The Chemorepellent, Netrin-1, Appears to Signal Through a Tyrosine Kinase in Tetrahymena thermophila

Netrin-1 is a pleiotropic peptide signaling molecule. Its most well-known role in vertebrate development is neuronal guidance. Depending upon the cell type and signal concentration gradient, netrin-1 may serve either as a chemoattractant, causing formation of axonal growth cones, or as a chemorepellent, causing growth cone collapse within the axon. Netrin-1 can bind to at least two types of receptors, and uses a variety of signaling proteins to convey its message. In some vertebrate cell types, the netrin-1 signal is G-protein mediated, while in other cell types, netrin signaling requires a tyrosine kinase or some other combination of kinases in order to signal. Tetrahymena thermophila are free-living, eukaryotic cells that can respond to chemoattractants and chemorepellents by moving toward attractants and away from repellents. By studying the behavior of these organisms, we have found that netrin-1 acts as a chemorepellent in T. thermophila. Response to netrin-1 is concentration dependent, with an EC100 of approximately 1 micromolar, and an EC50 of approximately 10 pM. Netrin-1 avoidance may be effectively eliminated by the addition of the broad-spectrum tyrosine kinase inhibitor, genistein, to the behavioral assay. The IC100 of genistein was approximately 75 micrograms/ml, while the IC50 of this compound was near 50 micrograms/ml. G-protein inhibitors, calcium chelators, and a number of other pharmacological inhibitors had no effect on netrin-1 signaling in this organism. These data show that netrin-1 is a chemorepellent in Tetrahymena thermophila and that netrin signaling appears to implicate a tyrosine kinase in this organism. Further studies will help us to determine whether genistein is specifically acting upon a tyrosine kinase pathway or whether the inhibition is occurring via some other genistein-mediated effect

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that de- fective KARS function is responsible for the phenotypes in these individuals. Conclusions: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease

Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

<p>Abstract</p> <p>Background</p> <p>Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in <it>S. aureus </it>strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (Dc^RS) <it>S. aureus </it>strains.</p> <p>Methods</p> <p>Transcriptional alterations in response to growth with diclofenac were measured using <it>S. aureus </it>gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry.</p> <p>Results</p> <p>Growth of <it>S. aureus </it>strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including <it>mepRAB </it>and a putative <it>emrAB/qacA</it>-family pump. Diclofenac up-regulated <it>sigB </it>(σ^B), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. <it>Staphylococcus aureus </it>microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σ^B-regulated. Diclofenac altered <it>S. aureus </it>susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to Dc^RSdid not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation.</p> <p>Conclusions</p> <p>The results of this study suggest that diclofenac influences antibiotic susceptibility in <it>S. aureus</it>, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.</p

Alterations In the Transciptome and Antibiotic Susceptibility of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e Grown In the Presence of Diclofenac

Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. Conclusions The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport

The clustering of galaxies in the SDSS-III Baryon Oscillation Spectroscopic Survey: measurements of the growth of structure and expansion rate at z=0.57 from anisotropic clustering

We analyze the anisotropic clustering of massive galaxies from the Sloan Digital Sky Survey III Baryon Oscillation Spectroscopic Survey (BOSS) Data Release 9 (DR9) sample, which consists of 264,283 galaxies in the redshift range 0.43 < z < 0.7 spanning 3,275 square degrees. Both peculiar velocities and errors in the assumed redshift-distance relation ("Alcock-Paczynski effect") generate correlations between clustering amplitude and orientation with respect to the line-of-sight. Together with the sharp baryon acoustic oscillation (BAO) standard ruler, our measurements of the broadband shape of the monopole and quadrupole correlation functions simultaneously constrain the comoving angular diameter distance (2190 +/- 61 Mpc) to z=0.57, the Hubble expansion rate at z=0.57 (92.4 +/- 4.5 km/s/Mpc), and the growth rate of structure at that same redshift (d sigma8/d ln a = 0.43 +/- 0.069). Our analysis provides the best current direct determination of both DA and H in galaxy clustering data using this technique. If we further assume a LCDM expansion history, our growth constraint tightens to d sigma8/d ln a = 0.415 +/- 0.034. In combination with the cosmic microwave background, our measurements of DA, H, and growth all separately require dark energy at z > 0.57, and when combined imply \Omega_{\Lambda} = 0.74 +/- 0.016, independent of the Universe's evolution at z<0.57. In our companion paper (Samushia et al. prep), we explore further cosmological implications of these observations.Comment: 19 pages, 11 figures, submitted to MNRAS, comments welcom

Netrin-1 Peptide Is a Chemorepellent in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation i s not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism

HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype.

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine SNPs associated with the extent of abdominal aortic calcification (n = 9,417) or descending thoracic aortic calcification (n = 8,422). Two genetic loci, HDAC9 and RAP1GAP, were associated with abdominal aortic calcification at a genome-wide level (P &lt; 5.0 × 10-8). No SNPs were associated with thoracic aortic calcification at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells promoted calcification and reduced contractility, while inhibition of HDAC9 in human aortic smooth muscle cells inhibited calcification and enhanced cell contractility. In matrix Gla protein-deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a previously unknown role for HDAC9 in the development of vascular calcification

Deciphering the genome structure and paleohistory of _Theobroma cacao_

We sequenced and assembled the genome of _Theobroma cacao_, an economically important tropical fruit tree crop that is the source of chocolate. The assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of them anchored on the 10 _T. cacao_ chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example flavonoid-related genes. It also provides a major source of candidate genes for _T. cacao_ disease resistance and quality improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten _T. cacao_ chromosomes were shaped from an ancestor through eleven chromosome fusions. The _T. cacao_ genome can be considered as a simple living relic of higher plant evolution

Competition for RISC binding predicts in vitro potency of siRNA

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients