The GLT‐1 (EAAT2; slc1a2) glutamate transporter is essential for glutamate homeostasis in the neocortex of the mouse

Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with [1‐¹³C]glucose and [1,2‐¹³C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and ¹³C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and ¹³C labeling of metabolites, glutamate level was decreased but labeling from [1‐¹³C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2‐¹³C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.

     

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.     

The Distinct Conformational Dynamics of K-Ras and H-Ras A59G

Ras proteins regulate signaling cascades crucial for cell proliferation and differentiation by switching between GTP- and GDP-bound conformations. Distinct Ras isoforms have unique physiological functions with individual isoforms associated with different cancers and developmental diseases. Given the small structural differences among isoforms and mutants, it is currently unclear how these functional differences and aberrant properties arise. Here we investigate whether the subtle differences among isoforms and mutants are associated with detectable dynamical differences. Extensive molecular dynamics simulations reveal that wild-type K-Ras and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. The crucial switch 1 and switch 2 regions along with loop 3, helix 3, and loop 7 contribute to this enhanced flexibility. Removing the gamma-phosphate of the bound GTP from the structure of A59G led to a spontaneous GTP-to-GDP conformational transition in a 20-ns unbiased simulation. The switch 1 and 2 regions exhibit enhanced flexibility and correlated motion when compared to non-transitioning wild-type H-Ras over a similar timeframe. Correlated motions between loop 3 and helix 5 of wild-type H-Ras are absent in the mutant A59G reflecting the enhanced dynamics of the loop 3 region. Taken together with earlier findings, these results suggest the existence of a lower energetic barrier between GTP and GDP states of the mutant. Molecular dynamics simulations combined with principal component analysis of available Ras crystallographic structures can be used to discriminate ligand- and sequence-based dynamic perturbations with potential functional implications. Furthermore, the identification of specific conformations associated with distinct Ras isoforms and mutants provides useful information for efforts that attempt to selectively interfere with the aberrant functions of these species.     

Longitudinal river zonation in the tropics: examples of fish and caddisflies from the endorheic Awash River,Ethiopia

Specific concepts of fluvial ecology are well studied in riverine ecosystems of the temperate zone but poorly investigated in the Afrotropical region. Hence, we examined the longitudinal zonation of fish and adult caddisfly (Trichoptera) assemblages in the endorheic Awash River (1,250 km in length), Ethiopia. We expected that species assemblages are structured along environmental gradients, reflecting the pattern of large-scale freshwater ecoregions. We applied multivariate statistical methods to test for differences in spatial species assemblage structure and identified characteristic taxa of the observed biocoenoses by indicator species analyses. Fish and caddisfly assemblages were clustered into highland and lowland communities, following the freshwater ecoregions, but separated by an ecotone with highest biodiversity. Moreover, the caddisfly results suggest separating the heterogeneous highlands into a forested and a deforested zone. Surprisingly, the Awash drainage is rather species-poor: only 11 fish (1 endemic, 2 introduced) and 28 caddisfly species (8 new records for Ethiopia) were recorded from the mainstem and its major tributaries. Nevertheless, specialized species characterize the highland forests, whereas the lowlands primarily host geographically widely distributed species. This study showed that a combined approach of fish and caddisflies is a suitable method for assessing regional characteristics of fluvial ecosystems in the tropics.

     

Effect of Symmetry Breaking on Plasmonic Coupling in Nanoring Dimers

Plasmonics - Breaking the morphological and compositional symmetries of metallic nanoparticle (NP) dimers provides a novel approach to modulate plasmon coupling between the NPs. In this study, we...     

β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA₁ are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA₁-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA₁. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.     

Maternal Complications and Women's Behavior in Seeking Care from Skilled Providers in North Gondar,Ethiopia

Background

Maternal complications are morbidities suffered during pregnancy through the postpartum period of 42 days. In Ethiopia, little is known about women''s experience of complications and their care-seeking behavior. This study attempted to assess experiences related to obstetric complication and seeking assistance from a skilled provider among women who gave birth in the last 12 months preceding the study.

