Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.     

Polymorphisms in human langerin affect stability and sugar binding activity

Langerhans cells are specialized skin dendritic cells that take up and degrade antigens for presentation to the immune system. Langerin, a cell surface C-type lectin of Langerhans cells, can be internalized and accumulates in Birbeck granules, subdomains of the endosomal recycling compartment that are specific to Langerhans cells. Langerin binds and mediates uptake and degradation of glycoconjugates containing mannose and related sugars. Analysis of the human genome has identified three single nucleotide polymorphisms that result in amino acid changes in the carbohydrate-recognition domain of langerin. The effects of the amino acid changes on the activity of langerin were examined by expressing each of the polymorphic forms. Expression of full-length versions of the four common langerin haplotypes in fibroblasts revealed that all of these forms can mediate endocytosis of neoglycoprotein ligands. However, sugar binding assays and differential scanning calorimetry performed on fragments from the extracellular domain showed that two of the amino acid changes reduce the affinity of the carbohydrate-recognition domain for mannose and decrease the stability of the extracellular domain. In addition, analysis of sugar binding by langerin containing the rare W264R mutation, previously identified in an individual lacking Birbeck granules, shows that this mutation abolishes sugar binding activity. These findings suggest that certain langerin haplotypes may differ in their binding to pathogens and thus might be associated with susceptibility to infection.     

Structure and function of wildfire and mountain pine beetle forest boundaries

Landscape boundaries, in particular those created by natural disturbances, are fundamental structures in landscape functioning. At the stand scale, forest boundaries show a wide range of characteristics varying from sharp to diffuse, wavy to straight, enclosed areas (patches) to open ones; all of these have different effects of forest vegetation. The objectives of this study were 1) to characterize the structure of forest boundaries (width, steepness, heterogeneity) and 2) to investigate forest vegetation functional response in the boundary zones created by wildfires and mountain pine beetle (MPB) outbreaks. We studied 11 mountain pine beetle and 7 wildfire boundary zones (4–7 y since disturbance) using field‐based two‐dimensional grids in southern central British Columbia (Canada). Boundary zone delineation was determined through spatially constrained clustering algorithms at three sampling unit resolutions (5×5 m, 10×10 m, and 20×20 m). Then, to characterize boundary width and shape, we developed two new boundary indexes (maximized and minimized boundary zones). The identified boundary widths ranged from 0 to 127 m with a mean width of 51 m at the 20×20 m resolution for both fire and MPB. Although the widths were comparable between disturbance types, fires generally had steeper boundaries (more pronounced) than MPB, largely due to higher peak tree mortality within the disturbances (89 vs 43%). Most of the forest vegetation response variables (understory vegetation diversity, plot‐level species richness, evenness, and multivariate community measures) in the boundary zones tended not to be intermediate in value between the intact forest and disturbance area, in spite of intermediate tree mortality. Tree mortality heterogeneity in the boundary zones was often highest in fires and was equal to the internal heterogeneity in MPB disturbances. Using historical natural disturbance patterns as a conservation strategy, this study proposes that forest management should create boundary complexity (width and shape), thereby creating landscape heterogeneity similar to landscapes influenced primarily by natural disturbances.     

Quantification of pelvic organ prolapse in mice: vaginal protease activity precedes increased MOPQ scores in fibulin 5 knockout mice

Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5(-/-) mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse.     

Ethanol preference in C. elegans

Caenorhabditis elegans senses multiple environmental stimuli through sensory systems and rapidly changes its behaviors for survival. With a simple and well-characterized nervous system, C. elegans is a suitable animal model for studying behavioral plasticity.
Previous studies have shown acute neurodepressive effects of ethanol on multiple behaviors of C. elegans similar to the effect of ethanol on other organisms. Caenorhabditis elegans also develops ethanol tolerance during continuous exposure to ethanol. In mammals, chronic ethanol exposure leads to ethanol tolerance as well as increased ethanol consumption. Ethanol preference is associated with the development of tolerance and may lead to the development of ethanol dependence.
In this study, we show that C. elegans is a useful model organism for studying chronic effects of ethanol, including the development of ethanol preference. We designed a behavioral assay for testing ethanol preference after prolonged ethanol exposure. Despite baseline aversive responses to ethanol, animals show ethanol preference after 4 h of pre-exposure to ethanol and exhibit significantly enhanced preference for ethanol after a lifetime of ethanol exposure. The cat-2 and tph-1 mutant animals have defects in the synthetic enzymes for dopamine and serotonin, respectively. These mutants are deficient in the development of ethanol preference, indicating that dopamine and serotonin are required for this form of behavioral plasticity.     

Context dependent substitution biases vary within the human genome

Background  

Models of sequence evolution typically assume that different nucleotide positions evolve independently. This assumption is widely appreciated to be an over-simplification. The best known violations involve biases due to adjacent nucleotides. There have also been suggestions that biases exist at larger scales, however this possibility has not been systematically explored.     

Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes

The success of cell-based tissue engineering approaches in restoring biological function will be facilitated by a comprehensive fundamental knowledge of the temporal evolution of the structure and properties of the newly synthesized matrix. Here, we quantify the dynamic oscillatory mechanical behavior of the engineered matrix associated with individual chondrocytes cultured in vitro for up to 28 days in alginate scaffolds. The magnitude of the complex modulus (|E*|) and phase shift (δ) were measured in culture medium using Atomic Force Microscopy (AFM)-based nanoindentation in response to an imposed oscillatory deformation (amplitude ～5 nm) as a function of frequency (f=1–316 Hz), probe tip geometry (2.5 μm radius sphere and 50 nm radius square pyramid), and in the absence and presence of growth factors (GF, insulin growth factor-1, IGF-1, and osteogenic protein-1, OP-1). |E*| for all conditions increased nonlinearly with frequency dependence approximately f^1/2 and ranged between ～1 and 25 kPa. This result, along with theoretical calculations of the characteristic poroelastic relaxation frequency, f_p, (～50–90 Hz) suggested that this time-dependent behavior was governed primarily by fluid flow-dependent poroelasticity, rather than flow-independent viscoelastic processes associated with the solid matrix. |E*(f)| increased, (f) decreased, and the hydraulic permeability, k, decreased with time in culture and with growth factor treatment. This trend of a more elastic-like response was thought to be associated with increased macromolecular biosynthesis, density, and a more mature matrix structure/organization.