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.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

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.

Meeting the nutrition challenge of stage 3 renal failure : considerations for nursing practice

Self-care management is needed for effective management of chronic kidney disease. The main aim for treatment or management of chronic kidney disease is to delay the worsening of kidney function, and to prevent or to manage the co-morbidities. Selfcare management is not easy, and patients will face many challenges, especially when they cannot get use to the new treatment plan. One of the challenges they face is dietary restriction, which is a very important aspect in any self-care management programme. Chronic kidney disease patients require a low-protein, low-sodium, low-potassium, and low-phosphorus diet. There are several strategies patients can undertake to ensure adherence, such as self-monitoring their dietary habits and type of food consumed using a food diary; involving social support, such as family members and spouse to help them to adhere to their diet restrictions; setting goals and providing positive reinforcement when they achieved the targeted goals; joining self-management programmes to equip themselves with the necessary skills so that they can better adhere to the treatment regimes, including diet restriction; and lastly, having the knowledge about their regime, and using this knowledge to help them understand and improve their adherence.

On food security and alternative food networks: Understanding and performing food security in the context of urban bias

This paper offers one explanation for the institutional basis of food insecurity in Australia, and argues that while alternative food networks and the food sovereignty movement perform a valuable function in building forms of social solidarity between urban consumers and rural producers, they currently make only a minor contribution to Australia’s food and nutrition security. The paper begins by identifying two key drivers of food security: household incomes (on the demand side) and nutrition-sensitive, ‘fair food’ agriculture (on the supply side). We focus on this second driver and argue that healthy populations require an agricultural sector that delivers dietary diversity via a fair and sustainable food system. In order to understand why nutrition-sensitive, fair food agriculture is not flourishing in Australia we introduce the development economics theory of urban bias. According to this theory, governments support capital intensive rather than labour intensive agriculture in order to deliver cheap food alongside the transfer of public revenues gained from rural agriculture to urban infrastructure, where the majority of the voting public resides. We chart the unfolding of the Urban Bias across the twentieth century and its consolidation through neo-liberal orthodoxy, and argue that agricultural policies do little to sustain, let alone revitalize, rural and regional Australia. We conclude that by observing food system dynamics through a re-spatialized lens, Urban Bias Theory is valuable in highlighting rural–urban socio-economic and political economy tensions, particularly regarding food system sustainability. It also sheds light on the cultural economy tensions for alternative food networks as they move beyond niche markets to simultaneously support urban food security and sustainable rural livelihoods.

Nursing fever management in adult oncology patients: A literature review

Fever associated with neutropenia, blood transfusion and disease processes is common in adult cancer patients. The literature indicates however that the aetiology, rationale and symptoms of fever are often misunderstood, resulting in fever management that is not evidence-based in this cohort. Thus in this review, an overview of fever, with a focus on fever in cancer contexts, is provided. Content includes an explanation of the therapeutic function of fever, an analysis of the physiological consequences of fever and an exploration of the aetiology of fever in cancer patients. Current guidelines for fever management in cancer patients and existing nursing practice are also discussed.

Doctors’ perspectives on law and life-sustaining treatment: Survey design and recruitment strategies for a challenging cohort

- Background Palliative medicine and other specialists play significant legal roles in decisions to withhold and withdraw life-sustaining treatment at the end of life. Yet little is known about their knowledge of or attitudes to the law, and the role they think it should play in medical practice. Consideration of doctors’ views is critical to optimizing patient outcomes at the end of life. However, doctors are difficult to engage as participants in empirical research, presenting challenges for researchers seeking to understand doctors’ experiences and perspectives. - Aims To determine how to engage doctors involved in end-of-life care in empirical research about knowledge of the law and the role it plays in medical practice at the end of life. - Methods Postal survey of all specialists in palliative medicine, emergency medicine, geriatric medicine, intensive care, medical oncology, renal medicine, and respiratory medicine in three Australian states: New South Wales, Victoria, and Queensland. The survey was sent in hard copy with two reminders and a follow up reminder letter was also sent to the directors of hospital emergency departments. Awareness was further promoted through engagement with the relevant medical colleges and publications in professional journals; various incentives to respond were also used. The key measure is the response rate of doctors to the survey. - Results Thirty-two percent of doctors in the main study completed their survey with response rate by specialty ranging from 52% (palliative care) to 24% (medical oncology). This overall response rate was twice that of the reweighted pilot study (16%). - Conclusions Doctors remain a difficult cohort to engage in survey research but strategic recruitment efforts can be effective in increasing response rate. Collaboration with doctors and their professional bodies in both the development of the survey instrument and recruitment of participants is essential.

