Aromatic Polyketide Production in <i>Cordyceps indigotica</i>, an Entomopathogenic Fungus, Induced by Exposure to a Histone Deacetylase Inhibitor

Cultivation of <i>Cordyceps indigotica</i>, an entomopathogenic fungus, in the presence of suberoyl bis-hydroxamic acid (an HDAC inhibitor) greatly activated its polyketide synthesis apparatus to afford six novel aromatic polyketides, indigotides C–F (<b>1</b>–<b>4</b>), 13-hydroxyindigotide A (<b>5</b>), and 8-<i>O</i>-methylindigotide B (<b>6</b>). The structures of these compounds were determined by NMR spectroscopic analyses. Among the compounds, indigotides C–E (<b>1</b>–<b>3</b>) possessed unprecedented dimeric polyketide frameworks possibly generated via a [4 + 2] cycloaddition or Michael type reaction

Establishment of <i>pten</i> knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

<div><p>Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The <i>PTEN</i> gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two <i>pten</i> genes, <i>ptena</i> and <i>ptenb</i>, similar to zebrafish. We established 16 <i>ptena</i> mutant lines and two <i>ptenb</i> mutant lines. Homozygous single <i>pten</i> mutants were found to be viable and fertile. In contrast, <i>pten</i> double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in <i>pten</i> dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of <i>pten</i> dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and <i>N</i>-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows <i>pten</i> dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.</p></div

Effects of rapamycin on <i>pten</i> dko embryos.

<p>(<b>A</b>) A total of 41 embryos obtained from <i>ptena</i>^+/−<i>ptenb</i>^−/− parents was treated with 5 μM rapamycin for 96 h beginning at 26 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. Eight of the 41 embryos had the <i>ptena</i>^−/−<i>ptenb</i>^−/− genotype (images 1–8), nine were <i>ptena</i>^+/−<i>ptenb</i>^−/−(image 9), and 10 were <i>ptena</i>^+/+<i>ptenb</i>^−/−(image 10). Partial Cuvierian ducts developed in seven of the eight dko embryos. X, no duct. (<b>C</b>) Snapshots from movies of the dko embryos shown in images 2 (upper) and 4 (lower) in (B). Blood cells can be seen flowing through the Cuvierian duct (upper) and the tail vascular duct (lower) indicated by the arrows. Times are in seconds.</p

PI3K-AKT signaling pathway activity in adult <i>pten</i> mutant medaka.

<p>Extracts (20 μg of protein) derived from the dorsal muscle of 7- to 10-mpf fish were subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of AKT as well as with those to α-tubulin (loading control). Each lane corresponds to an individual. The pAKT/AKT ratio for individual fish of the indicated <i>pten</i> genotypes was determined by densitometry. There were another two independent replications that showed much the same results.</p

Characterization of medaka PTEN genes and design of TALEN constructs.

<p><b>(A</b>) RT-PCR analysis of total RNA isolated from whole embryos of medaka was performed with two sets of primers designed to amplify almost the entire open reading frames of <i>ptena</i> (lane 1) and <i>ptenb</i> (lane 2). The arrow indicates the specific amplification products, with the faster-migrating bands being found to represent technical artifacts by sequencing analysis. Lane M, molecular size markers. (<b>B</b>) Products of medaka <i>ptena</i> and <i>ptenb</i> predicted from the sequences of the amplified cDNAs and database information. Arrowheads labeled #1 and #2 indicate the localization of the TALEN target sequences shown in (C). (<b>C</b>) TALEN target sites in <i>ptena</i> and <i>ptenb</i>.</p

Effects of LY294002 on vascular development and blood cell flow in <i>pten</i> dko embryos.

<p>(<b>A</b>) Wild-type or <i>pten</i> dko embryos were exposed (or not) to 15 μM LY294002 for 48 h beginning at 30 or 48 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. The dko embryos treated with LY294002 developed partial Cuvierian ducts (4 of 4 treated at 30 hpf, and 2 of 3 treated at 48 hpf). The dko embryos not exposed to the drug (2/2) did not manifest vasculogenesis. X, no duct. (<b>C</b>) Snapshots from a movie of a 4-dpf dko embryo that had been treated with LY294002 for 48 h beginning at 30 hpf. Blood cells can be seen flowing through the Cuvierian duct (arrow). Times are indicated in seconds.</p

Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.

<p>Mutation efficiency for <i>pten</i> TALEN constructs revealed by analysis of G0 medaka embryos.</p

Generation of Strong Acid Sites on Yttrium-Doped Tetragonal ZrO₂‑Supported Tungsten Oxides: Effects of Dopant Amounts on Acidity, Crystalline Phase, Kinds of Tungsten Species, and Their Dispersion

A tungsten oxide catalysts supported on yttrium- or ytterbium-doped crystalline zirconium oxide (WO_<i>x</i>/Y­(Yb)-ZrO₂) with different dopant concentrations but the same tungsten loadings and calcination temperatures were prepared to investigate the mechanism responsible for the strong solid acidity of so-called tungstated zirconia. The surface acid–base properties of WO_<i>x</i>/Y­(Yb)-ZrO₂ and the support were characterized by catalyses of <i>n</i>-butane skeletal isomerization, alkylation of anisole with benzyl alcohol, and 2-butanol decomposition. The structural characterization and quantitative analysis were conducted by XRD, XAFS, and UV–Vis techniques. Crystalline zirconium oxides were effective as the starting material for tungsten–zirconium-based strong solid acid catalysts instead of amorphous Zr­(OH)_<i>x</i> if the crystalline form was tetragonal. Tetragonal-phase Y­(Yb)-ZrO₂ substitutional solid solutions with dopant concentrations of 3–5 mol % was a suitable support for stabilization of active small WO₃ clusters. Tungsten species aggregated to form inactive large WO₃ particles on the monoclinic ZrO₂ polymorph with Y-dopant amount less than 2 mol % even though the surface density of tungsten species and calcination temperature were the same as those for the active catalysts. Inactive yttrium–tungsten binary oxides formed when the yttrium doping level exceeded 10 mol %. Intrinsic role of crystalline phase of tetragonal zirconia on generating the strong solid acidity is confirmed to stabilize WO₃ small clusters without aggregation

Activity time budget of red-legged kittiwakes during foraging trips as revealed by GPS loggers and accelerometers.

<p>^a Generalized linear mixed model (a binomial distribution) with likelihood ratio test was used. Trip identity and bird identity were included as fixed factor and random effect, respectively. See "Statistics" in the "<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138850#sec002" target="_blank">Materials and Methods</a>".</p><p>Activity time budget of red-legged kittiwakes during foraging trips as revealed by GPS loggers and accelerometers.</p

Behavioral patterns of red-legged kittiwakes as revealed by accelerometers.

<p>Panel A shows a time series of depth, heave acceleration, body angle, dominant amplitude of the heave acceleration, dominant cycle of the heave acceleration, and behavioral categories determined by accelerometry (the colors correspond to each behavioral category). Panel B shows enlarged records from panel A.</p