Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea.

Aureins are a family of peptides (13-25 residues), some of which possess potent antimicrobial and anti-cancer properties, which have been classified into 5 subgroups based upon primary structural similarities. They were originally isolated from the defensive skin secretions of the closely related Australian bell frogs, Litoria aurea and Litoria raniformis, and of the 23 aurein peptides identified, 10 are common to both species. Using a recently developed technique, we have constructed a cDNA library from the defensive secretion of the green and golden bell frog, L. aurea, and successfully cloned a range of aurein precursor transcripts containing entire open-reading frames. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of aurein. The deduced precursor structures for the most active aureins (2.2 and 3.1) confirmed the presence of a C-terminal amidation motif whereas that of aurein 5.3 did not. Processed peptides corresponding in molecular mass to aureins 2.2, 2.3, 2.5, 3.1 and 5.3 were identified in the same secretion sample using LC/MS. The application of this technique thus permits parallel peptidomic and transcriptomic analyses on the same lyophilized skin secretion sample circumventing sacrifice of specimens of endangered herpetofauna.

An Unkind Cut: Circumcision, Death and the Impact of Irish Health Policy on Asylum Seekers

Healthcare and the wider social determinants of health are the keystone of a number of complex progressive social justice issues that evoke complex emotions. As the demography of Ireland rapidly changes, the practices and expectations of some asylum seekers presents new opportunities for the providers of health service provision and reform. This paper looks at some of the emotions evoked in health care issues and draws on observations and interviews from empirical fieldwork carried out for the Health Research Board. The research was conducted both in the Adelaide and Meath Hospital, incorporating the National Children’s Hospital, Tallaght and in a number of refugee reception centres in Ireland. At one level honouring faith choices within a healthcare setting is a societal acknowledgement made to people at their most vulnerable, that the potent and cathartic transformative rituals they value are significant in mediating and managing their emotions - at another level, it is a practical and a symbolic communication of a statutory commitment to inter-culturalism and community cohesion..

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

Translating ‘gaytown’: the collision of global and local in bringing Australian queer play Bison to Belfast.

Sydney playwright Lachlan Philpott’s Bison (2000/2009) is immersed in a sweaty, summery Antipodean scene of bronzed and toned bodies. It is located in the flora and fauna of gum trees and biting ants. Yet, despite this, it could be argued that at its heart it is not a specifically Australian site, but an all-too translatable scene that seems to be played out in gay clubs, bars, chatrooms and saunas around the Western world: men repeating patterns, looking for sex or love; checking out bodies, craving perfection; avoiding, and occasionally seeking, disease. At least, that was my assumption when I decided to direct the play in Belfast, Northern Ireland, in 2009. Philpott came to Belfast to workshop the play with the actors and, as a group, we restructured the play and tried to find a way to ‘de-Australianise’ it without necessarily placing it in a new geographical place - Northern Ireland - through linguistic clues in the text. As Philpott put it: ‘Let’s not make this play about Belfast or Sydney or London or anywhere because it is not a fair reflection of these scenes. Maybe we should just identify the generic elements of this world and then make Bison a play that reflects gaytown – because the rituals are all the same in Western society’. The experience of doing the play in Belfast made clear, however, that ideas of a global gay identity/experience –though highly marketed – fail to account for the vastly different situations of embodied gay experience. And the Northern Irish gay experience, while it has imported the usual ‘generic’ tropes of gayness, sits within a specific cultural context in which the farsighted legislation on equality for gays (imposed by either London or the EU) vastly outstrips wider societal thinking. For many in Northern Ireland, erstwhile MP Iris Robinson’s comments about homosexuality being an ‘abomination’ were a reason to support her, rather than to reject her. For me, the comments were the catalyst to doing Bison in Belfast.

Music, Magic, and the Mythic: The Dynamics of Visual and Aural Discourse in Souleymane Cissé’s Yeelen

The 1980s saw a wave of African films that aimed to represent, on both local and international screens, a sophisticated pre-colonial Africa, thus debunking notions of the continent as primitive. Toward this aim the films inscribed the conventions of oral performance within their visual styles, denying spectator identification with the protagonists and emphasising the presence of the narrator. However, some critics argued that these films exoticised Africa, while their use of oral performance’s distancing effect echoed the ‘scientific’ distance structured by the ethnographic film, in which African societies were represented as ‘the other’. Souleymane Cissé’s Yeelen exemplifies this tension, transposing into cinematic form oral storytelling techniques in the depiction of a power struggle within the covert cult of the komo, a Bambara initiation society unfamiliar to most non-Bambara viewers. This paper demonstrates how the film negotiates this tension via music, which interpellates the international spectator by eliciting a greater identification with the protagonists than that determined at a visual level, while encoding a verisimilitude to rituals that may otherwise be read as the superstitious practices of ‘the other’. In this way, music and image in Yeelen operate as parallel, though often overlapping, discourses, bridging the gap between the film’s culturally specific narrative and formal components, and its international spectators.

Limnonectins: A new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog – the Fujian large-headed frog, Limnonectes fujianensis – and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 µM) and are devoid of haemolytic activity at concentrations up to 160 µM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data

pcDNA3.1tdTomato is superior to pDsRed2-N1 for optical fluorescence imaging in the F344/AY-27 rat model of bladder cancer

PURPOSE: Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment.

MATERIALS AND METHODS: AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems.

RESULTS: AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells.

CONCLUSIONS: In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.

Prediction-Based Power-Performance Adaptation of Multithreaded Scientific Codes

Computing has recently reached an inflection point with the introduction of multicore processors. On-chip thread-level parallelism is doubling approximately every other year. Concurrency lends itself naturally to allowing a program to trade performance for power savings by regulating the number of active cores; however, in several domains, users are unwilling to sacrifice performance to save power. We present a prediction model for identifying energy-efficient operating points of concurrency in well-tuned multithreaded scientific applications and a runtime system that uses live program analysis to optimize applications dynamically. We describe a dynamic phase-aware performance prediction model that combines multivariate regression techniques with runtime analysis of data collected from hardware event counters to locate optimal operating points of concurrency. Using our model, we develop a prediction-driven phase-aware runtime optimization scheme that throttles concurrency so that power consumption can be reduced and performance can be set at the knee of the scalability curve of each program phase. The use of prediction reduces the overhead of searching the optimization space while achieving near-optimal performance and power savings. A thorough evaluation of our approach shows a reduction in power consumption of 10.8 percent, simultaneous with an improvement in performance of 17.9 percent, resulting in energy savings of 26.7 percent.

The geometry of unstented and stented pig common carotid artery bypass grafts

The long-term success of arterial bypass grafting with autologous saphenous veins is limited by neointimal hyperplasia (NIH), which seemingly develops preferentially at sites where hydrodynamic wall shear is low. Placement of a loose-fitting, porous stent around end-to-end, or end-to-side, autologous saphenous vein grafts on the porcine common carotid artery has been found significantly to reduce NIH, but the mechanism is unclear. In a preliminary study, we implanted autologous saphenous vein grafts bilaterally on the common carotid arteries of pigs, placing a stent around one graft and leaving the contralateral graft unstented. At sacrifice 1 month post implantation, the grafts were pressure fixed in situ and resin casts were made. Unstented graft geometry was highly irregular, with non-uniform dilatation, substantial axial lengthening, curvature, kinking, and possible long-pitch helical distortion. In contrast, stented grafts showed no major dilatation, lengthening or curvature, but there was commonly fine corrugation, occasional slight kinking or narrowing of segments, and possible long-pitch helical distortion. Axial growth of grafts against effectively tethered anastomoses could account for these changes. CFD studies are planned, using 3D MR reconstructions, on the effects of graft geometry on the flow. Abnormality of the flow could favour the development of vascular pathology, including NIH.

Optimization of Weighted Finite State Transducer for Speech Recognition

There is considerable interest in creating embedded, speech recognition hardware using the weighted finite state transducer (WFST) technique but there are performance and memory usage challenges. Two system optimization techniques are presented to address this; one approach improves token propagation by removing the WFST epsilon input arcs; another one-pass, adaptive pruning algorithm gives a dramatic reduction in active nodes to be computed. Results for memory and bandwidth are given for a 5,000 word vocabulary giving a better practical performance than conventional WFST; this is then exploited in an adaptive pruning algorithm that reduces the active nodes from 30,000 down to 4,000 with only a 2 percent sacrifice in speech recognition accuracy; these optimizations lead to a more simplified design with deterministic performance.

Ireland's Decade of Commemoration: A Round Table

Beginning with last year’s centennial of the passage of the Third Home Rule Bill, Ireland commenced an extraordinary “Decade of Commemorations,” during which the entire island will recall the anniversaries of crucial historic events: the Dublin Lock-out, the Easter Rising, the “Ulster Sacrifice” of the Somme, Partition, and the Irish Civil War, to name a few. The high-profile public history that will be crafted to mark these events is likely to set the received narrative of the events for another century—and, in turn, will enter the interdisciplinary intellectual project of Irish Studies itself. With the special assistance of Dr. Mike Cronin, New Hibernia Review gathered four scholars (two historians, a literary scholar, and a social anthropologist) to discuss the implications, opportunities, and perils of the Decade of Commemorations. They conducted their discussion by e-mail over the summer of 2013.

Politically sensitive encounters: Ethnography, access and the benefits of ‘hanging out’

Negotiating politically sensitive research environments requires both a careful consideration of the methods involved, and a great deal of personal resolve. In drawing upon two distinct yet comparable fieldwork experiences this paper champions the benefits of ethnographic methods in seeking to gain positionality and research legitimacy amongst those identified as future research participants. The authors explore and discuss their use of the ethnographic concept of ‘hanging out’ in politically sensitive environments when seeking to negotiate access to potentially hard to reach participants living in challenging research environments. Through an illustrative examination of their experiences in researching commemorative rituals in Palestine and mental health in a Northern Irish prison, both authors reflect upon their use of hanging out when seeking to break down barriers and gain acceptance amongst their target research participants. Their involvement in a range of activities, not directly related to the overall aims of the research project, highlight a need for qualitative researchers to adopt a flexible research design, one that embraces serendipitous or chance encounters, when seeking to gain access to hard to reach research participants or when issues of researcher legitimacy are particularly pronounced such as is the case in politically sensitive research environments.