The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including 18O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

SUMMARY RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.

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Flocculation studies of precipitated calcium carbonate induced by cationic polyacrylamides (C-PAMs) were carried out using light diffraction scattering (LDS). The effect of both polymer charge density and concentration on the flocculation process and on flocs density was investigated. As expected, results show that high charge density C-PAM induces flocculation by bridging and patching mechanisms simultaneously, while medium charge density C-PAM acts mainly according to the bridging mechanism. Consequently, the mass fractal dimensions of the flocs produced by high charge density C-PAM are higher. Results also show the effect of flocculant concentration: flocculation rate decreases and denser flocs are obtained as flocculant concentration increases. The results obtained so far allowed a preliminary quantitative evaluation of flocculation kinetics. In the flocculation curve, two regions corresponding to different kinetics were identified: a first region dominated by particle aggregation and a second region dominated by flocs stabilization. Therefore, LDS is considered a useful tool to evaluate flocculants performance. A strategy was developed that resulted in the use of LDS to retrieve, in a single test, information on the evolution with time of flocs dimension and structure, flocs resistance and flocculation kinetics. All the tests were performed under turbulent conditions similar to the ones prevailing in process equipment.

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

In papermaking, mill water closure may result in a significant increase of inorganic salts in the white water. The effect of these contaminants on the performance of retention aids was evaluated, in this study, through flocculation of Precipitated Calcium Carbonate (PCC) with three very high molecular weight cationic polyacrylamides (C-PAM) of medium charge density and with different degrees of branching. Furthermore, flocs resistance and reflocculation capacity was also investigated when 2 different types of shear forces were applied. Tests were carried out in distilled water and in industrial water containing a high concentration of cationic compounds. The flocculation, deflocculation and reflocculation processes were monitored by evaluating particle size distribution using a light diffraction spectroscopy technique. Additionally to the effect of the cationic content of the medium, the influence of the flocculant dosage and degree of polymer branching were also studied. The effect of these parameters on the flocs structure was estimated by determining both the mass fractal dimension and the scattering exponent of the aggregates. The results show that the presence of inorganic salts affects significantly the performance of the polyelectrolytes. The flocculation kinetics is faster but the required flocculant dosage is higher when the suspending medium is industrial water. The cationic entities affect also the flocs structure because they reduce the reconformation of the polymer during flocculation. Additionally, in industrial water, flocs become more resistant and this effect is more pronounced as the flocculant branching decreases. In the case of the linear polymer, this effect is not so obvious because reconformation is less pronounced due to its molecular structure. Reflocculation capacity of flocs is very reduced both in distilled and industrial water.

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation.