Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award     

Environmental responses of plants and ecosystems as predictors of the impact of global change

An understanding of plant responses to fluctuations in environment is critical to predictions of plant and ecosystem responses to climate change. In the northern hemisphere, the northern limits of distribution of major biomes are probably determined by the tolerance of their dominant physiognomic types (e.g., deciduous hardwood trees) to minimum winter temperatures and can thus be predicted from long-term patterns of temperature fluctuations. At a more detailed level, the responses of functional groups of plants to altered climate can be predicted from their known responses to fluctuations in soil resources (nutrients and water) and the expected effect of climatic change on these soil resources. Laboratory and field experiments demonstrate the feasibility of this approach.     

Population density and genetic diversity are positively correlated in wild felids globally

Aim

Insights into the biological and evolutionary traits of species, and their ability to cope with global changes, can be gained by studying genetic diversity within species. A cornerstone hypothesis in evolutionary and conservation biology suggests that genetic diversity decreases with decreasing population size, however, population size is difficult to estimate in threatened species with large distribution ranges, and evidence for this is limited to few species. To address this gap, we tested this hypothesis across multiple closely related species at a global scale using population density which is a more accessible measure.

Location

Global.

Time Period

Contemporary.

Major Taxa Studied

Wild felids in their natural habitats.

Methods

We obtained data from published estimates of population density assessed via camera trap and within-population genetic diversity generated from microsatellite markers on 18 felid species across 41 countries from 354 studies. We propose a novel method to standardize population density estimates and to spatially join data using K-means clustering. Linear mixed-effect modelling was applied to account for confounding factors such as body mass, generation length and sample size used for the genetic estimates.

Results

We found a significant positive correlation between population density and genetic diversity, particularly observed heterozygosity and allelic richness. While the confounding factors did not affect the main results, long generation length and large sample size were significantly associated with high genetic diversity. Body mass had no effect on genetic diversity, likely because large-bodied species were over-represented in our data sets.

Main Conclusions

Our study emphasizes how recent demographic processes shape neutral genetic diversity in threatened and small populations where extinction vortex is a risk. Although caution is needed when interpreting the small population density effect in our findings, our methodological framework shows promising potential to identify which populations require actions to conserve maximal genetic variation.     

In Vitro and Molecular Docking Evaluation of Larvicidal Effects of Essential Oils of Five Aromatic Plants on the Fall Armyworm Spodoptera frugiperda JE. Smith (Lepidoptera: Noctuidae) from Ivory Coast

Faced with the serious consequences resulting from the abusive and repeated use of synthetic chemicals, today rethinking crop protection is more than necessary. It is in this context that the essential oils of the Lamiaceae Ocimum gratissimum and Ocimum canum, the Poaceae Cymbopogon citratus and nardus and a Rutaceae Citrus sp. of known chemical compositions were experimented. The evaluation of the larvicidal potential of the essential oils was done by the method of topical application of the test solutions, on the L1−L2 stage larvae from the first generation of S. frugiperda obtained after rearing in an air-conditioned room. Lethal concentrations (LC₁₀, LC₅₀ and LC₉₀) were determined after 48 h. After assessing the larvicidal potential of essential oils, molecular docking was carried out to study protein-ligand interactions and their propensity to bind to insect enzyme sites (AChE). The essential oil of O. gratissimum was the most effective with the lowest lethal concentrations (LC₁₀=0.91 %, LC₅₀=1.91 % and LC₉₀=3.92 %). The least toxic oil to larvae was Citrus sp. (LC₁₀=5.44 %, LC₅₀=20.50 % and LC₉₀=77.41 %). Molecular docking revealed that p-cymene and thymol from O. gratissimum essential oil are structurally similar and bind to the AChE active site via predominantly hydrophobic interactions and a H-bond with Tyr374 in the case of thymol. The essential oil of O. gratissimum constitutes a potential candidate for the development of biological insecticides for the fight against insect pests and for the protection of the environment.     

Novel Miscanthus hybrids: Modelling productivity on marginal land in Europe using dynamics of canopy development determined by light interception

New biomass crop hybrids for bioeconomic expansion require yield projections to determine their potential for strategic land use planning in the face of global challenges. Our biomass growth simulation incorporates radiation interception and conversion efficiency. Models often use leaf area to predict interception which is demanding to determine accurately, so instead we use low-cost rapid light interception measurements using a simple laboratory-made line ceptometer and relate the dynamics of canopy closure to thermal time, and to measurements of biomass. We apply the model to project the European biomass potentials of new market-ready hybrids for 2020–2030. Field measurements are easier to collect, the calibration is seasonally dynamic and reduces influence of weather variation between field sites. The model obtained is conservative, being calibrated by crops of varying establishment and varying maturity on less productive (marginal) land. This results in conservative projections of miscanthus hybrids for 2020–2030 based on 10% land use conversion of the least (productive) grassland and arable for farm diversification, which show a European potential of 80.7–89.7 Mt year⁻¹ biomass, with potential for 1.2–1.3 EJ year⁻¹ energy and 36.3–40.3 Mt year⁻¹ carbon capture, with seeded Miscanthus sacchariflorus × sinensis displaying highest yield potential. Simulated biomass projections must be viewed in light of the field measurements on less productive land with high soil water deficits. We are attempting to model the results from an ambitious and novel project combining new hybrids across Europe with agronomy which has not been perfected on less productive sites. Nevertheless, at the time of energy sourcing issues, seed-propagated miscanthus hybrids for the upscaled provision of bioenergy offer an alternative source of renewable energy. If European countries provide incentives for growers to invest, seeded hybrids can improve product availability and biomass yields over the current commercial miscanthus variety.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.