Tissue section AFM: In situ ultrastructural imaging of native biomolecules

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.     

Social Eavesdropping: A Game-Theoretic Analysis

Here we extend the classic Hawk-Dove model of animal conflict to allow for continuous variation in fighting strengths. Whereas the winner of a fight is chosen at random in the discrete game, in our continuous game, the winner of any fight is the stronger individual, and costs are higher for more evenly matched opponents. We identify the evolutionary stable strength threshold beyond which an animal should be prepared to engage in aggressive behaviour and show that this threshold increases with variance in fighting strength when the costs of aggression are insensitive to the level of strength asymmetry, but decreases with variance when the costs are sensitive to the level of asymmetry. In contrast to the classic discrete game, population-wide aggressive behaviour occurs only when the costs of fighting are zero. It is now known that animals can eavesdrop on the outcome of contests between neighbours and modify their behaviour towards observed winners and losers. We therefore further extend our model to allow for social eavesdropping within networks comprising three individuals. Whereas earlier work showed that eavesdropping increases the frequency of mutually aggressive contests in the discrete game by enhancing the value of victory, here we show that aggression thresholds in the continuous game are always higher with eavesdropping than without it: for sufficiently weak animals, avoiding the costs of challenging an observed winner over-rides the potential benefit of winning, so that eavesdropping reduces the frequency of aggressive encounters. Thus, even though strength is not directly observable, information is extracted from the variation in fighting ability that the classic Hawk-Dove game ignores.     

Ontogenetic shift in diet of a large elapid snake is facilitated by allometric change in skull morphology

Evolutionary Ecology - As snakes are limbless, gape-limited predators, their skull is the main feeding structure involved in prey handling, manipulation and feeding. Ontogenetic changes in prey...     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Why are defensive toxins so variable? An evolutionary perspective

Defensive toxins are widely used by animals, plants and micro-organisms to deter natural enemies. An important characteristic of such defences is diversity both in the quantity of toxins and the profile of specific defensive chemicals present. Here we evaluate evolutionary and ecological explanations for the persistence of toxin diversity within prey populations, drawing together a range of explanations from the literature, and adding new hypotheses. We consider toxin diversity in three ways: (1) the absence of toxicity in a proportion of individuals in an otherwise toxic prey population (automimicry); (2) broad variation in quantities of toxin within individuals in the same population; (3) variation in the chemical constituents of chemical defence. For each of these phenomena we identify alternative evolutionary explanations for the persistence of variation. One important general explanation is diversifying (frequency- or density-dependent) selection in which either costs of toxicity increase or their benefits decrease with increases in the absolute or relative abundance of toxicity in a prey population. A second major class of explanation is that variation in toxicity profiles is itself nonadaptive. One application of this explanation requires that predator behaviour is not affected by variation in levels or profiles of chemical defence within a prey population, and that there are no cost differences between different quantities or forms of toxins found within a population. Finally, the ecology and life history of the animal may enable some general predictions about toxin variation. For example, in animals which only gain their toxins in their immature forms (e.g. caterpillars on host plants) we may expect a decline in toxicity during adult life (or at least no change). By contrast, when toxins are also acquired during the adult form, we may for example expect the converse, in which young adults have less time to acquire toxicity than older adults. One major conclusion that we draw is that there are good reasons to consider within-species variation in defensive toxins as more than mere ecological noise. Rather there are a number of compelling evolutionary hypotheses which can explain and predict variation in prey toxicity.     

An Analysis of Vegetation Stripe Formation in Semi-Arid Landscapes

Vegetation stripes (“tiger bush”) are a characteristic feature of semi-arid environments. The stripes typically lie along the contours of gentle slopes, and some authors report a gradual uphill migration. A previous mathematical model (Klausmeier, Science, 284:1826, 1999) has shown that this phenomenon can be explained relatively simply by the downhill flow of rainwater coupled with the diffusive spread of the plant population. This paper presents a detailed analysis of pattern formation in the Klausmeier model. The author derives formulae for the wavelength and migration speed of the predicted patterns, and systematically investigates how these depend on model parameters. The results make new predictions and suggest possible approaches to testing the model.     

Hiding in plain sight

Although the principles of disruptive colouration are widely believed to explain a variety of animal colour patterns, there has been no field evidence that it works to reduce the detection rates of natural prey. In a recent paper, Cuthill et al. successfully address this shortfall, separating the benefits of background matching from those of disruptive colouration. Their results provide the first definitive field support for this long-recognized phenomenon and suggest several new avenues of research.     

Do unprofitable prey evolve traits that profitable prey find difficult to exploit?

Prey that are unprofitable to attack (for example, those containing noxious chemicals) are often conspicuously patterned and move in a slower and more predictable manner than species lacking these defences. Contemporary theories suggest these traits have evolved as warning signals because they can facilitate both associative and discriminative avoidance learning in predators. However, it is unclear why these particular traits and not others have tended to evolve in unprofitable prey. Here we show using a signal detection model that unprofitable prey will evolve conspicuous colours and patterns partly because these characteristics cannot readily evolve in profitable prey without close mimicry. The stability of this signal is maintained through the costs of dishonesty in profitable prey. Indeed, unprofitable prey will sometimes evolve a conspicuous form to reduce mimetic parasitism, even in the unlikely event that this form can be more closely mimicked. This is one of the first mathematical models of the evolution of warning signals to allow for the possibility of mimicry, yet our analyses suggest it may offer a general explanation as to why warning signals take the form that they do. Warning signals and mimicry may therefore be more closely related than is currently supposed.