Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Attaching-effacing bacteria in animals

Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man. In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens. In other host species, AE bacteria are of less certain significance. With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer. The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion. The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the "Locus for Enterocyte Effacement". It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some A-EEC strains. Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria. This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated. Reference is made to human pathogens where appropriate. The focus is mainly on natural colonization and disease, but complementary experimental data are also included. (C) 2004 Elsevier Ltd. All rights reserved.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Theology and politics in Richard Hooker's thought

Although Richard Hooker’s private attitudes were clericalist and authoritarian, his constitutional theory subordinated clergymen to laymen and monarchy to parliamentary statute. This article explains why his political ideas were nonetheless appropriate to his presumed religious purposes. It notes a very intimate connection between his teleological conception of a law and his hostility towards conventional high Calvinist ideas about predestination. The most significant anomaly within his broadly Aristotelian world-view was his belief that politics is nothing but a means to cope with sin. This too can be linked to his religious ends, but it creates an ambiguity that made his doctrines usable by Locke.

‘Did Ronald McDonald also tend to scare you as a child?’: working to emplace consumption, commodities, and citizen-students in a large classroom setting

So-called ‘radical’ and ‘critical’ pedagogy seems to be everywhere these days on the landscapes of geographical teaching praxis and theory. Part of the remit of radical/critical pedagogy involves a de-centring of the traditional ‘banking’ method of pedagogical praxis. Yet, how do we challenge this ‘banking’ model of knowledge transmission in both a large-class setting and around the topic of commodity geographies where the banking model of information transfer still holds sway? This paper presents a theoretically and pedagogically driven argument, as well as a series of practical teaching ‘techniques’ and tools—mind-mapping and group work—designed to promote ‘deep learning’ and a progressive political potential in a first-year large-scale geography course centred around lectures on the Geographies of Consumption and Material Culture. Here students are not only asked to place themselves within and without the academic materials and other media but are urged to make intimate connections between themselves and their own consumptive acts and the commodity networks in which they are enmeshed. Thus, perhaps pedagogy needs to be emplaced firmly within the realms of research practice rather than as simply the transference of research findings.

Chaos of Wolbachia Sequences Inside the Compact Fig Syconia of Ficus benjamina (Ficus: Moraceae)

Figs and fig wasps form a peculiar closed community in which the Ficus tree provides a compact syconium (inflorescence) habitat for the lives of a complex assemblage of Chalcidoid insects. These diverse fig wasp species have intimate ecological relationships within the closed world of the fig syconia. Previous surveys of Wolbachia, maternally inherited endosymbiotic bacteria that infect vast numbers of arthropod hosts, showed that fig wasps have some of the highest known incidences of Wolbachia amongst all insects. We ask whether the evolutionary patterns of Wolbachia sequences in this closed syconium community are different from those in the outside world. In the present study, we sampled all 17 fig wasp species living on Ficus benjamina, covering 4 families, 6 subfamilies, and 8 genera of wasps. We made a thorough survey of Wolbachia infection patterns and studied evolutionary patterns in wsp (Wolbachia Surface Protein) sequences. We find evidence for high infection incidences, frequent recombination between Wolbachia strains, and considerable horizontal transfer, suggesting rapid evolution of Wolbachia sequences within the syconium community. Though the fig wasps have relatively limited contact with outside world, Wolbachia may be introduced to the syconium community via horizontal transmission by fig wasps species that have winged males and visit the syconia earlier.

The look: style, technology and televisuality in the New Who

This chapter explores the distinctive qualities of the Matt Smith era Doctor Who, focusing on how dramatic emphases are connected with emphases on visual style, and how this depends on the programme's production methods and technologies. Doctor Who was first made in the 1960s era of live, studio-based, multi-camera television with monochrome pictures. However, as technical innovations like colour filming, stereo sound, CGI and post-production effects technology have been routinely introduced into the programme, and now High Definition (HD) cameras, they have given Doctor Who’s creators new ways of making visually distinctive narratives. Indeed, it has been argued that since the 1980s television drama has become increasingly like cinema in its production methods and aesthetic aims. Viewers’ ability to view the programme on high-specification TV sets, and to record and repeat episodes using digital media, also encourage attention to visual style in television as much as in cinema. The chapter evaluates how these new circumstances affect what Doctor Who has become and engages with arguments that visual style has been allowed to override characterisation and story in the current Doctor Who. The chapter refers to specific episodes, and frames the analysis with reference to earlier years in Doctor Who’s long history. For example, visual spectacle using green-screen and CGI can function as a set-piece (at the opening or ending of an episode) but can also work ‘invisibly’ to render a setting realistically. Shooting on location using HD cameras provides a rich and detailed image texture, but also highlights mistakes and especially problems of lighting. The reduction of Doctor Who’s budget has led to Steven Moffat’s episodes relying less on visual extravagance, connecting back both to Russell T. Davies’s concern to show off the BBC’s investment in the series but also to reference British traditions of gritty and intimate social drama. Pressures to capitalise on Doctor Who as a branded product are the final aspect of the chapter’s analysis, where the role of Moffat as ‘showrunner’ links him to an American (not British) style of television production where the preservation of format and brand values give him unusual power over the look of the series.

