AM-879, a PPARy non-agonist and Ser273 phosphorylation blocker, promotes insulin sensitivity without adverse effects in mice.

Obesity is one of the main risk factors for type 2 diabetes, and peroxisome proliferator-activated receptor γ (PPARγ) is considered a promising pathway on insulin sensitivity and adipose tissue metabolism. The search for molecules acting as insulin sensitizers have increased, especially for molecules that block PPARγ-Ser273 phosphorylation, without reaching full agonism. We evaluated the in vivo effects of AM-879, a PPARγ non-agonist, and found that AM-879 exerts different effects in mice depending on the dose. At lower doses, this ligand decreased BAT, increased leptin and Crh expression. However, at a higher dose, it promoted improvement on insulin sensitivity, ameliorates expression of metabolism-related genes, decreased the expression of genes related to liver toxicity, maintaining body weight and adipocyte size. These results present a new lead molecule to ameliorates insulin resistance and confirm AM-879 as a PPARγ non-agonist which blocks Ser273 phosphorylation as a good strategy to modulate insulin sensitivity without developing the adverse effects promoted by PPARγ full agonists.

Bioactive compounds and antibacterial activities in crystallized honey liquefied with ultrasound.

The effect of ultrasound on the crystal size, phenols, flavonoids, Maillard products and antibacterial activity of crystallized honeys was studied. Three multifloral honeys (M), one monofloral (MO) and one honeydew (HD) honey were used. Ultrasound was performed at 42 kHz for different times (0, 5, 10 and 15 min). The antibacterial activities were tested against Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. In all honeys, the parameters analyzed had significant differences ((P < 0.05)). After 15 min of ultrasound the HD had increments of 44 mg of gallic acid/100 g of honey in phenols, and some M showed increase in flavonoids (5.64 mg of quercitin /100 g of honey) and improvement in inhibition against Salmonella typhimurium was 13.1%. In some honeys the correlation between phenols or flavonoids and antibacterial activity were significant ((P < 0.05)). No correlation was found between Maillard products and antibacterial activity. The ultrasound treatment effect on the crystal size, phenols, flavonoid, Maillard products, and antibacterial activity of crystallized honeys were different in each honey.

Exploring the mechanism of PPARγ phosphorylation mediated by CDK5.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation.

Long-term exercise training prevents mammary tumorigenesis-induced muscle wasting in rats through the regulation of TWEAK signalling.

AIM: Exercise training has been suggested as a non-pharmacological approach to prevent skeletal muscle wasting and improve muscle function in cancer cachexia. However, little is known about the molecular mechanisms underlying such beneficial effect. In this study, we aimed to, firstly, examine the contribution of TWEAK signalling to cancer-induced skeletal muscle wasting and, secondly, evaluate whether long-term exercise alters TWEAK signalling and prevents muscle wasting. METHODS: Female Sprague-Dawley rats were randomly assigned to control and exercise groups. Fifteen animals from each group were exposed to N-Methyl-N-nitrosourea carcinogen. Animals in exercise groups were submitted to moderate treadmill exercise for 35 weeks. After the experimental period, animals were killed and gastrocnemius muscles were harvested for morphological and biochemical analysis. RESULTS: We verified that exercise training prevented tumour-induced TWEAK/NF-κB signalling in skeletal muscle with a beneficial impact in fibre cross-sectional area and metabolism. Indeed, 35 weeks of exercise training promoted the upregulation of PGC-1α and oxidative phosphorylation complexes. This exercise-induced muscle remodelling in tumour-bearing animals was associated with less malignant mammary lesions. CONCLUSION: Data support the benefits of an active lifestyle for the prevention of muscle wasting secondary to breast cancer, highlighting TWEAK/NF- κB signalling as a potential therapeutic target for the preservation of muscle mass.

Identification of a new hormone-binding site on the surface of thyroid hormone receptor.

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors involved in cell differentiation, growth, and homeostasis. Although X-ray structures of many nuclear receptor ligand-binding domains (LBDs) reveal that the ligand binds within the hydrophobic core of the ligand-binding pocket, a few studies suggest the possibility of ligands binding to other sites. Here, we report a new x-ray crystallographic structure of TR-LBD that shows a second binding site for T3 and T4 located between H9, H10, and H11 of the TRα LBD surface. Statistical multiple sequence analysis, site-directed mutagenesis, and cell transactivation assays indicate that residues of the second binding site could be important for the TR function. We also conducted molecular dynamics simulations to investigate ligand mobility and ligand-protein interaction for T3 and T4 bound to this new TR surface-binding site. Extensive molecular dynamics simulations designed to compute ligand-protein dissociation constant indicate that the binding affinities to this surface site are of the order of the plasma and intracellular concentrations of the thyroid hormones, suggesting that ligands may bind to this new binding site under physiological conditions. Therefore, the second binding site could be useful as a new target site for drug design and could modulate selectively TR functions.

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange.

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.

Influence of an aerobic fungus grown on solid culture on ruminal degradability and on a mixture culture of anaerobic cellulolytic bacteria.

In this work, the effect of a solid fungal culture of Aspergillus niger (An) grown on coffee pulp on the in situ ruminal degradability (RD) of corn stover was evaluated. In addition, the effect of its extracts on the in vitro dry matter disappearance (IVDMD) and on a mixed culture of anaerobic cellulolytic bacteria (MCACB) was also investigated. The solid ferment was a crude culture of An, grown on coffee pulp. Regarding in situ RD, a significant difference (p < 0.05) was found between treatment with 200 g/day of the solid culture and control (no solid culture added) on dry matter, crude protein and neutral detergent fibre on RD. All the water extracts (pH 4, 7 and 10) enhanced IVDMD and stimulated the cellulolytic activity on a MCACB. Ultrafiltration results showed that active compounds with a molecular weight lower than 30 kDa were responsible for the effect on MCACB. Such results suggest that the effects of the solid An culture in RD are related to the presence of water soluble compounds having a molecular weight lower than 30 kDa.

Transfontanelle ultrasonography in infants: comparative study with cranial computed tomography.

The findings of transfontanelle ultrasonography, undertaken in 36 infants with clinical evidence of central nervous system (CNS) disorders, were compared with the findings of cranial computed tomography (CT). Considering the CT scan findings as gold standard, cranial ultrasonography had a sensitivity of 82 per cent and a specificity of 75 per cent in identifying cerebral pathology. The positive predictive value was 92 per cent and negative predictive value was 45 per cent.