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Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment
- Xochitl C Morgan†1,
- Timothy L Tickle†1, 2,
- Harry Sokol†3, 4, 5,
- Dirk Gevers2,
- Kathryn L Devaney3, 4,
- Doyle V Ward2,
- Joshua A Reyes1,
- Samir A Shah6,
- Neal LeLeiko6,
- Scott B Snapper7,
- Athos Bousvaros7,
- Joshua Korzenik3, 7,
- Bruce E Sands8,
- Ramnik J Xavier2, 3, 4 and
- Curtis Huttenhower1, 2Email author
DOI: 10.1186/gb-2012-13-9-r79
© Morgan et al.; licensee BioMed Central Ltd. 2012
Received: 16 April 2012
Accepted: 26 September 2012
Published: 26 September 2012
The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis result from alterations in intestinal microbes and the immune system. However, the precise dysfunctions of microbial metabolism in the gastrointestinal microbiome during IBD remain unclear. We analyzed the microbiota of intestinal biopsies and stool samples from 231 IBD and healthy subjects by 16S gene pyrosequencing and followed up a subset using shotgun metagenomics. Gene and pathway composition were assessed, based on 16S data from phylogenetically-related reference genomes, and associated using sparse multivariate linear modeling with medications, environmental factors, and IBD status.
Firmicutes and Enterobacteriaceae abundances were associated with disease status as expected, but also with treatment and subject characteristics. Microbial function, though, was more consistently perturbed than composition, with 12% of analyzed pathways changed compared with 2% of genera. We identified major shifts in oxidative stress pathways, as well as decreased carbohydrate metabolism and amino acid biosynthesis in favor of nutrient transport and uptake. The microbiome of ileal Crohn's disease was notable for increases in virulence and secretion pathways.
This inferred functional metagenomic information provides the first insights into community-wide microbial processes and pathways that underpin IBD pathogenesis.
Inflammatory bowel disease (IBD), a chronic and relapsing inflammatory condition of the gastrointestinal (GI) tract, is intimately linked to the microbial communities of the human gut. Although it is now widely accepted that IBD results from altered interactions between gut microbes and the intestinal immune system, the precise nature of the intestinal microbiota dysfunction in IBD remains to be elucidated [1]. IBD further includes two main subtypes, ulcerative colitis (UC) and Crohn's disease (CD), which each include distinct microbial perturbations and tissue localizations. The former is confined to the colon, while the latter may affect any part of the digestive tract, with unclear implications for microbial involvement or causality [2]. In particular, the microbial mechanisms and metabolism underlying the role of the GI microbiome in IBD onset and its alteration in the course of active treatment and recovery are still unknown.
In the last decade, advances in DNA sequencing have allowed exploration of the 40% of the gut microbiome that is still uncultured [3], setting the stage for investigation of the IBD microbiome. The GI microbiome of healthy humans is dominated by four major bacterial phyla: Firmicutes, Bacteroidetes, and to a lesser degree Proteobacteria and Actinobacteria [4, 5]. Many studies have observed imbalances or dysbioses in the GI microbiomes of IBD patients [6–13]; in both CD and UC patients, there is decreased biodiversity, a lower proportion of Firmicutes, and an increase in Gammaproteobacteria [14]. In CD, proportions of the Clostridia are altered: the Roseburia and Faecalibacterium genera of the Lachnospiracae and Ruminococcaceae families are decreased, whereas Ruminococcus gnavus increases [15–17]. Specific features of UC-associated dysbiosis are less described, although increased sulfate-reducing Deltaproteobacteria have been reported [18, 19]. These studies have described typical changes in composition of the IBD gut community, but the functional roles of these organisms - or of the entirety of a dysbiotic community - remain less clear.
The normal gut microbiome exhibits tremendous functional diversity encoded by a collection of bacterial genes numbering more than 100 times the human gene set [4, 20]. Thus, the genomic potential of the human microbiome is far greater than that of its host, and treatments, diets, or medications that affect the host will also likely affect the microbiome. A primary example of the importance of the microbiome to host health is in the digestion of dietary fiber, which is used by the microbiota of the lower GI tract as their main source of energy [21]. Fibrolytic bacteria degrade polysaccharides into smaller carbohydrates, which are then fermented into short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. Butyrate in particular is a major source of energy for colonocytes, but all three of these have demonstrated immunomodulatory properties [22–27]. In addition to these metabolic functions, many genetic studies in IBD have highlighted the central role of host-microbe interactions in IBD pathogenesis [1, 28–30]. Specific host pathways linked to microbial response in IBD include T-cell activation, the IL-23/T helper 17 pathway, autophagy [31], and Paneth cell function [32]. Together, these results support the centrality of host-microbiota crosstalk for gut homeostasis and in turn the role of dysfunctional crosstalk between the host and GI microbiome in IBD.
Little work has yet bridged the gap between IBD pathogenesis in a human host, individual microbes, and alterations in metabolism of the GI microbial community in IBD. Few studies of the IBD gut microbiome have investigated microbiome function [33], and these have not systematically accounted for the influences of treatments and environmental factors. We have thus analyzed the GI microbiomes of 121 CD patients, 75 UC patients, and 27 healthy controls using a novel multivariate metagenomic analysis pipeline specifically accounting for environmental factors (including treatment, age, and tobacco use). In addition to assessing microbiome composition, we have analyzed the inferred metagenome as determined from phylogenetically-associated reference genomes, including metabolic modules and pathways also associated with disease status and with environmental factors such as medications and smoking. Not only were these GI microbiomes characterized by shifts in bacterial populations during disease as previously described, but these dysbioses were highly functionally coordinated. Cross-species enrichments included mucin metabolism and redox tolerance by means of glutathione transport, cysteine biosynthesis, and riboflavin metabolism. Conversely, processes linked broadly to clades IV and XIVa Clostridia were depleted, particularly short chain fatty acid production. Dysbioses in IBD are correspondingly not simply structural changes in the gut microbiota, but are instead associated with major impairments in many fundamental microbial metabolic functions with potential impact on the host.
Characteristics of patients in this study
Female gender (n)
59.5% (72)
49.3% (37)
55.6% (15)
62.5% (5)
Age (lower 95%-upper 95%)
37.3 (34.3-40.3)
41.1 (37.4-44.9)
35.1 (29.1-41.2)
26.9 (13.4-40.3)
Smoker (n)
63.6% (77)
57.3% (43)
85.2% (23)
75.0% (6)
24.8% (30)
40% (30)
11.1% (3)
12.5% (1)
10.7% (13)
2.7% (2)
0% (0)
12.5% (1)
0.8% (1)
0% (0)
3.7% (1)
0% (0)
Stool (n)
51.2% (62)
64% (48)
66.7% (18)
100% (8)
Biopsy (n)
48.8% (59)
36% (27)
33.3% (9)
0% (0)
Active disease (n)a
26.4% (32)
29.3% (22)
0% (0)
0% (0)
Ileal (n)
35.5% (43)
Mesalamine (n)
55.4% (67)
77.3% (58)
0% (0)
75.0% (6)
Steroids (n)
31.4% (38)
37.3% (28)
0% (0)
50% (4)
Immunosuppressant (n)b
38.8% (47)
16% (12)
0% (0)
0% (0)
Antibiotics (n)
12.4% (15)
13.3% (10)
0% (0)
12.5% (1)
Assessing significant covariation of microbiome structure with host IBD status, treatment, and environment
An important consideration that informed the remainder of our analysis, and which is often overlooked in studies of the microbiome, was the consistent covariation among disease status, aspects of subject environment, and microbiome structure. For example, the factor most associated with changes in microbiome composition was not disease but whether the sample origin was stool or biopsy. Biopsy location induced minor changes in microbiome composition (Additional files 2, 3 and 4) relative to the extreme differences between stool and biopsy communities, in agreement with previous studies [36, 37]. In this cohort, iCD was always represented by biopsy, whereas 18.4% of non-iCD and 36% of UC samples were biopsies. iCD was also associated with greater likelihood of immunosuppressant treatment: iCD, non-iCD, and UC patients were treated by immunosuppressants in 74.4%, 19.2% and 16% of samples, respectively. In contrast, non-iCD and UC cases were more likely to be treated with mesalamine or antibiotics: mesalamine was used for 30.2% of iCD samples, 69.2% of non-iCD samples, and 77.3% of UC samples, while antibiotics were used in 2.3% of iCD, 17.9% of non-iCD, and 13.3% of UC samples. These associations lead to a range of non-independent covariates. Although disease activity may influence microbiome composition, after adjusting for the other factors, it was not independently associated with a specific shift in the microbiome composition in our analysis, and there were no significant (P < 0.01) associations between microbiome composition and gender (Additional file 5).
