Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12 000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.

Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin.

SummaryBackground Fine-needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20-40% of cases the FNAB result is uncertain. Objective We investigated whether a search for the oncogenes RET/PTC, TRK and BRAF V600E in thyroid aspirates could refine an

Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF V600E ), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF V600E significantly correlated with absence of node metastasis. Conclusions: BRAF V600E is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.European Journal of Endocrinology 154 341-348

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS(-/-) mice. IEC p53 levels and apoptosis were reduced in IL-10(-/-) colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF-treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF-treated CUC patients.