Kinetic microplate assay for determining immobilized antimicrobial peptide activity

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations. Furthermore, the method yields information regarding sublethal concentrations not realized in the traditional assays.     

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.     

How Prevalent Is Object-Based Attention?

Previous research suggests that visual attention can be allocated to locations in space (space-based attention) and to objects (object-based attention). The cueing effects associated with space-based attention tend to be large and are found consistently across experiments. Object-based attention effects, however, are small and found less consistently across experiments. In three experiments we address the possibility that variability in object-based attention effects across studies reflects low incidence of such effects at the level of individual subjects. Experiment 1 measured space-based and object-based cueing effects for horizontal and vertical rectangles in 60 subjects comparing commonly used target detection and discrimination tasks. In Experiment 2 we ran another 120 subjects in a target discrimination task in which rectangle orientation varied between subjects. Using parametric statistical methods, we found object-based effects only for horizontal rectangles. Bootstrapping methods were used to measure effects in individual subjects. Significant space-based cueing effects were found in nearly all subjects in both experiments, across tasks and rectangle orientations. However, only a small number of subjects exhibited significant object-based cueing effects. Experiment 3 measured only object-based attention effects using another common paradigm and again, using bootstrapping, we found only a small number of subjects that exhibited significant object-based cueing effects. Our results show that object-based effects are more prevalent for horizontal rectangles, which is in accordance with the theory that attention may be allocated more easily along the horizontal meridian. The fact that so few individuals exhibit a significant object-based cueing effect presumably is why previous studies of this effect might have yielded inconsistent results. The results from the current study highlight the importance of considering individual subject data in addition to commonly used statistical methods.     

The Ecology of fear: host foraging behavior varies with the spatio-temporal abundance of a dominant ectoparasite

Prey engage in myriad behaviors to avoid predation, and these indirect effects of predators on their prey are often measured by the amount of food abandoned by a forager (the "giving-up density", or GUD) in a given habitat. Recent evidence suggests that hosts may engage in comparable behaviors to avoid exposure to parasites. We investigated changes in local foraging and regional space use by mammal hosts for the lone star tick (Amblyomma americanum), using GUDs as an indicator of the perceived risk of parasitism. At eight study sites at the Tyson Research Center (Eureka, MO), we placed two feeding trays, one on the ground and one at 1.5 m height in a tree, in order to assess how the emergence of ground-dwelling ticks affected foraging by several mammal species both locally (between the two GUD stations) and regionally (among the eight sites, mean distance 1064 m apart). Though GUDs did not differ between the ground and tree GUD stations, we did find that greater amounts of food were "given-up" at sites with higher abundances of ticks. This increase in food abandonment suggests that hosts respond to the risk of parasitism and alter their space use accordingly, potentially affecting a cascade of other ecological interactions across large spatial scales.     

The Lung Microbiome in Moderate and Severe Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by incompletely reversible airflow obstruction. Bacterial infection of the lower respiratory tract contributes to approximately 50% of COPD exacerbations. Even during periods of stable lung function, the lung harbors a community of bacteria, termed the microbiome. The role of the lung microbiome in the pathogenesis of COPD remains unknown. The COPD lung microbiome, like the healthy lung microbiome, appears to reflect microaspiration of oral microflora. Here we describe the COPD lung microbiome of 22 patients with Moderate or Severe COPD compared to 10 healthy control patients. The composition of the lung microbiomes was determined using 454 pyrosequencing of 16S rDNA found in bronchoalveolar lavage fluid. Sequences were analyzed using mothur, Ribosomal Database Project, Fast UniFrac, and Metastats. Our results showed a significant increase in microbial diversity with the development of COPD. The main phyla in all samples were Actinobacteria, Firmicutes, and Proteobacteria. Principal coordinate analyses demonstrated separation of control and COPD samples, but samples did not cluster based on disease severity. However, samples did cluster based on the use of inhaled corticosteroids and inhaled bronchodilators. Metastats analyses demonstrated an increased abundance of several oral bacteria in COPD samples.     

Roles of Eph receptors and ephrins in neural crest pathfinding

Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 μm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications for a functioning sense organ in an animal that is supposed to be blind. In this study, the organization and development of the dopaminergic system have been examined in the retinae of embryonic, larval, and adult I. kohtaoensis, by using an antiserum against tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, and an antiserum against dopamine itself. Labeled somata are situated in the inner nuclear layer and in the ganglion cell layer. Dopamine-positive fibers form a dense diffuse plexus, that covers the whole inner plexiform layer, whereas tyrosine hydroxylase-immunoreactive processes show a tendency to arborize in a stratified manner. Tyrosine-hydroxylase-immunolabeled fibers can occasionally be observed in the optic nerve head of larval stages. During ontogenesis and larval development, the distribution of transmitter-expressing cells changes and their number decreases, but no general degeneration of the visual system is detectable. Adult Ichthyophis still have retinal transmitters, indicating that the eyes, although obviously playing a minor role in a subterranean ecological niche, retain all the elements of functioning sense organs. Received: 20 November 1996 / Accepted: 23 February 1997     

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.     

Conserved features and evolutionary shifts of the EDA signaling pathway involved in vertebrate skin appendage development

It is widely accepted that evolutionary changes in conserved developmental signaling pathways play an important role in morphological evolution. However, few in silico studies were interested in tracking such changes in a signaling pathway. The Ectodysplasin (EDA) pathway provides an opportunity to fill this gap because it is involved in vertebrate skin appendage development such as scales, teeth, hair, and feathers that take an obvious part in the adaptation of species to their environment. We benefited from the large amount of genomic data now available to explore the evolution of the upstream genes of the EDA pathway. In mammals, these genes are eda (encoding 2 ligands, EDA-A1 and EDA-A2), edar (EDA-A1 receptor), edaradd (EDA receptor [EDAR] adapter), xedar (EDA-A2 receptor), and troy (a XEDAR-related receptor). We show that the evolution of EDA pathway genes combines both strongly conserved features and evolutionary shifts. These shifts are found at different signaling levels (from the ligand to intracellular signaling) and at different taxonomic levels (class, suborder, and genera). Although conserved features likely participate to the similarities found in the early development of vertebrate skin appendages, these shifts might account for innovations and specializations. Moreover, our study demonstrates that we can now benefit from the large number of sequenced vertebrate genomes to explore the evolution of specific signaling pathways and thereby to open new perspectives for developmental biology and evolutionary developmental biology.