Development of non-weak link bulk YBCO grain boundaries for high magnetic field eegimeering applications

A non-weak link joining technique has been developed for YBCO pseudo-crystals fabricated by seeded peritectic solidification based on the formation of a liquid phase which segregates from the platelet boundaries at temperatures above = 920 °C. Electrical and magnetic measurements on these boundaries suggest that their irreversibility field can be as high as 7 T at 77 K in fully oxygenated pseudo-crystals joined along their crystallographic ab-planes which is comparable to the irreversibility behaviour of the adjacent YBCO grains. © 1999 IEEE.

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

DNA double strand break repair: a radiation perspective

SIGNIFICANCE:
Ionizing radiation (IR) can induce a wide range of unique deoxyribonucleic acid (DNA) lesions due to the spatiotemporal correlation of the ionization produced. Of these, DNA double strand breaks (DSBs) play a key role. Complex mechanisms and sophisticated pathways are available within cells to restore the integrity and sequence of the damaged DNA molecules.
RECENT ADVANCES:
Here we review the main aspects of the DNA DSB repair mechanisms with emphasis on the molecular pathways, radiation-induced lesions, and their significance for cellular processes.
CRITICAL ISSUES:
Although the main characteristics and proteins involved in the two DNA DSB repair processes present in eukaryotic cells (homologous recombination and nonhomologous end-joining) are reasonably well established, there are still uncertainties regarding the primary sensing event and their dependency on the complexity, location, and time of the damage. Interactions and overlaps between the different pathways play a critical role in defining the repair efficiency and determining the cellular functional behavior due to unrepaired/miss-repaired DNA lesions. The repair pathways involved in repairing lesions induced by soluble factors released from directly irradiated cells may also differ from the established response mechanisms.
FUTURE DIRECTIONS:
An improved understanding of the molecular pathways involved in sensing and repairing damaged DNA molecules and the role of DSBs is crucial for the development of novel classes of drugs to treat human diseases and to exploit characteristics of IR and alterations in tumor cells for successful radiotherapy applications.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

The role of ATM in the cell cycle control of V(D)J recombination

Lymphocyte development requires the assembly of diversified antigen receptor complexes generated by the genetically programmed V(D)J recombination event. Because germline DNA is cut, introducing potentially dangerous double-stranded breaks (DSBs) and rearranged prior to repair, its activity is limited to the non-cycling stages of the cell cycle, G0/G1. The potential involvement of a key mediator, Ataxia Telangiectasia Mutated or ATM, in the DNA damage response (DDR) and cell cycle checkpoints has been implicated in recombination, but its role is not fully understood. Thymic lymphomas from ATM deficient mice contain clonal chromosomal translocations involving the T-cell antigen receptor (TCR). A previous report found ATM and its downstream target p53 associated with V(D)J intermediates, suggesting the DDR senses recombination. In this study, we sought to understand the role of ATM in V(D)J recombination. Developing thymocytes from ATM deficient mice were analyzed according to the cell cycle to detect V(D)J intermediates. Examination of all TCR loci in the non-cycling (G0/G1) and cycling (S/G2/M) fractions revealed the persistence of intermediates in ATM deficient thymocytes, contrary to the wild-type in which intermediates are found only during G0/G1. Further analysis found no defect in end-joining of intermediates, nor were they detected in developed T-cells. Based upon the presence of persisting intermediates, the recombination initiating nuclease Rag-2 was examined; strict regulation limits it to G 0/G1. Rag-2 regulation was not affected by an ATM deficiency as Rag-2 expression remained contained within G0/G 1, indicating recombination is not continuous. To determine if an ATM deficiency affects recognition of V(D)J breaks, sites of recombination identified by a TCR locus or Rag expression were analyzed according to co-localization with a DDR factor phosphorylated immediately after DNA damage, phosphorylated H2AX (γH2AX). No differences in co-localization were found between the wild-type and ATM deficiency, demonstrating ATM deficient lymphocytes retain the ability to recognize DSBs. Together, these results suggest ATM is necessary in the cell cycle regulation of recombination but not essential for the identification of V(D)J breaks. ATM ensures the containment of intermediates within G0/G1 and maintains genomic stability of developing lymphocytes, emphasizing its fundamental role in preventing tumorigenesis.^

End-User Development Success Factors and their Application to Composite Web Development Environments

