Identifying the Computational Requirements of an Integrated Top-Down-Bottom-Up Model for Overt Visual Attention within an Active Vision System

Computational visual attention systems have been constructed in order for robots and other devices to detect and locate regions of interest in their visual world. Such systems often attempt to take account of what is known of the human visual system and employ concepts, such as ‘active vision’, to gain various perceived advantages. However, despite the potential for gaining insights from such experiments, the computational requirements for visual attention processing are often not clearly presented from a biological perspective. This was the primary objective of this study, attained through two specific phases of investigation: 1) conceptual modeling of a top-down-bottom-up framework through critical analysis of the psychophysical and neurophysiological literature, 2) implementation and validation of the model into robotic hardware (as a representative of an active vision system). Seven computational requirements were identified: 1) transformation of retinotopic to egocentric mappings, 2) spatial memory for the purposes of medium-term inhibition of return, 3) synchronization of ‘where’ and ‘what’ information from the two visual streams, 4) convergence of top-down and bottom-up information to a centralized point of information processing, 5) a threshold function to elicit saccade action, 6) a function to represent task relevance as a ratio of excitation and inhibition, and 7) derivation of excitation and inhibition values from object-associated feature classes. The model provides further insight into the nature of data representation and transfer between brain regions associated with the vertebrate ‘active’ visual attention system. In particular, the model lends strong support to the functional role of the lateral intraparietal region of the brain as a primary area of information consolidation that directs putative action through the use of a ‘priority map’.     

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.     

Inter‐α‐inhibitor deficiency in the mouse is associated with alterations in anxiety‐like behavior,exploration and social approach

In recent years, several genome‐wide association studies have identified candidate regions for genetic susceptibility in major mood disorders. Most notable are regions in a locus in chromosome 3p21, encompassing the genes NEK4‐ITIH1‐ITIH3‐ITIH4. Three of these genes represent heavy chains of the composite protein inter‐α‐inhibitor (IαI). In order to further establish associations of these genes with mood disorders, we evaluated behavioral phenotypes in mice deficient in either Ambp/bikunin, which is necessary for functional ITIH1 and ITIH3 complexes, or in Itih4, the gene encoding the heavy chain Itih4. We found that loss of Itih4 had no effect on the behaviors tested, but loss of Ambp/bikunin led to increased anxiety‐like behavior in the light/dark and open field tests and reduced exploratory activity in the elevated plus maze, light/dark preference and open field tests. Ambp/bikunin knockout mice also exhibited a sex‐dependent exaggeration of acoustic startle responses, alterations in social approach during a three‐chamber choice test, and an elevated fear conditioning response. These results provide experimental support for the role of ITIH1/ITIH3 in the development of mood disorders.     

Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay

Background

Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Methodology/Principal Findings

Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC₅₀ of 2 µM.

Conclusions/Significance

The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.     

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Motile bacteria usually rely on external apparatus like flagella or pili, but gliding bacteria do not rely on obvious surface appendages for their movement. This study shows that bacteria in the phylum Bacteroidetes use proton-dependent motors to power protein secretion and gliding motility.     

Excess of rare amino acid polymorphisms in the Toll-like receptor 4 in humans

The Toll-like receptor 4 protein acts as the transducing subunit of the lipopolysaccharide receptor complex and assists in the detection of Gram-negative pathogens within the mammalian host. Several lines of evidence support the view that variation at the TLR4 locus may alter host susceptibility to Gram-negative infection or the outcome of infection. Here, we surveyed TLR4 sequence variation in the complete coding region (2.4 kb) in 348 individuals from several population samples; in addition, a subset of the individuals was surveyed at 1.1 kb of intronic sequence. More than 90% of the chromosomes examined encoded the same structural isoform of TLR4, while the rest harbored 12 rare amino acid variants. Conversely, the variants at silent sites (intronic and synonymous positions) occur at both low and high frequencies and are consistent with a neutral model of mutation and random drift. The spectrum of allele frequencies for amino acid variants shows a significant skew toward lower frequencies relative to both the neutral model and the pattern observed at linked silent sites. This is consistent with the hypothesis that weak purifying selection acted on TLR4 and that most mutations affecting TLR4 protein structure have at least mildly deleterious phenotypic effects. These results may imply that genetic variants contributing to disease susceptibility occur at low frequencies in the population and suggest strategies for optimizing the design of disease-mapping studies.     

The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural beta-hairpin motif

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable beta-hairpin, incorporating a type VIb beta-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 A over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein beta-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide's solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation approximately 0.7 A) but also for the side-chains. This isolated beta-hairpin retains the biologically active "canonical conformation" typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this beta-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this beta-hairpin and its biological activity is discussed.