Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

The importance of human FcyRI in mediating protection to malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria.

Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling

BACKGROUND/AIMS: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. METHODS: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. RESULTS: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. CONCLUSION: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

Identification of hypothalamic genes implicated in the development of obesity in Psammomys obesus using differential display PCR

The hypothalamus is a key central controller of energy homeostasis and is the source and/or site of action of many neuropeptides involved in this process. The aim of this study was to isolate hypothalamic genes differentially expressed between lean and obese Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. Differential display PCR was used to compare hypothalamic gene expression profiles of lean and healthy, obese and hyperinsulinemic, and obese, diabetic P. obesus in both the fed and fasted states. We conducted differential display with 180 separate primer combinations to amplify approximately 9000 expressed transcripts. Sixty differentially expressed bands were excised. Taqman PCR was performed on 36 of these transcripts to confirm differential gene expression in a larger sample population. Of these 36 transcripts, 9 showed homology to known genes, and 27 were considered to be novel sequences. Gene expression profiles for two of these genes are presented here. In conclusion, differential display PCR was successfully used to isolate several transcripts that may be involved in the central regulation of energy balance. We are currently conducting numerous studies to further investigate the role of these genes in the development of obesity in P. obesus.

It's not only rock and roll: rockumentary, direct cinema and performative display

The documentary form commonly referred to as rockumentary has become, since its inception in the early 1960s, a staple of American direct cinema. In keeping with its associations with observational direct cinema, rockumentary emphasizes showing over telling; that is, rockumentary privileges the visual capacities of documentary over patterns of exposition. While the ‘documentary display’ of rockumentary is comparable to certain features of  the early ‘cinema of attractions’ it exceeds such features in its focus on  performance. Typically, an emphasis within documentary theory on unmediated and unreconstructed access to the real as the basis of documentary film has not admitted a place for notions of performance before the camera. Rockumentary, with its relentless foregrounding of the performing body and the performance of musicians, revises this  understanding. This essay examines rockumentary within the context of direct cinema as a mode centred on a documentary performative display as it operates within selected works from the 1960s to the present. The film theorist Brian Winston has claimed that ‘[d]irect cinema made the rock performance/tour movie into the most popular and commercially viable documentary form thus far.’ The inverse of this assessment may be closer to the mark: the rockumentary turned direct cinema into a commercially and widely available form, one which the rockumentary has at times returned to and superseded in its scopic attention to performative display.

Cultured muscle cells display defects of mitochondrial myopathy ameliorated by anti-oxidants

The mitochondrial DNA A3243G mutation causes neuromuscular disease. To investigate the muscle-specific pathophysiology of mitochondrial disease, rhabdomyosarcoma transmitochondrial hybrid cells (cybrids) were generated that retain the capacity to differentiate to myotubes. In some cases, striated muscle-like fibres were formed after innervation with rat embryonic spinal cord. Myotubes carrying A3243G mtDNA produced more reactive oxygen species than controls, and had altered glutathione homeostasis. Moreover, A3243G mutant myotubes showed evidence of abnormal mitochondrial distribution, which was associated with down-regulation of three genes involved in mitochondrial morphology, Mfn1, Mfn2 and DRP1. Electron microscopy revealed mitochondria with ultrastructural abnormalities and paracrystalline inclusions. All these features were ameliorated by anti-oxidant treatment, with the exception of the paracrystalline inclusions. These data suggest that rhabdomyosarcoma cybrids are a valid cellular model for studying muscle-specific features of mitochondrial disease and that excess reactive oxygen species production is a significant contributor to mitochondrial dysfunction, which is amenable to anti-oxidant therapy.

