A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

On display : Ravensthorpe Wildflower Show and the assembly of place

Each September since 1983 in the rural Shire of Ravensthorpe, Western Australia, volunteers collect samples of up to 700 wildfl ower species which are then displayed in the Ravensthorpe Senior Citizens Centre from 9.00 am to 4.00 pm daily over a two-week period. This chapter offers an ethnographic interpretation of this enduring annual event focusing on the 25th show held in 2007. The study contributes to understanding the complex and nuanced role of local wildflower shows in shaping and supporting rural senses of place and of community. Importantly, this particular type of festival, and more specifically this local instance, foregrounds a less-remarked aspect of festivals, namely the (re)production and celebration of place-specific knowledge through validations of, and interconnections between, scientific flower classification and emotive experience. This feature, encapsulated in Laurel Lamperd’s poem above, invites consideration of the ways in which local place knowledge and the simultaneous (re)production of ‘place’ are constituted by a complex layering of rational, objective ways of knowing and those which emphasize emotions, aesthetics and memories. This rural wildflower show not only mobilises both the rational and the emotional in ‘making sense of the world’ for local residents and for tourists, but also offers insights into the production of place as constituted in and through relations between humans and non-human life forms (Cloke & Jones, 2001; Conradson, 2005; see also Chapter 6).

Hip and knee kinematics display complex and time-varying sagittal kinematics during repetitive stepping: Implications for design of a functional fatigue model of the knee extensors and flexors

The validity of fatigue protocols involving multi-joint movements, such as stepping, has yet to be clearly defined. Although surface electromyography can monitor the fatigue state of individual muscles, the effects of joint angle and velocity variation on signal parameters are well established. Therefore, the aims of this study were to i) describe sagittal hip and knee kinematics during repetitive stepping ii) identify periods of high inter-trial variability and iii) determine within-test reliability of hip and knee kinematic profiles. A group of healthy men (N = 15) ascended and descended from a knee-high platform wearing a weighted vest (10%BW) for 50 consecutive trials. The hip and knee underwent rapid flexion and extension during step ascent and descent. Variability of hip and knee velocity peaked between 20-40% of the ascent phase and 80-100% of the descent. Significant (p<0.05) reductions in joint range of motion and peak velocity during step ascent were observed, while peak flexion velocity increased during descent. Healthy individuals use complex hip and knee motion to negotiate a knee-high step with kinematic patterns varying across multiple repetitions. These findings have important implications for future studies intending to use repetitive stepping as a fatigue model for the knee extensors and flexors.

Nanofabrication of single-crystalline flat-panel display microemitters : a plasma-building unit approach

This contribution is focused on plasma-enhanced chemical vapor deposition systems and their unique features that make them particularly attractive for nanofabrication of flat panel display microemitter arrays based on ordered patterns of single-crystalline carbon nanotip structures. The fundamentals of the plasma-based nanofabrication of carbon nanotips and some other important nanofilms and nanostructures are examined. Specific features, challenges, and potential benefits of using the plasma-based systems for relevant nanofabrication processes are analyzed within the framework of the "plasma-building unit" approach that builds up on extensive experimental data on plasma diagnostics and nanofilm/nanostructure characterization, and numerical simulation of the species composition in the ionized gas phase (multicomponent fluid models), ion dynamics and interaction with ordered carbon nanotip patterns, and ab initio computations of chemical structure of single crystalline carbon nanotips. This generic approach is also applicable for nanoscale assembly of various carbon nanostructures, semiconductor quantum dot structures, and nano-crystalline bioceramics. Special attention is paid to most efficient control strategies of the main plasma-generated building units both in the ionized gas phase and on nanostructured deposition surfaces. The issues of tailoring the reactive plasma environments and development of versatile plasma nanofabrication facilities are also discussed.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

Solution-processed dual-gate polymer field-effect transistors for display applications

We describe the advantages of dual-gate thin-film transistors (TFTs) for display applications. We show that in TFTs with active semiconductor layers composed of diketopyrrolopyrrole-naphthalene copolymer, the on-current is increased, the off-current is reduced, and the sub-threshold swing is improved compared to single-gate devices. Charge transport measurements in steady-state and under non-quasi-static conditions reveal the reasons for this improved performance. We show that in dual-gate devices, a much smaller fraction of charge carriers move in slow trap states. We also compare the activation energies for charge transport in the top-gate and bottom-gate configurations.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Author response to : Brough et al. in response to the recently published article : Lottering,N., MacGregor,D.M.,Barry,M.D.,Reynolds,M.S., Gregory,L.S., 2014. Introducing standardized protocols for anthropological measurement of virtual sub-adult crania using computed tomography 2(1),34–38

Firstly, we would like to thank Ms. Alison Brough and her colleagues for their positive commentary on our published work [1] and their appraisal of our utility of the “off-set plane” protocol for anthropometric analysis. The standardized protocols described in our manuscript have wide applications, ranging from forensic anthropology and paleodemographic research to clinical settings such as paediatric practice and orthopaedic surgical design. We affirm that the use of geometrically based reference tools commonly found in computer aided design (CAD) programs such as Geomagic Design X® are imperative for more automated and precise measurement protocols for quantitative skeletal analysis. Therefore we stand by our recommendation of the use of software such as Amira and Geomagic Design X® in the contexts described in our manuscript...

Can a text message a week improve breastfeeding?

Background Breastfeeding is recognised as the optimal method for feeding infants with health gains made by reducing infectious diseases in infancy; and chronic diseases, including obesity, in childhood, adolescence and adulthood. Despite this, exclusivity and duration in developed countries remains resistant to improvement. The objectives of this research were to test if an automated mobile phone text messaging intervention, delivering one text message a week, could increase “any” breastfeeding rates and improve breastfeeding self-efficacy and coping. Methods Women were eligible to participate if they were: over eighteen years; had an infant less than three months old; were currently breastfeeding; no diagnosed mental illness; and used a mobile phone . Women in the intervention group received MumBubConnect, a text messaging service with automated responses delivered once a week for 8 weeks. Women in the comparison group received their usual care and were sampled two years after the intervention group. Data collection included online surveys at two time points, week zero and week nine, to measure breastfeeding exclusivity and duration, coping, emotions, accountability and self-efficacy. A range of statistical analyses were used to test for differences between groups. Hierarchical regression was used to investigate change in breastfeeding outcome, between groups, adjusting for co-variates. Results The intervention group had 120 participants at commencement and 114 at completion, the comparison group had 114 participants at commencement and 86 at completion. MumBubConnect had a positive impact on the primary outcome of breastfeeding behaviors with women receiving the intervention more likely to continue exclusive breastfeeding; with a 6% decrease in exclusive breastfeeding in the intervention group, compared to a 14% decrease in the comparison group (p < 0.001). This remained significant after controlling for infant age, mother’s income, education and delivery type (p = 0.04). Women in the intervention group demonstrated active coping and were less likely to display emotions-focussed coping (p < .001). There was no discernible statistical effect on self-efficacy or accountability. Conclusions A fully automated text messaging services appears to improve exclusive breastfeeding duration. The service provides a well-accepted, personalised support service that empowers women to actively resolve breastfeeding issues. Trial registration Australian New Zealand Clinical Trials Registry: ACTRN12614001091695.

Reterritorializing multimodal design in the classroom through a socially mediated Indigenous heritage

National or International Significance Flows of cultural heritage in textual practices are vital to sustaining Indigenous communities - a national and international priority (Commonwealth of Australia, 2011). Indigenous heritage, whether passed on by oral tradition or ubiquitous social media, can be seen as a "conversation between the past and the future" (Fairclough, 2012, p. xv). Indigenous heritage involves appropriating memories within a cultural flow to pass on a spiritual legacy. This presentation reports ethnographic research of social media practices in a small independent Aboriginal school in Southeast Queensland, Australia that is resided over by the Yuggera elders and an Aboriginal principal. Quality of Research The purpose was to rupture existing notions of white literacies in schools, and to deterritorialize the uses of digital media by dominant cultures in the public sphere. Examples of learning experiences included the following: i. Integrating Indigenous language and knowledge into media text production; ii. Classroom visits from Indigenous elders; and iii. Publishing oral histories through digital scrapbooking. The program aligned with the Australian National Curriculum English (ACARA, 2014), which mandates the teaching of multimodal text creation. Data sources included a class set of digital scrapbooks collaboratively created in a preparatory-one primary classroom. The digital scrapbooks combined digitally encoded words, images of material artifacts, and digital music files. A key feature of the writing and digital design task was to retell and digitally display and archive a cultural narrative of significance to the Indigenous Australian community and its memories and material traces of the past for the future. Data analysis of the students' digital stories involved the application of key themes of negotiated, material, and digitally mediated forms of heritage practice. It drew on Australian Indigenous research by Keddie et al. (2013) to guard against the homogenizing of culture that can arise from a focus on a static view of culture. The interpretation of findings located Indigenous appropriation of social media within broader racialized politics that enables Indigenous literacy to be understood as a dynamic, negotiated, and transgenerational flows of practice. It demonstrates that Indigenous children's use of media production reflects "shifting and negotiated identities" in response to changing media environments that can function to sustain Indigenous cultural heritages (Appadurai, 1696, p. xv). Impact on practice, policy or theory The findings are important for teachers at a time when Aboriginal and Torres Strait Islander Histories and Cultures is a cross-curricular policy priority in the Australian Curriculum (ACARA, 2014). The findings show how curriculum policies can be applied to classroom practice in ways that are epistemologically consistent with Indigenous ways of knowing and being. Theoretically, it demonstrates how the children's experiences of culture are layered over time, as successive generations inherit, interweave, and hear others' cultural stories or maps. Practically, recommendations are provided for an approach to appropriating social media in schools that explicitly attends to the dynamic nature of Indigenous practices, negotiated through intercultural constructions and flows, and opening space for a critical anti-racist approach to multimodal text production. Timeliness The research is timely in the context of the accessibility and role of digital and multimodal forms of communication, including for Aboriginal and Torres Strait Islander communities.