The second largely independent factor influencing microbiome composition was age, itself negatively associated with smoking (Figure 1; Additional file 1). Twenty-four (10.4%) of the available subjects were less than 18 years of age and 26 were 60 years or older. Aging is associated with continual changes in the microbiome, primarily a gradual decrease in Bifidobacterium as observed here (Additional file 6) and by others [38, 39]. After observing these overall patterns of covariation among disease, treatment, environment, and gut microbiome composition, we continued our analysis only after assessing the significance of microbiome-disease associations in a multivariate manner to account for host environment and treatment.
Microbial clades differentially abundant specifically in IBD include Roseburia, the Ruminococcaceae, and the Enterobacteriaceae
The Ruminococcaceae, which are acetate producers [43], were decreased in CD, while the Leuconostocaceae, which produce acetate and lactate [44], were decreased in UC. The only major clade with a significant increase in abundance specific to CD was the Enterobacteriaceae, specifically Escherichia/Shigella. This family has been previously implicated in intestinal inflammation [6, 45–47].
Crohn's disease with ileal involvement presents a distinct microbiome phenotype including reduced Faecalibacterium, and Odoribacter is reduced both in iCD and in pancolonic UC
In CD patients with ileal involvement, sequences of the Ruminococcaceae family and of Faecalibacterium in particular were dramatically reduced compared to other subjects (Figure 2a), confirming previous studies [48, 49]. Faecalibacterium prausnitzii, the only cultured representative of Faecalibacterium, is able to metabolize both diet-derived polysaccharides and host-derived substrates such as N-acetyl glucosamine from intestinal mucus [50]. It is also a major butyrate producer and exhibits anti-inflammatory effects in a colitis setting [51]. The Ruminococcaceae represent the first step of microbiome-linked carbohydrate metabolism, as they degrade several types of polysaccharides in the lower GI tract, including starch, cellulose, and xylan [21]. The Roseburia genus, which is significantly reduced in all IBD patients (including iCD), and the Ruminococcaceae are further functionally connected in that the latter consume hydrogen and produce acetate that can be utilized by Roseburia to produce butyrate [41, 43]. Consistent reductions in all of these clades may thus have functional consequences on the ability of the host to repair the epithelium and to regulate inflammation.
The genera Escherichia/Shigella (indistinguishable as a 16S-based phylotype) were particularly highly enriched in iCD (q < 0.2; Additional file 1) above their general overabundance in CD patients. Lipopolysaccharide produced by Gram-negative bacteria such as Escherichia coli is a canonical microbe-associated molecular pattern, known to activate toll-like receptor 4 (TLR4) signaling [52] and thus trigger inflammatory cascades. TLR4 expression is highly up-regulated in the intestinal epithelium of IBD patients [53], and mutations in TLR4 are associated with both CD and UC [54]. Previous culture-based studies have found that E. coli, specifically E. coli exhibiting pathogen-like behaviors such as adhesion and invasiveness [55], are more frequently cultured from iCD biopsies, and culture-independent studies have found an enrichment in E. coli that contain virulence-associated genes in iCD [6]. This suggests that CD-involved ileum is a favorable milieu for establishment of E. coli with pathobiont features, which may have implications for IBD exacerbations and its chronicity. An inflamed ileum may furnish a specialized niche permissive for microbes with enhanced fitness in inflamed conditions.
The most severe form of UC is pancolitis, in which UC affects the entire colon; this condition is associated with greatly increased risk of colon cancer [56]. Patients with pancolitis did not harbor a clear specificity in their dysbiosis. However, both these patients and iCD patients had a reduced abundance of the Odoribacter genus, which belongs to the Porphyromonadaceae family and to the Bacteroidetes phylum. As Odoribacter splanchnus is a known producer of acetate, propionate, and butyrate [57], decreased Odoribacter may affect host inflammation via reduced SCFA availability.
Microbiome composition is also strongly associated with subject age, treatment, smoking, and sample biogeography
Critical to determining causality in links between IBD and the gut microbiome, IBD treatments were also associated with alterations in microbiome composition. Mesalamine (5-aminosalicylic acid) is a bowel-specific aminosalicylate drug. Although its exact mode of action is unknown, it is thought to act as an antioxidant and to decrease intestinal inflammation, in part by peroxisome proliferator-activated receptor-γ (PPARγ) activation and inhibition of NFκB and pro-inflammatory eicosanoid production. Here, its use was linked to strong reductions in Escherichia/Shigella (> 100% of average abundance, q < 0.04; Additional file 7), in agreement with a recent study [59]. Both 5-aminosalicylic acid and immunosuppressant treatment were associated with modest increases in Enterococcus, the only genus perturbed in immunosuppressant-treated patients with low false discovery rate (also > 100% of average abundance, q < 0.09).
Antibiotics were among the strongest factors associated with a reduction in ecological diversity (Figure 2b). Many individual clades were greatly reduced or nearly absent after administration of antibiotics, including the Collinsella, Dorea, Butyricicoccus, Subdoligranulum, and Acetivibrio (all q < 0.2; Additional file 1). These genera are predominantly from the Clostridiales order, Gram-positive and anaerobic bacteria that are targeted by the antibiotics commonly used in IBD, such as ciprofloxacin and metronidazole.
Smoking is likely the best-known environmental factor that impacts IBD [60]. It is associated with increased risk of CD and is conversely protective towards developing UC [61]. The only common organism to which tobacco usage was linked in these individuals was Anaerostipes (Firmicutes phylum), which decreased (> 60% average abundance, q < 0.15; Figure 3) in current or former tobacco users, beyond any change due solely to smokers' higher average age. The Anaerostipes genera can utilize lactate to produce butyrate [62], which is beneficial to colonic health.
Finally, as previously mentioned, samples of the stool as opposed to mucosal biopsies differed strongly in microbiome composition (Additional file 2). More than 70 clades were significantly over- or under-enriched in stool samples relative to biopsies at q < 0.2. This effect extended to entire phyla, as the Firmicutes were approximately twofold more abundant in stool (Additional file 1). Microbial habitat dictates the composition of microbial communities [36]; in the GI tract, this has been suggested to occur on biogeographical scales of intestinal regions [37, 63] or even millimeters apart [64, 65], and luminal/mucosal differences may be further perturbed by bowel preparation prior to colonoscopy [66]. The data did not suggest that the luminal and mucosal communities were independent; rather, all 14 clades significantly associated with IBD retained the same trend when stratified by sample origin (Additional file 8). The fecal microbiome appeared to convey a consistent but numerically transformed function of mucosal communities, both of which shifted in composition in association with host environment, treatment, and disease.
In a closer analysis of intestinal biogeography as reflected by biopsies drawn from distinct regions, differences in most clades were modest and correlated largely with previously described changes in pH (Additional files 2, 3 and 9) [67]. The clades with the largest regional changes included the Roseburia and Ruminococcaceae, with lower abundance in the low-pH terminal ileum, transverse, and right colon; Alistipes, following a similar pattern; and the Fusobacteria and Enterobacteriaceae, with an opposite pattern of somewhat increased abundance in the ileum and right colon. Particularly as the former have also been associated with the colorectal cancer microenvironment in previous work [68, 69], it is of note that these variations in the microbiota with respect to biogeography and pH are similar to those we observed with respect to IBD and potentially redox status as detailed below.
The metagenomic abundances of microbial metabolic pathways are more consistently perturbed in IBD than are organismal abundances
