Discovery of fused bicyclic agonists of the orphan G-protein coupled receptor GPR119 with in vivo activity in rodent models of glucose control

We herein outline the design of a new series of agonists of the pancreatic and GI-expressed orphan G-protein coupled receptor GPR119, a target that has been of significant recent interest in the field of metabolism, starting from our prototypical agonist AR231453. A number of key parameters were improved first by incorporation of a pyrazolopyrimidine core to create a new structural series and secondly by the introduction of a piperidine ether group capped with a carbamate. Chronic treatment with one compound from the series, 3k, showed for the first time that blood glucose and glycated hemoglobin (HbA1c) levels could be significantly reduced in Zucker Diabetic Fatty (ZDF) rats over several weeks of dosing. As a result of these and other data described here, 3k (APD668, JNJ-28630368) was the first compound with this mechanism of action to be progressed into clinical development for the treatment of diabetes.     

Sub-lethal effects of moxidectin on the Neotropical dung beetle Onthophagus landolti Harold (Coleoptera: Scarabaeinae)

Macrocyclic lactones can have adverse effects in dung beetles exposed to manure containing them. An assessment of the survival and fertility of adult Onthophagus landolti Harold fed with manure from cows treated with moxidectin was performed, as well as the emergence rate of the imagoes from the masses. Three cows (Bos indicus x B. taurus) were subcutaneously injected with 1% moxidectin (0.2 mg kg^?1 b.w.) and another three were injected with 10% moxidectin (1.0 mg/kg^?1 b.w.). Manure was collected from these animals one day prior to moxidectin administration, five days post-treatment in the 1% and 10% treatments, and fourteen days in the 10% treatment. Four bioassays were done: a control using manure without moxidectin; 1% moxidectin at five days post-treatment; 10% moxidectin at five days post-treatment; and 10% moxidectin at fourteen days post-treatment. In each replicate, for each pair of adult O. landolti was daily fed with 30 g manure according to the treatments. No lethal effects were observed in any of the four treatments. Sub-lethal effects (P < .05) were present in the 10% moxidectin treatments at five and fourteen days post-treatment. Fecundity was reduced by 78.2% at five days and 54.9% at fourteen days, and imago emergence was negatively affected at both times. Current moxidectin application methods may have negative effects on the environmental services provided by dung beetles, and therefore need to be modified to minimize any impacts they might have on these vital members of tropical livestock systems.     

SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.     

Trunk neural crest has skeletogenic potential

During early vertebrate development, neural crest cells emerge from the dorsal neural tube, migrate into the periphery, and form a wide range of derivatives. There is, however, a significant difference between the cranial and trunk neural crest with respect to the diversity of cell types that each normally produces. Thus, while crest cells from all axial levels form neurons, glia, and melanocytes, the cranial crest additionally generates skeletal derivatives such as bone and cartilage; trunk crest cells are generally thought to lack skeletogenic potential. Here, we show, however, that if avian trunk neural crest cells are cultured in appropriate media, they form both bone and cartilage cells, and if placed into the developing head, they contribute to cranial skeletal components. Thus, the neural crest from all axial levels can generate the full repertoire of crest derivatives. The skeletogenic potential of the trunk neural crest is significant, as it was likely realized in early vertebrates, which had extensive postcranial exoskeletal coverings.     

Prevalence dependence in model goodness measures with special emphasis on true skill statistics

It has long been a concern that performance measures of species distribution models react to attributes of the modeled entity arising from the input data structure rather than to model performance. Thus, the study of Allouche et al. (Journal of Applied Ecology, 43, 1223, 2006) identifying the true skill statistics (TSS) as being independent of prevalence had a great impact. However, empirical experience questioned the validity of the statement. We searched for technical reasons behind these observations. We explored possible sources of prevalence dependence in TSS including sampling constraints and species characteristics, which influence the calculation of TSS. We also examined whether the widespread solution of using the maximum of TSS for comparison among species introduces a prevalence effect. We found that the design of Allouche et al. (Journal of Applied Ecology, 43, 1223, 2006) was flawed, but TSS is indeed independent of prevalence if model predictions are binary and under the strict set of assumptions methodological studies usually apply. However, if we take realistic sources of prevalence dependence, effects appear even in binary calculations. Furthermore, in the widespread approach of using maximum TSS for continuous predictions, the use of the maximum alone induces prevalence dependence for small, but realistic samples. Thus, prevalence differences need to be taken into account when model comparisons are carried out based on discrimination capacity. The sources we identified can serve as a checklist to safely control comparisons, so that true discrimination capacity is compared as opposed to artefacts arising from data structure, species characteristics, or the calculation of the comparison measure (here TSS).     

