Metabolic Reconstruction for Metagenomic Data and Its Application to the Human Microbiome

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/human​n. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.National Institutes of Health (U.S.) (U54HG004968

Vanadate prevents glucocorticoid-induced apoptosis of osteoblasts in vitro and osteocytes in vivo.

Skeletal mass is maintained by a balance between formation and resorption, cell proliferation and apoptosis. In vitro, glucocorticoids (GCs) decrease extracellular signal-regulated kinases (ERK) activation by mitogens, thus inhibiting osteoblast proliferation. Both ERK activity and proliferation are restored by co-treatment with the protein tyrosine phosphatase inhibitor, vanadate. Since ERK signalling may also be anti-apoptotic, we explored the effects of vanadate on GC-induced apoptosis in vitro and in vivo. Apoptosis in MBA-15.4 pre-osteoblasts increased from 6 h and remained up to eightfold higher through 6 days of 10(- 6) M dexamethasone (Dex) treatment. Co-incubation with 10(- 7) M vanadate markedly reduced apoptosis at all time points. Vanadate also prevented GC-induced poly-ADP-ribose polymerase cleavage. We assessed the transcriptional profiles of seven anti-apoptotic proteins (Bcl-2, Bcl-X(L), inhibitors of apoptosis protein-1 (IAP-1), IAP-2, X-linked IAP (XIAP), Fas-associated death-domain-like IL-1beta-converting enzyme-inhibitory protein (FLIP(Long)) and FLIP(Short)) in osteoblasts subjected to various stimuli using real-time quantitative PCR. Although these anti-apoptotic genes responded to different mitogenic conditions, Dex failed to repress their expression, and in fact significantly up-regulated Bcl-X(L), IAP-2 and XIAP. Dex may therefore induce apoptosis by up-regulating pro-apoptotic gene expression. We have previously demonstrated that rats treated with GC develop low formation osteoporosis (bone histomorphometry and DEXA) and skeletal fragility (breaking strength) that were largely prevented by co-treatment with vanadate. We report here that vertebrae from rats treated with 3.5 mg/kg per day methylprednisolone for 9 weeks showed increased incidence of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling-positive apoptotic osteocytes, which was reduced by vanadate co-treatment. We conclude that vanadate prevents GC-induced apoptosis of pre-osteoblasts in vitro and osteocytes in vivo, and this may contribute to its bone-sparing effects in vivo

Expression of the pituitary transcription factor Ptx-1, but not that of the trans-activating factor prop-1, is reduced in human corticotroph adenomas and is associated with decreased alpha-subunit secretion.

We have studied the expression of the pituitary transcription factors Ptx-1 and Prop-1 in a series of 34 pituitary adenomas fully characterized for in vitro hormone secretion and histological staining. In studies involving mammalian cell lines, the pituitary transcription factor Ptx-1 has been shown to be a pituitary hormone panactivator, whereas more recent studies have shown that it plays an important role in alpha-subunit gene expression. Its expression has not been examined previously in human pituitary adenomas characterized by in vitro hormone secretory profiles. Of the 34 pituitary adenomas studied, Ptx-1 expression was reduced by more than 50% compared to that of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase in the 6 corticotroph adenomas, which also had significantly reduced alpha-subunit production (all 6 tumors secreting < or =0.5 ng/24 h). Mutations of the pituitary transcription factor Prop-1, which is responsible for the syndrome of Ames dwarfism in mice, are being increasingly recognized as a cause of combined pituitary hormone deficiency in humans, although ACTH deficiency has been described only once. Prop-1 expression was detected in all 34 pituitary adenomas, including 6 corticotroph adenomas and 5 gonadotroph adenomas. The expression of Prop-1 has not been described previously in these cell phenotypes

Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway.

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway

Leptin in pituitary adenomas--a novel paracrine regulatory system.

A growing number of physiological and pathophysiological processes have been shown to be influenced by leptin apart from its first recognised role as a modulator of hypothalamic appetite and weight control centers. We investigated the presence and pattern of distribution of leptin mRNA and the mRNA of the long isoform of the leptin receptor in the normal pituitary and in different types of pituitary adenomas. We also studied leptin secretion from human pituitary tumors in culture, and the in vitro pituitary hormone release following stimulation with human leptin. Leptin mRNA expression was detected at a low level of expression in 50% of tumors but in none of the normal pituitaries. By immunohistochemistry, leptin was present in occasional scattered cells in the normal pituitary and in pituitary tumors. The leptin receptor long isoform was detected in the majority (65%) of pituitary tumors and in all normal pituitaries. It did not segregate with any particular tumor type, and varying levels of expression were detected between the tissues studied. 34% of pituitary adenomas showed leptin release into the incubation media during in vitro culture. Leptin mRNA, the mRNA of the long isoform of the receptor, or in vitro leptin release, did not correlate with tumor type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumors caused stimulation of FSH and a-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. As the co-localisation of ACTH and leptin in corticotroph cells was previously suggested, we investigated whether in vivo ACTH release is accompanied by a simultaneous plasma leptin level rise (i) in peripheral plasma samples after food intake-induced ACTH rise in healthy obese and nonobese individuals and (ii) in petrosal sinus samples after CRH injection in Cushing's disease patients. Our data suggest that a rise in ACTH levels is not accompanied by detectable rise in leptin levels in peripheral and in petrosal sinus blood samples. In summary, leptin is synthesized and stored within the pituitary and may modulate other pituitary hormone secretion, although probably it does not contribute to plasma leptin level changes. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function