Applicability of control materials to support gene promoter characterization and expression in engineered cells using digital PCR

The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology “toolkit” of methods and materials to support the current standardization efforts

Wellness through a comprehensive Yogic breathing program – A controlled pilot trial

<p>Abstract</p> <p>Background</p> <p>Increasing rates of psychosocial disturbances give rise to increased risks and vulnerability for a wide variety of stress-related chronic pain and other illnesses. Relaxation exercises aim at reducing stress and thereby help prevent these unwanted outcomes. One of the widely used relaxation practices is yoga and yogic breathing exercises. One specific form of these exercises is Sudarshan Kriya and related practices (SK&P) which are understood to have favourable effects on the mind-body system. The goal of this pilot study was to design a protocol that can investigate whether SK&P can lead to increased feeling of wellness in healthy volunteers.</p> <p>Methods</p> <p>Participants were recruited in a small university city in Sweden and were instructed in a 6-day intensive program of SK&P which they practiced daily for six weeks. The control group was instructed to relax in an armchair each day during the same period. Subjects included a total of 103 adults, 55 in the intervention (SK&P) group and 48 in the control group. Various instruments were administered before and after the intervention. Hospital Anxiety Depression Scale measured the degree of anxiety and depression, Life Orientation Test measured dispositional optimism, Stress and Energy Test measured individual's energy and stress experiences. Experienced Deviation from Normal State measured the experience of altered state of consciousness.</p> <p>Results</p> <p>There were no safety issues. Compliance was high (only 1 dropout in the SK&P group, and 5 in the control group). Outcome measures appeared to be appropriate for assessing the differences between the groups. Subjective reports generally correlated with the findings from the instruments. The data suggest that participants in the SK&P group, but not the control group, lowered their degree of anxiety, depression and stress, and also increased their degree of optimism (ANOVA; p < 0.001). The participants in the yoga group experienced the practices as a positive event that induced beneficial effects.</p> <p>Conclusion</p> <p>These data indicate that the experimental protocol that is developed here is safe, compliance level is good, and a full scale trial is feasible. The data obtained suggest that adult participants may improve their wellness by learning and applying a program based on yoga and yogic breathing exercises; this can be conclusively assessed in a large-scale trial.</p> <p>Trial Registration</p> <p>Australian Clinical Trial Registry ACTRN012607000175471.</p

Increased expression of the ubiquitin – proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-κB

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκBα levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκBα, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκBα from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK

Ablative therapy for people with localised prostate cancer : a systematic review and economic evaluation

The research reported in this issue of the journal was funded by the HTA programme as project number 10/136/01. The contractual start date was in April 2012. The draft report began editorial review in October 2013 and was accepted for publication in April 2014. The authors have been wholly responsible for all data collection, analysis and interpretation, and for writing up their work. The HTA editors and publisher have tried to ensure the accuracy of the authors’ report and would like to thank the reviewers for their constructive comments on the draft document. However, they do not accept liability for damages or losses arising from material published in this report. Acknowledgements We thank l the people recruited from the local UCAN for providing valuable consumer insight and advice through their participation as members of the project focus group: - Mark Emberton (Professor of Interventional Oncology), Damian Greene (consultant urologist), Axel Heidenreich (Professor and Director of Department of Urology), Christoph von Klot (specialist in brachytherapy), Roger Kockelbergh (BAUS chairman and Clinical Director of Urology) and Axel Merserburger (Deputy Clinical Director of Urology and Urologic Oncology) for providing their clinical expertise as members of the project advisory group - Edgar Paez (consultant urologist) and Gill Lawrence (Head of Radiotherapy Physics) for providing a list of staff time by grade and specialty involved in EBRT - Debbie Bennett (Radiotherapy Service Manager) for providing estimates for the expected number of uses for EBRT - Ian Pedley (clinical director/clinical oncologist) and Gill Lawrence for providing a list of all resource inputs relevant to brachytherapy - Steve Locks (Consultant Clinical Scientist in Radiotherapy) for providing a list of reusable equipment and consumables used during brachytherapy, along with their unit costs - Sue Asterling (urology research nurse) and Mark Kelly (Acting Divisional General Manager – Theatres) for providing a list of all resource inputs relevant to cryotherapy - Lara Kemp for providing secretarial support. The Health Services Research Unit is core funded by the Chief Scientist Office of the Scottish Government Health Directorates.Peer reviewedPublisher PD

Increasing the Depth of Current Understanding: Sensitivity Testing of Deep-Sea Larval Dispersal Models for Ecologists

