Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

An overview of physical growth and maturation

An understanding of physical growth and maturation is relevant to many disciplines, including exercise and sport science, anthropology, human biology, medicine, psychology and education. Growth and maturation is governed by a complex interaction between genetic and environmental factors. There is increasing evidence that physical activity plays an important role in normal growth, development, health and well-being of children and youth, however, caution is required in the activity setting so that growth and maturation is not jeopardised. To appreciate the impact of physical activity and/or exercise on growth and maturation, a thorough understanding of the general principles of auxology is useful. Following an introduction to terminology, an overview of physical growth and development is provided in the context of morphological changes. Detailed information is provided regarding individual variability in growth and development along with sexual dimorphism. A small degree of sexual dimorphism exists at birth however striking differences develop during the pubertal years. Sexual dimorphism in body composition is largely regulated by endocrine factors with critical roles played by growth hormone and gonadal steroids.

In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU

Purpose: One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens–induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses.---------- Methods: A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. ---------- Results: In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P ≤ 0.05). ---------- Conclusions: Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

Rethinking public service media and citizenship: digital strategies for news and current affairs at Australia’s Special Broadcasting Service

This article considers the concept of media citizenship in relation to the digital strategies of the Special Broadcasting Service (SBS). At SBS, Australia’s multicultural public broadcaster, there is a critical appraisal of its strategies to harness user-created content (UCC) and social media to promote greater audience participation through its news and current affairs Web sites. The article looks at the opportunities and challenges that user-created content presents for public service media organizations as they consolidate multiplatform service delivery. Also analyzed are the implications of radio and television broadcasters’ moves to develop online services. It is proposed that case study methodologies enable an understanding of media citizenship to be developed that maintains a focus on the interaction between delivery technologies, organizational structures and cultures, and program content that is essential for understanding the changing focus of 21st-century public service media.

Copyrights and creative commons licensing : pedagogical innovation in a higher education media literacy classroom

This article examines the role of copyrights in contemporary media literacies. It argues that, provided they are ethical, young people’s engagement with text should occur in environments that are as free from restriction as possible. Discussion of open culture ecologies and the emergent education commons is followed by a theorisation of both literacy and copyrights education as forms of epistemology: that is, as effects of knowledge producing discourses and practices. Because Creative Commons licenses respect and are based on existing copyright laws, a brief overview of traditional copyrights for educators is first provided. We then describe the voluntary Creative Commons copyright licensing framework (“some rights reserved”) as an alternative to conventional “all rights reserved” models. This is followed by an account of a series of workshop activities on copyrights and Creative Commons conducted by the authors in the media literacy classes of a preservice teacher education program in Queensland, Australia. It provides one example of a practical program on critical copyrights approaches, which may be adapted and used by other school and higher education institutions.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

'Radiant with possibility': involving young people in creating a vision for the future of their community

This paper argues that young people need to be given the opportunity to recognise the interaction between their own understandings of the world as it is now and the vision of what it might become. To support this argument, we discuss an urban planning project, known as the Lower Mill Site Project, which involved active participation of high school students from the local community. The outcomes of this project demonstrate the positive contributions young people can make to the process of urban redevelopment, the advantages of using a participatory design approach, and the utopian possibilities that can emerge when young people are invited to be part of an intergenerational community project.

Randomised controlled trial of an automated, interactive telephone intervention to improve type 2 diabetes self-management (Telephone-Linked Care Diabetes Project) : study protocol

Background: An estimated 285 million people worldwide have diabetes and its prevalence is predicted to increase to 439 million by 2030. For the year 2010, it is estimated that 3.96 million excess deaths in the age group 20-79 years are attributable to diabetes around the world. Self-management is recognised as an integral part of diabetes care. This paper describes the protocol of a randomised controlled trial of an automated interactive telephone system aiming to improve the uptake and maintenance of essential diabetes self-management behaviours. ---------- Methods/Design: A total of 340 individuals with type 2 diabetes will be randomised, either to the routine care arm, or to the intervention arm in which participants receive the Telephone-Linked Care (TLC) Diabetes program in addition to their routine care. The intervention requires the participants to telephone the TLC Diabetes phone system weekly for 6 months. They receive the study handbook and a glucose meter linked to a data uploading device. The TLC system consists of a computer with software designed to provide monitoring, tailored feedback and education on key aspects of diabetes self-management, based on answers voiced or entered during the current or previous conversations. Data collection is conducted at baseline (Time 1), 6-month follow-up (Time 2), and 12-month follow-up (Time 3). The primary outcomes are glycaemic control (HbA1c) and quality of life (Short Form-36 Health Survey version 2). Secondary outcomes include anthropometric measures, blood pressure, blood lipid profile, psychosocial measures as well as measures of diet, physical activity, blood glucose monitoring, foot care and medication taking. Information on utilisation of healthcare services including hospital admissions, medication use and costs is collected. An economic evaluation is also planned.---------- Discussion: Outcomes will provide evidence concerning the efficacy of a telephone-linked care intervention for self-management of diabetes. Furthermore, the study will provide insight into the potential for more widespread uptake of automated telehealth interventions, globally.

