Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich Ataxia

Copyright © 2014 Anjomani Virmouni 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.This article has been made available through the Brunel Open Access Publishing Fund.Background - Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats). Methodology/Principal Findings - We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R. Conclusions/Significance - Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.European Union, Ataxia UK and FARA

Analysis of 17,576 Potentially Functional SNPs in Three Case–Control Studies of Myocardial Infarction

Myocardial infarction (MI) is a common complex disease with a genetic component. While several single nucleotide polymorphisms (SNPs) have been reported to be associated with risk of MI, they do not fully explain the observed genetic component of MI. We have been investigating the association between MI and SNPs that are located in genes and have the potential to affect gene function or expression. We have previously published studies that tested about 12,000 SNPs for association with risk of MI, early-onset MI, or coronary stenosis. In the current study we tested 17,576 SNPs that could affect gene function or expression. In order to use genotyping resources efficiently, we staged the testing of these SNPs in three case–control studies of MI. In the first study (762 cases, 857 controls) we tested 17,576 SNPs and found 1,949 SNPs that were associated with MI (P<0.05). We tested these 1,949 SNPs in a second study (579 cases and 1159 controls) and found that 24 SNPs were associated with MI (1-sided P<0.05) and had the same risk alleles in the first and second study. Finally, we tested these 24 SNPs in a third study (475 cases and 619 controls) and found that 5 SNPs in 4 genes (ENO1, FXN (2 SNPs), HLA-DPB2, and LPA) were associated with MI in the third study (1-sided P<0.05), and had the same risk alleles in all three studies. The false discovery rate for this group of 5 SNPs was 0.23. Thus, we have identified 5 SNPs that merit further examination for their potential association with MI. One of these SNPs (in LPA), has been previously shown to be associated with risk of cardiovascular disease in other studies

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress

3D Volume Reconstruction by Serially Acquired 2D Slices Using a Distance Transform-Based Global Cost Function

Abstract. An accurate, computationally eÆcient and fully-automated algorithm for the alignment of 2D serially acquired sections forming a 3D volume is presented. The method accounts for the main shortcomings of 3D image alignment: corrupted data (cuts and tears), dissimilarities or discontinuities between slices, missing slices. The approach relies on the optimization of a global energy function, based on the object shape, measuring the similarity between a slice and its neighborhood in the 3D volume. Slice similarity is computed using the distance transform measure in both directions. No particular direction is privileged in the method avoiding global osets, biases in the estimation and error prop-agation. The method was evaluated on real images (medical, biological and other CT scanned 3D data) and the experimental results demon-strated the method&apos;s accuracy as reconstuction errors are less than 1 degree in rotation and less than 1 pixel in translation.

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions

Data Reduction and Error Analysis for the Physical Sciences

ABSTRACT Polycrystalline thin films (PTF) of p-WSe2, p-WS2, and p-MoSe2 have been prepared and characterized with respect to their photoelectrochemical properties, p-WS2 showed the highest open-circuit photovoltages and the highest conversion efficiencies in various redox couples. In addition, the band structure of all the films has been determined experimentally and compared to those reported for single crystals. Over the last two decades a great deal of interest has developed in the area of photoelectrochemistry, particularly in the application of photoelectrochemical systems to the problem of solar energy conversion and storage. The interest is to develop new energy sources to supplement and eventually replace fossil fuels. The first photoelectrochemical experiment was performed in 1839 by Becquerel (1), who demonstrated that a voltage and current are generated when a silver chloride electrode, immersed in an electrolytic solution and connected to a counterelectrode, is illuminated. Although the concept of a semiconductor did not exist at that time, it is now clear that the electrode which Becquerel used had semiconducting properties. In 1955, Brattain and Garett (2) used germanium as the first semiconductor electrode in photoelectrochemistry. Since then, the knowledge of semiconductors has grown steadily. Fujishima and Honda (3) were the first to point out the potential application of photoelectrochemical systems for solar energy conversion and storage. They demonstrated that the photo-oxidation of water to 02 was possible by utilizing an n-type semiconducting titanium dioxide photoanode. Since then, there has been a large and rapidly growing international interest in the study of photoelectrochemistry of semiconductors (4). The effective use of solar energy in photovoltaic or photoelectrochemical applications depends in part on the development of materials that can show high conversion efficiencies and long-term stability under operation. In ad-*Electrochemical Society Active Member. **Electrochemical Society Student Member. dition, the desirable materials should have a bandgap that closely matches the solar spectrum and be made of readily available and inexpensive materials. We have focused our attention on the transition metal dichalcogenides (e.g., WSe2, WS2, MoSe2, and others), also known as layered or d-d semiconductors. Tributsch&apos;s (5, 6) pioneering work on the use of these materials has stimulated intensive research in this area, and single Crystals of a number of materials have been studied extensively in both aqueous and nonaqueous solvents and in photovoltaic and photoelectrosynthetic cells. The advantages of using these materials are that they have bandgaps (1.1-1.6 eV) that closely match the solar spectrum and exhibit high conversion efficiencies as single crystals. In addition, they can achieve long-term stability due to the fact that the transitions are localized in the nonbonding d orbitals of the metal. These materials consist of metal dichalcogenide sandwiches (e.g., Se-W-Se) held together by van der Waals forces. The fact that there is strong covalent bonding within the layers, but only weak interactions between layers, makes these materials highly anisotropic in their properties. For example, the surface parallel to the C axis (IIC) is more conducting than the surface perpendicular to the C axis (• Therefore, edges and surface imperfections on the surface parallel to the C axis act as efficient recombination centers for photogenerated carriers or products (7

Inter-MAR Association Contributes to Transcriptionally Active Looping Events in Human β-globin Gene Cluster

Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the β-globin gene cluster, it is unclear that how these MAR elements are involved in regulating β-globin genes expression. Here, we report the identification of a new MAR element at the LCR(locus control region) of human β-globin gene cluster and the detection of the inter-MAR association within the β-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in β-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the α-like globin genes and β-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure

Global Assessment of Extinction Risk to Populations of Sockeye Salmon Oncorhynchus nerka

BACKGROUND: Concern about the decline of wild salmon has attracted the attention of the International Union for the Conservation of Nature (IUCN). The IUCN applies quantitative criteria to assess risk of extinction and publishes its results on the Red List of Threatened Species. However, the focus is on the species level and thus may fail to show the risk to populations. The IUCN has adapted their criteria to apply to populations but there exist few examples of this type of assessment. We assessed the status of sockeye salmon Oncorhynchus nerka as a model for application of the IUCN population-level assessments and to provide the first global assessment of the status of an anadromous Pacific salmon. METHODS/PRINCIPAL FINDINGS: We found from demographic data that the sockeye salmon species is not presently at risk of extinction. We identified 98 independent populations with varying levels of risk within the species' range. Of these, 5 (5%) are already extinct. We analyzed the risk for 62 out of 93 extant populations (67%) and found that 17 of these (27%) are at risk of extinction. The greatest number and concentration of extinct and threatened populations is in the southern part of the North American range, primarily due to overfishing, freshwater habitat loss, dams, hatcheries, and changing ocean conditions. CONCLUSIONS/SIGNIFICANCE: Although sockeye salmon are not at risk at the species-level, about one-third of the populations that we analyzed are at risk or already extinct. Without an understanding of risk to biodiversity at the level of populations, the biodiversity loss in salmon would be greatly underrepresented on the Red List. We urge government, conservation organizations, scientists and the public to recognize this limitation of the Red List. We also urge recognition that about one-third of sockeye salmon global population diversity is at risk of extinction or already extinct