Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies

Intracellular reactive oxygen species (ROS) production is essential to normal cell function. However, excessive ROS production causes oxidative damage and cell death. Many pharmacological compounds exert their effects on cell cycle progression by changing intracellular redox state and in many cases cause oxidative damage leading to drug cytotoxicity. Appropriate measurement of intracellular ROS levels during cell cycle progression is therefore crucial in understanding redox-regulation of cell function and drug toxicity and for the development of new drugs. However, due to the extremely short half-life of ROS, measuring the changes in intracellular ROS levels during a particular phase of cell cycle for drug intervention can be challenging. In this article, we have provided updated information on the rationale, the applications, the advantages and limitations of common methods for screening drug effects on intracellular ROS production linked to cell cycle study. Our aim is to facilitate biomedical scientists and researchers in the pharmaceutical industry in choosing or developing specific experimental regimens to suit their research needs.

Cross-sector surveys assessing perceptions of key stakeholders towards barriers, concerns and facilitators to the appropriate use of adaptive designs in confirmatory trials

Background Appropriately conducted adaptive designs (ADs) offer many potential advantages over conventional trials. They make better use of accruing data, potentially saving time, trial participants, and limited resources compared to conventional, fixed sample size designs. However, one can argue that ADs are not implemented as often as they should be, particularly in publicly funded confirmatory trials. This study explored barriers, concerns, and potential facilitators to the appropriate use of ADs in confirmatory trials among key stakeholders. Methods We conducted three cross-sectional, online parallel surveys between November 2014 and January 2015. The surveys were based upon findings drawn from in-depth interviews of key research stakeholders, predominantly in the UK, and targeted Clinical Trials Units (CTUs), public funders, and private sector organisations. Response rates were as follows: 30(55 %) UK CTUs, 17(68 %) private sector, and 86(41 %) public funders. A Rating Scale Model was used to rank barriers and concerns in order of perceived importance for prioritisation. Results Top-ranked barriers included the lack of bridge funding accessible to UK CTUs to support the design of ADs, limited practical implementation knowledge, preference for traditional mainstream designs, difficulties in marketing ADs to key stakeholders, time constraints to support ADs relative to competing priorities, lack of applied training, and insufficient access to case studies of undertaken ADs to facilitate practical learning and successful implementation. Associated practical complexities and inadequate data management infrastructure to support ADs were reported as more pronounced in the private sector. For funders of public research, the inadequate description of the rationale, scope, and decision-making criteria to guide the planned AD in grant proposals by researchers were all viewed as major obstacles. Conclusions There are still persistent and important perceptions of individual and organisational obstacles hampering the use of ADs in confirmatory trials research. Stakeholder perceptions about barriers are largely consistent across sectors, with a few exceptions that reflect differences in organisations’ funding structures, experiences and characterisation of study interventions. Most barriers appear connected to a lack of practical implementation knowledge and applied training, and limited access to case studies to facilitate practical learning. Keywords: Adaptive designs; flexible designs; barriers; surveys; confirmatory trials; Phase 3; clinical trials; early stopping; interim analyses

Copyright exceptions and contract in the UK: the impact of recent amendments

In June and October 2014 a number of amendments to the Copyright, Designs and Patents Act 1988 came into effect, introducing new copyright exceptions and amending several existing exceptions. Whilst following recent judicial review the private copying exception has been quashed, many of the remaining new exceptions significantly alter the relationship between copyright exceptions and contract, making contractual terms unenforceable where those terms restrict users from taking advantage of an exception. This paper explores the rationale for the UK amendments and considers whether the changes, as implemented, prevent rightsholders from contracting out of exceptions and whether they increase the clarity of the copyright system.