Experimentally driven atomistic model of 1,2 Polybutadiene

We present an efficient method of combining wide angle neutron scattering data with detailed atomistic models, allowing us to perform a quantitative and qualitative mapping of the organisation of the chain conformation in both glass and liquid phases. The structural refinement method presented in this work is based on the exploitation of the intrachain features of the diffraction pattern and its intimate linkage with atomistic models by the use of internal coordinates for bond lengths, valence angles and torsion rotations. Atomic connectivity is defined through these coordinates that are in turn assigned by pre-defined probability distributions, thus allowing for the models in question to be built stochastically. Incremental variation of these coordinates allows for the construction of models that minimise the differences between the observed and calculated structure factors. We present a series of neutron scattering data of 1,2 polybutadiene at the region 120-400K. Analysis of the experimental data yield bond lengths for C-C and C=C of 1.54Å and 1.35Å respectively. Valence angles of the backbone were found to be at 112° and the torsion distributions are characterised by five rotational states, a three-fold trans-skew± for the backbone and gauche± for the vinyl group. Rotational states of the vinyl group were found to be equally populated, indicating a largely atactic chan. The two backbone torsion angles exhibit different behaviour with respect to temperature of their trans population, with one of them adopting an almost all trans sequence. Consequently the resulting configuration leads to a rather persistent chain, something indicated by the value of the characteristic ratio extrapolated from the model. We compare our results with theoretical predictions, computer simulations, RIS models and previously reported experimental results.

The timing and societal synchronisation of energy demand

It is increasingly important to know about when energy is used in the home, at work and on the move. Issues of time and timing have not featured strongly in energy policy analysis and in modelling, much of which has focused on estimating and reducing total average annual demand per capita. If smarter ways of balancing supply and demand are to take hold, and if we are to make better use of decarbonised forms of supply, it is essential to understand and intervene in patterns of societal synchronisation. This calls for detailed knowledge of when, and on what occasions many people engage in the same activities at the same time, of how such patterns are changing, and of how might they be shaped. In addition, the impact of smart meters and controls partly depends on whether there is, in fact scope for shifting the timing of what people do, and for changing the rhythm of the day. Is the scheduling of daily life an arena that policy can influence, and if so how? The DEMAND Centre has been linking time use, energy consumption and travel diary data as a means of addressing these questions and in this working paper we present some of the issues and results arising from that exercise.

Visceral behaviours and leadership: a dark side of boardroom life?

Purpose – Characteristics of leaders whose behaviour is visceral include taking action based on instinct rather than intellect and exhibiting coarse, base and often negative emotions. Despite the challenge of precisely defining the nature of visceral behaviour, the purpose of this paper is to provide insight into this less attractive side of boardroom life. Design/methodology/approach – Following a literature review of the research into the negative behaviour leaders exhibit, the paper highlights four forms of visceral behaviour based on focused and intimate qualitative case studies involving the experiences of those on the receiving end of that behaviour within a boardroom context. Findings – Based on interviews with an international sample of five chief executive officers (CEOs), plus three subordinates with substantial profit and loss responsibility, the study reveals a distinctly human experience from which no one is exempt. The idiosyncratic nature of the visceral behaviour experienced resulted in each study participant's unique experience. The authors conclude that leaders need to adopt specific measures in order to control and reduce the darker human tendencies. Research limitations/implications – The experiences of study participants are presented in four case studies, providing insight into their experiences whilst also protecting their identity. The study participants were drawn from a sample of companies operating globally within a single sector of the manufacturing industry. The concepts the authors present require validating in other organisations with different demographic profiles. Originality/value – The paper presents a model based on two dimensions – choice and level of mastery – that provides the reader with insight into the forms of visceral behaviour to which leaders succumb. Insight enables us to offer managers strategic suggestions to guard against visceral behaviour and assist them in mitigating its worst aspects, in both those with whom they work and themselves.

