Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis c than the IL28b (IFN lambda 3) genotype

Background: Therapy of chronic hepatitis C (CHC) with pegIFNa/ribavirin achieves sustained virologic response (SVR) in ~55%. Pre-activation of the endogenous interferon system in the liver is associated non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. Methods: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. Results: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. Conclusions: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Genome-wide association study of metabolic traits reveals novel gene-metabolite-disease links.

Metabolic traits are molecular phenotypes that can drive clinical phenotypes and may predict disease progression. Here, we report results from a metabolome- and genome-wide association study on (1)H-NMR urine metabolic profiles. The study was conducted within an untargeted approach, employing a novel method for compound identification. From our discovery cohort of 835 Caucasian individuals who participated in the CoLaus study, we identified 139 suggestively significant (P<5×10(-8)) and independent associations between single nucleotide polymorphisms (SNP) and metabolome features. Fifty-six of these associations replicated in the TasteSensomics cohort, comprising 601 individuals from São Paulo of vastly diverse ethnic background. They correspond to eleven gene-metabolite associations, six of which had been previously identified in the urine metabolome and three in the serum metabolome. Our key novel findings are the associations of two SNPs with NMR spectral signatures pointing to fucose (rs492602, P = 6.9×10(-44)) and lysine (rs8101881, P = 1.2×10(-33)), respectively. Fine-mapping of the first locus pinpointed the FUT2 gene, which encodes a fucosyltransferase enzyme and has previously been associated with Crohn's disease. This implicates fucose as a potential prognostic disease marker, for which there is already published evidence from a mouse model. The second SNP lies within the SLC7A9 gene, rare mutations of which have been linked to severe kidney damage. The replication of previous associations and our new discoveries demonstrate the potential of untargeted metabolomics GWAS to robustly identify molecular disease markers.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

A novel gene expression signature in peripheral blood mononuclear cells for early detection of colorectal cancer.

BACKGROUND: Early detection and treatment of colorectal adenomatous polyps (AP) and colorectal cancer (CRC) is associated with decreased mortality for CRC. However, accurate, non-invasive and compliant tests to screen for AP and early stages of CRC are not yet available. A blood-based screening test is highly attractive due to limited invasiveness and high acceptance rate among patients. AIM: To demonstrate whether gene expression signatures in the peripheral blood mononuclear cells (PBMC) were able to detect the presence of AP and early stages CRC. METHODS: A total of 85 PBMC samples derived from colonoscopy-verified subjects without lesion (controls) (n = 41), with AP (n = 21) or with CRC (n = 23) were used as training sets. A 42-gene panel for CRC and AP discrimination, including genes identified by Digital Gene Expression-tag profiling of PBMC, and genes previously characterised and reported in the literature, was validated on the training set by qPCR. Logistic regression analysis followed by bootstrap validation determined CRC- and AP-specific classifiers, which discriminate patients with CRC and AP from controls. RESULTS: The CRC and AP classifiers were able to detect CRC with a sensitivity of 78% and AP with a sensitivity of 46% respectively. Both classifiers had a specificity of 92% with very low false-positive detection when applied on subjects with inflammatory bowel disease (n = 23) or tumours other than CRC (n = 14). CONCLUSION: This pilot study demonstrates the potential of developing a minimally invasive, accurate test to screen patients at average risk for colorectal cancer, based on gene expression analysis of peripheral blood mononuclear cells obtained from a simple blood sample.

A simple genetic basis for complex social behaviour mediates widespread gene expression differences.

A remarkable social polymorphism is controlled by a single Mendelian factor in the fire ant Solenopsis invicta. A genomic element marked by the gene Gp-9 determines whether workers tolerate one or many fertile queens in their colony. Gp-9 was recently shown to be part of a supergene with two nonrecombining variants, SB and Sb. SB/SB and SB/Sb queens differ in how they initiate new colonies, and in many physiological traits, for example odour and maturation rate. To understand how a single genetic element can affect all these traits, we used a microarray to compare gene expression patterns between SB/SB and SB/Sb queens of three different age classes: 1-day-old unmated queens, 11-day-old unmated queens and mated, fully reproductive queens collected from mature field colonies. The number of genes that were differentially expressed between SB/SB and SB/Sb queens of the same age class was smallest in 1-day-old queens, maximal in 11-day-old queens and intermediate in reproductive queens. Gene ontology analysis showed that SB/SB queens upregulate reproductive genes faster than SB/Sb queens. For all age classes, genes inside the supergene were overrepresented among the differentially expressed genes. Consistent with the hypothesized greater number of transposons in the Sb supergene, 13 transposon genes were upregulated in SB/Sb queens. Viral genes were also upregulated in SB/Sb mature queens, consistent with the known greater parasite load in colonies headed by SB/Sb queens compared with colonies headed by SB/SB queens. Eighteen differentially expressed genes between reproductive queens were involved in chemical signalling. Our results suggest that many genes in the supergene are involved in regulating social organization and queen phenotypes in fire ants.