Considering only the contrast between IBD (CD or UC) and healthy subjects, 24 of 200 (12%) total metabolic modules were differentially abundant at q < 0.2. This is in stark contrast to the microbial shifts discussed above, in which only 6 of 263 (2%) genus-level clades reached this significance threshold. Even in the absence of metagenomic or metatranscriptomic data and only leveraging the genes and pathways in reference genomes associated with these communities, changes in microbial function were more consistent than changes in community structure. This has been noted in environmental communities [72] and suggested with respect to obesity and other biometrics [73, 74], but to date it has not been reported for disease-linked dysbioses or IBD.
We validated these functional shifts by shotgun metagenomic sequencing of the small subset of available samples with appropriate stool DNA, seven healthy controls and four CD patients (Additional file 14). These were sequenced to a shallow depth averaging 119 meganucleotides per sample of 150-nucleotide paired-end Illumina MiSeq reads, reducing our effective limit of detection but otherwise providing close agreement with inferred metabolic shifts in the IBD metagenome. Of the modules highlighted below and in Figure 5, one (cobalamin biosynthesis) fell below the limit of detection, and the remaining six retained the expected trend of over- or under-enrichment in Crohn's disease, as did additional processes detailed below, including glycolysis and bacterial secretion.
Amino acid biosynthesis and carbohydrate metabolism are reduced in the IBD microbiome in favor of nutrient uptake
We observed that even basic GI microbiome metabolism was altered in both UC and CD. Amino acid metabolism showed major perturbation: genes for the metabolism and biosynthesis of nearly all amino acids (particularly histidine and lysine) decreased in abundance (Figure 4), while arginine, histidine, and lysine transport (Figure 5) gene abundance increased. In iCD we also observed a decrease in glutamine-related functional modules, which would lead to a lower amount of glutamate required for gamma-aminobutyric acid, ornithine, and arginine biosynthesis; abundance of all three of these modules also decreased. In marked contrast to the other amino acids, genes for metabolism of the sulfur-containing amino acid cysteine significantly increased in abundance, with even greater increase in iCD. This corresponded with an overrepresentation of genes related to sulfate transport in UC and CD (Figure 5), and in increase in sulfur and nitrogen metabolism in CD (Figure 4).
CD was associated with increased abundance of many genes related to carbohydrate transport (Figure 5). There were large increases in pentose phosphate pathway and fructose/mannose metabolism gene abundance in iCD (Figure 4), which were accompanied by increase in carbohydrate metabolism, but they were not significant in UC and CD. In addition, iCD showed increased abundance of transporter genes for glucose, hexoses, maltose, and mono-, di-, and oligosaccharides (Figure 5). We observed a decrease in both butanoate and propanoate metabolism in iCD (Figure 4), suggesting a potential decrease in SCFA production by the microbiome, possibly due to the observed decrease in Roseburia and Faecalibacterium.
We saw an increase in glutathione transport gene abundance in UC and CD (Figure 5) and an increase in glutathione metabolism gene abundance in UC. Glutathione is a tripeptide of cysteine and glutamate, synthesized by Proteobacteria and a few streptococci and enterococci [75], which allows bacteria to maintain homeostasis during oxidative or acid stress. Inflammatory cascades include production of highly reactive oxygen and nitrogen metabolites, which are greatly increased in active IBD [76]. Lamina propia monocytes also release homocysteine during inflammation, which further contributes to oxidative stress; IBD is associated with higher levels of both mucosal and serum homocysteine [77]. Thus, the increases in sulfate transport, cysteine metabolism, and glutathione metabolism may reflect a mechanism by which the gut microbiome addresses the oxidative stress caused by inflammation.
Extreme functional shifts in iCD include changes in redox metabolism, enrichment of signaling/secretion, and suggest a 'pathobiont-like' invasive metagenome
CD with ileal involvement exhibited specific dysfunction at the module level. It was associated with an increase in several modules involved in glycolysis and carbohydrate transport and metabolism (Figure 5). Conversely, iCD exhibited lower abundance of genes involved in lipid metabolism and catabolism, confirming a major imbalance in energy metabolism. We observed a global decrease in nicotinamide, purine, and pyrimidine nucleotide biosynthesis modules in iCD, CD, and UC (Figure 5).
There was a decrease in vitamin biosynthesis associated with iCD, but increases in thiamine and particularly riboflavin metabolism modules (Figure 4). Interestingly, this pathway is fed by the pentose phosphate pathway, which was also overrepresented in iCD. Riboflavin is necessary for regenerating oxidized glutathione back to its reduced form, and is thus essential for pH and oxidative stress homeostasis, as is NADPH, a product of the pentose phosphate pathway. Metabolism of the sulfur-containing amino acids cysteine and methionine was increased in iCD, in marked contrast to the IBD-associated decreases in the non-sulfur-containing amino acids such as lysine and glutamine. As homocysteine is easily convertible to methionine, this may indicate a further mechanism of maintaining redox homeostasis. Alternatively, this may be connected to the iCD-specific increase in carbohydrate metabolism, as cysteine may be metabolized to pyruvate.
Finally, genes involved in pathogenesis processes, such as secretion systems and adherence/invasion, were overrepresented in iCD (Figure 4). For example, genes involved in the shigellosis pathway were more abundant in CD, and type II secretion genes were more abundant in iCD. Type II secretion is involved in the secretion of cell wall-degrading enzymes [78] and the secretion of toxins such as heat-labile enterotoxin, similar to cholera toxin [79]. These functions are typical of pathobiont adherent-invasive E. coli, which have been observed to increase in iCD in our own study and others [6, 55]. This may be associated with tissue damage, either primarily as a result of toxin secretion, or secondarily as a result of stimulated cytokine production. This tissue destruction is a likely source of metabolites for microbial overgrowth, selecting for auxotrophic specialists able to thrive in this environment and resulting in the microbiome-wide loss of basic biosynthetic processes (Figures 4 and 5). This would in turn lead to further tissue breakdown, bacterial overgrowth, and community structural and functional dysbiosis.
The GI microbiome influences dietary energy extraction, immune system development, vitamin production, and drug metabolism, yet most molecular and metabolic functions of the bacteria of the GI microbiome are uncharacterized [20]. To gain insight into the functional consequences of IBD-associated dysbiosis, we used a novel approach pairing microbial community 16S gene sequence profiles with information from the closest available whole-genome sequences. This defined an inferred metagenome and thus complement of metabolic functional modules for each microbiome in this study. This allowed us to identify unique functional perturbations in the microbiomes of IBD patients. Interestingly, although we identified only nine changes in bacterial clades that associated with UC (of 350 total, 2.6%), we identified 21 statistically significant differences in functional pathways and metabolic modules (of 295, 7.1%); this pattern held for CD and iCD function as well. This underscores the fact that phylogenetically diverse changes in the composition of the GI microbiome can be functionally coordinated and lead to major modifications in the metabolic potential of the microbiota.
The microbial metabolic information available in this study represents only one step in the functional investigation of the IBD microbiota, as it is an accurate but approximate inference using prior knowledge of microbial genomes. The metagenomes inferred from our 16S data were supported by shotgun sequencing of a subset of samples, providing one confirmation that they were representative of community functional capability. As sequencing costs continue to fall, rich metagenomic data for dozens or hundreds of samples will further improve our ability to resolve species-level gene function in communities. Of course, a community expresses only a variable subset of its functional capability at any given time, in response to environmental stimuli. Thus, metatranscriptomic, proteomic, and metabolomic data will continue to add to our understanding of which of a community's potential functions are most strongly affecting the host during inflammatory disease.
