Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

A community based field research project investigating anaemia amongst young children living in rural Karnataka, India: a cross sectional study

<p>Abstract</p> <p>Background</p> <p>Anaemia is an important problem amongst young children living in rural India. However, there has not previously been a detailed study of the biological aetiology of this anaemia, exploring the relative contributions of iron, vitamin B12, folate and Vitamin A deficiency, inflammation, genetic haemoglobinopathy, hookworm and malaria. Nor have studies related these aetiologic biological factors to household food security, standard of living and child feeding practices. Barriers to conducting such work have included perceived reluctance of village communities to permit their children to undergo venipuncture, and logistical issues. We have successfully completed a community based, cross sectional field study exploring in detail the causes of anaemia amongst young children in a rural setting.</p> <p>Methods and design</p> <p>A cross sectional, community based study. We engaged in extensive community consultation and tailored our study design to the outcomes of these discussions. We utilised local women as field workers, harnessing the capacity of local health workers to assist with the study. We adopted a programmatic approach with a census rather than random sampling strategy in the village, incorporating appropriate case management for children identified to have anaemia. We developed a questionnaire based on existing standard measurement tools for standard of living, food security and nutrition. Specimen processing was conducted at the Primary Health Centre laboratory prior to transport to an urban research laboratory.</p> <p>Discussion</p> <p>Adopting this study design, we have recruited 415 of 470 potentially eligible children who were living in the selected villages. We achieved support from the community and cooperation of local health workers. Our results will improve the understanding into anaemia amongst young children in rural India. However, many further studies are required to understand the health problems of the population of rural India, and our study design and technique provide a useful demonstration of a successful strategy.</p

Self-assembled monolayers of alendronate on Ti6Al4V alloy surfaces enhance osteogenesis in mesenchymal stem cells

Phosphonates have emerged as an alternative for functionalization of titanium surfaces by the formation of homogeneous self-assembled monolayers (SAMs) via Ti-O-P linkages. This study presents results from an investigation of the modification of Ti6Al4V alloy by chemisorption of osseoinductive alendronate using a simple, effective and clean methodology. The modified surfaces showed a tailored topography and surface chemistry as determined by SEM microscopy and RAMAN spectroscopy. X-ray photoelectron spectroscopy revealed that an effective mode of bonding is created between the metal oxide surface and the phosphate residue of alendronate, leading to formation of homogenous drug distribution along the surface. In-vitro studies showed that alendronate SAMs induce differentiation of hMSC to a bone cell phenotype and promote bone formation on modified surfaces. Here we show that this novel method for the preparation of functional coatings on titanium-based medical devices provides osseoinductive bioactive molecules to promote enhanced integration at the site of implantation

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Effect of fenugreek (Trigonella foenum-graecum L.) intake on glycemia: A meta-analysis of clinical trials

10.1186/1475-2891-13-7Nutrition Journal131

Global gene expression analyses of bystander and alpha particle irradiated normal human lung fibroblasts: Synchronous and differential responses

<p>Abstract</p> <p>Background</p> <p>The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is now well established. It raises concerns for the interpretation of risks arising from exposure to low doses of ionizing radiation. However, the regulatory mechanisms involved in the bystander response have not been well elucidated. To provide insight into the signaling pathways responding in bystanders, we have measured global gene expression four hours after bystander and direct alpha particle exposure of primary human lung fibroblasts.</p> <p>Results</p> <p>Although common p53-regulated radiation response genes like <it>CDKN1A </it>were expressed at elevated levels in the directly exposed cultures, they showed little or no change in the bystanders. In contrast, genes regulated by NFκB, such as <it>PTGS2 </it>(cyclooxygenase-2), <it>IL8 </it>and <it>BCL2A1</it>, responded nearly identically in bystander and irradiated cells. This trend was substantiated by gene ontology and pathway analyses of the microarray data, which suggest that bystander cells mount a full NFκB response, but a muted or partial p53 response. In time-course analyses, quantitative real-time PCR measurements of <it>CDKN1A </it>showed the expected 4-hour peak of expression in irradiated but not bystander cells. In contrast, <it>PTGS2, IL8 </it>and <it>BCL2A1 </it>responded with two waves of expression in both bystander and directly irradiated cells, one peaking at half an hour and the other between four and six hours after irradiation.</p> <p>Conclusion</p> <p>Two major transcriptional hubs that regulate the direct response to ionizing radiation are also implicated in regulation of the bystander response, but to dramatically different degrees. While activation of the p53 response pathway is minimal in bystander cells, the NFκB response is virtually identical in irradiated and bystander cells. This alteration in the balance of signaling is likely to lead to different outcomes in irradiated cells and their bystanders, perhaps leading to greater survival of bystanders and increased risk from any long-term damage they have sustained.</p

A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone

<p>Abstract</p> <p>Background</p> <p>Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.</p> <p>Methods</p> <p>Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.</p> <p>Results</p> <p>CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER^-PR^-Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.</p> <p>Conclusions</p> <p>The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.</p

Small molecule compounds targeting the p53 pathway: are we finally making progress?

Loss of function of p53, either through mutations in the gene or through mutations to other members of the pathway that inactivate wild-type p53, remains a critically important aspect of human cancer development. As such, p53 remains the most commonly mutated gene in human cancer. For these reasons, pharmacologic activation of the p53 pathway has been a highly sought after, yet unachieved goal in developmental therapeutics. Recently progress has been made not only in the discovery of small molecules that target wild-type and mutant p53, but also in the initiation and completion of the first in-human clinical trials for several of these drugs. Here, we review the current literature of drugs that target wild-type and mutant p53 with a focus on small-molecule type compounds. We discuss common means of drug discovery and group them according to their common mechanisms of action. Lastly, we review the current status of the various drugs in the development process and identify newer areas of p53 tumor biology that may prove therapeutically useful

QCD and strongly coupled gauge theories : challenges and perspectives

We highlight the progress, current status, and open challenges of QCD-driven physics, in theory and in experiment. We discuss how the strong interaction is intimately connected to a broad sweep of physical problems, in settings ranging from astrophysics and cosmology to strongly coupled, complex systems in particle and condensed-matter physics, as well as to searches for physics beyond the Standard Model. We also discuss how success in describing the strong interaction impacts other fields, and, in turn, how such subjects can impact studies of the strong interaction. In the course of the work we offer a perspective on the many research streams which flow into and out of QCD, as well as a vision for future developments.Peer reviewe