The Future Internet is expected to be composed of a mesh of interoperable Web services accessed from all over the Web. This approach has not yet caught on since global user-service interaction is still an open issue. Successful composite applications rely on heavyweight service orchestration technologies that raise the bar far above end-user skills. The weakness lies in the abstraction of the underlying service front-end architecture rather than the infrastructure technologies themselves. In our opinion, the best approach is to offer end-to-end composition from user interface to service invocation, as well as an understandable abstraction of both building blocks and a visual composition technique. In this paper we formalize our vision with regard to the next-generation front-end Web technology that will enable integrated access to services, contents and things in the Future Internet. We present a novel reference architecture designed to empower non-technical end users to create and share their own self-service composite applications. A tool implementing this architecture has been developed as part of the European FP7 FAST Project and EzWeb Project, allowing us to validate the rationale behind our approach.

A component- and connector-based approach for end-user composite web applications development

Enabling real end-user development is the next logical stage in the evolution of Internet-wide service-based applications. Successful composite applications rely on heavyweight service orchestration technologies that raise the bar far above end-user skills. This weakness can be attributed to the fact that the composition model does not satisfy end-user needs rather than to the actual infrastructure technologies. In our opinion, the best way to overcome this weakness is to offer end-to-end composition from the user interface to service invocation, plus an understandable abstraction of building blocks and a visual composition technique empowering end users to develop their own applications. In this paper, we present a visual framework for end users, called FAST, which fulfils this objective. FAST implements a novel composition model designed to empower non-programmer end users to create and share their own self-service composite applications in a fully visual fashion. We projected the development environment implementing this model as part of the European FP7 FAST Project, which was used to validate the rationale behind our approach.

Break-induced replication: A review and an example in budding yeast

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the LEU2 gene at one end was transformed into a strain in which the 5′ end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2+. Ade1+ Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2(Cdk1) provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Physical and Genetic Analysis of the CUP1 Tandem Array in the Yeast Saccharomyces cerevisiae

The genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations.

In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.

In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis.

The research described in Chapter 2 is published in 2014.

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information

Background The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. Results In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. Conclusion A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Community participation in the development of digital radio : the Australian experience

The participation of the community broadcasting sector in the development of digital radio provides a potentially valuable opportunity for non-market, end user-driven experimentation in the development of these new services in Australia. However this development path is constrained by various factors, some of which are specific to the community broadcasting sector and others that are generic to the broader media and communications policy, industrial and technological context. This paper filters recent developments in digital radio policy and implementation through the perspectives of community radio stakeholders, obtained through interviews, to describe and analyse these constraints. The early stage of digital community radio presented here is intended as a baseline for tracking the development of the sector as digital radio broadcasting develops. We also draw upon insights from scholarly debates about citizens media and participatory culture to identify and discuss two sets of opportunities for social benefit that are enabled by the inclusion of community radio in digital radio service development. The first arises from community broadcasting’s involvement in the propagation of the multi-literacies that drive new digital economies, not only through formal and informal multi- and trans-media training, but also in the ‘co-creative’ forms of collaborative and participatory media production that are fostered in the sector. The second arises from the fact that community radio is uniquely placed — indeed charged with the responsibility — to facilitate social participation in the design and operation of media institutions themselves, not just their service outputs.

The QUT innovation space : a trans-disciplinary learning environment for entrepreneurship education : final report