Methods

This study was a cross-sectional survey of women who gave birth within one year preceding the study regardless of their delivery place. The study was carried out in six selected districts in North Gondar Zone, Amhara Region. Data was collected house-to-house in 12 selected clusters (kebeles) using a pretested Amharic questionnaire. During the survey, 1,668 women were interviewed. Data entry was done using Epi Info version 3.5.3 and was exported to SPSS for analysis. Logistic regression was applied to control confounders.

Results

Out of the total sample, 476 women (28.5%, 95% CI: 26.4%, 30.7%) reported some kind of complication. The most common complications reported were; excessive bleeding and prolonged labor that occurred mostly at the time of delivery and postpartum period. Out of the total women who faced complications, 248 (52.1%, 95% CI: 47.6%, 56.6%) sought assistance from a skilled provider. Inability to judge the severity of morbidities, distance/transport problems, lack of money/cost considerations and use of traditional options at home were the major reasons for not seeking care from skilled providers. Belonging to a wealthier quintile, getting antenatal care from a skilled provider and agreement of a woman in planning for possible complications were significantly associated with seeking assistance from a skilled provider.

Conclusion

Nearly half of the women who faced complications did not use skilled providers at the time of obstetric complications. Cognitive, geographic, economic and cultural barriers were involved in not using skilled maternal care.     

Effect of mustard green manure and dried plant residue on chickpea wilt (Fusarium oxysporum f.sp. ciceris)

Pot experiments were carried out in the green house at Amhara Regional Agriculture Research Institute (ARARI) Bahirdar, Ethiopia, to evaluate the potential of Brassica carinata cultivars, namely Holleta-l, S-67 and Yellow Dodola in 2007 and 2008. The effect of B. carinata (Ethiopian mustard) cultivars Holleta-1, S-67 and Yellow Dodola as green manure and Holleta-1 as dried plant residue on chickpea fusarium wilt (Fusarium oxysporum f.sp. ciceris) was studied. Six rates of green manure and dried plant residue (0, 20, 40, 60, 80 and 100 g) each per kg of pathogen-infested soil were used in the experiments. Infested soil without B. carinata cultivars amendment as a control and susceptible check variety JG-62 without amendment was used in the experiments. In the experiments, the treatments were arranged in randomised complete block design in three replications and repeated twice. Data on seedling emergence, wilt incidence, fresh weight and dry weight were collected. The amendments of infested soil with B. carinata cultivars green manure and dried plant residue reduced the incidence of chickpea fusarium wilt. The incorporation of the green manure Holleta-1, S-67 and Yellow Dodola at 20–100 g/kg of infested soil was effective in reducing wilt incidences on chickpea. However, the incorporation of Yellow Dodola at 80 and 100 g green manure per kg of infested soil were the best combination in reducing significantly wilt incidence. The application of the dried plant residue at 20–100 g/kg of infested soil was effective in reducing wilt incidences on chickpea. However when applied dried plant residue at 60, 80 and 100 g green manure per kg of infested soil were better in reducing wilt incidence as compared to 20 and 40 g/kg of infested soil. The three cultivars green manure incorporated at different level of doses affected the influence of fusarium wilt on the fresh and dry weight respectively. The use of Holleta-1 green manure at 20–100 g/kg of infested soil significantly reduced disease incidence in the range of 20.0–33.3%. Green manure amendment S-67 significantly reduced disease incidence in the range of 20.0–46.6%. Yellow Dodola reduce disease incidence with 26.7–60%. The dried plant residue incorporated at different level influence fusarium wilt. The application of Holleta-1 dried plant residue at 20–100 g/kg of infested soil reduced disease incidence in the range 20.0–26.7%. The results imply the potential of using B. carinata green manure and dried plant residue as cultural management components in chickpea fusarium wilt disease management.     

Novel allosteric sites on Ras for lead generation

Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs.