feast, Guided Lantern Tour and dance. Be sure to visit "Whoville" and the Artists Co-op.
65,000 Christmas lights will be lit from 6 till 10 pm every evening from November 22nd until January 6th. There's something for everyone this Holiday Season so make plans now to attend this years' Sherbrooke Village Old Fashioned Christmas!
For reservations or more information call 1-888-743-7845, visit our website oldfashionedchristmas.sherbrookevillage.ca/ or Facebook page: facebook.com/ofc.at.sv/


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New laws are increasing parental leave entitlements. Find out what impact it will have on your business.
When: 1 April 2016.
What: Parental leave payments are increasing from 16 to 18 weeks.
The new law extends parental leave payments to more workers, including:
Previously only biological or formal adoptive parents could claim parental leave payments. The law change extends this to others who take on the permanent care of children aged under six, eg home-for-life and whāngai arrangements.
Eligible parents of premature babies will receive parental leave payments for longer.
Staff who've worked for an employer between six and 12 months will be entitled to six months' parental leave. This includes the 18-week paid period plus eight weeks of unpaid leave.
The changes also make it easier for parents to stay connected to the workforce. Current rules stop anyone on parental leave from returning to work for training or planning days. This will change to give workers on parental leave more flexibility.
Why: The aim is to let parents and primary carers take more time off work to spend time with the new additions to their families, while also staying in touch with work.
What you'll need to do: If a member of your staff is about to become a parent or primary carer, make sure you both know what they're entitled to. Even if this isn't the case, staff may appreciate hearing the details from you.
Modernising parental leave (external link) — Ministry of Business, Innovation and Employment
Applying for parental leave: Information for employees and employers (external link) — Employment New Zealand


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Do I Need a Driving License for a Motability Car?
You do not need to drive if you take out a lease agreement on a Motability car. You may nominate up to two drivers (friends or relatives) and parents may apply for a lease agreement on behalf of a child aged 3 or above. Designated drivers must be exempt from major driving convictions and disqualifications, in the 5 years prior to taking out the agreement. Under 25's and holders of provisional licenses may have restrictions.
For more information on the Motability scheme you can visit our dedicated page, or please call 0333 323 7070 to find out more.Motability Offers
Frequently Asked Questions
Get the answers to the Motability questions we get asked the mostView FAQs
Have you lost your allowance
All the information you need to support you through the transitionHelp & Guidance
Sell Your Car
We'll buy your car, even if you don't purchase from us. Let us value your carFind Out More
Find Your Motability Retailer
Locate your nearest Motability retailer, where our specialists are on hand to helpFind A Retailer


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Looking at my inbox, I saw a message with "Wais P" and "NSFW" in the subject and had to take a look. The hook on this song turns one man's adversity into another man's slang. By the way, this is the safe-for-work version for all you cats on the West Coast or those of us on the East still grinding in the office. Check out the NSFW version here. Read More
Harlem rapper Trevell Coleman, aka G. Dep, confesses to murder during a robbery gone wrong back in 1993. Read More


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We offer three levels of membership, bringing together some of Australia's top female executives, CEOs, business leaders and entrepreneurs just like you. Women who are;
- committed to building their businesses or careers
- wanting to fast track their growth and personal success
- ready and willing to ask for and receive support
- understand the importance of accessing continuous learning at a professional learning to achieve mastery in what they do
- willing to challenge the status quo
- looking to surround themselves with the best in their respective businesses
- interested in intelligent, commercially savvy conversations and exploring new ways to solve business challenges
- understand the opportunity that exists when you bring together collective knowledge and wisdom.
CONNECT, INSPIRE and SUCCEED membership levels are designed to offer increasing mentoring, business development, networking and training opportunities. Depending on where you are in your corporate or entrepreneurial journey, there is a membership level to suit your individual needs.
Some of our key events and benefits;
- Mentoring – as an LBD member you will receive mentoring in the moment with our CEO, Paula Kensington. The SUCCEED membership will give you greater exposure and time with Paula during workshops and quarterly IMMERSION – the perfect way to develop a laser sharp focus on key goals and success strategies
- The Hub – a library of expertise to support business, professional & personal growth – videos, podcasts and resources from subject matter experts and business leaders
- Exclusive Networking Dinner Series – facilitated dinners in private dining rooms for up to 16 members giving you the platform to discuss in confidence your challenges and opportunities, to share ideas and to learn from your business peers
- Networking Events – network and learn – connect over a glass of wine and hear the real stories behind success as we put members and guests under the spotlight – the perfect mix of networking, learning and the opportunity to build business connections for your future growth and success
- Business Immersion Days – four times a year and for a full day, step away from working in your business and work on your business or career – learn from our CEO and leadership experts, prepare for the future and develop new skills, tools and strategies – highly interactive our Business Immersion Days give you the time to work strategically on your career and business goals.
- Annual Retreat – once a year you will have the opportunity to join us on an exclusive retreat with your fellow members – be inspired, be re-energised, be motivated to commit and engage with your personal and business success goals
- Closed Facebook member only group – restricted to LBDGroup members only, the closed Facebook page allows you to connect, share and seek advise in a private forum
- Kick start your network – an introductory phone call to establish your immediate business needs and receive a personal introduction to three members of LBDGroup
|$150 p/month
|$395 p/month
|$645 p/month
|Monthly Dinner in Sydney, Brisbane, Melbourne, London or Singapore
|Access to member-only Facebook Group
|Weekly Member Newsletter
|Access to Monthly Member Webinars
|Bi-Monthly Breakfast Think Tanks
|Quarterly Business Book direct to your door
|Quarterly Business Immersion Days
|2x Financial Strategy Days per year
|2x Cocktail Conversation Events per year
|Invitation to LBD Annual Retreat
To book your membership CLICK HERE
All previous packages conditions and inclusions will be honoured, and cut over to new packages will be discussed with all members at their renewal dates.
* Prices exclude GST


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Blue Enchantress® Hydrangea
Hydrangea macrophylla 'Monmar' Plant Patent #25,209
Sku #9275
Striking ruby-black stems support big mophead flowers on this exquisite reblooming hydrangea. A superb border shrub for color throughout summer; blue in acidic soils and pink in more alkaline soils. Flowers age to a vintage cream-splashed green color. Prized for cut or dried flower arrangements. Deciduous.
Light Needs
Water NeedsWater regularly to maintain evenly moist soil - weekly, or more often.
USDA Zone ?There are very few rules in gardening, but perhaps the most important one is to work within your zone. Choosing plants that grow and thrive in your particular climate is the very first step toward having a beautiful garden. Learn More
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Note: This plant is currently NOT for sale. This page is preserved for informational use
Key Feature
Showy Repeat Bloomer
Light Needs
Partial shade to partial sun
Landscape Uses
Watering Needs:Water regularly to maintain evenly moist soil - weekly, or more often.
Botanical Pronunciation:hy-DRAIN-juh mak-roh-FIL-uh
Growth habit:Rounded
Growth rate:Fast
Average landscape size:Rounded growth; quickly reaches 3 to 5 ft. tall and wide.
Special features:Attracts Butterflies, Easy Care, Fast Growing, Gift Plant
Foliage color:Green
Blooms:Spring, repeating through summer.
Flower color:Blue
Flower attributesFlowers for Cutting, Repeat Flowering, Showy Flowers
Garden styleContemporary, Cottage
Patent Act:Asexual reproduction of plants protected by the Plant Patent Act is prohibited during the life of the patent.
Design IdeasThis stunning beauty is a collector's dream and a must for every garden. A superb shrub for accent, specimen, or massed in a foundation or border. Pairs beautifully with partial sun to shade-loving annuals or perennials.
Companion PlantsAzalea (Azalea); Holly (Ilex); Hosta (Hosta); Rose of Sharon (Hibiscus); Sweetspire (Itea)
Care InformationProvide organically rich, well-drained soil. Apply mulch to conserve moisture and protect in harsh winters. Water deeply, regularly in first growing season to establish extensive root system. Soil and water pH influences bloom color; consult garden center for suitable amendments. Feed in early spring. Deadhead spent blooms; prune dead or weak stems as needed.
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This Plant's Growing Zones: 4 - 9


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From Team TME - Have a Happy and Safe Fourth of July!


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