Changes in predictor influence with time and with vegetation type identity in a post-abandonment situation

Variation in the explanatory potential of separate variable groups describing past vegetation patterns and/or abiotic environment were investigated using data from two points in time for three vegetation types, which are variants of the steppe grassland component of the forest steppe biome. First, we compared the predictive power of environmental variables assessed by the performance of generalised linear models (GLMs) explaining the distribution of three vegetation types in 1988 and 2002. Second, we fitted models based on conceptual hypotheses to explain vegetation distribution in 2002. Specifically, we wanted to examine, whether (i) current abiotic topo-environment, (ii) past neighbourhood configuration, or (iii) historical vegetation patterns or a combination of these determine best the current distribution of vegetation types. We developed basic predictor sets for each hypothesis, and using GLMs we tested to what degree these predictor sets were capable of explaining the currently observed patterns of individual vegetation types. We compared model accuracy by AUC and TSS values. Predictive performance of models changed both with time and with vegetation type. The analyses of changes over time showed that two of the three vegetation types had come closer to an equilibrium with abiotic conditions, while the third had moved farther away from equilibrium. Knowledge of past conditions was sufficient to predict the distribution of one of the three investigated vegetation types alone, thus no topo-environmental predictors were needed to successfully predict this type. The other two vegetation types were best explained by current topo-environmental predictors. We conclude that historical conditions clearly improve predictive models, though there may be variation in their contribution to models of different vegetation types and this may depend on how far vegetation types are from an equilibrium state.     

Conservation of the salt overly sensitive pathway in rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.     

Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells

Around 30% of exocytosis events recorded by amperometry at carbon fiber microelectrodes exhibit a pre-spike feature (PSF) termed a “foot”. This wave is associated with the release of the neurotransmitters via a transitory fusion pore, whilst the large, main exocytotic spike is due to complete release. The amperometric data reported herein were obtained using bovine chromaffin cells stimulated with either potassium or barium ions, two commonly-employed elicitors of exocytosis. Identical trends are observed with both activators: (i) they induce the same ratio (close to 30%) of events with a foot in the population of amperometric spikes, and (ii) spikes with a foot can be divided into two primary categories, depending on the temporal variation of the current wave (viz. as a ramp, or a ramp followed by a plateau). Correlations between the characteristics of the whole current spike, and of its observed foot, have been sought; such analyses demonstrate that the maximum current of both foot and spike signals are highly correlated, but, in contrast, the integrated charges of both are poorly correlated. Moreover, the temporal duration of the PSF is fully uncorrelated with any parameter pertaining to the main current spike. On the basis of these reproducible observations, it is hypothesized that the characteristics (dimensions and topology, at least) of each secretory vesicle determine the probability of formation of the fusion pore and its maximum size, whilst molecular factors of the cell membrane control its duration, and, consequently, the amount delivered prior to the massive exocytosis of catecholamines observed as a spike in amperometry.     

Acanthocheilonema sabanicolae n. sp. (Filarioidea: Onchocercidae) from the savanna armadillo (Dasypus sabanicola) in Venezuela, with comments on the genus Acanthocheilonema

A new species of Acanthocheilonema, Acanthocheilonema sabanicolae n. sp., is described from the subcutaneous tissues of the savanna armadillo (Dasypus sabanicola) in Venezuela. The adults (females 25-30.6 mm long by 61-78 micron wide; males 10.9-15.3 mm long by 42-44 micron wide) and the microfilariae (112-131 micron long by 3-5 micron wide) are the smallest within the genus. The microfilaria, a skin dweller, assumes a characteristic C-shaped curve, when fixed in 2% formalin, which serves to distinguish A. sabanicolae from others in the genus. Moreover, the spicules are notably smaller than any others in the Acanthocheilonema. Acanthocheilonema sabanicolae is the first species in this genus to be described from edentates, and, along with A. reconditum of dogs and A. pricei of opossums, is the third species of Acanthocheilonema to be reported from South America. Morphological features, vectors, and vertebrate hosts of the genus Acanthocheilonema are reviewed.     

The Trypanosoma cruzi nucleolus: a morphometrical analysis of cultured epimastigotes in the exponential and stationary phases

Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To establish cellular parameters for studying ribosome biogenesis in T. cruzi, a morphometric analysis of the nucleoli of cultured cells in the exponential and stationary phases was conducted. Electron micrograph-based measurements of nuclear sections from independent cells demonstrated that the nucleolar area is over twofold higher in exponentially growing cells, as compared with epimastigotes in the stationary phase. The granular component of the nucleoli of actively growing cells was the main structural element. Cycloheximide moderately reduced the apparent size of the nucleoli without an apparent disruption of their architecture. Our results provide a firm basis for the establishment of an experimental model to study the organization of the nucleolus during the growth and development of T. cruzi.