The absent body: representations of dying early modern women in a selection of seventeenth-century diaries

This article seeks to explore the absence of the body in the depiction of dying women in a selection of seventeenth-century diaries. It considers the cultural forces that made this absence inevitable, and the means by which the physical body was replaced in death by a spiritual presence. The elevation of a dying woman from physical carer to spiritual nurturer in the days before death ensured that gender codes were not broken. The centrality of the body of the dying woman, within a female circle of care and support, was paradoxically juxtaposed with an effacement of the body in descriptions of a good death. In death, a woman might achieve the stillness, silence and compliance so essential to perfect early modern womanhood, and retrospective diary entries can achieve this ideal by replacing the body with images that deflect from the essential physicality of the woman.

Molecular species delimitation of a symbiotic fig-pollinating wasp species complex reveals extreme deviation from reciprocal partner specificity

Background: Symbiotic relationships have contributed to major evolutionary innovations, the maintenance of fundamental ecosystem functions, and the generation and maintenance of biodiversity. However, the exact nature of host/symbiont associations, which has important consequences for their dynamics, is often poorly known due to limited understanding of symbiont taxonomy and species diversity. Among classical symbioses, figs and their pollinating wasps constitute a highly diverse keystone resource in tropical forest and savannah environments. Historically, they were considered to exemplify extreme reciprocal partner specificity (one-to-one host-symbiont species relationships), but recent work has revealed several more complex cases. However, there is a striking lack of studies with the specific aims of assessing symbiont diversity and how this varies across the geographic range of the host. Results: Here, we use molecular methods to investigate cryptic diversity in the pollinating wasps of a widespread Australian fig species. Standard barcoding genes and methods were not conclusive, but incorporation of phylogenetic analyses and a recently developed nuclear barcoding gene (ITS2), gave strong support for five pollinator species. Each pollinator species was most common in a different geographic region, emphasising the importance of wide geographic sampling to uncover diversity, and the scope for divergence in coevolutionary trajectories across the host plant range. In addition, most regions had multiple coexisting pollinators, raising the question of how they coexist in apparently similar or identical resource niches. Conclusion: Our study offers a striking example of extreme deviation from reciprocal partner specificity over the full geographical range of a fig-wasp system. It also suggests that superficially identical species may be able to co-exist in a mutualistic setting albeit at different frequencies in relation to their fig host’s range. We show that comprehensive sampling and molecular taxonomic techniques may be required to uncover the true structure of cryptic biodiversity underpinning intimate ecological interactions.

Effects of allometry, productivity and lifestyle on rates and limits of body size evolution

Body size affects nearly all aspects of organismal biology, so it is important to understand the constraints and dynamics of body size evolution. Despite empirical work on the macroevolution and macroecology of minimum and maximum size, there is little general quantitative theory on rates and limits of body size evolution. We present a general theory that integrates individual productivity, the lifestyle component of the slow–fast life-history continuum, and the allometric scaling of generation time to predict a clade's evolutionary rate and asymptotic maximum body size, and the shape of macroevolutionary trajectories during diversifying phases of size evolution. We evaluate this theory using data on the evolution of clade maximum body sizes in mammals during the Cenozoic. As predicted, clade evolutionary rates and asymptotic maximum sizes are larger in more productive clades (e.g. baleen whales), which represent the fast end of the slow–fast lifestyle continuum, and smaller in less productive clades (e.g. primates). The allometric scaling exponent for generation time fundamentally alters the shape of evolutionary trajectories, so allometric effects should be accounted for in models of phenotypic evolution and interpretations of macroevolutionary body size patterns. This work highlights the intimate interplay between the macroecological and macroevolutionary dynamics underlying the generation and maintenance of morphological diversity.

Leveraging everyday technology for people living with dementia: a case study

– The purpose of this paper is to present the self-described “journey” of a person with dementia (Brian; author 3) in his re-learning of old technologies and learning of new ones and the impact this had on his life. Design/methodology/approach – This is a single case study detailing the participant's experiences collaborating with a researcher to co-create methods of facilitating this learning process, which he documented in the form of an online blog and diary entries. These were analysed using NVivo to reveal the key themes. Findings – Brian was able to relearn previously used technologies and learn two new ones. This lead to an overarching theme of positive outlook on life supported by person-centredness, identity and technology, which challenged negative perceptions about dementia. Research limitations/implications – The paper provides an example of how learning and technology improved the life of one person with dementia. By sharing the approach the authors hope to encourage others to embrace the challenge of designing and developing innovative solutions for people with a dementia diagnosis by leveraging both current mainstream technology and creating novel bespoke interventions for dementia. Originality/value – The personal perspective of a person with dementia and his experiences of (re-) learning provide a unique insight into the impact of technology on his life.