Experiments inside a box lead to out-of-the-box ideas on cellular organization

Microtubules are biopolymers that assemble from tubulin dimers into hollow tubes and play an important role in cellular organization. Their fascinating properties and variety of functions, like for example chromosome segregation, sperm propagation and polarity establishment, have made them a popular subject of study. In this perspective I focus on the contribution of minimal in vitro systems to our understanding of microtubule organization within the physical confinement of a cell.     

Chloroplast transit peptide prediction: a peek inside the black box

Previous work in predicting protein localization to the chloroplast organelle in plants led to the development of an artificial neural network-based approach capable of remarkable accuracy in its prediction (ChloroP). A common criticism against such neural network models is that it is difficult to interpret the criteria that are used in making predictions. We address this concern with several new prediction methods that base predictions explicitly on the abundance of different amino acid types in the N-terminal region of the protein. Our successful prediction accuracy suggests that ChloroP uses little positional information in its decision-making; an unexpected result given the elaborate ChloroP input scheme. By removing positional information, our simpler methods allow us to identify those amino acids that are useful for successful prediction. The identification of important sequence features, such as amino acid content, is advantageous if one of the goals of localization predictors is to gain an understanding of the biological process of chloroplast localization. Our most accurate predictor combines principal component analysis and logistic regression. Web-based prediction using this method is available online at http://apicoplast.cis.upenn.edu/pclr/.     

Digging deep to open the white black box of snow root phenology

Snow roots are specialized structures recently discovered in the Caucasian alpine snow-bed plant Corydalis conorhiza. They form extensive networks that grow into snow packs against gravity, most probably to gather nitrogen from snow. Here we test the hypothesis that snow roots are true winter organs, i.e., they should already start growth early in winter to lay down the infrastructure for N capture from snow packs well before their melt-out. This would require winter surface and soil temperatures continuously close to or above freezing. Excavations of snow roots from snow packs in January and May, accompanied by temperature recordings and anatomical observations, supported our hypothesis. These findings complete the annual cycle of snow root phenology. They also emphasize the evolutionary and ecological significance of these specialized winter organs. Moreover, their likely association with a particular abiotic temperature and snow regime will facilitate the search for snow roots in other species.     

Self-interested partner selection can lead to the emergence of fairness

The theory of biological markets and competitive altruism contends that competitive partner selection is favorable to the selection of prosocial behaviors in social evolution. The current study provides an empirical assessment of this theory based on a laboratory experiment with human subjects using the Ultimatum game. The experimental results show that more generous proposers and more tolerant responders are preferred as partners. This indicates that subjects tend to choose partners in a manner that coincides with their own interests. In competitive partner selection, partner preferences driven by self-interest nevertheless generate an assortative pairing structure that prompts players to behave fairly in the game. The study shows that a free market of partner selection, plus the type of partner preferences driven by self-interests, can facilitate the emergence of fairness in social exchange.     

Alternate succession of steps can lead to the folding of a multidomain oligomeric protein

The beta 2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured forms of beta 2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of beta chains. We describe the kinetics of regain of a variety of physical, functional, and immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded beta 2. It is shown that whereas identical molecular events occur with the same kinetics, the two folding pathways are different, and involve different structural intermediates.     

Using skimmed milk agar to functionally screen a gut metagenomic library for proteases may lead to false positives

AIMS: Functional screens using skimmed-milk agar to obtain protease activity is a common approach. The aim of this study was to determine the efficacy of this screen to obtain protease activity from a metagenomic library. METHODS AND RESULTS: A distal gut metagenomic library was functionally screened using a skimmed-milk agar. The functional screen provided 231 clones generating the characteristic clear halo indicative of protease production. Clone analysis revealed that they were not protease-positive, but expressed glycosidic hydrolases and produced acid, which was responsible for the clear halos. CONCLUSIONS: The current skimmed-milk agar method to obtain proteases is not sufficiently robust to provide a definitive screen. Other- non-protease activities will also give the same clear halo and these would be interpreted as protease positive clones without further analysis. Hence a more robust buffered medium or a specific protein should be used. SIGNIFICANCE AND IMPACT OF THE STUDY: Functional screens are a powerful approach to obtaining enzymes from large metagenomic libraries and proteases are a particularly interesting target. The skimmed-milk agar is not sufficiently robust to ensure that only proteases are isolated and in order to save time and money this study has shown that better designed media can aid in the process.     

Gene expression profiling: opening the black box of plant ecosystem responses to global change

The use of genomic techniques to address ecological questions is emerging as the field of genomic ecology. Experimentation under environmentally realistic conditions to investigate the molecular response of plants to meaningful changes in growth conditions and ecological interactions is the defining feature of genomic ecology. Because the impact of global change factors on plant performance are mediated by direct effects at the molecular, biochemical, and physiological scales, gene expression analysis promises important advances in understanding factors that have previously been consigned to the 'black box' of unknown mechanism. Various tools and approaches are available for assessing gene expression in model and nonmodel species as part of global change biology studies. Each approach has its own unique advantages and constraints. A first generation of genomic ecology studies in managed ecosystems and mesocosms have provided a testbed for the approach and have begun to reveal how the experimental design and data analysis of gene expression studies can be tailored for use in an ecological context.     