Alternatively, the increased biosynthesis of cysteine (a precursor of glutathione) and of glutathione transport modules may speak to the microbiome's response to the oxidative stress (high levels of reactive oxygen and nitrogen species) of the inflamed IBD gut [76]. In support of this concept, we found that riboflavin metabolism, which is required to convert glutathione between its oxidized and reduced forms, is increased in iCD. Furthermore, the pentose phosphate pathway, which produces the NADPH also required for glutathione reduction, is increased as well. Recent studies have shown that redox stress allows Salmonella to use ethanolamine as a carbon source [80] and allows enterohemorrhagic E. coli to use it as a nitrogen source [81], thus conferring a competitive advantage to these microbes. This raises the interesting possibility that E. coli or related species in IBD may be highly represented because they gain a competitive advantage from oxidative stress and are better able to compensate for it with glutathione production.
In both UC and CD, there were decreases in the biosynthesis of lysine, arginine, and histidine in favor of transport in both UC and CD; a further decrease in tryptophan metabolism was associated with iCD. The data showed additional broad decreases in many essential processes, such as cobalamin synthesis, purine and pyrimidine biosythesis, lipid catabolism, and phospholipid metabolism, as well as marked increases in transport. This overall decrease in abundance of genes for amino acid and nucleotide biosynthesis bears striking resemblance to the lifestyle of highly symbiotic bacteria that are intrinsically auxotropic and also of some pathobionts (Figure 6). One such example are segmented filamentous bacteria (SFB), a symbiont that belongs to the Candidadatus Arthromitis, a sub-group of clade I (sensu stricto) Clostridia. A recently sequenced SFB genome lacked genes for nucleotide biosynthesis as well as nearly all vitamins and amino acids [82, 83]. SFB are often abundant in the rodent terminal ileum and are responsible for the maturation of Th17 cells [84], which play an important role in CD-associated inflammation [85]. To date, neither SFB nor phylogenetically related sequences have been observed in humans [82, 86]; this was also true in our data (zero 16S sequences with > 90% identity to X77814 SFB). However, a functional trend similar to SFB was observed in these IBD community metagenomes, as biosynthetic mechanisms throughout central carbon metabolism, amino acid biosynthesis, and nucleotide maintenance were all reduced (Figures 4 and 5), hinting that humans may host functional equivalents of SFB-like pathobionts that increase in IBD but are not phylogenetically close to Candidatus Arthromitis. Host tissue destruction, either inflammation-mediated or bacterially mediated, would provide a ready nutrient source (Figure 6).
The data presented here show that IBD and iCD in particular are associated with a dysbiosis characterized by changes in Firmicutes and Proteobacteria phyla. Environmental factors and, notably, treatments were also associated with independent changes in the GI microbiome; these must be taken into account during future studies of the microbiota in IBD. These perturbations in bacterial composition, although modest, were associated with major perturbations of GI microbiome function, which revolved around metabolism in the presence of oxidative stress and perturbed nutrient availability during tissue damage. Further studies, particularly including transcriptomic, proteomic, or metabolomic characterization, longitudinal data, and dietary metadata, will be needed to additionally define the consequences of the IBD-associated microbiome dysfunction on the host and the specific mechanisms by which they are carried out or regulated by the microbiota.
Materials and methods
The OSCCAR and PRISM cohorts
The Ocean State Crohn's and Colitis Area Registry (OSCCAR) is a state-based, prospective inception cohort of IBD patients that was designed to study the epidemiology of IBD, to determine the incidence of IBD in Rhode Island, and to extrapolate these rates to the general population of the United States. The diverse population of over 1 million, limited geographic range, and well-circumscribed gastroenterology community of Rhode Island were ideal circumstances for establishing a prospective inception cohort of IBD patients. All but one of the 98 gastroenterologists/colorectal surgeons in Rhode Island agreed to refer patients to OSCCAR, and 11 gastroenterologists practicing in Massachusetts just over the Rhode Island border also agreed to refer their newly diagnosed IBD patients who resided in Rhode Island. Enrollment began 1 January 2008. All Rhode Island residents with a newly confirmed diagnosis of CD, UC, or indeterminate colitis were eligible for inclusion (within 12 months from diagnosis). Ethnic background of the subjects was not available for consideration in the analysis, and indeterminate colitis patients were analyzed only for other metadata and not for IBD diagnosis. Diagnosis of CD, UC, or indeterminate colitis was made by endoscopic, pathologic, or radiographic findings according to the criteria of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) IBD Genetics Consortium. OSCCAR research protocols were reviewed and approved by three institutional review boards (Lifespan (#0214-07), the Partners Human Research Committee (#2007-P-001705), and the Program for the Protection of Human Subjects/Mount Sinai School of Medicine (#11-01479)), and all experiments adhered to the regulations of these review boards. Informed consent and HIPAA (Health Insurance Portability and Accountability Act) authorization were obtained from each subject prior to study participation. Individuals diagnosed with IBD prior to the study start date, pregnant women, those unwilling to provide informed consent for study participation, and those who were prisoners at the time of diagnosis were not permitted to enroll.
The Prospective Registry in IBD Study at MGH (PRISM) is a referral center-based, prospective cohort of IBD patients. Enrollment began 1 January 2005. Patients aged 18 years and older with a diagnosis of CD or UC based upon standard endoscopic, radiographic, and histologic criteria were eligible to participate. Controls consisted of healthy patients aged 18 years and older, from whom biopsies were obtained during colonoscopies performed for screening purposes.
Patients were excluded from the healthy volunteer group for current acute illness, if awaiting transplant, or if chronically ill (for example, renal failure, diabetes, congestive heart failure). During routine colonoscopies, subjects were offered the opportunity to donate biopsy samples. After sampling, intestinal biopsies were stored in 5% glycerol at -80°C until DNA extraction. Stool samples were kept at 4°C for less than 24 h before storage at -80°C until DNA extraction. PRISM research protocols were reviewed and approved by the Partners Human Research Committee (#2004-P-001067), and all experiments adhered to the regulations of this review board.
DNA extractions
DNA from stool and biopsy samples was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Inc., Valencia, CA, USA) according to manufacturer's instructions and as described previously [87]. The manufacturer's protocol was altered to accommodate larger stool volumes and to improve homogenization using bead-beating at several steps: a) a minimum of 2 ml of Buffer ASL and 300 mg of stool was used in the protocol; b) a ratio of 700 μl of Buffer ASL per 100 mg of stool weight was used for larger volumes using no more than 1,500 mg of stool and 10.5 ml of Buffer ASL; c) following the addition of Buffer ASL to each sample (step 2), 0.70 mm Garnet Beads (MO BIO Laboratories, Inc., Carlsbad, CA, USA) were added to the suspension and vortexed for 10 seconds; d) a second bead-beating was performed following the heating of the suspension (step 3) in 0.1 mm Glass Bead Tubes (MO BIO Laboratories, Inc.), and vortexed for 10 minutes.
Amplification and 454 sequencing of the 16S gene
The 16S gene dataset consists of 454 FLX Titanium sequences spanning the V3 to V5 variable regions. Detailed protocols used for 16S amplification and sequencing are available on the Human Microbiome Project Data Analysis and Coordination Center website [88]. In brief, genomic DNA was subjected to 16S amplifications using primers designed incorporating the FLX Titanium adapters and a sample barcode sequence, allowing directional sequencing covering variable regions V5 to partial V3 (primers: 357F 5'-CCTACGGGAGGCAGCAG-3' and 926R 5' CCGTCAATTCMTTTRAGT-3'). PCR mixtures (25 μl) contained 10 ng of template, 1× Easy A reaction buffer (Stratagene, La Jolla, CA, USA), 200 mM of each dNTP (Stratagene), 200 nM of each primer, and 1.25 U AccuPrime hifi cloning enzyme (Invitrogen, Carlsbad, CA, USA). The cycling conditions for the V3-V5 consisted of an initial denaturation of 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 40 s, annealing at 50°C for 30 s, extension at 72°C for 5 minutes and a final extension at 72°C for 7 minutes. Amplicons were confirmed on 1.2% Flash Gels (Lonza, Rockland, ME, USA), purified with AMPure XP DNA purification beads (Beckman Coulter, Danvers, MA, USA) according to the manufacturer, and eluted in 25 μl of 1× low TE buffer (pH 8.0). Amplicons were quantified on Agilent Bioanalyzer 2100 DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and pooled in equimolar concentration. Emulsion PCR and sequencing were performed according to the manufacturer's specifications.
