Electrosensitive spatial vectors in elasmobranch fishes: implications for source localization

The electrosense of sharks and rays is used to detect weak dipole-like bioelectric fields of prey, mates and predators, and several models propose a use for the detection of streaming ocean currents and swimming-induced fields for geomagnetic orientation. We assessed pore distributions, canal vectors, complementarity and possible evolutionary divergent functions for ampullary clusters in two sharks, the scalloped hammerhead (Sphyrna lewini) and the sandbar shark (Carcharhinus plumbeus), and the brown stingray (Dasyatis lata). Canal projections were determined from measured coordinates of each electrosensory pore and corresponding ampulla relative to the body axis. These species share three ampullary groups: the buccal (BUC), mandibular (MAN) and superficial ophthalmic (SO), which is subdivided into anterior (SOa) and posterior (SOp) in sharks. The stingray also has a hyoid (HYO) cluster. The SOp in both sharks contains the longest (most sensitive) canals with main projections in the posterior-lateral quadrants of the horizontal plane. In contrast, stingray SO canals are few and short with the posterior-lateral projections subsumed by the HYO. There was strong projection coincidence by BUC and SOp canals in the posterior lateral quadrant of the hammerhead shark, and laterally among the stingray BUC and HYO. The shark SOa and stingray SO and BUC contain short canals located anterior to the mouth for detection of prey at close distance. The MAN canals of all species project in anterior or posterior directions behind the mouth and likely coordinate prey capture. Vertical elevation was greatest in the BUC of the sandbar shark, restricted by the hammerhead cephalofoil and extremely limited in the dorsoventrally flattened stingray. These results are consistent with the functional subunit hypothesis that predicts specialized ampullary functions for processing of weak dipole and geomagnetic induced fields, and provides an anatomical basis for future experiments on central processing of different forms of relevant electric stimuli.     

Image-based screening identifies novel roles for IkappaB kinase and glycogen synthase kinase 3 in axonal degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.     

Positive Reinforcement Mediated by Midbrain Dopamine Neurons Requires D1 and D2 Receptor Activation in the Nucleus Accumbens

The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.     

2-Aminoimidazoles inhibitors of TGF-β receptor 1

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-βR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-β-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.     

Skittle: A 2-Dimensional Genome Visualization Tool

Background  

It is increasingly evident that there are multiple and overlapping patterns within the genome, and that these patterns contain different types of information - regarding both genome function and genome history. In order to discover additional genomic patterns which may have biological significance, novel strategies are required. To partially address this need, we introduce a new data visualization tool entitled Skittle.     

School-based participatory health education for malaria control in Ghana: engaging children as health messengers

Background

Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target.

Methods

A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob.

Results

Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules.

Conclusions

High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should primarily induce strongly adhering, high avidity antibodies whose association rate constant is less important than their dissociation rate constant.     

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.     

Extracellular functions of galectin-3

Galectin-3 has been suspected of modulating cell to extracellular matrix interactions in a novel fashion ever since it was first described. However, the rapid accumulation of research data in just the last 8 years alone has completely changed our perspective of this multifunctional protein. Its chimeric nature (consists of carbohydrate recognition and collagen like domains) somehow makes it suited to interact with a plethora of interesting extracellular matrix proteins some of which might enable it to cross the plasma membrane despite its lack of appropriate signal peptides. It is now becoming established as a mediator of signal transduction events on the cell surface as well as a mediator of a variety of extra-cellular processes such as kidney development, angiogenesis, neuronal functions, tumor metastasis, autoimmune disorders, endocytosis and possibly exocytosis. Nevertheless, it still retains its unique position as a mediator/modulator of cell to extracellular matrix adhesive interactions. Cells, particularly epithelial cells which lack galectin-3 expression, interact poorly with their extracellular matrices. In some of these processes, it functions as a matricellular protein, displaying both pro- and anti-adhesive properties.