3-(Pyridin-2-yl-ethynyl)benzamide metabotropic glutamate receptor 5 negative allosteric modulators: hit to lead studies

A series of 3-(pyridin-2-yl-ethynyl)benzamide negative allosteric modulators of the metabotropic glutamate receptor 5 (mGluR5 NAMs) have been prepared. Starting from HTS hit 1 (IC₅₀: 926 nM), potent mGluR5 NAMs showing excellent potencies (IC₅₀s <50 nM) and good physicochemical profiles were prepared by monitoring LipE values. One compound 26 showed excellent mGluR5 binding (K_i: 21 nM) and antagonism (IC₅₀: 8 nM), an excellent rat PK profile (CL: 12 mL/min/kg, %F: 85) and showed oral activity in a mouse 4-Plate Behavioral model of anxiety (MED: 30 mpk) and a mouse Stress Induced Hyperthermia model of anxiety (MED 17.8 mpk).     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Adhesiveness and surface patterns of eggs in neotropical freshwater teleosts

Eggs of 23 Characiformes and eight Siluriformes, belonging to nine families with diverse reproductive behaviour, were ultrastructurally analysed. The migratory species exhibited non-adhesive eggs, whereas, most of the sedentary species presented some degree of egg adhesiveness. Among the Characiformes, non-adhesive eggs showed zona radiata with pore-canals or a fibrillar net at the surface; weakly adhesive eggs presented only zona radiata with pore-canals while adhesive eggs exhibited zona radiata with apparatus like globules, filaments, villi or honeycomb-like pores depending on the systematic group. The 'jelly' coat is strongly related to the Siluriformes eggs apparently without relationship with adhesiveness. A micropylar disc was present in adhesive eggs of a few species of both Characiformes and Siluriformes. Some patterns were characteristic of the animal pole, others of the vegetal pole, and others were common to both poles. The radial ridges converging to the micropyle in Astyanax bimaculatus lacustris appear to be related to fertilization. In general, egg surface structures in the Characiformes varied according to the genus, whereas all Siluriformes showed a similar egg surface pattern, regardless of the group analysed. Multivariate analysis allowed the identification of eight clusters among the Characiformes and three among the Siluriformes showing relationships between reproductive style and egg characteristics. It is suggested that egg surface and adhesiveness may be related to reproductive patterns and to phylogenesis.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.     

Antibody-neutralizing activity against all urogenital Chlamydia trachomatis serovars in Chlamydia suis-infected pigs

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.     

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A New 4D Trajectory-Based Approach Unveils Abnormal LV Revolution Dynamics in Hypertrophic Cardiomyopathy

The assessment of left ventricular shape changes during cardiac revolution may be a new step in clinical cardiology to ease early diagnosis and treatment. To quantify these changes, only point registration was adopted and neither Generalized Procrustes Analysis nor Principal Component Analysis were applied as we did previously to study a group of healthy subjects. Here, we extend to patients affected by hypertrophic cardiomyopathy the original approach and preliminarily include genotype positive/phenotype negative individuals to explore the potential that incumbent pathology might also be detected. Using 3D Speckle Tracking Echocardiography, we recorded left ventricular shape of 48 healthy subjects, 24 patients affected by hypertrophic cardiomyopathy and 3 genotype positive/phenotype negative individuals. We then applied Generalized Procrustes Analysis and Principal Component Analysis and inter-individual differences were cleaned by Parallel Transport performed on the tangent space, along the horizontal geodesic, between the per-subject consensuses and the grand mean. Endocardial and epicardial layers were evaluated separately, different from many ecocardiographic applications. Under a common Principal Component Analysis, we then evaluated left ventricle morphological changes (at both layers) explained by first Principal Component scores. Trajectories’ shape and orientation were investigated and contrasted. Logistic regression and Receiver Operating Characteristic curves were used to compare these morphometric indicators with traditional 3D Speckle Tracking Echocardiography global parameters. Geometric morphometrics indicators performed better than 3D Speckle Tracking Echocardiography global parameters in recognizing pathology both in systole and diastole. Genotype positive/phenotype negative individuals clustered with patients affected by hypertrophic cardiomyopathy during diastole, suggesting that incumbent pathology may indeed be foreseen by these methods. Left ventricle deformation in patients affected by hypertrophic cardiomyopathy compared to healthy subjects may be assessed by modern shape analysis better than by traditional 3D Speckle Tracking Echocardiography global parameters. Hypertrophic cardiomyopathy pathophysiology was unveiled in a new manner whereby also diastolic phase abnormalities are evident which is more difficult to investigate by traditional ecocardiographic techniques.     

Absolute Configuration Assignment of a Paraconic Acid Derivative via Vibrational Circular Dichroism Spectroscopy and Density Functional Theory Calculation

Density functional theory calculation of the vibrational circular dichroism spectrum was used to assign the absolute configuration of an all‐carbon quaternary β‐stereocenter of a γ‐butyrolactone recently synthesized through an asymmetric organocatalytic tandem aldol/lactonization sequence. Comparison with the experimental spectrum is satisfactory, on account of the fact that spectroscopic features are weak due to the presence of multiple conformers. As a result, the (R) absolute configuration was assigned to the (+) optical isomer. Chirality 28:110–115, 2016. © 2015 Wiley Periodicals, Inc.