Processing sequencing samples
Sequences were processed in a data curation pipeline implemented in MOTHUR [89], which removed sequences from the analysis if they were less than 200 nucleotides or greater than 600 nucleotides, had a low read quality score (< 25), contained ambiguous characters, had a non-exact barcode match, or had more than 4 mismatches to the reverse primer sequences (926R). Remaining sequences were assigned to samples based on barcode matches, and barcode and primer sequences were then trimmed. Chimeric sequences were identified using the ChimeraSlayer [90] algorithm, and reads were classified with the MSU RDP classifier v2.2 [91] using the taxonomy maintained at the Ribosomal Database Project (RDP 10 database, version 6). Sequencing depth after processing averaged 2,860 (standard deviation 1,730) reads per sample.
Metagenome inference from microbiome composition
for GG tree nodes g and h separated by phylogenetic branch length dist(g, h) and annotated with KO genome vectors and .
Using this vector representation of genomes, the abundance of an individual gene family (KO) i in a community due to the presence of a specific phylotype g is the product of the corresponding gene count and the measured abundance of phylotype g. Therefore, the total relative abundance of each KO was estimated for each sample by adding the individual contributions of all phylotypes present in the sample. Using this method, we inferred the functional composition for each sampled community. The inference process's accuracy was validated by comparing inferred KO abundances in 16S datasets from the Human Microbiome Project with their metagenomically sequenced counterparts (Additional file 15).
Metabolic pathway reconstruction
Inferred per-community gene (KO) abundances were subsequently reconstructed into microbial pathway relative abundances using HUMAnN, the Human Microbiome Project metabolic reconstruction pipeline [71]. KOs were grouped into pathways represented as gene sets using HUMAnN, which chooses pathways by maximum parsimony using MinPath [93] and computes each pathway's relative abundance as a smoothed average over all genes within it, taking into account outliers and gap filling. We ran HUMAnN three times to reconstruct three complementary types of pathways from these genes: small metabolic modules (using KEGG's conjunctive normal form logic), large metabolic pathways, and Gene Ontology terms (using annotation-to-KO mappings from nine well-characterized KEGG microbes: ban, cje, cpe, eco, nse, pae, sce, son, and vch). For each of these three types of pathway, HUMAnN input the inferred relative abundances of all genes in each sample, and output the relative abundances of pathways within the sample. Subsequent analysis handled these sample-by-pathway relative abundances in the same manner as sample-by-clade microbial abundances.
Significant associations of microbial clades and pathways with sample metadata
Inverse Simpson diversity, Chao1 richness (using the R fossil package), and Pielou evenness were calculated for clade abundance, KEGG pathway and module abundance, and Gene Ontology term abundance [94–97]. Next, data were pre-processed for quality control before modeling. Clinical metadata were removed when more than 10% of data were missing, or when they did not vary in value over the available samples. Clades, pathways, and features of very low abundance (< 0.001 in ≥ 90% of samples) and feature outliers outside of the lower or upper outer fence (3× interquartile range) were removed. Missing data were imputed for significance testing with the mean abundance of the sample; missing factor metadata were imputed with a 'NA' factor level using the na.gam.replace function from the R package [98]. Unless stated otherwise, all subsequent analyses and calculations were performed using these processed data. After processing, 228 and 231 samples passed quality control for clade abundance and functional abundance analyses, respectively.
where p are the clinical metadata selected from boosting.
Within each metadatum/clade association independently, multiple comparisons over factor levels were adjusted using a Bonferonni correction; multiple hypothesis tests over all clades and metadata were adjusted to produce a final Benjamini and Hochberg false discovery rate [100]. Unless otherwise indicated, significant association was considered below a q-value threshold of 0.25; the KEGG pathway sulfur metabolism (ko00920) had an average q-value of 0.26 for association with Crohn's disease. Multiple factor analysis was performed to visualize the relationships within heterogeneous factor data as well as with a select group of taxa found to be significantly associated with metadata (using the FactoMineR R package [101]). Total abundances and significant associations between metadata, taxa, and functions are listed in Additional files 1 and 11.
Sequence alignment for segmented filamentous bacteria
To determine whether SFB were present in samples, three sequences of SFB (X80834, X87244, and X77814) from three species (chicken, rat, and mouse) were aligned by blastn, using both a 20 and 15 seed word. No sequences were found with ≥ 95% identity over an alignment length of at least 100 nucleotides. The average sequence length from the study was 435 nucleotides.
Shotgun metagenomic sequencing
To provide internal validation of inferred microbial community gene and pathway compositions, stool DNA from seven healthy controls and four CD patients was subjected to metagenomic shotgun sequencing. Libraries were constructed with the Illumina Nextera XT kit and sequenced on an Illumina MiSeq using 2 × 150 bp paired-end sequencing according to the manufacturer's instructions. This resulted in sequencing depths ranging from 3.9 to 270 meganucleotides, average 119 meganucleotides, from which microbial community function was determined with HUMAnN [71] as described above.
Sequence accession numbers and availability
Sequences generated in this study are publicly available (NCBI BioProject ID numbers 82111 and 175224).
Crohn's disease
inflammatory bowel disease
- iCD:
ileal Crohn's disease
Kyoto Encyclopedia of Genes and Genomes
KEGG ortholog
Ocean State Crohn's and Colitis Area Registry
Prospective Registry in IBD Study at MGH
Ribosomal Database Project
short-chain fatty acid
segmented filamentous bacteria
toll-like receptor
ulcerative colitis.
We thank Wendy Garrett, Patrick Veiga, Ken Croitoru, and Mark Silverberg for their valuable comments on this manuscript and study, as well as Bruce Birren and the Broad Genome Sequencing and Analysis Program. This work was funded in part by grants from the French Society of Gastroenterology, the Bettencourt Schueller Foundation, the Philippe Foundation, and the Arthur Sachs Scholarship (HS); NIH (NIHU54HG004969) and the Crohn's and Colitis Foundation of America (DG and DVW); the philanthropic support of the MacInnes and Rasmussen families; NIH 1R21HD058828-01A1 to NL; and NIH 1R01HG005969, NSF DBI-1053486, and ARO W911NF-11-1-0473 and W911NF-11-1-0429 to CH. Dr. Sands acknowledges NIH (7 R21 DK078555), Centers for Disease Control and Prevention (5 U01 DP002676, 5 U01 DP000340), and the Crohn's and Colitis Foundation of America
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Yeah, I got the action figures. I'm not a fan of the exagerrated charicatures but they are the REAL Vice cops so I had to get them. It would be cool if Sideshow or Hot Toys made 12" figures. I'd buy them in a.... if you'll excuse the bad Don Johnson song... Heartbeat.
I can't stand anything associated with the movie. It was a mistake and I knew it when I heard they were filming it without DJ or PMT. I do not like re-imaginings in any form (don't get me started on Galactica, don't even mention it) but there is no way they could have continued where the series ended (you'll see why). It would have to have been an alternate reality storyline and would definitely have to address the fact that Crockett and Tubbs are two aging Vice cops that had been on the force for most of their careers and were tired of dealing with the corrupt judicial system and were beginning to enforce their own system of personal justice (that they could get away with). I don't think I could buy that Sonny's son followed in his footsteps. Caroline took him away from Sonny because she did not want him exposed to that lifestyle so I think he would have chosen a different career, maybe even became a football player that Sonny wanted to be before he injured himself. The problem with most of these franchises being made into films is that they waited 20 years to make them. I wanted to see Vice made into a feature back in the early 90's and reunite the cast. It would have been an epic along the lines as Heat. Instead, we get two contemporary clowns "posing" as Crocket and Tubbs in name only and Michael Mann ripping off his own cliches. Thank God for DVD. Now, if they would just hurry up and release the rest of them.