Impact of chemotherapy on cancer-related fatigue and cytokines in 1312 patients: A systematic review of quantitative studies

Purpose of review: Cancer-related fatigue (CRF) is the most common psychosomatic distress experienced by cancer patients before, during and after chemotherapy. Its impact on functional status and Health Related Quality of Life is a great concern among patients, healthcare professionals and researchers. The primary objective of this systematic review is to determine whether the different chemotherapies affect the association of CRF with individual pro- and anti-inflammatory cytokines. The PRISMA statement guideline has been followed to systematically search and screen article from PubMed and Embase. Recent findings: This review has examined 14 studies which included a total of 1312 patients. These studies assayed 20 different kinds of cytokines. The cytokines interleukin-6, interleukin-1RA, TGF-[beta] and sTNF-R2 were associated with CRF in patients receiving anthracycline-based chemotherapy. However, only interleukin-13 was identified in the taxane-based chemotherapy. Similarly, different sets of cytokines were linked with CRF in patients with chemotherapy regimens containing platinum, cyclophosphamides, topotecan or bleomycin. Summary: This review has identified that cytokines are differentially linked with CRF according to the various types of chemotherapy regimens.

e-SURAKSHAK: A Cyber-Physical Healthcare System with Service Oriented Architecture

In this paper we present the design of ``e-SURAKSHAK,'' a novel cyber-physical health care management system of Wireless Embedded Internet Devices (WEIDs) that sense vital health parameters. The system is capable of sensing body temperature, heart rate, oxygen saturation level and also allows noninvasive blood pressure (NIBP) measurement. End to end internet connectivity is provided by using 6LoWPAN based wireless network that uses the 802.15.4 radio. A service oriented architecture (SOA) 1] is implemented to extract meaningful information and present it in an easy-to-understand form to the end-user instead of raw data made available by sensors. A central electronic database and health care management software are developed. Vital health parameters are measured and stored periodically in the database. Further, support for real-time measurement of health parameters is provided through a web based GUI. The system has been implemented completely and demonstrated with multiple users and multiple WEIDs.

Práticas de Saúde de Agentes Comunitários no Médio Paraíba/RJ: do institucionalizado à realidade

Esta pesquisa teve como objetivo geral, analisar as práticas de saúde desenvolvidas por ACS, em consonância com os documentos determinados pelo Ministério da Saúde, na ESF na Região do Médio Paraíba, Estado do Rio de Janeiro. Este estudo corresponde ao desdobramento da pesquisa Perfil dos agentes comunitários de saúde do Estado do Rio de Janeiro: análise comparativa entre municípios e regiões em articulação com o estudo Saberes e Práticas na Atenção Primária à Saúde: diálogos entre usuários e profissionais de saúde no Estado do Rio de Janeiro. Trata-se de um estudo descritivo, de natureza qualitativa e teve como abordagem teórico-filosófico, a hermenêutica-dialética. Teve como cenários os municípios de Piraí, Porto Real, Pinheiral, Rio Claro e Rio das Flores. Os sujeitos corresponderam a 53 ACS atuantes nas UBSF dos municípios selecionados. Os instrumentos utilizados para a coleta de dados foram: a observação assistemática, o questionário semiestruturado e o grupo focal. Para a análise dos dados foi utilizada a hermenêutica-dialética proposta por Minayo. Através dos resultados alcançados foi possível realizar uma breve caracterização dos sujeitos provenientes do questionário e a análise do grupo focal desdobrando-se em três categorias. A primeira categoria focou nas práticas de saúde dos ACS que colaboram com a gestão do cuidado. A segunda buscou analisar as práticas de atenção à saúde dos ACS. E a terceira categoria direcionou-se para as práticas educativas dos ACS. Entendemos as práticas de saúde dos ACS acontecem de forma conectada. Pois no cotidiano das práticas não percebemos que as mesmas são realizadas concomitantemente preservando a ideologia da integralidade do cuidado aos usuários da ESF. Encontramos que a maioria dessas práticas de saúde está de acordo com o preconizado pelo MS, porém, identificamos no cotidiano das práticas dos ACS, as que não são abordadas pelos respectivos documentos, como a entrega de recados, a entrega de medicamentos e a atuação na farmácia e recepção das unidades. Assim, podemos afirmar de antemão, que as práticas de saúde dos ACS dos municípios estudados contribuem para a consolidação da Estratégia Saúde da Família no interior do Estado do Rio de janeiro. E além do mais, indicam possíveis desdobramentos para a firmação do modelo da Vigilância da Saúde na Atenção Básica à Saúde.

Physicians in leadership: the association between medical director involvement and staff-to-patient ratios

In a hospital environment that demands a careful balance between commercial and clinical interests, the extent to which physicians are involved in hospital leadership varies greatly. This paper assesses the influence of the extent of this involvement on staff-to-patient ratios. Using data gathered from 604 hospitals across Germany, this study evidences the positive relationship between a full-time medical director (MD) or heavily involved part-time MD and a higher staff-to-patient ratio. The data allows us to control for a range of confounding variables, such as size, rural/urban location, ownership structure, and case-mix. The results contribute to the sparse body of empirical research on the effect of clinical leadership on organizational outcomes.

Memoria-proyecto Unidad de Hospitalización “A” de cirugía ortopédica y rehabilitación neurológica

Este Trabajo Fin de Máster (TFM) propone la elaboración de una memoria proyecto de la unidad de hospitalización A de un centro monográfico de Cirugía Ortopédica y Traumatología (COT) integrado en un hospital general de nivel terciario. Se trata de una memoria con carácter retrospectivo que abarca desde Enero de 2013 hasta Diciembre de 2015. Una memoria que recoge un análisis de la situación junto con todos los aspectos de la nueva organización de la unidad y con propuestas de mejora futuras. Se realiza un análisis externo del modelo organizativo del sistema sanitario regional, del hospital general, de su centro monográfico de COT y de la unidad la cual está integrada en el mismo. El análisis interno ha supuesto una descripción pormenorizada de la unidad, tanto de espacio físico y recursos materiales como de recursos humanos y actividad asistencial junto con los cambios realizados en este periodo y las necesidades que se objetivan para los próximos años y que formarán el eje principal de la parte del proyecto de la presente memoria. Tras ambos análisis se realiza una matriz DAFO, método de ordenación de la información en cuatro categorías básicas: Debilidad, Amenaza, Fortaleza y Oportunidad, a partir del cual se iniciará un proceso de identificación de propuestas de mejora de cara a plantear acciones que se deberían poner en marcha para aprovechar las oportunidades detectadas y permitieran prepararse contra las amenazas teniendo conciencia de las debilidades y fortalezas.

New public management of Higher education, Foucault and power

The theory of New Public Management (NPM) suggest that one of the features of advanced liberal rule is the tendency to define social, economic and political issues as problems to be solved through management. This paper argues that the restructuring of Higher Education (HE) in many Western countries since the 1980s has involved a shift from an emphasis on administration and policy to one of its efficient management. Utilising Foucault’s concept of governmentality rather than the liberal discourse of management as a politically neutral technology, managerialism can be seen as a newly emergent and increasingly rationalised disciplinary regime of governmentalising practices in advanced liberalism. As such the contemporary University as an institution governed by NPM can be demonstrated to have emerged not as the direct outcome of democratic policies that have rationalised its activities (so that the emancipatory aims of personal development, an educated workforce and of true research can be fully realised), nor can it be understood as the instrument through which individuals or self-realising classes are defeated through the calculations of the state acting on behalf of economic interests, rather it can be seen as the contingent and intractable outcome of the complex power/knowledge relations of advanced liberalism. I analyse the interlocking of the ‘tutor-subject’ and ‘student-subject’ as a local enacting of policy discourse informed by the NPM of HE that reshapes subjectivity and retunes the relationship between tutor and student. I put forward suggestions for how resistance to these new modes of disciplinary subjectification can be enacted.

Estimating the economic loss of recent North Atlantic fisheries management

It is accepted that world’s fisheries are not generally exploited at their biological or their economic optimum. Most fisheries assessments focus on the biological capacity of fish stocks to respond to harvesting and few have attempted to estimate the economic efficiency at which ecosystems are exploited. The latter is important as fisheries contribute considerably to the economic development of many coastal communities. Here we estimate the overall potential economic rent for the fishing industry in the North Atlantic to be B€ 12.85, compared to current estimated profits of B€ 0.63. The difference between the potential and the net profits obtained from North Atlantic fisheries is therefore B€ 12.22. In order to increase the profits of North Atlantic fisheries to a maximum, total fish biomass would have to be rebuilt to 108 Mt (2.4 times more than present) by reducing current total fishing effort by 53%. Stochastic simulations were undertaken to estimate the uncertainty associated with the aggregate bioeconomic model that we use and we estimate the economic loss NA fisheries in a range of 2.5 and 32 billion of euro. We provide economic justification for maintaining or restoring fish stocks to above their MSY biomass levels. Our conclusions are consistent with similar global scale studies.