Talkin' up sport and gender : Three Australian Aboriginal women speak

This paper presents the perspectives from three Aboriginal women on body image, sport and physical activity within Australian contemporary society. It draws on a range of literature along with artworks from visual artist Pamela Croft.

Informational city

Many cities worldwide face the prospect of major transformation as the world moves towards a global information order. In this new era, urban economies are being radically altered by dynamic processes of economic and spatial restructuring. The result is the creation of ‘informational cities’ or its new and more popular name, ‘knowledge cities’. For the last two centuries, social production had been primarily understood and shaped by neo-classical economic thought that recognized only three factors of production: land, labor and capital. Knowledge, education, and intellectual capacity were secondary, if not incidental, factors. Human capital was assumed to be either embedded in labor or just one of numerous categories of capital. In the last decades, it has become apparent that knowledge is sufficiently important to deserve recognition as a fourth factor of production. Knowledge and information and the social and technological settings for their production and communication are now seen as keys to development and economic prosperity. The rise of knowledge-based opportunity has, in many cases, been accompanied by a concomitant decline in traditional industrial activity. The replacement of physical commodity production by more abstract forms of production (e.g. information, ideas, and knowledge) has, however paradoxically, reinforced the importance of central places and led to the formation of knowledge cities. Knowledge is produced, marketed and exchanged mainly in cities. Therefore, knowledge cities aim to assist decision-makers in making their cities compatible with the knowledge economy and thus able to compete with other cities. Knowledge cities enable their citizens to foster knowledge creation, knowledge exchange and innovation. They also encourage the continuous creation, sharing, evaluation, renewal and update of knowledge. To compete nationally and internationally, cities need knowledge infrastructures (e.g. universities, research and development institutes); a concentration of well-educated people; technological, mainly electronic, infrastructure; and connections to the global economy (e.g. international companies and finance institutions for trade and investment). Moreover, they must possess the people and things necessary for the production of knowledge and, as importantly, function as breeding grounds for talent and innovation. The economy of a knowledge city creates high value-added products using research, technology, and brainpower. Private and the public sectors value knowledge, spend money on its discovery and dissemination and, ultimately, harness it to create goods and services. Although many cities call themselves knowledge cities, currently, only a few cities around the world (e.g., Barcelona, Delft, Dublin, Montreal, Munich, and Stockholm) have earned that label. Many other cities aspire to the status of knowledge city through urban development programs that target knowledge-based urban development. Examples include Copenhagen, Dubai, Manchester, Melbourne, Monterrey, Singapore, and Shanghai. Knowledge-Based Urban Development To date, the development of most knowledge cities has proceeded organically as a dependent and derivative effect of global market forces. Urban and regional planning has responded slowly, and sometimes not at all, to the challenges and the opportunities of the knowledge city. That is changing, however. Knowledge-based urban development potentially brings both economic prosperity and a sustainable socio-spatial order. Its goal is to produce and circulate abstract work. The globalization of the world in the last decades of the twentieth century was a dialectical process. On one hand, as the tyranny of distance was eroded, economic networks of production and consumption were constituted at a global scale. At the same time, spatial proximity remained as important as ever, if not more so, for knowledge-based urban development. Mediated by information and communication technology, personal contact, and the medium of tacit knowledge, organizational and institutional interactions are still closely associated with spatial proximity. The clustering of knowledge production is essential for fostering innovation and wealth creation. The social benefits of knowledge-based urban development extend beyond aggregate economic growth. On the one hand is the possibility of a particularly resilient form of urban development secured in a network of connections anchored at local, national, and global coordinates. On the other hand, quality of place and life, defined by the level of public service (e.g. health and education) and by the conservation and development of the cultural, aesthetic and ecological values give cities their character and attract or repel the creative class of knowledge workers, is a prerequisite for successful knowledge-based urban development. The goal is a secure economy in a human setting: in short, smart growth or sustainable urban development.

Deterring drug drivers : a study into the initial impact of oral random roadside drug testing in Queensland

Objective: The global implementation of oral random roadside drug testing is relatively limited, and correspondingly, the literature that focuses on the effectiveness of this intervention is scant. This study aims to provide a preliminary indication of the impact of roadside drug testing in Queensland. Methods: A sample of Queensland motorists’ (N= 922) completed a self-report questionnaire to investigate their drug driving behaviour, as well as examine the perceived affect of legal sanctions (certainty, severity and swiftness) and knowledge of the countermeasure on their subsequent offending behaviour. Results: Analysis of the collected data revealed that approximately 20% of participants reported drug driving at least once in the last six months. Overall, there was considerable variability in respondent’s perceptions regarding the certainty, severity and swiftness of legal sanctions associated with the testing regime and a considerable proportion remained unaware of testing practices. In regards to predicting those who intended to drug driving again in the future, perceptions of apprehension certainty, more specifically low certainty of apprehension, were significantly associated with self-reported intentions to offend. Additionally, self-reported recent drug driving activity and frequent drug consumption were also identified as significant predictors, which indicates that in the current context, past behaviour is a prominent predictor of future behaviour. To a lesser extent, awareness of testing practices was a significant predictor of intending not to drug drive in the future. Conclusion: The results indicate that drug driving is relatively prevalent on Queensland roads, and a number of factors may influence such behaviour. Additionally, while the roadside testing initiative is beginning to have a deterrent impact, its success will likely be linked with targeted intelligence-led implementation in order to increase apprehension levels as well as the general deterrent effect.

Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay

Reviewing the available literature, one could conclude that marrow-derived mesenchymal stem cells (BMSCs) are the ‘gold standard’ source for bone tissue engineering applications, due to their multilineage differentiation potential and easy accessibility. However, comprehensive studies comparing their osteogenic potential with bone-derived osteoblasts (OBs) to justify the preferred application of BMSCs based on performance are few. To address these shortfalls, in the present study, ovine BMSCs and OBs seeded onto scaffolds were characterized in vitro and transplanted subcutaneously into NOD/SCID mice in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7). It was hypothesized that cell origin, ossification type and degree of vascularization and ossification depends on the nature and commitment of transplanted cells and stimulating growth factors, such as rhBMP-7. After retrieval, specimens were analysed by biomechanical testing, µCT analysis, scanning electron microscopy/energy-dispersive X-ray spectroscopy and histo- and immunohistochemistry for osteocalcin, type II collagen and BrdU. The results showed a high degree of cell survival and proliferation ectopically, resulting in active contribution to endochondral osteogenesis. When compared to BMSCs, OBs showed a higher degree of bone deposition while OB-derived bone was of higher maturation. Stimulation with rhBMP-7 increased the rate of bone synthesis for both BMSCs and OBs, additionally promoting neovascularization and osteoclast activity. These results suggest that the origin and commitment of transplanted cells highly influence the type and degree of ossification, that rhBMP-7 represents a powerful adjuvant for bone tissue-engineering applications, and that mature bone is an adequate alternative cell source for bone tissue-engineering applications.

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

The delta-phase spectrum with application to voice activity detection and speaker recognition

For several reasons, the Fourier phase domain is less favored than the magnitude domain in signal processing and modeling of speech. To correctly analyze the phase, several factors must be considered and compensated, including the effect of the step size, windowing function and other processing parameters. Building on a review of these factors, this paper investigates a spectral representation based on the Instantaneous Frequency Deviation, but in which the step size between processing frames is used in calculating phase changes, rather than the traditional single sample interval. Reflecting these longer intervals, the term delta-phase spectrum is used to distinguish this from instantaneous derivatives. Experiments show that mel-frequency cepstral coefficients features derived from the delta-phase spectrum (termed Mel-Frequency delta-phase features) can produce broadly similar performance to equivalent magnitude domain features for both voice activity detection and speaker recognition tasks. Further, it is shown that the fusion of the magnitude and phase representations yields performance benefits over either in isolation.

Estimates of crowding in long-term care: Comparing two approaches

Objective: Because studies of crowding in long-term care settings are lacking, the authors sought to: (1) generate initial estimates of crowding in nursing homes and assisted living facilities; and (2) evaluate two operational approaches to its measurement. ----- ----- Background: Reactions to density and proximity are complex. Greater density intensifies people's reaction to a situation in the direction (positive or negative) that they would react if the situation were to occur under less dense conditions. People with dementia are especially reactive to the environment. ----- ----- Methods: Using a cross-sectional correlational design in nursing homes and assisted living facilities involving 185 participants, multiple observations (N = 6,455) of crowding and other environmental variables were made. Crowding, location, and sound were measured three times per observation; ambiance was measured once. Data analyses consisted of descriptive statistics, t-tests, and one-way analysis of variance. ----- ----- Results: Crowding estimates were higher for nursing homes and in dining and activity rooms. Crowding also varied across settings and locations by time of day. Overall, the interaction of location and time affected crowding significantly (N = 5,559, df [47, 511], F = 105.69, p < .0001); effects were greater within location-by-hour than between location-by-hour, but the effect explained slightly less variance in Long-Term Care Crowding Index (LTC-CI) estimates (47.41%) than location alone. Crowding had small, direct, and highly significant correlations with sound and with the engaging subscale for ambiance; a similar, though inverse, correlation was seen with the soothing subscale for ambiance. ----- ----- Conclusions: Crowding fluctuates consistent with routine activities such as meals in long-term care settings. Furthermore, a relationship between crowding and other physical characteristics of the environment was found. The LTC-CI is likely to be more sensitive than simple people counts when seeking to evaluate the effects of crowding on the behavior of elders-particularly those with dementia-in long-term care settings. aging in place.