SHIMMER (1.0): a novel mathematical model for microbial and biogeochemical dynamics in glacier forefield ecosystems

SHIMMER (Soil biogeocHemIcal Model for Microbial Ecosystem Response) is a new numerical modelling framework designed to simulate microbial dynamics and biogeochemical cycling during initial ecosystem development in glacier forefield soils. However, it is also transferable to other extreme ecosystem types (such as desert soils or the surface of glaciers). The rationale for model development arises from decades of empirical observations in glacier forefields, and enables a quantitative and process focussed approach. Here, we provide a detailed description of SHIMMER, test its performance in two case study forefields: the Damma Glacier (Switzerland) and the Athabasca Glacier (Canada) and analyse sensitivity to identify the most sensitive and unconstrained model parameters. Results show that the accumulation of microbial biomass is highly dependent on variation in microbial growth and death rate constants, Q10 values, the active fraction of microbial biomass and the reactivity of organic matter. The model correctly predicts the rapid accumulation of microbial biomass observed during the initial stages of succession in the forefields of both the case study systems. Primary production is responsible for the initial build-up of labile substrate that subsequently supports heterotrophic growth. However, allochthonous contributions of organic matter, and nitrogen fixation, are important in sustaining this productivity. The development and application of SHIMMER also highlights aspects of these systems that require further empirical research: quantifying nutrient budgets and biogeochemical rates, exploring seasonality and microbial growth and cell death. This will lead to increased understanding of how glacier forefields contribute to global biogeochemical cycling and climate under future ice retreat.

Influence of implant positioning in extraction sockets on osseointegration: histomorphometric analyses in dogs

Aim To evaluate the influence of implant positioning into extraction sockets on osseointegration. Material and methods Implants were installed immediately into extraction sockets in the mandibles of six Labrador dogs. In the control sites, the implants were positioned in the center of the alveolus, while in the test sites, the implants were positioned 0.8 mm deeper and more lingually. After 4 months of healing, the resorptive patterns of the alveolar crest were evaluated histomorphometrically. Results All implants were integrated in mineralized bone, mainly composed of mature lamellar bone. The alveolar crest underwent resorption at the control as well as at the test sites. After 4 months of healing, at the buccal aspects of the control and test sites, the location of the implant rough/smooth limit to the alveolar crest was 2 +/- 0.9 mm and 0.6 +/- 0.9 mm, respectively (P < 0.05). At the lingual aspect, the bony crest was located 0.4 mm apically and 0.2 mm coronally to the implant rough/smooth limit at the control and test sites, respectively (NS). Conclusions From a clinical point of view, implants installed into extraction sockets should be positioned approximately 1 mm deeper than the level of the buccal alveolar crest and in a lingual position in relation to the center of the alveolus in order to reduce or eliminate the exposure above the alveolar crest of the endosseous (rough) portion of the implant. To cite this article:Caneva M, Salata LA, de Souza SS, Baffone G, Lang NP, Botticelli D. Influence of implant positioning in extraction sockets on osseointegration: histomorphometric analyses in dogs.Clin. Oral Impl. Res. 21, 2010; 43-49.

Exercise improves cardiovascular control in a model of dislipidemia and menopause

The present study investigated the effects of exercise training on arterial pressure, baroreflex sensitivity, cardiovascular autonomic control and metabolic parameters on female LDL-receptor knockout ovariectomized mice. Mice were divided into two groups: sedentary and trained. Trained group was submitted to an exercise training protocol. Blood cholesterol was measured. Arterial pressure (AP) signals were directly recorded in conscious mice. Baroreflex sensitivity was evaluated by tachycardic and bradycardic responses to AP changes. Cardiovascular autonomic modulation was measured in frequency (FFT) and time domains. Maximal exercise capacity was increased in trained as compared to sedentary group. Blood cholesterol was diminished in trained mice (191 +/- 8 mg/dL) when compared to sedentary mice (250 +/- 9 mg/dL, p<0.05). Mean AP and HR were reduced in trained group (101 +/- 3 mmHg and 535 +/- 14 bpm, p<0.05) when compared with sedentary group (125 +/- 3 mmHg and 600 +/- 12 bpm). Exercise training induced improvement in bradycardic reflex response in trained animals (-4.24 +/- 0.62 bpm/mmHg) in relation to sedentary animals (-1.49 +/- 0.15 bpm/mmHg, p<0.01); tachycardic reflex responses were similar between studied groups. Exercise training increased the variance (34 +/- 8 vs. 6.6 +/- 1.5 ms(2) in sedentary, p<0.005) and the high-frequency band (HF) of the pulse interval (IP) (53 +/- 7% vs. 26 +/- 6% in sedentary, p<0.01). It is tempting to speculate that results of this experimental study might represent a rationale for this non-pharmacological intervention in the management of cardiovascular risk factors in dyslipidemic post-menopause women. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

