Quantification of gammaH2AX foci in response to ionising radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX(1). Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)(2). Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB(2,3). This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning approximately 2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete gammaH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy(2). The loss of gammaH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary(4-8). The disappearence of gammaH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C(5,6). Further, removal of gammaH2AX by redistribution involving histone exchange with H2A.Z has been implicated(7,8). Importantly, the quantitative analysis of gammaH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of gammaH2AX foci in gamma-irradiated adherent human keratinocytes(9)

Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.

Quantitation of gammaH2AX foci in tissue samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

Geochemistry-based coral palaeoclimate studies and the potential of ‘non-traditional’ (non-massive Porites) corals : recent developments and future progression

Understanding the natural variability of the Earth's climate system and accurately identifying potential anthropogenic influences requires long term, geographically distributed records of key climate indicators, such as temperature and precipitation that extend prior to the last 400. years of the Holocene. Reef corals provide an excellent source of high resolution climate records, and importantly represent the tropical marine environment where palaeoclimate data are urgently required. Recent decades have seen significant improvement in our understanding of coral biomineralisation, the associated uptake of geochemical proxies and methods of identifying and understanding the effects of both early and late, post depositional diagenetic alteration. These processes all have significant implications for interpreting geochemical proxies relevant to palaeoclimatic reconstructions. This paper reviews the current 'state of the art' in terms of coral based palaeoclimate reconstructions and highlights a key remaining problem. The majority of coral based palaeoclimate research has been derived from massive colonies of Porites. However, massive Porites are not globally abundant and may not provide material of a particular age of interest in those regions where they are present. Therefore, there is great potential for alternate coral genera to act as complimentary climate archives. While it remains critical to consider five key factors - vital effects, differential growth morphologies, geochemical heterogeneity in the skeletal ultrastructure, transfer equation selection and diagenetic screening of skeletal material - in order to allow the highest level of accuracy in coral palaeoclimate reconstructions, it is also important to develop alternate taxa for palaeoclimate studies in regions where Porites colonies are absent or rare. Currently as many as nine genera other than Porites have proven at least limited utility in palaeothermometry, most of which are found in the Atlantic/Caribbean region where massive Porites do not exist. Even branching taxa such as Acropora have significant potential to preserve environmental archives. Increasing this capability will greatly expand the number of potential geochemical archives available for longer term temporal records of palaeoclimate.

The effectiveness of dietary counselling versus oral nutrition supplements in improving nutritional intake in malnourished patients with Chronic Obstructive Pulmonary Disease (COPD)

The evidence for nutritional support in COPD is almost entirely based on oral nutritional supplements (ONS) yet despite this dietary counseling and food fortification (DA) are often used as the first line treatment for malnutrition. This study aimed to investigate the effectiveness of ONS vs. DA in improving nutritional intake in malnourished outpatients with COPD. 70 outpatients (BMI 18.4 SD 1.6 kg/m2, age 73 SD 9 years, severe COPD) were randomised to receive a 12-week intervention of either ONS or DA (n 33 ONS vs. n 37 DA). Paired t-test analysis revealed total energy intakes significantly increased with ONS at week 6 (+302 SD 537 kcal/d; p = 0.002), with a slight reduction at week 12 (+243 SD 718 kcal/d; p = 0.061) returning to baseline levels on stopping supplementation. DA resulted in small increases in energy that only reached significance 3 months post-intervention (week 6: +48 SD 623 kcal/d, p = 0.640; week 12: +157 SD 637 kcal/d, p = 0.139; week 26: +247 SD 592 kcal/d, p = 0.032). Protein intake was significantly higher in the ONS group at both week 6 and 12 (ONS: +19.0 SD 25.0 g/d vs. DA: +1.0 SD 13.0 g/d; p = 0.033 ANOVA) but no differences were found at week 26. Vitamin C, Iron and Zinc intakes significantly increased only in the ONS group. ONS significantly increased energy, protein and several micronutrient intakes in malnourished COPD patients but only during the period of supplementation. Trials investigating the effects of combined nutritional interventions are required.

Entrepreneurship Programs and the Modern University

Entrepreneurship Programs and the Modern University is an excellent resource for those who intend to set up an entrepreneurship program or centre, and arguably even more so for those who wish to coordinate or expand such activities into an effective "entrepreneurship eco-system" across the university and its external stakeholders. Morris, Kuratko, and Cornwall's intention in writing this book is to "advocate a more strategic and integrative framework for building the curricular, co-curricular, research, community engagement, and infrastructure components of a program" (p. xii).