In the current climate of global economic volatility, there are increasing calls for training in enterprising skills and entrepreneurship to underpin the systemic innovation required for even medium-term business sustainability. The skills long-recognised as the essential for entrepreneurship now appear on the list of employability skills demanded by industry. The QUT Innovation Space (QIS) was an experiment aimed at delivering entrepreneurship education (EE), as an extra-curricular platform across the university, to the undergraduate students of an Australian higher education institute. It was an ambitious project that built on overseas models of EE studied during an Australian Learning and Teaching Council (ALTC) Teaching Fellowship (Collet, 2011) and implemented those approaches across an institute. Such EE approaches have not been attempted in an Australian university. The project tested resonance not only with the student population, from the perspective of what worked and what didn’t work, but also with every level of university operations. Such information is needed to inform the development of EE in the Australian university landscape. The QIS comprised a physical co-working space, virtual sites (web, Twitter and Facebook) and a network of entrepreneurial mentors, colleagues, and students. All facets of the QIS enabled connection between like-minded individuals that underpins the momentum needed for a project of this nature. The QIS became an innovation community within QUT. This report serves two purposes. First, as an account of the QIS project and its evolution, the report serves to identify the student demand for skills and training as well as barriers and facilitators of the activities that promote EE in an Australian university context. Second, the report serves as a how-to manual, in the tradition of many tomes on EE, outlining the QIS activities that worked as well as those that failed. The activities represent one measure of QIS outcomes and are described herein to facilitate implementation in other institutes. The QIS initially aimed to adopt an incubation model for training in EE. The ‘learning by doing’ model for new venture creation is a highly successful and high profile training approach commonly found in overseas contexts. However, the greatest demand of the QUT student population was not for incubation and progression of a developed entrepreneurial intent, but rather for training that instilled enterprising skills in the individual. These two scenarios require different training approaches (Fayolle and Gailly, 2008). The activities of the QIS evolved to meet that student demand. In addressing enterprising skills, the QIS developed the antecedents of entrepreneurialism (i.e., entrepreneurial attitudes, motivation and behaviours) including high-level skills around risk-taking, effective communication, opportunity recognition and action-orientation. In focusing on the would-be entrepreneur and not on the (initial) idea per se, the QIS also fostered entrepreneurial outcomes that would never have gained entry to the rigid stage-gated incubation model proposed for the original QIS framework. Important lessons learned from the project for development of an innovation community include the need to: 1. Evaluate the context of the type of EE program to be delivered and the student demand for the skills training (as noted above). 2. Create a community that builds on three dimensions: a physical space, a virtual environment and a network of mentors and partners. 3. Supplement the community with external partnerships that aid in delivery of skills training materials. 4. Ensure discovery of the community through the use of external IT services to deliver advertising and networking outlets. 5. Manage unrealistic student expectations of billion dollar products. 6. Continuously renew and rebuild simple activities to maintain student engagement. 7. Accommodate the non-university end-user group within the community. 8. Recognise and address the skills bottlenecks that serve as barriers to concept progression; in this case, externally provided IT and programming skills. 9. Use available on-line and published resources rather than engage in constructing project-specific resources that quickly become obsolete. 10. Avoid perceptions of faculty ownership and operate in an increasingly competitive environment. 11. Recognise that the continuum between creativity/innovation and entrepreneurship is complex, non-linear and requires different training regimes during the different phases of the pipeline. One small entity, such as the QIS, cannot address them all. The QIS successfully designed, implemented and delivered activities that included events, workshops, seminars and services to QUT students in the extra-curricular space. That the QIS project can be considered successful derives directly from the outcomes. First, the QIS project changed the lives of emerging QUT student entrepreneurs. Also, the QIS activities developed enterprising skills in students who did not necessarily have a business proposition, at the time. Second, successful outcomes of the QIS project are evidenced as the embedding of most, perhaps all, of the QIS activities in a new Chancellery-sponsored initiative: the Leadership Development and Innovation Program hosted by QUT Student Support Services. During the course of the QIS project, the Brisbane-based innovation ecosystem underwent substantial change. From a dearth of opportunities for the entrepreneurially inclined, there is now a plethora of entities that cater for a diversity of innovation-related activities. While the QIS evolved with the landscape, the demand endpoint of the QIS activities still highlights a gap in the local and national innovation ecosystems. The freedom to experiment and to fail is not catered for by the many new entities seeking to build viable businesses on the back of the innovation push. The onus of teaching the enterprising skills, which are the employability skills now demanded by industry, remains the domain of the higher education sector.

Molecular characterisation of dicot-infecting mastreviruses from Australia

Monocotyledonous and dicotyledonous plant infecting mastreviruses threaten various agricultural systems throughout Africa, Eurasia and Australasia. In Australia three distinct mastrevirus species are known to infect dicotyledonous hosts such as chickpea, bean and tobacco. Amongst 34 new "dicot-infecting" mastrevirus full genome sequences obtained from these hosts we discovered one new species, four new strains, and various variants of previously described mastrevirus species. Besides providing additional support for the hypothesis that evolutionary processes operating during dicot-infecting mastrevirus evolution (such as patterns of pervasive homologous and non-homologous recombination, and strong purifying selection acting on all genes) have mostly mirrored those found in their monocot-infecting counterparts, we find that the Australian dicot-infecting viruses display patterns of phylogeographic clustering reminiscent of those displayed by monocot infecting mastrevirus species such as Panicum streak virus and Maize streak virus.