Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Characterization and modeling of monocyte-derived macrophages after spinal cord injury

Spinal cord injury (SCI) elicits a neuroinflammatory reaction dominated by microglia and monocyte-derived macrophages (MDM). Because MDM do not infiltrate the spinal cord until days after injury, it may be possible to control whether they differentiate into neuroprotective or neurotoxic effector cells. However, doing so will require better understanding of the factors controlling MDM differentiation and activation. Our goal was to develop an in vitro model of MDM that is relevant in the context of SCI. This tool would allow future studies to define mechanisms and intracellular signaling pathways that are associated with MDM-mediated neuroprotection or neurotoxicity. We first characterized SCI-induced cytokine expression in MDM using laser capture microdissection and real-time PCR. Based on this data, we assessed which easily procurable primary macrophage subset would mimic this phenotype in vitro. We established the baseline and inductive potential of resident peritoneal, thioglycollate-elicited peritoneal and bone marrow-derived macrophages (BMDM) at the molecular, cellular and functional level. Of these cells, only BMDM retained the phenotypic, molecular and functional characteristics of MDM that infiltrate the injured spinal cord. Thus, peripheral macrophages should not be used interchangeably in vitro to model the functional consequences of the MDM response elicited by SCI.     

Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103–Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103–Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.     

Happy Chickens Lay Tastier Eggs: Motivations for Buying Free-range Eggs in Australia

Recent public interest in so-called “ethical” food production, and in particular the welfare of intensively housed farm animals, has been linked to an increase in sales of free-range eggs in several countries including Australia. Animal activist groups around the world have campaigned for the abolition of caged-egg production, retailers and large food companies are now sourcing less of these products, and governments in various locales have placed restrictions on caged-egg production. In addition, the recent focus on food production and preparation in popular culture including books, films, and television has made these practices, including those associated with eggs, more transparent to mainstream audiences. Previous studies have examined consumers’ willingness-to-pay for free-range eggs, and community attitudes to animal welfare, but there has been little qualitative work that unpacks a key assumption which underlies much discussion of these issues: that free-range egg purchases are primarily or solely linked to consumers’ desires to have egg production systems changed from intensive to free-range. This paper analyses qualitative research undertaken in Australia that explores consumers’ motivations for buying free-range (or cage-free) eggs, which was part of a larger study examining ethical foods. Qualitative analysis of focus groups and interviews involving 73 participants revealed that free-range and cage-free eggs are perceived as being better quality, more nutritious, and safer, and having better sensory characteristics, than caged eggs. In response to open-ended questions, free-range and cage-free eggs were mentioned much more frequently than free-range meats, and were described as easy to identify and affordable, compared with other products with humane production claims. Several participants even had begun keeping their own hens in order to have an alternative to purchasing caged (or expensive free-range) eggs. Although caged-egg production was described by many participants as cruel, the desire to purchase free-range eggs was more often described in connection to efforts to avoid “industrialized” food than in relation to taking a stance on the issue of caged-hen welfare.     

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout

We have investigated the morphological effects of a genetic locus, Pgm1- t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.      

Re-thinking organisms: The impact of databases on model organism biology

Community databases have become crucial to the collection, ordering and retrieval of data gathered on model organisms, as well as to the ways in which these data are interpreted and used across a range of research contexts. This paper analyses the impact of community databases on research practices in model organism biology by focusing on the history and current use of four community databases: FlyBase, Mouse Genome Informatics, WormBase and The Arabidopsis Information Resource. We discuss the standards used by the curators of these databases for what counts as reliable evidence, acceptable terminology, appropriate experimental set-ups and adequate materials (e.g., specimens). On the one hand, these choices are informed by the collaborative research ethos characterising most model organism communities. On the other hand, the deployment of these standards in databases reinforces this ethos and gives it concrete and precise instantiations by shaping the skills, practices, values and background knowledge required of the database users. We conclude that the increasing reliance on community databases as vehicles to circulate data is having a major impact on how researchers conduct and communicate their research, which affects how they understand the biology of model organisms and its relation to the biology of other species.     

Regulation of hESC Research in Australia: Promises and Pitfalls for Deliberative Democratic Approaches

This paper considers the legislative debates in Australia that led to the passage of the Research Involving Human Embryos Act (Cth 2002) and the Prohibition of Human Cloning Act (Cth 2002). In the first part of the paper, we discuss the debate surrounding the legislation with particular emphasis on the ways in which demands for public consultation, public debate and the education of Australians about the potential ethical and scientific impact of human embryonic stem cells (hESC) research were deployed, and the explicit and implicit framing of the scope of public consultation. We then ask whether, given the calls for public consultations, debate and understanding, current work in democratic theory could be helpful in analysing the process of policy-making in these areas. In particular, we canvass the literature relating to aggregative and deliberative models of democracy for processes that support the legitimacy of policy. We identify features of the debate that reflect the appeal of deliberative approaches as well as some of the possible hurdles or limitations to developing deliberative democratic approaches to policy in ethically contentious areas.     

Not just about “the science”: science education and attitudes to genetically modified foods among women in Australia

Previous studies investigating attitudes to genetically modified (GM) foods suggest a correlation between negative attitudes and low levels of science education, both of which are associated with women. In a qualitative focus group study of Australian women with diverse levels of education, we found attitudes to GM foods were part of a complex process of making “good” food decisions, which included other factors such as locally produced, fresh/natural, healthy and nutritious, and convenient. Women involved in GM crop development and those with health science training differed in how they used evidence to categorize GM foods. Our findings contribute to a deeper understanding of how GM food, and the role of science and technology in food production and consumption more broadly, is understood and discussed amongst diverse “publics” and across different “sciences,” and to research related to deepening public engagement at the intersection of science and values.     

The Scope of Public Discourse Surrounding Proposition 71: Looking Beyond the Moral Status of the Embryo

Human embryonic stem cell research has generated considerable discussion and debate in bioethics. Bioethical discourse tends to focus on the moral status of the embryo as the central issue, however, and it is unclear how much this reflects broader community values and beliefs related to stem cell research. This paper presents the results of a study which aims to identify and classify the issues and arguments that have arisen in public discourse associated with one prominent policy episode in the United States: the 2004 Californian Stem Cell Research and Cures Initiative (also known as Proposition 71). The findings show that public discourse about Proposition 71 is characterised by a broader range of issues than those usually addressed in scholarly publications and public policy documents. While attention to the moral status of the embryo is an important issue in stem cell research, making it the main focus of public discourse has a polarising effect. This also limits opportunities to identify shared values, understand how political alliances are forged, and develop social consensus. Implications for future research and policy are discussed.