Implementing ICT in Schools in a Developing Country : A Fijian Experience

Significant advances have been made towards the United Nations Millennium Development Goals on universal education. While there has been an increase in the number of children who now attend primary school, the quality of education remains an issue in many countries. Knowledge and the ability to use ICT are considered to be mandatory for citizens of this century. Information and Communication Technology (ICT) is becoming increasingly more common in classrooms in developed countries. However, ICT use is often beyond the reach of many school communities in developing countries. While supporting these developing countries through donations of technology is a start, there is an equal if not a greater need to build teacher capacity so that the resources are effective in classrooms. The Share, Engage and Educate (SEE) project is about creating educational opportunities for learners in developing countries using ICT. Through the efforts of volunteers, the project provides technological resources to schools and engages teachers in activities that develop their capacity. The SEE project has adopted a four-phase model for ICT integration: objectives, implementation, feedback, and reflection. This chapter reports upon the project’s ongoing implementation phase in Fiji, including the approach taken to build teacher capacity and the identification of factors which have impacted upon the project’s success.

Three-dimensional geological modelling of the Galilee and central Eromanga basins, Australia: New insights into aquifer/aquitard geometry and potential influence of faults on inter-connectivity

Study region The Galilee and Eromanga basins are located in central Queensland, Australia. Both basins are components of the Great Artesian Basin which host some of the most significant groundwater resources in Australia. Study focus This study evaluates the influence of regional faults on groundwater flow in an aquifer/aquitard interbedded succession that form one of the largest Artesian Basins in the world. In order to assess the significance of regional faults as potential barriers or conduits to groundwater flow, vertical displacements of the major aquifers and aquitards were studied at each major fault and the general hydraulic relationship of units that are juxtaposed by the faults were considered. A three-dimensional (3D) geological model of the Galilee and Eromanga basins was developed based on integration of well log data, seismic surfaces, surface geology and elevation data. Geological structures were mapped in detail and major faults were characterised. New hydrological insights for the region Major faults that have been described in previous studies have been confirmed within the 3D geological model domain and a preliminary assessment of their hydraulic significance has been conducted. Previously unknown faults such as the Thomson River Fault (herein named) have also been identified in this study.

The effect of the ecstasy 'come-down' on the diagnosis of ecstasy dependence

Background: The existence of an ecstasy dependence syndrome is controversial. We examined whether the acute after-effects of ecstasy use (i.e., the “come-down”) falsely lead to the identification of ecstasy withdrawal and the subsequent diagnosis of ecstasy dependence. Methods: The Structured Clinical Interview for DSM-IV-TR Disorders: Research Version (SCID-RV) was administered to 214 Australian ecstasy users. Ecstasy withdrawal was operationalized in three contrasting ways: (i) as per DSM-IV criteria; (ii) as the expected after effects of ecstasy (a regular come-down); or (iii) as a substantially greater or longer come-down than on first use (intense come-down). These definitions were validated against frequency of ecstasy use, readiness to change and ability to resist the urge to use ecstasy. Confirmatory factor analyses were used to see how they aligned with the overall dependence syndrome. Results: Come-down symptoms increased the prevalence of withdrawal from 1% (DSM-IV criterion) to 11% (intense come-downs) and 75% (regular come-downs). Past year ecstasy dependence remained at 31% when including the DSM-IV withdrawal criteria and was 32% with intense come-downs, but increased to 45% with regular come-downs. Intense come-downs were associated with lower ability to resist ecstasy use and loaded positively on the dependence syndrome. Regular come-downs did not load positively on the ecstasy dependence syndrome and were not related to other indices of dependence. Conclusion: The acute after-effects of ecstasy should be excluded when assessing ecstasy withdrawal as they can lead to a false diagnosis of ecstasy dependence. Worsening of the ecstasy come-down may be a marker for dependence.

A conceptual approach to track design changes within a multi-disciplinary BIM environment

Successful management of design changes is critical for the efficient delivery of construction projects. Building Information Modeling (BIM) is envisioned to play an important role in integrating design, construction and facility management processes through coordinated changes throughout the project life-cycle. BIM currently provides significant benefits in coordinating changes across different views in a single model, and identifying conflicts between different discipline-specific models. However, current BIM tools provide limited support in managing changes across several discipline-specific models. This paper describes an approach to represent, coordinate, and track changes within a collaborative multi-disciplinary BIM environment. This approach was informed by a detailed case study of a large, complex, fast-tracked BIM project where we investigated numerous design changes, analyzed change management processes, and evaluated existing BIM tools. Our approach characterises design changes in an ontology to represent changed component attributes, dependencies between components, and change impacts. It explores different types of dependencies amongst different design changes and describes how a graph based approach and dependency matrix could assist with automating the propagation and impact of changes in a BIM-based project delivery process.