6-Sulfatoxymelatonin as a predictor of clinical outcome in depressive patients

Objectives This study established the value of the 6-sulfatoxymelatonin (aMT6s) urine concentration as a predictor of the therapeutic response to noradrenaline reuptake inhibitors in depressive patients. Methods Twenty-two women aged 18-60 years were selected. Depressive symptoms were assessed by using the Hamilton Depression Scale. Urine samples were collected at 0600-1200 h, 1200-1800 h, 1800-2400 h, and 2400-0600 h intervals, 1 day before and 1 day after starting on the nortriptyline treatment. Urine aMT6s concentration was analyzed by a one-way analysis of variance/Bonferroni test. Spearman`s rank correlation coefficient was used to analyze the correlation between depressive symptoms after 2 weeks of antidepressant treatment and the increase in aMT6s urine concentration. Results Higher and lower size effect groups were compared by independent Student`s t-tests. At baseline, the 2400- to 0600-h interval differed from all other intervals presenting a significantly higher aMT6s urine concentration. A significant difference in aMT6s urine concentrations was found 1 day after treatment in all four intervals. Higher size effect group had lower levels of depressive symptoms 2 weeks after the treatment. A positive correlation between depressive symptoms and the delta of aMT6s in the 2400-0600h interval was observed. Conclusion Our results reinforce the hypothesis that aMT6s excretion is a predictor of clinical outcome in depression, especially in regard to noradrenaline reuptake inhibitors. Copyright (C) 2011 John Wiley & Sons, Ltd.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Myeloperoxidase as Inflammatory Marker of Periodontal Disease: Experimental Study in Rats

Rationale: Previous studies have used myeloperoxidase (MPO) as an inflammatory marker to estimate the accumulation of neutrophils in inflamed regions. Objective: The aim of this experimental study was to quantify the levels of MPO related to experimental periodontal disease in rats. Methods: Periodontal disease was induced in a group of rats using placement of a ligature around molar teeth. A group of rats without ligature placement served as a control. Measurements were made on the 3rd, 7th, 15th and 30th day from baseline. Gingival tissues were taken for quantification of MPO levels by ELISA. Results: The rats with induced periodontal disease showed statistically higher MPO levels (p 0.05) when compared to control rats. A significant increase in the levels of MPO released on days 7 and 30 was observed, with higher levels in the group with induced periodontitis. Conclusion: The levels of MPO were found to be higher in rats with induced periodontal disease, confirming the hypothesis that MPO may serve as an inflammatory marker for periodontitis.

Genetic Algorithm for the Determination of Linear Viscoelastic Relaxation Spectrum from Experimental Data

Conventional procedures employed in the modeling of viscoelastic properties of polymer rely on the determination of the polymer`s discrete relaxation spectrum from experimentally obtained data. In the past decades, several analytical regression techniques have been proposed to determine an explicit equation which describes the measured spectra. With a diverse approach, the procedure herein introduced constitutes a simulation-based computational optimization technique based on non-deterministic search method arisen from the field of evolutionary computation. Instead of comparing numerical results, this purpose of this paper is to highlight some Subtle differences between both strategies and focus on what properties of the exploited technique emerge as new possibilities for the field, In oder to illustrate this, essayed cases show how the employed technique can outperform conventional approaches in terms of fitting quality. Moreover, in some instances, it produces equivalent results With much fewer fitting parameters, which is convenient for computational simulation applications. I-lie problem formulation and the rationale of the highlighted method are herein discussed and constitute the main intended contribution. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 113: 122-135, 2009

Orbital-polarization terms: From a phenomenological to a first-principles description of orbital magnetism in density-functional theory

Phenomenological orbital-polarizition (OP) terms have been repeatedly introduced in the single-particle equations of spin-density-functional theory, in order to improve the description of orbital magnetic moments in systems containing transition metal ions. Here we show that these ad hoc corrections can be interpreted as approximations to the exchange-correlation vector potential A(xc) of current-density functional theory (CDFT). This connection provides additional information on both approaches: phenomenological OP terms are connected to first-principles theory, leading to a rationale for their empirical success and a reassessment of their limitations and the approximations made in their derivation. Conversely, the connection of OP terms with CDFT leads to a set of simple approximations to the CDFT potential A(xc), with a number of desirable features that are absent from electron-gas-based functionals. (C) 2008 Wiley Periodicals, Inc.

