Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting

Designing minimal effective normative systems with the help of lightweight formal methods

Normative systems (i.e., a set of rules) are an important approach to achieving effective coordination among (often an arbitrary number of) agents in multiagent systems. A normative system should be effective in ensuring the satisfaction of a desirable system property, and minimal (i.e., not containing norms that unnecessarily over-constrain the behaviors of agents). Designing or even automatically synthesizing minimal effective normative systems is highly non-trivial. Previous attempts on synthesizing such systems through simulations often fail to generate normative systems which are both minimal and effective. In this work, we propose a framework that facilitates designing of minimal effective normative systems using lightweight formal methods. Given a minimal effective normative system which coordinates many agents must be minimal and effective for a small number of agents, we start with automatically synthesizing one such system with a few agents. We then increase the number of agents so as to check whether the same design remains minimal and effective. If it is, we manually establish an induction proof so as to lift the design to an arbitrary number of agents

Detrended fluctuation analysis as a statistical tool to monitor the climate

Detrended fluctuation analysis is used to investigate power law relationship between the monthly averages of the maximum daily temperatures for different locations in the western US. On the map created by the power law exponents, we can distinguish different geographical regions with different power law exponents. When the power law exponents obtained from the detrended fluctuation analysis are plotted versus the standard deviation of the temperature fluctuations, we observe different data points belonging to the different climates, hence indicating that by observing the long-time trends in the fluctuations of temperature we can distinguish between different climates.Comment: 8 pages, 4 figures, submitted to JSTA

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

Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus

© 2013 Al-Mahdawi 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.Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP

MutLα heterodimers modify the molecular phenotype of Friedreich ataxia

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA), the most common autosomal recessive ataxia disorder, is caused by a dynamic GAA repeat expansion mutation within intron 1 of FXN gene, resulting in down-regulation of frataxin expression. Studies of cell and mouse models have revealed a role for the mismatch repair (MMR) MutS-heterodimer complexes and the PMS2 component of the MutLα complex in the dynamics of intergenerational and somatic GAA repeat expansions: MSH2, MSH3 and MSH6 promote GAA repeat expansions, while PMS2 inhibits GAA repeat expansions. Methodology/Principal Findings: To determine the potential role of the other component of the MutLα complex, MLH1, in GAA repeat instability in FRDA, we have analyzed intergenerational and somatic GAA repeat expansions from FXN transgenic mice that have been crossed with Mlh1 deficient mice. We find that loss of Mlh1 activity reduces both intergenerational and somatic GAA repeat expansions. However, we also find that loss of either Mlh1 or Pms2 reduces FXN transcription, suggesting different mechanisms of action for Mlh1 and Pms2 on GAA repeat expansion dynamics and regulation of FXN transcription. Conclusions/Significance: Both MutLα components, PMS2 and MLH1, have now been shown to modify the molecular phenotype of FRDA. We propose that upregulation of MLH1 or PMS2 could be potential FRDA therapeutic approaches to increase FXN transcription. © 2014 Ezzatizadeh et al.This article has been made available through the Brunel Open Access Publishing Fund

The building up of the disk galaxy M33 and the evolution of the metallicity gradient

The evolution of radial gradients of metallicity in disk galaxies and its relation with the disk formation are not well understood. Theoretical models of galactic chemical evolution make contrasting predictions about the time evolution of metallicity gradients. To test chemical evolution models and trace the star formation and accretion history of low luminosity disk galaxies we focus on the Local Group galaxy M33. We analyze O/H and S/H abundances in planetary nebulae, H{\sc ii} regions, and young stars, together with known [Fe/H] abundances in the old stellar population of M33. With a theoretical model, we follow the time evolution of gas (diffuse and condensed in clouds), stars, and chemical abundances in the disk of M33, assuming that the galaxy is accreting gas from an external reservoir. Our model is able to reproduce the available observational constraints on the distribution of gas and stars in M33 and to predict the time evolution of several chemical abundances. In particular, we find that a model characterized by a continuous infall of gas on the disk, at a rate of $\dot M_{\rm inf}\approx 1$ $M_\odot$ yr$^{-1}$, almost constant with time, can also account for the relatively high rate of star formation and for the shallow chemical gradients. Supported by a large sample of high resolution observations for this nearby galaxy, we conclude that the metallicity in the disk of M33 has increased with time at all radii, with a continuous flattening of the gradient over the last $\sim 8$ Gyr.Comment: 16 pages, 11 figures, A&A accepte