subset: commoncrawl


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[QUOTE=HNTFSH;1100464]Assume you mean training an upland flushing dog. Do that and the test will be easy. Takes lotsa birds. Find a game farm after you review the process.
Upland training has no real "formula" for the number of birds used in the training of a solid hunt test dog. However from a practical standpoint when starting a project dog of my own easily i would budget minimally 100 pigeons per year to reach the Master level . That being about 8 pigeons per month on the average. In additon possible some pheasants at appropriate stages of training. Personally I not much of a fan of chukars for my own dogs and like quail do not feel they are appropriate for spaniels.
How those birds are used and when is the essence of spaniel training whether for test or trial


text: So far no one can tell me if a Gibson is considered a long box or a short. The two bottom calls are what I've entered at Nationals as a Box Call Set in the hunting calls. The problem is no one can tell me if a Gibson is a long or short box? I talked with Karen Cavender who registers all the calls for the contest, to see if I can enter them as a set or if I need to enter them as single calls? She forwarded my question onto thhe chairman of the call committee and he couldn't answere it either. He sent me an email back and said they would check it out and get back to me. He got back to me and said he ran out of people to ask. There has never been a Gibson entered at Nationals. [:D] So now we are down to what does it measure? The long box on top is 12" with a 6-1/2" cut out, the Gibson is 10" with a 6" cut out, the Turpin is 8" with a 5-5/8 cut out, a Gibson sits right in between. So I'm still waiting to find out what it is considered? At Midwest I entered it as a long box? So what do you guys think?


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- Model
- Internet/Catalog:
- 204625012
Sheila Golden grew up in New York, attending the School of Visual Arts and the New School of the Social Reserach in New York City, then worked in graphic design and illustration. She studied film animation, lithography and ceramics, and received her bacherlor's degree in fine art. While living in California, Sheila started teaching classes on watercolor collage. Sheila has combined her multiple skills and talents to translate her love of nature into painting and collage. Her original watercolors and watercolor collages are exhibited in numeroud juried art shows around the country and her work is represented in private as well as corporate collections in major cities.
- This ready to hang, gallery-wrapped art piece features pink and yellow poppies
- Giclee (jee-clay) is an advanced printmaking process for creating high quality fine art reproductions, the attainable excellence that giclee printmaking affords makes the reproduction virtually indistinguishable from the original piece
- Artist: shelia golden
- Subject: still life
- Style: contemporary
- Product type: gallery-wrapped canvas art
- Made in USA
- Art Classification :
- Contemporary Looks
- Artist's name :
- Sheila Golden
- Artwork name :
- Garden Poppies
- Deco Style :
- Contemporary
- Decor Product Type :
- Wall Decor
- Hanging method :
- Hooks
- Indoor/Outdoor :
- Indoor
- Limited edition :
- No
- Number of pieces included :
- 1
- Print Product Type :
- Frameless
- Product Depth (in.) :
- 2
- Product Height (in.) :
- 35
- Product Weight (lb.) :
- 11
- Product Width (in.) :
- 47
- Returnable :
- 90-Day
- Wall Decor Product Type :
ReviewsNo Reviews
Shipping & Delivery OptionsShipping options for [zipcode] (change)
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Anyway, the good news is that we have taken our own way, with already code being contributed for debian installers, endorsing sponsors, wonderful work of Alex Neudorf for Gtk and OpenOffice integration into KDE. UserLinux does not look to be moving that fast :-)
So all in all, it looks quite good for KDE's future in enterprise work. And we still have the possibility of returning to UserLinux of Bruce ever changes its mind.


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- April 19: Woman Punctured in the Throat by Exploding Takata Airbag news | 11 days ago
- April 8: Honda Pilots Recalled to Replace Gas Tanks recalls | 22 days ago
- April 7: Honda Recalls 2003 Accord to Replace Takata Airbags recalls | 23 days ago
NHTSA — Latches/Locks/Linkages Problems
2007 Honda CR-V (Page 1 of 11)
CarComplaints.com Notes: Honda redesigned the CR-V for the 2007 model year to kick off the 3rd-generation of the popular SUV. Unfortunately that redesign came with malfunctioning door locksand shoddy tires that wear out fast.
2.1hardly worth mentioning
- Crashes / Fires:
- 0 / 0
- Injuries / Deaths:
- 0 / 0
- Average Mileage:
- 73,261 miles
About These NHTSA Complaints:
This data is from the NHTSA — the US gov't agency tasked with vehicle safety. Complaints are spread across multiple & redundant categories, & are not organized by problem.
So how do you find out what problems are occurring? For this NHTSA complaint data, the only way is to read through the comments below. Any duplicates or errors? It's not us.
accessories - exterior problem


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Top Stories Search: ROMENA, Conflict, General Assembly
2 results
25 September 2013 - 12:48pmThis morning (25 September), the United Nations Secretary General and Humanitarian Chief, as well as Ministers, directors of UN agencies and others will gather in the margins of the UN General Assembly to discuss the political, economic and security situation in Yemen. Ahead of this gathering of the...
23 September 2013 - 9:22amDozens of heads of state along with the UN Secretary-General, Government Ministers, leaders of UN agencies and civil society and other dignitaries are gathering in New York for the 68th Session of the United Nation's General Assembly. The annual event – a series of meetings starting on 24...


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We've moved!
The alumni website can now be found at www.nottingham.ac.uk/alumni
There's a whole world of contacts out therefor you as a member of The Universityof Nottingham alumni community.
Use our site to start your own special interest groups
Starting/supporting groups About our alumni networks Facebook groups/page
Lenton & Wortley Hall Class of 2005, 10 Year Anniversary Reunion DinnerIf it has been 10 years since you started your University adventure at Lenton and Wortley Hall, join us to celebrate your accomplishments over the last decade, catch up with peers and enjoy an evening on University grounds......more
More events.
Find out about volunteering and contributing to your University
The Campaign Office How can I contribute? Giving something back
Alumni Exchange Blog - Alumni Life E-newsletter Impact Campaign Annual Review University Annual Report


