JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

Direct evidence for the existence of dairying farms in prehistoric Central Europe (4th millennium BC)

The molecular and isotopic chemistry of organic residues from archaeological potsherds was used to obtain further insight into the dietary trends and economies at the Constance lake-shore Neolithic settlements. The archaeological organic residues from the Early Late Neolithic (3922-3902 BC) site Hornstaad-Hornle IA/Germany are, at present, the oldest archaeological samples analysed at the Institute of Mineralogy and Geochemistry of the University of Lausanne. The approach includes 13C/12C and 15N/14N ratios of the bulk organic residues, fatty acids distribution and 13C/12C ratios of individual fatty acids. The results are compared with those obtained from the over 500 years younger Neolithic (3384-3370 BC) settlement of Arbon Bleiche 3/Switzerland and with samples of modern vegetable oils and fat of animals that have been fed exclusively on C3 forage grasses. The overall fatty acid composition (C9 to C24 range, maximizing at C14 and C16), the bulk 13C/12C and 15N/14N ratios (delta13C, delta15N) and the 13C/12C ratios of palmitic (C16:0), stearic (C18:0) and oleic acids (C18:1) of the organic residues indicate that most of the studied samples (25 from 47 samples and 5 from 41 in the delta13C18:0 vs. delta13C16:0 and delta13C18:0 vs. delta13C18:1 diagrams, respectively) from Hornstaad-Hornle IA and Arbon Bleiche 3 sherds contain fat residues of pre-industrial ruminant milk, and young suckling calf/lamb adipose. These data provide direct proof of milk and meat (mainly from young suckling calves) consumption and farming practices for a sustainable dairying in Neolithic villages in central Europe around 4000 BC.dagger

Direct comparison of 3D spiral vs. Cartesian gradient-echo coronary magnetic resonance angiography.

While 3D thin-slab coronary magnetic resonance angiography (MRA) has traditionally been performed using a Cartesian acquisition scheme, spiral k-space data acquisition offers several potential advantages. However, these strategies have not been directly compared in the same subjects using similar methodologies. Thus, in the present study a comparison was made between 3D coronary MRA using Cartesian segmented k-space gradient-echo and spiral k-space data acquisition schemes. In both approaches the same spatial resolution was used and data were acquired during free breathing using navigator gating and prospective slice tracking. Magnetization preparation (T(2) preparation and fat suppression) was applied to increase the contrast. For spiral imaging two different examinations were performed, using one or two spiral interleaves, during each R-R interval. Spiral acquisitions were found to be superior to the Cartesian scheme with respect to the signal-to-noise ratio (SNR) and contrast-to-noise-ratio (CNR) (both P < 0.001) and image quality. The single spiral per R-R interval acquisition had the same total scan duration as the Cartesian acquisition, but the single spiral had the best image quality and a 2.6-fold increase in SNR. The double-interleaf spiral approach showed a 50% reduction in scanning time, a 1.8-fold increase in SNR, and similar image quality when compared to the standard Cartesian approach. Spiral 3D coronary MRA appears to be preferable to the Cartesian scheme. The increase in SNR may be "traded" for either shorter scanning times using multiple consecutive spiral interleaves, or for enhanced spatial resolution.

The underestimation of compliance to direct help requests explained by adaptive sampling

Helping behavior is any intentional behavior that benefits another living being or group (Hogg & Vaughan, 2010). People tend to underestimate the probability that others will comply with their direct requests for help (Flynn & Lake, 2008). This implies that when they need help, they will assess the probability of getting it (De Paulo, 1982, cited in Flynn & Lake, 2008) and then they will tend to estimate one that is actually lower than the real chance, so they may not even consider worth asking for it. Existing explanations for this phenomenon attribute it to a mistaken cost computation by the help seeker, who will emphasize the instrumental cost of “saying yes”, ignoring that the potential helper also needs to take into account the social cost of saying “no”. And the truth is that, especially in face-to-face interactions, the discomfort caused by refusing to help can be very high. In short, help seekers tend to fail to realize that it might be more costly to refuse to comply with a help request rather than accepting. A similar effect has been observed when estimating trustworthiness of people. Fetchenhauer and Dunning (2010) showed that people also tend to underestimate it. This bias is reduced when, instead of asymmetric feedback (getting feedback only when deciding to trust the other person), symmetric feedback (always given) was provided. This cause could as well be applicable to help seeking as people only receive feedback when they actually make their request but not otherwise. Fazio, Shook, and Eiser (2004) studied something that could be reinforcing these outcomes: Learning asymmetries. By means of a computer game called BeanFest, they showed that people learn better about negatively valenced objects (beans in this case) than about positively valenced ones. This learning asymmetry esteemed from “information gain being contingent on approach behavior” (p. 293), which could be identified with what Fetchenhauer and Dunning mention as ‘asymmetric feedback’, and hence also with help requests. Fazio et al. also found a generalization asymmetry in favor of negative attitudes versus positive ones. They attributed it to a negativity bias that “weights resemblance to a known negative more heavily than resemblance to a positive” (p. 300). Applied to help seeking scenarios, this would mean that when facing an unknown situation, people would tend to generalize and infer that is more likely that they get a negative rather than a positive outcome from it, so, along with what it was said before, people will be more inclined to think that they will get a “no” when requesting help. Denrell and Le Mens (2011) present a different perspective when trying to explain judgment biases in general. They deviate from the classical inappropriate information processing (depicted among other by Fiske & Taylor, 2007, and Tversky & Kahneman, 1974) and explain this in terms of ‘adaptive sampling’. Adaptive sampling is a sampling mechanism in which the selection of sample items is conditioned by the values of the variable of interest previously observed (Thompson, 2011). Sampling adaptively allows individuals to safeguard themselves from experiences they went through once and turned out to lay negative outcomes. However, it also prevents them from giving a second chance to those experiences to get an updated outcome that could maybe turn into a positive one, a more positive one, or just one that regresses to the mean, whatever direction that implies. That, as Denrell and Le Mens (2011) explained, makes sense: If you go to a restaurant, and you did not like the food, you do not choose that restaurant again. This is what we think could be happening when asking for help: When we get a “no”, we stop asking. And here, we want to provide a complementary explanation for the underestimation of the probability that others comply with our direct help requests based on adaptive sampling. First, we will develop and explain a model that represents the theory. Later on, we will test it empirically by means of experiments, and will elaborate on the analysis of its results.

Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Direct numerical simulations of interface dynamics to link capillary pressure and total surface energy

The flow of two immiscible fluids through a porous medium depends on the complex interplay between gravity, capillarity, and viscous forces. The interaction between these forces and the geometry of the medium gives rise to a variety of complex flow regimes that are difficult to describe using continuum models. Although a number of pore-scale models have been employed, a careful investigation of the macroscopic effects of pore-scale processes requires methods based on conservation principles in order to reduce the number of modeling assumptions. In this work we perform direct numerical simulations of drainage by solving Navier-Stokes equations in the pore space and employing the Volume Of Fluid (VOF) method to track the evolution of the fluid-fluid interface. After demonstrating that the method is able to deal with large viscosity contrasts and model the transition from stable flow to viscous fingering, we focus on the macroscopic capillary pressure and we compare different definitions of this quantity under quasi-static and dynamic conditions. We show that the difference between the intrinsic phase-average pressures, which is commonly used as definition of Darcy-scale capillary pressure, is subject to several limitations and it is not accurate in presence of viscous effects or trapping. In contrast, a definition based on the variation of the total surface energy provides an accurate estimate of the macroscopic capillary pressure. This definition, which links the capillary pressure to its physical origin, allows a better separation of viscous effects and does not depend on the presence of trapped fluid clusters.

Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells.

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Total synthesis of (-)-isoavenaciolide

An enantioselective approach to (-)-isoavenaciolide was achieved starting from 1- undecyn-3-ol. The synthesis relied upon the preparation of a chiral 4-silyloxy-2-alkenylborane by hydroboration of a protected 2,3-allenol and subsequent stereoselective addition to 2- thiophenecarboxaldehyde

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Synthesis of F-18-labeled cyclic-[RGDfK] peptides for PET imaging of angiogenesis

Objectives: αvβ3 integrin is of great interest for tumor targeting because of its high concentration in tumor tissue. It recognizes ligands containing an arginine-glycine-aspartate motif (RGD), and a number of RGD-containing peptides have been developed as PET imaging probes of angiogenesis. We synthesized a series of 18F-labeled cyclic-[RGDfK] peptides for in vivo imaging of αvβ3 expression. Our F-18 labeled prosthetic groups were attached to the αvβ3 ligand via click chemistry, and the reaction conditions (time, temperature, solvent and pH) were optimized by using single modified amino acids.Methods: Seven amino acids were selected considering their different biochemical properties (polarity, total charge, presence of aromatic ring and heteroatom). All the amino acids were modified by the introduction of azido moiety to allow the interaction with alkyne prosthetic groups. Once the conditions of the click chemistry were optimized, the prosthetic groups were also coupled with the cyclic-[RGDfK] exhibiting an azido function. 4- Trimethylammonium-nitrobenzene triflate was used as precursor for the radiosynthesis of the prosthetic groups. The fluorination was carried out with K2CO3/K2.2.2 in CH3CN at 95 oC, and the nitro group was reduced with NaBH4 and Pd/C in MeOH. The resulting 18F-aniline was subsequently coupled to alkynoic acids to yield the final F-18 labeled prosthetic groups. Finally, the prosthetic groups were attached to the peptides via Huisgen's cycloaddition. Figure 1. F-18 labeled αvβ3 ligand.Results: Our new prosthetic groups were successfully clicked to the modified amino acids and to the cyclic- [RGDfK], and the reactions were almost quantitative within 1 to 3.5 h. The pH of the reaction did not influence the reaction kinetic and yield. The four steps of the F-18 labeling were completely automated providing the final products in quantities and yields practical for PET imaging. IC50 values of our ligands for αvβ3 and α5β1 demonstrated a high selectivity of our compounds towards αvβ3, as well as the negligible effect of the prosthetic groups on the affinity of the ligand to its receptor, as confirmed by the prediction of the molecular modeling.Conclusions: We have successfully synthesized novel F-18 labeled prosthetic groups, as well as novel PET imaging probes of αvβ3 expression. The reaction conditions of the Huisgen's cycloaddition were optimized with selected modified amino acids, and subsequently transposed to the cyclic-[RGDfK] peptide. IC50 data demonstrate that our 18F-labeled ligands were selective for αvβ3. In vivo microPET/CT studies in tumor bearing mice are underway.