Time clustered sampling can inflate the inferred substitution rate in foot-and-mouth disease virus analyses

With the emergence of analytical software for the inference of viral evolution, a number of studies have focused on estimating important parameters such as the substitution rate and the time to the most recent common ancestor (tMRCA) for rapidly evolving viruses. Coupled with an increasing abundance of sequence data sampled under widely different schemes, an effort to keep results consistent and comparable is needed. This study emphasizes commonly disregarded problems in the inference of evolutionary rates in viral sequence data when sampling is unevenly distributed on a temporal scale through a study of the foot-and-mouth (FMD) disease virus serotypes SAT 1 and SAT 2. Our study shows that clustered temporal sampling in phylogenetic analyses of FMD viruses will strongly bias the inferences of substitution rates and tMRCA because the inferred rates in such data sets reflect a rate closer to the mutation rate rather than the substitution rate. Estimating evolutionary parameters from viral sequences should be performed with due consideration of the differences in short-term and longer-term evolutionary processes occurring within sets of temporally sampled viruses, and studies should carefully consider how samples are combined

Asteroseismology of the Hyades red giant and planet host epsilon Tauri

Asteroseismic analysis of solar-like stars allows us to determine physical parameters such as stellar mass, with a higher precision compared to most other methods. Even in a well-studied cluster such as the Hyades, the masses of the red giant stars are not well known, and previous mass estimates are based on model calculations (isochrones). The four known red giants in the Hyades are assumed to be clump (core-helium-burning) stars based on their positions in colour-magnitude diagrams, however asteroseismology offers an opportunity to test this assumption. Using asteroseismic techniques combined with other methods, we aim to derive physical parameters and the evolutionary stage for the planet hosting star epsilon Tau, which is one of the four red giants located in the Hyades. We analysed time-series data from both ground and space to perform the asteroseismic analysis. By combining high signal-to-noise (S/N) radial-velocity data from the ground-based SONG network with continuous space-based data from the revised Kepler mission K2, we derive and characterize 27 individual oscillation modes for epsilon Tau, along with global oscillation parameters such as the large frequency separation and the ratio between the amplitude of the oscillations measured in radial velocity and intensity as a function of frequency. The latter has been measured previously for only two stars, the Sun and Procyon. Combining the seismic analysis with interferometric and spectroscopic measurements, we derive physical parameters for epsilon Tau, and discuss its evolutionary status.Comment: 13 pages, 13 figures, 4 tables, accepted for publication in Astronomy & Astrophysic

The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others

Pathway engineering in yeast for synthesizing the complex polyketide bikaverin

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast

SAT 1 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 1 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

<i>dN/dS</i> ratios for all datasets.

<p>Single likelihood ancestor counting (SLAC) P < 0.1. Serotype sequences constituted 221 amino acids (SAT 1), 216 amino acids (SAT 2).</p><p><i>dN/dS</i> ratios for all datasets.</p

SAT 2 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 2 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

A novel method of comparing laser trim pattern geometries of thin film resistors

We present the results from stable isotope labeled precursor feeding studies combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry for the identification of labeled polyketide (PK) end-products. Feeding experiments were performed with ¹³C₈-6-methylsalicylic acid (6-MSA) and ¹³C₁₄-YWA1, both produced in-house, as well as commercial ¹³C₇-benzoic acid and ²H₇-cinnamic acid, in species of <i>Fusarium, Byssochlamys, Aspergillus</i>, and <i>Penicillium</i>. Incorporation of 6-MSA into terreic acid or patulin was not observed in any of six evaluated species covering three genera, because the 6-MSA was shunted into (2<i>Z</i>,4<i>E</i>)-2-methyl-2,4-hexadienedioic acid. This indicates that patulin and terreic acid may be produced in a closed compartment of the cell and that (2<i>Z</i>,4<i>E</i>)-2-methyl-2,4-hexadienedioic acid is a detoxification product toward terreic acid and patulin. In <i>Fusarium</i> spp., YWA1 was shown to be incorporated into aurofusarin, rubrofusarin, and antibiotic Y. In <i>A. niger</i>, benzoic acid was shown to be incorporated into asperrubrol. Incorporation levels of 0.7–20% into the end-products were detected in wild-type strains. Thus, stable isotope labeling is a promising technique for investigation of polyketide biosynthesis and possible compartmentalization of toxic metabolites