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An integral part of many Christian tourists' visit to Israel is the religious and spiritual experience. Pilgrimages to the main ritual sites, churches, monasteries and historical sites are central to the itinerary of a Christian tour of the Holy Land. To heighten this experience, it is worth trying out alternative, less well known accommodation options, such as staying at one of the Christian hostels located in seven Israeli cities. Most of these hostels are adjacent to monasteries or churches.
Christian hostels were first built in Israel over 1,000 years ago. The Crusaders, who ruled this country at that time, sought accommodation for the Christian pilgrims who came to tour the Holy Land. The greatest number of Christian hostels was built in the late 19th and early 20th centuries, when there was a constant stream of pilgrims, mainly from Europe. During that period a total of over 1,000 beds were added to several hostels, mostly in Jerusalem. There are now 38 Christian hostels in Israel, from Tiberias in the north, to Mt. Tavor, Nazareth, Haifa, Acre, Petakh Tikva, Tel Aviv and Jerusalem.
The hostels belong to the various Christian groups and are open to the general public, regardless of religion or race, though visitors must obviously respect and comply with the rules of each place. These hostels are simple and modest. Some are located in buildings with interesting architecture with well-tended gardens and an atmosphere of sanctity that complements the visitor's spiritual experience. They offer good value for money, as the price range is reasonable and the enjoyment guaranteed.
The Christian Information Center in Israel


text: Currently I'm in the process of constructing the frame work for a large Hoth Rebel base. The size should be around 7 ft high, & 7 ft wide by close to 8 ft long. The base will have multiple levels, an elevator, two large hanger bays, a hoth cantina, a medical center, a command center, with many ladders, catwalks & several other misc items as well. Building supplies are lots of 2X3X8's, a wooded boxed crate used for shipping snow blowers, plywood, plaster cloth, chicken wire, led lights & several house hold items that you would not believe will be incorporated in the base. I will try to include as many figures/vehicles as possible (including prequel) into this base when it's complete. It should take several months to complete & I shall posts some pics tonight of some of the progress I've had in the last two weeks.


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By Tiernan Ray
Shares of Apple (AAPL) are up $4.53, or 0.9%, at $524.21, reversing losses in early trading, as Carl Icahn just before the opening bell tweeted about a new letter to Apple shareholders saying that he no longer sees any reason to press for his proposal, which is on the slate to be voted on at Apple's annual meeting this month, that Apple do an immediate $50 billion buyback of shares.
Icahn's letter follows rejection by Institutional Shareholder Services of his proposal over the weekend, as reported by Reuters's Olivia Oran.
Icahn was also up against the objection of Scott Stringer, New York City's comptroller, who also publicly opposed the plan. Stringer went on CNBC to talk with David Faber this morning shortly after Icahn's latest announcement.
Stringer, who has to think about the needs of New York City's pension fund, remarked of Icahn's proposal "I thought it was risky and unnecessary," especially given Apple already has a $100 billion dividend plus buyback payout plan set up over the course of three years.
"As someone who represents hundreds of thousands of retirees, we wanted to make sure by representing the company, and not Carl Icahn, that we were thinking long term," said Stringer.
"Why Mr. Icahn thought we needed $50 billion on top of that [$100 billion plan], caused us concern. I have to protect the pensions of working people. I'm not sure Mr. Icahn's proposal benefitted anyone but him."
Stringer went on, "We need to give them [Apple] a cushion so that they can innovate."
Icahn's letter reads,
Dear Fellow Apple Shareholders,
While we are disappointed that last night ISS recommended against our proposal, we do not altogether disagree with their assessment and recommendation in light of recent actions taken by the company to aggressively repurchase shares in the market.
In their recommendation, ISS points out, and we agree, that "on the spectrum of options for allocating capital, the board appears to have been sluggish only in returning excess cash to shareholders," and even though the company has in place "one of the largest buybacks in history" we agree with ISS that this effort seems "like bailing with a leaky bucket" when "given the scale of the company's cash reserves."
That being said, we also agree with ISS's observation, taking into account that the company recently repurchased in "two weeks alone" $14 billion worth in shares, that "for fiscal 2014, it appears on track to repurchase at least $32 billion in shares." Our proposal, as ISS points out, "thus effectively only asks the board to spend another $18 billion on repurchases in the current year."
As Tim Cook describes them, these recent actions taken by the company to repurchase shares have been both "opportunistic" and "aggressive" and we are supportive. In light of these actions, and ISS's recommendation, we see no reason to persist with our non-binding proposal, especially when the company is already so close to fulfilling our requested repurchase target.
Furthermore, in light of Tim Cook's confirmed plan to launch new products in new categories this year (in addition to an exciting product roadmap with respect to new products in existing categories), we are extremely excited about Apple's future. Additionally, we are pleased that Tim and the board have exhibited the "opportunistic" and "aggressive" approach to share repurchases that we hoped to instill with our proposal. It is our expectation that Tim and the board continue to exhibit this behavior as fiduciaries to the shareholders since they clearly seem to agree that our company continues to be extremely undervalued, and we all share a common optimism with respect to the company's bright long term future.
Sincerely yours,
Carl C. Icahn


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HAppy Birthday #1.
HAppy Birthday #1.
thanks Chux Turbo LBC Bobafrett Mtriv73 Rjarvis JF96 JT JMG FB Rogue2 Tycho Slicker Deoxy Caesar JontheJedi JJReason Brandon Solo JMS UK for great deals.
SSG Pro Football Pick em and Bowl Pick em Champ 2006. 2007 NCAA Bracket Champ
#24 - Gone but not forgotten
Yeah!!! HBJT!!!
Happy Birthday O Supreme One!
Treat your stepmother with respect Pantera, or you'll be sleeping in the streets!!
Happy Birthday. We're all chipping in and buying you a case of Head Cheese.
Yo momma. That's right, I said "yo momma".
Happy Birthday!
Good Traders/Sellers: Cameo, Darth Cruel, Sith Killer 99, JJReason, icatch9, ChasingJediDogma, AT-AT Man, JediMasterGuyute, Brainiak76, JangoFett96, njscollectibles, Vulcantouch, AC Pin, TheDarthVader, msjedi, DarthQuack, Roojay
see now if we were allowed to link images that woulda looked nice
excellent traders; Val Da Car, Dark Marble, Commtech and info from KidHuman
Looking for: Master Replicas Darth Maul .45 scale full lightsaber & a 12" Grievous
Thanks gang! I'm old now, so if you hear creaking, that's either my walker or as Jira might put it, my achin' bones Ani.
(I wasn't going to be online today, but got all huffy about something from the Transformers movie and had to post right away, then saw this thread and had to drop by. )
Darth Vader is becoming the Mickey Mouse of Star Wars.
Kylo Ren - came from Space Brooklyn, although he moved to Space Williamsburg before it was trendy.
The use of a lightsaber does not make one a Jedi, it is the ability to not use it.
JT's sooo cool that he's got tennis balls on the front legs of his walker.
*Doctor Evil voice * Happy birthday #2 *end Doctor Evil voice*
Up, up, and OKAAAAY!!!


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Main Content
- Water-resistant leather strap
- Leather wrapped outsole with non-marking molded rubber traction areas
- Compression-molded footbed
- Quality embroidery
- Quick-drying jersey strap lining and neoprene backer
- Debossed graphics
- Genuine leather upper
- Imported
- Rubber soles
- Made in the USA
- Brand: OluKai
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Special event items are produced by manufacturers only after the outcome of a game or event. These are advanced sale items and will ship immediately after they are received in our warehouse.
Manufacturer direct items are shipped directly from the manufacturer. These items are not available for international or expedited shipping.
Customized items can be personalized with options such as your name, your favorite number, and/or designs. Some options may be limited by league rules.