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.

Historielärares ämnesförståelse : Centrala begrepp i historielärares förståelse av skolämnet historia

This thesis focuses on four different aspects of history teachers’ comprehensiveunderstanding of the school subject history. More specifically, the aim is tostudy the comprehension of the subject as perceived by individual historyteachers. Special emphasis is placed on identifying the concepts of the field ofhistory that are central to the teachers’ understanding of the school subject history.The first aspect studied is the teachers’ biographical changes. In a life historyperspective it seems as if the teachers’ subject conception changes from anunproblematic and tentative approach to a more complex and confident understandingof the subject. The second aspect treated is the rationale behind theirgrasp of the purpose and content of the subject. Three major positions areidentified, namely educational (bildung) orientation, critical orientation, andidentity orientation.The third aspect studied is the teachers’ interpretation of a curriculumnew to them. The teachers placed the curriculum in the field of tension betweenan education policy position, emphasizing more precise knowledge, onthe one hand, and a history science position, emphasizing concepts of historicalconsciousness. The fourth aspect studied is five different conceptual tools displayedin the teachers’ remarks on having completed the teaching of a newcourse. These are termed ‘history as narrative’, ‘history as time-space’, ‘historyas explanation’, ‘history as perspective taking’, and ‘history as skills’At the general level the study shows not only that subject conception is ofimportance to the teachers’ understanding of their obligation as teachers of historybut also how it is formed and constantly transformed by many differentfactors. In this process it is clear that the concepts used by the teachers, althoughvariously defined, can be seen as specific to the school subject historyand essential to the construction of history as a school subject.

The effectiveness of proactive telephone support provided to breastfeeding mothers of preterm infants : study protocol for a randomized controlled trial

Background: Although breast milk has numerous benefits for infants' development, with greater effects in those born preterm (at < 37 gestational weeks), mothers of preterm infants have shorter breastfeeding duration than mothers of term infants. One of the explanations proposed is the difficulties in the transition from a Neonatal Intensive Care Unit (NICU) to the home environment. A person-centred proactive telephone support intervention after discharge from NICU is expected to promote mothers' sense of trust in their own capacity and thereby facilitate breastfeeding. Methods/design: A multicentre randomized controlled trial has been designed to evaluate the effectiveness and cost-effectiveness of person-centred proactive telephone support on breastfeeding outcomes for mothers of preterm infants. Participating mothers will be randomized to either an intervention group or control group. In the intervention group person-centred proactive telephone support will be provided, in which the support team phones the mother daily for up to 14 days after hospital discharge. In the control group, mothers are offered a person-centred reactive support where mothers can phone the breastfeeding support team up to day 14 after hospital discharge. The intervention group will also be offered the same reactive telephone support as the control group. A stratified block randomization will be used; group allocation will be by high or low socioeconomic status and by NICU. Recruitment will be performed continuously until 1116 mothers (I: 558 C: 558) have been included. Primary outcome: proportion of mothers exclusively breastfeeding at eight weeks after discharge. Secondary outcomes: proportion of breastfeeding (exclusive, partial, none and method of feeding), mothers satisfaction with breastfeeding, attachment, stress and quality of life in mothers/partners at eight weeks after hospital discharge and at six months postnatal age. Data will be collected by researchers blind to group allocation for the primary outcome. A qualitative evaluation of experiences of receiving/providing the intervention will also be undertaken with mothers and staff. Discussion: This paper presents the rationale, study design and protocol for a RCT providing person-centred proactive telephone support to mothers of preterm infants. Furthermore, with a health economic evaluation, the cost-effectiveness of the intervention will be assessed. Trial registration: NCT01806480