Larval dispersal is an important ecological process of great interest to conservation and the establishment of marine protected areas. Increasing numbers of studies are turning to biophysical models to simulate dispersal patterns, including in the deep-sea, but for many ecologists unassisted by a physical oceanographer, a model can present as a black box. Sensitivity testing offers a means to test the models' abilities and limitations and is a starting point for all modelling efforts. The aim of this study is to illustrate a sensitivity testing process for the unassisted ecologist, through a deep-sea case study example, and demonstrate how sensitivity testing can be used to determine optimal model settings, assess model adequacy, and inform ecological interpretation of model outputs. Five input parameters are tested (timestep of particle simulator (TS), horizontal (HS) and vertical separation (VS) of release points, release frequency (RF), and temporal range (TR) of simulations) using a commonly employed pairing of models. The procedures used are relevant to all marine larval dispersal models. It is shown how the results of these tests can inform the future set up and interpretation of ecological studies in this area. For example, an optimal arrangement of release locations spanning a release area could be deduced; the increased depth range spanned in deep-sea studies may necessitate the stratification of dispersal simulations with different numbers of release locations at different depths; no fewer than 52 releases per year should be used unless biologically informed; three years of simulations chosen based on climatic extremes may provide results with 90% similarity to five years of simulation; and this model setup is not appropriate for simulating rare dispersal events. A step-by-step process, summarising advice on the sensitivity testing procedure, is provided to inform all future unassisted ecologists looking to run a larval dispersal simulation

Hypoxia and oxidative stress in breast cancer: Tumour hypoxia – therapeutic considerations

Conclusive research has shown that regions of acute/chronic hypoxia, which exist within the majority of solid tumours, have a profound influence on the therapeutic outcome of cancer chemotherapy and radiotherapy and are a strong prognostic factor of disease progression and survival. A strong argument therefore exists for assessing the hypoxic fraction of tumours, prior to patient treatment, and to tailor this treatment accordingly. Tumour hypoxia also provides a powerful physiological stimulus that can be exploited as a tumour-specific condition, allowing for the rationale design of hypoxia-activated anticancer drugs or novel hypoxia-regulated gene therapy strategies

Model-Derived Dispersal Pathways from Multiple Source Populations Explain Variability of Invertebrate Larval Supply

Background: Predicting the spatial and temporal patterns of marine larval dispersal and supply is a challenging task due to the small size of the larvae and the variability of oceanographic processes. Addressing this problem requires the use of novel approaches capable of capturing the inherent variability in the mechanisms involved. Methodology/Principal Findings: In this study we test whether dispersal and connectivity patterns generated from a biophysical model of larval dispersal of the crab Carcinus maenas, along the west coast of the Iberian Peninsula, can predict the highly variable daily pattern of wind-driven larval supply to an estuary observed during the peak reproductive season (March–June) in 2006 and 2007. Cross-correlations between observed and predicted supply were significant (p,0.05) and strong, ranging from 0.34 to 0.81 at time lags of 26 to+5 d. Importantly, the model correctly predicted observed cross-shelf distributions (Pearson r = 0.82, p,0.001, and r = 0.79, p,0.01, in 2006 and 2007) and indicated that all supply events were comprised of larvae that had been retained within the inner shelf; larvae transported to the outer shelf and beyond never recruited. Estimated average dispersal distances ranged from 57 to 198 km and were only marginally affected by mortality. Conclusions/Significance: The high degree of predicted demographic connectivity over relatively large geographic scales is consistent with the lack of genetic structuring in C. maenas along the Iberian Peninsula. These findings indicate that the dynamic nature of larval dispersal can be captured by mechanistic biophysical models, which can be used to provid

Development of a multi-dimensional measure of resilience in adolescents: the Adolescent Resilience Questionnaire

Background: The concept of resilience has captured the imagination of researchers and policy makers over the past two decades. However, despite the ever growing body of resilience research, there is a paucity of relevant, comprehensive measurement tools. In this article, the development of a theoretically based, comprehensive multidimensional measure of resilience in adolescents is described.Methods: Extensive literature review and focus groups with young people living with chronic illness informed the conceptual development of scales and items. Two sequential rounds of factor and scale analyses were undertaken to revise the conceptually developed scales using data collected from young people living with a chronic illness and a general population sample.Results: The revised Adolescent Resilience Questionnaire comprises 93 items and 12 scales measuring resilience factors in the domains of self, family, peer, school and community. All scales have acceptable alpha coefficients. Revised scales closely reflect conceptually developed scales.Conclusions: It is proposed that, with further psychometric testing, this new measure of resilience will provide researchers and clinicians with a comprehensive and developmentally appropriate instrument to measure a young person&rsquo;s capacity to achieve positive outcomes despite life stressors.<br /

Recruitment Constraints in Singapore's Fluted Giant Clam (Tridacna squamosa) Populations - A Dispersal Model Approach

10.1371/journal.pone.0058819PLoS ONE83