Poetics of the uncanny : Jean Painlevé, documentary display, and documentary theory

In Grierson's theory of documentary, films of the so-called cinema of attractions were relegated to the position of 'lower forms' in contrast to his description of documentary as a form of civic pedagogy. The modes and forms of a cinema of attractions, which form what is here referred to as the practice of documentary display, did not disappear with the ascendency of Griersonian documentary; instead such approaches continued to inform avant-garde film. This paper examines the connections between nonfiction documentations and avant-garde film- and the resultant implications of this meeting for documentary film theory- through an analysis of the film and critical writings of Jean Painlevé (1902-1989). Painlevé, whom, according to Bazin, 'occupies a singular and privileged place in French cinema', informed his understandings of documentary through contact with, among others, Buñuel, Man Ray, Bataille, Eisenstein, and Grierson. The analysis of Painlevé's surrealist documentary display and his extensive body of critical literature on documentary foregrounds overlooked documentary works and constructs, or resurrects, ignored perspectives on documentary film as a way of extending and revising documentary film theory and the documentary canon.

From city symphony to global city film : documentary display and the corporeal

In the introduction to his history of the relationship between the body and the city in Western civilisation, Richard Sennett includes an anecdote about attending a cinema in New York. Sennett uses the story of watching film as a way of commenting on the place of the body and senses within urban settings and is concerned to document 'physical sensations in urban space' as a way of addressing what he sees as the 'tactile sterility which afflicts the urban environment.'[1] While Sennett's work performs an important task by drawing attention to various historical conditions implicated in urban and metropolitan experience, it is possible to rework the categories he deploys - bodies, the city, and film - into a very different argument concerning representations of the city. Indeed the three categories coalesce in the so-called city film - works which include the 'city symphony' of the 1920s and subsequent documentary representations of urban spaces, among them the New York City films of the 1940s and 1950s, and films of non-Western cities produced in the decades from the 1960s to the present - within which the city is realised through a focus on people.

Cinema of (coming) attractions : Bruce Conner's a movie and documentary display

Bruce Conner's film 'Movie' (1958) constructs an argument concerning popular media and history within and through a radical juxtaposition of images to release new and productive meanings. It also comments on the media's representation of history as violence and analyses the consequences of aggression and violence.

Documentary display : re-viewing nonfiction film and video

Not all documentary films and videos are sober depictions of the real world. Documentary representations can present expressive, entertaining and spectacular images. This book examines such innovative approaches as they occur within the process of 'documentary display' - a practice which emphasises the visual attractions of documentary representation. Works of documentary display explore modes of exhibitionistic 'showing' in which sensation is frequently the vehicle of cognition and knowledge. Such a display is analysed within the popular and prominent forms of found-footage film, 'rockumentary', the city film, nonfiction surf film and video, and certain views of natural science topics. This accessible and informed study - with its focus on entertaining, popular, spectacular and sensational froms of nonfiction representation - makes an important contribution to theoretical analyses of documentary film and video

The impact of computer display height and desk design on 3D posture during information technology work by young adults

Computer display height and desk design to allow forearm support are two critical design features of workstations for information technology tasks. However there is currently no 3D description of head and neck posture with different computer display heights and no direct comparison to paper based information technology tasks. There is also inconsistent evidence on the effect of forearm support on posture and no evidence on whether these features interact. This study compared the 3D head, neck and upper limb postures of 18 male and 18 female young adults whilst working with different display and desk design conditions. There was no substantial interaction between display height and desk design. Lower display heights increased head and neck flexion with more spinal asymmetry when working with paper. The curved desk, designed to provide forearm support, increased scapula elevation/protraction and shoulder flexion/abduction.

2. Scanning and processing 3D objects for web display

We present a case study of scanning 3D objects for the purposes of education and public information. We begin by describing the original design of a 3D scanning system now in use in Cairo’s Egyptian Museum. The system captures both the geometry and surface color and detail of museum artifacts. We report on the experience using the system in the museum setting, and how practical problems with the system were addressed. We present samples of how the processed 3D data will be used on a web site designed to communicate Egyptian culture.We present a case study of scanning 3D objects for the purposes of education and public information. We begin by describing the original design of a 3D scanning system now in use in Cairo’s Egyptian Museum. The system captures both the geometry and surface color and detail of museum artifacts. We report on the experience using the system in the museum setting, and how practical problems with the system were addressed. We present samples of how the processed 3D data will be used on a web site designed to communicate Egyptian culture.