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League Two round-up
Crawley hit four as Bradford come back in the final minute
By Hayley Paterson. Last Updated: 03/09/11 6:24pm
Connell: His late brace against Rotherham snatches the win for Swindon Town
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Two late Alan Connell strikes helped Swindon to come from behind and beat League Two leaders Rotherham.
The Millers took an early lead when former Town loan star Alex Revell guided a header past Mattia Lanzano.
Matt Ritchie equalised for the hosts, his third of the campaign, but following the interval Rotherham were back in front again when an unmarked Revell nodded home a Marcus Marshall free-kick.
Despite being 2-1 down, Swindon were by far the better of the two and when Rotherham's Danny Schofield received his second booking the ten men were punished as Connell levelled the score with his first touch.
Connell then connected with a Ritchie cross to put the Robins in front for the first time in the game which was again marred when Alberto Comazzi was given his marching orders for a second yellow after tripping Brett Williams.
Despite the two sides being a man down it was the home side that came away with the crucial three points in what has been a colourful week for Robins manager Paolo Di Canio after his Carling Cup bust-up with striker Leon Clarke.
Swindon 3 Rotherham 2
AFC Wimbledon 3 Port Vale 2
Aldershot 1 Cheltenham 0
Barnet 0 Accrington 0
Burton 2 Plymouth 1
Crawley 4 Bristol Rovers 1
Crewe 3 Oxford 1
Gillingham 0 Shrewsbury 1
Hereford 1 Dagenham & Redbridge 0
Morecambe 1 Bradford 1
Southend 2 Northampton 2
Torquay 3 Macclesfield 0
Crawley piled the pressure on Rotherham's early kick-off defeat with a comfortable win over Bristol Rovers.
Tyrone Barnett fired home just before the quarter hour mark and Ben Smith's header 14 minutes later made it an uphill task for Rovers.
Delightful free-kick
The goal of the game came in the 74th minute as Matthew Tubbs curled in a delightful free-kick after Sergio Torres was fouled on the edge of the box.
Rovers did respond in the form of Lee Brown with ten minutes remaining but it was no more than consolation as Barnett netted his second of the game for a comfortable 4-1 victory. That win sees Crawley level on points with Scott's side and third-placed Morecambe.
Morecambe dropped two points when Bradford's Ross Hannah swept in a late equaliser to cancel out unlikely scorer Izak Reid's opener.
Reid's goal was just his second in three years but Hannah's effort brought the Bantams fans to their feet.
Bottom of the table Plymouth continued their miserable run of form with another loss, this time at the hands of Burton.
Albion's Justin Richards struck in the first half and a 57th minute strike by Calvin Zola was enough for the hosts to condemn Argyle's fortunes further with Will Atkinson's late tap-in not enough for Peter Reid's side.
Hereford moved out of the drop zone with a dramatic winner from Sam Winnall in the 85th minute against Dagenham and Redbridge.
The Bulls, who were second from bottom before the tie, moved up one spot for their first win of the season.
Christian Jolley netted in emphatic fashion with minutes to spare to snatch a 3-2 win for Terry Brown's AFC Wimbledon over Port Vale.
Jack Midson had put the Dons ahead five minutes before the half-time whistle but a minute after the restart Louis Dodds curled a clinical effort into the top corner.
A superb strike from Gareth Gwillim across the goalkeeper looked to have claimed three points for the Dons, however, a late Sam Morsy equaliser then looked like both sides would share the spoils.
However Jolley had other ideas and in the dying minutes sent the home side's crowd into jubilation.
Nicky Wroe smashed home a last-minute spot-kick to give Shrewsbury a much-needed win as they faced high-flying Gillingham.
Wroe had to retake his penalty after the referee felt there was too much activity in the box, however he still kept his nerve to net in the 90th minute.
Aldershot's Mark Rankine headed home in the 72nd minute to beat Cheltenham 1-0 at the Recreation Ground.
Both he and Danny Hylton rose highest for the header, however Rankine just got there before his strike partner.
Southend and Northampton played out an entertaining 2-2 draw at Roots Hall.
Bilel Mohsni broke the deadlock in the 27th minute and Southend went 2-0 up through Ryan Hall.
However the Cobblers responded a minute later through Adebayo Akinfenwa and then Arron Davies five minutes after to share the spoils.
Torquay smashed three past Macclesfield thanks to Brian Saah's header, a well-worked Chris McPhee finish in his 50th game for the club and Eunan O'Kane killed off Town in the final minutes.
Shaun Miller bagged a brace for Crewe as Alexandra thumped Oxford at home 3-1.
The striker fired his first on the half-hour mark and again seven minutes later as United looked dead and buried early doors.
Ajay Leitch-Smith hampered any sign of a comeback with Alexandra's third as Liam Davis' nod in the 77th minute was not enough for Chris Wilder's outfit.
Barnet and Accrington played out a 0-0 draw despite Stanley being reduced to nine men in what was the only goalless encounter in League Two.
Charlie Barnett was given his second yellow just before the interval and Kevin Long later joined him after receiving a straight red in the 90th minute.


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Conference: Gluten-Free awareness Tour
The Gluten-Free Awareness Tour is heading west to San Francisco! Our event will be on September 21st and 22nd, from 10 am-3 pm. If you like to eat gluten-free food or have celiac disease, we have the perfect event for you. Our stop in San Francisco will include dozens of gluten-free food and beverage samples from local and national gluten-free food manufacturers, live chef demos and presentations from celiac disease experts.
San Francisco's own Lisa Lam, a specialist in Acupuncture and Chinese medicine will be giving a fascinating speech concerning demystifying Chinese medicine and celiac disease. Ravi Harryram, chef at the World Gardens Cafe, will be cooking up something delicious during his chef demo. Amy Fothergill, author of "The Warm Kitchen" and creator of "The Family Chef" blog will be discussing gluten-free food preparation. Join us for a day of gluten-free fun and food and help us on our mission to make the world a more gluten-free friendly place, one city at a time.
Adult admission is $10 and a two-day pass for $15. Children under 12 are admitted free.


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Manufacturer: Sonomax Manufacturer: Genius 2 Results
Genius GHP-i200 20Hz~20KHz Earbud Headphones
Genius GHP-i200 Stylish In-Earphones for iPod. Excellent Sound Performance in a Stylish Design. The stylish GHP-i200 in-earphones from Genius provide the most comfortable wearing comfort with three sizes of ear cushions. Featured with a miniaturized, advanced-technology 9mm driver, GHP-i200 delivers an immersive listening experience while jogging, exercising, or taking a trip. GHP-i200 delivers...
Sonomax EERS PCS-250 Custom Fit Dual Driver In-Ear Headphones with Inline Microphone
Premium dual drivers provide a faithful reproduction of the original audio recording


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Renault is the latest marque to outline plans for joining the small urban crossover segment.
The French marque has announced its new Captur model, which it says will deliver the styling and driving position of an SUV, the cabin space and modular interior of an MPV, and drive with the agility of a compact hatchback - all within a compact overall length of 4.12 metres.
The Captur will compete in the same market as the upcoming Ford EcoSport and Peugeot 2008 as well as the Nissan Juke.
Evolving from a 2011 motor show concept using the Captur name, the production version uses the Renault Nissan Alliance B-platform (in common with the Clio III and Nissan Juke) and introduces the latest step in Renault's design offensive, under the leadership of Laurens Van den Acker.
The production car takes its inspiration from the Captur concept car.
It boasts compact dimensions, raised ground clearance, wide wheels and sill guards and will be easy to customise.
Renault Captur also boasts the appointments and comfort refinements expected of an MPV, such as a raised driving position, large boot, modular interior and innovative stowage solutions.
Petrol and diesel engines will be available with the latest Renault technologies and will return CO2 emissions starting from as low as 96g/km.
The Captur will make its debut at the Geneva Motor Show in March.


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I decorated this paper mache box sometime ago. I like everything that are swirly and I love this swirl stitching pattern. I made a card using this swirl here. However, when I tried then on the lid of the paper mache box, I wasn't really happy with the result. I think because they are too close together.
I stamped the Fancy Flourish from Paper Trey Ink around the box using Elegant Eggplant ink from SU and added small quilling flower to the center.
Thanks for looking!