Clinton, Bush, and Obama : changing policy and rhetoric in the United States Annual Trafficking in Persons Report

This chapter charts the political transitions in the anti-trafficking agenda and rhetoric of the U.S. Government across three Presidential administrations through a detailed examination of the annual Trafficking in Persons reports released by the Office to Monitor and Combat Trafficking in Persons between 2001 and 2012. We argue that the transitions in language and focus reflect key tensions that have dominated trafficking discourse throughout the Clinton, Bush and Obama Presidencies. These fissures include debate over law enforcement versus rights-based frameworks, competing approaches on victim protection and identification, and ongoing disputes about the relationship between prostitution and human trafficking.

How does the use of a reflective journal enhance students’ critical thinking about complexity?

Introduction to Youth Services is a second year Social Work and Human Services unit. In this unit a reflective writing task was introduced to assess students’ reflections on an ongoing tutorial discussion to which they contributed. The discussion was based on a fictional young person each tutorial group ‘worked with’ across eight weeks of a semester. In developing the process and the criteria for the reflective journal, the ideas raised by the Teaching and Assessing Reflective Learning (TARL) in Higher Education project (see Chap. 2) were utilised, scaffolding the work with resources and submission of a draft. The students were also invited to choose the form of reflective process they used, it could be a written journal but did not need to be. The evidence exemplified that a reflective journal is an effective tool for students to record their developing understanding regarding the concept that issues people experience are complex and compounding. Importantly, it was also a useful vehicle for students to begin to consider the impacts of their own and others’ values and beliefs on their response to the issues raised within the case discussion. The reflective journal also helped participants to consider how this learning contributes to the ongoing development of their professional practice framework.

Staged homicides: An examination of common features of faked burglaries, suicides, accidents and car accidents

A staged crime scene involves deliberate alteration of evidence by the offender to simulate events that did not occur for the purpose of misleading authorities (Geberth, 2006; Turvey, 2000). This study examined 115 staged homicides from the USA to determine common elements; victim and perpetrator characteristics; and specific features of different types of staged scenes. General characteristics include: multiple victims and offenders; a previous relationship be- tween parties involved; and victims discovered in their own home, often by the offender. Staged scenes were separated by type with staged burglaries, suicides, accidents, and car accidents examined in more detail. Each type of scene displays differently with separate indicators and common features. Features of staged burglaries were: no points of entry/exit staged; non-valuables taken; scene ransacking; offender self- injury; and offenders bringing weapons to the scene. Features of staged suicides included: weapon arrangement and simulating self-injury to the victim; rearranging the body; and removing valuables. Examples of elements of staged accidents were arranging the implement/weapon and re- positioning the deceased; while staged car accidents involved: transporting the body to the vehicle and arranging both; mutilation after death; attempts to secure an alibi; and clean up at the primary crime scene. The results suggest few staging behaviors are used, despite the credibility they may have offered the façade. This is the first peer-reviewed, published study to examine the specific features of these scenes, and is the largest sample studied to date.

Designing a digital experience for young children with developmental disabilities

This paper reports on the development of a playful digital experience, Anim-action, designed for young children with developmental disabilities. This experience was built using the Stomp platform, a technology designed specifically to meet the needs of people with intellectual disability through facilitating whole body interaction. We provide detail on how knowledge gained from key stakeholders informed the design of the application and describe the design guidelines used in the development process. A study involving 13 young children with developmental disabilities was conducted to evaluate the extent to which Anim-action facilitates cognitive, social and physical activity. Results demonstrated that Anim-action effectively supports cognitive and physical activity. In particular, it promoted autonomy and encouraged problem solving and motor planning. Conversely, there were limitations in the system’s ability to support social interaction, in particular, cooperation. Results have been analyzed to determine how design guidelines might be refined to address these limitations.

The private problem with public service: Rural teachers in educational markets

Staffing rural and remote schools is an important policy issue for the public good. This paper examines the private issues it also poses for teachers with families working in these communities, as they seek to reconcile careers with educational choices for children. The paper first considers historical responses to staffing rural and remote schools in Australia, and the emergence of neoliberal policy encouraging marketisation of the education sector. We report on interviews about considerations motivating household mobility with 11 teachers across regional, rural and remote communities in Queensland. Like other middle-class parents, these teachers prioritised their children’s educational opportunities over career opportunities. The analysis demonstrates how teachers in rural and remote communities constitute a special group of educational consumers with insider knowledge and unique dilemmas around school choice. Their heightened anxieties around school choice under neoliberal policy are shown to contribute to the public issue of staffing rural and remote schools.