Increased coupling between subpopulations in a spatially structured environment can lead to population outbreaks

Destruction and fragmentation of habitats is widely considered as a major threat to biological diversity. A theoretical framework aimed at understanding and predicting species responses to these destructive processes is still lacking, however. In this paper, the species dynamics in a spatially structured, two-habitat, patchy environment is considered subject to changes in individual migration intensity, i.e. coupling between the habitats. The subpopulation dynamics inside each habitat is assumed to be bistable but with different parameter values. By using space-discrete/continuous metapopulation dynamic models and computer simulations, we show that there can be two principally different regimes of metapopulation dynamics. With increasing intensity in the interplay between subpopulations, the total abundance can either gradually decrease or experience a sudden burst-like increase. This result is shown to be robust to the choice of mathematical models (discrete or continuous). Particularly, both the "self-excitation" and "self-inhibition" regimes of the metapopulation system are robust to variation in habitat size; however, when one of the habitats is much smaller than the other, the "self-excitation" regime can give way to the "self-inhibition" regime and vice versa.     

Simple assumptions on age composition lead to erroneous conclusions on the nature of density dependence in age-structured populations

Ecologists have debated the nature of density dependence in natural populations for decades, and efforts to detect density dependence from time series of abundance data have paralleled these debates. Yet due to the correlative nature of time series data, these undertakings have been statistically problematic. Most analyses of density dependence have focused on simple population models (i.e., non-overlapping generations), but in reality most vertebrates exhibit more complex life histories, and this complexity has been incorporated into population models in a variety of ways. Unfortunately, adding complexity to population models can further exacerbate efforts to detect density dependence. We examined the effect of adding age structure when inadequate data exist in support; to demonstrate this effect, we adopted Pacific salmon (Oncorhynchus spp.) as our study organism. Most salmon populations are semelparous and have variable age at maturity. Salmon populations (and many other fish species populations) are typically modeled in terms of numbers of recruits arising from spawners in a given brood year. Recruits are enumerated as they return as adults to spawn, and proper assignment of recruits to brood year requires age information. Unfortunately, while adult counts are common, detailed age information is not. A common practice is to apply long-term averages of age composition to returning adults to "reconstruct" time series of recruits. Here, by conducting simulations and analyzing data from natural populations, we demonstrated that this practice leads to a biased portrayal of density dependence by overestimating recruits from small spawning classes and underestimating recruits from large spawning classes. Also, productivity was overestimated and variance was underestimated, which could lead to overly optimistic predictions of extinction risk or overharvesting.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Mutual benefit can promote the evolution of preferential interactions and in this way can lead to the evolution of true altruism

We analyse the evolution of the assortment of encounters through active choice of companions among individuals that interact cooperatively in a situation of mutual benefit. Using a simple mathematical model, we show that mutual benefit can favour the evolution of a preference to interact with individuals that are similar to themselves with respect to an arbitrary tag even when both the preference and the tag depend on two independent and unlinked genes. Two necessary requisites to obtain this result are: (i) a small population or a large subdivided metapulation and (ii) an asymmetry between partners in such a way that one of them (donor) proposes the cooperation and elects the partner, whereas the other (receiver) never rejects the offer. We also show that mutual benefit can be the starting point for the evolution of altruistic behaviours as long as there are preferential interactions. This requires that the tag used in the election of partners is the altruistic or selfish behaviour itself.     

Foraging in a pathogen reservoir can lead to local host population extinction: a case study of a Lepidoptera-virus interaction

In 1990, natural infestations of the polyphagous vapourer moth, Orgyia antiqua (Lepidoptera: Lymantriidae) in lodgepole pine plantations in northern Scotland, were studied to ascertain the role of host foraging behaviour on the prevalence of nucleopolyhedrovirus (NPV; Baculoviridae) infection in the population. Aerial dispersal of early instar larvae (L1–L3) from the tree canopy onto heather foliage at the forest understorey, with subsequent relocation back onto the tree as late-instar larvae (L4–L6) appeared to play a significant role in the development of a widespread virus epizootic in which approximately 80% of L4–L6 individuals succumbed to disease. Bioassays of foliage 1 year later showed that the distribution of NPV followed a pronounced vertical gradient through the forest canopy culminating in high concentrations of virus in the forest understorey. Experimental systems comprising potted pine trees positioned above heather bases showed that NPV infections could be acquired by early stage larvae following dispersal from the tree and feeding on the undercanopy vegetation, then translocated to the tree component for secondary transmission to susceptible tree-feeding individuals. Behavioural studies indicated that the tendency for first-, second- and third-instar larvae to disperse to the understorey was probably not influenced by larval density on the tree but was strongly dependent on larval instar. In contrast, the tendency for larvae to relocate from the understorey heather to the tree was affected by both larval density and larval instar, suggesting that both these factors may significantly affect virus acquisition, translocation and transmission in the host population. In the present study, the heather understorey appeared to act as a pathogen reservoir in which virus could persist between host generations. Spatial heterogeneity in virus distribution combined with host foraging behaviour (dispersal and feeding) resulted in the pathogen playing a major role in host population dynamics over an extended time period (3 years). The reservoir theory is supported by the observation that similar dynamics were not observed in O. antiqua populations at neighbouring sites which lacked understorey food plants. Received: 8 June 1998 / Accepted: 5 October 1998