Astraodoric Acids A–D: New Lanostane Triterpenes from Edible Mushroom <i>Astraeus odoratus</i> and Their Anti-<i>Mycobacterium tuberculosis</i> H₃₇Ra and Cytotoxic Activity

Tuberculosis (TB) is one of the chronic infectious diseases caused by <i>Mycobacterium tuberculosis</i> that causes about 2–3 million deaths per year. Isoniazid and rifampicin are examples of first line drugs used for TB treatment; however, they are potentially hepatotoxic. More effective and safer drugs are urgently needed, especially from natural products. Basidiomycete mushrooms are known as important sources of pharmaceutically active metabolites including an anti-TB agent. In this work, the chemical constituents of the edible mushroom <i>Astraeus odoratus</i> were isolated and investigated for antibacterial activity against <i>M. tuberculosis</i> H₃₇Ra. The cytotoxic activity against cancerous cell lines was also evaluated. Four new lanostane triterpenes, astraodoric acids A–D, and new 5-hydroxyhypaphorine have been isolated together with four known compounds. The structures were elucidated by NMR spectroscopic methods, HR-ESI-MS results, and X-ray crystallographic analysis. Astraodoric acids A and B exhibited moderate antibacterial (MICs of 50 and 25 μg/mL) and cytotoxic activities (IC₅₀ values of 34.69 and 18.57 μg/mL against KB and 19.99 and 48.35 μg/mL against NCI-H187), respectively. The results of this study show that <i>A. odoratus</i> could be a significant natural source for safer antitubercular and anticancer agents

Maximum likelihood tree from ITS dataset identifies <i>Astraeus sirindhorniae</i> as a distinct species of <i>Astraeus</i>.

<p>Numbers above branches identify the statistics bootstrap percentages (bold text, before forward slash) and Bayesian posterior probabilities (normal text, after forward slash) for that branch. Maximum likelihood bootstraps from 1000 iterations. Bayesian posterior probabilities from 1000 iterations (1 million runs sampling every 1000^th iteration).</p

<i>Astraeus sirindhorniae</i>.

<p>Exoperidium layers (A–B). (A) exoperidial subpellis, bar = 5 µm. (B) exoperidial subpellis (innermost), bar = 10 µm. (C) rhizomorph hyphae with clamp connection (arrowhead), bar = 5 µm. (D) capillitium hyphae displaying continuous lumen (arrowhead) and basidiospore (arrow), bar = 5 µm. (E–F) spore ornamentation demonstrated coalescent spines in groups, bar = 1 µm. A–D magnification at 1,000×.</p

<i>Astraeus sirindhorniae</i>.

<p>(A) immature basidiomes, bar = 60 mm. (B) short stipitate endoperidium (arrowhead), bar = 3 mm. (C) fibrillose endoperidium (arrowhead), bar = 3 mm. (D) gleba colour become umber to date- brown when mature (arrowhead), bar = 10 mm. (E) complex outer peridium, bar = 3 mm.</p

List of specimens in this study.

<p>Notes:</p>a<p>New species described in Phosri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone.0071160-Phosri3" target="_blank">[32]</a>: <i>A. smithii</i> and ASTRAE-86 is the holotype.</p>b<p>New species described in Phosri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone.0071160-Phosri3" target="_blank">[32]</a>: <i>A. telleriae</i> and ASTRAE-87 is the holotype.</p><p>Specimen codes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone-0071160-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071160#pone-0071160-g003" target="_blank">3</a>. All specimens from Phu Khieo Wildlife Sanctuary abbreviated as PKWS.</p

Maximum likelihood tree from a multigene dataset reveals the placement of <i>Astraeus sirindhorniae</i> within the Sclerodermatineae.

<p>Thick vertical black bars identify root branch for the taxonomic lineage indicated by the adjacent label. Numbers above branches identify the statistics bootstrap percentages (bold text, before forward slash) and Bayesian posterior probabilities (normal text, after forward slash) for that branch. Maximum likelihood bootstraps from 1000 iterations. Bayesian posterior probabilities from 1000 iterations (1 million runs sampling every 1000^th iteration).</p

Fungi in Thailand: A Case Study of the Efficacy of an ITS Barcode for Automatically Identifying Species within the Annulohypoxylon and Hypoxylon Genera

<div><p>Thailand, a part of the Indo-Burma biodiversity hotspot, has many endemic animals and plants. Some of its fungal species are difficult to recognize and separate, complicating assessments of biodiversity. We assessed species diversity within the fungal genera <em>Annulohypoxylon</em> and <em>Hypoxylon</em>, which produce biologically active and potentially therapeutic compounds, by applying classical taxonomic methods to 552 teleomorphs collected from across Thailand. Using probability of correct identification (PCI), we also assessed the efficacy of automated species identification with a fungal barcode marker, ITS, in the model system of <em>Annulohypoxylon</em> and <em>Hypoxylon</em>. The 552 teleomorphs yielded 137 ITS sequences; in addition, we examined 128 GenBank ITS sequences, to assess biases in evaluating a DNA barcode with GenBank data. The use of multiple sequence alignment in a barcode database like BOLD raises some concerns about non-protein barcode markers like ITS, so we also compared species identification using different alignment methods. Our results suggest the following. (1) Multiple sequence alignment of ITS sequences is competitive with pairwise alignment when identifying species, so BOLD should be able to preserve its present bioinformatics workflow for species identification for ITS, and possibly therefore with at least some other non-protein barcode markers. (2) Automated species identification is insensitive to a specific choice of evolutionary distance, contributing to resolution of a current debate in DNA barcoding. (3) Statistical methods are available to address, at least partially, the possibility of expert misidentification of species. Phylogenetic trees discovered a cryptic species and strongly supported monophyletic clades for many <em>Annulohypoxylon</em> and <em>Hypoxylon</em> species, suggesting that ITS can contribute usefully to a barcode for these fungi. The PCIs here, derived solely from ITS, suggest that a fungal barcode will require secondary markers in <em>Annulohypoxylon</em> and <em>Hypoxylon</em>, however. The URL <a href="http://tinyurl.com/spouge-barcode">http://tinyurl.com/spouge-barcode</a> contains computer programs and other supplementary material relevant to this article.</p> </div

PCIs for each of four alignment types and two types of sequence distance.

<p>The error bars indicate 95% confidence intervals, as calculated by the Wilson score interval <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054529#pone.0054529-Wilson1" target="_blank">[46]</a>. The four alignment types used (indicated by different colors at the bottom) were multiple sequence alignment (which imposes an implicit pairwise global alignment on each pair of sequences), and global, semi-global, and local pairwise alignment. The two types of sequence distance used for each alignment method were alignment distance and evolutionary distance. (In fact, for a fixed alignment type and dataset, all evolutionary distances produced the same PCI as p-distance.) The green bars give the value of the barcode gap PCI.</p

Morphological characteristics of <i>Annulohypoxylon</i> and <i>Hypoxylon</i> species found in Thailand.

<p>Stromata (a–p); perithecial structure (q–s); ascospore shapes (t–z); perispore dehiscence (v, y, z). (a) <i>Annulohypoxylon stygium</i> SUT058, (b) <i>A. purpureonitens</i> H125, (c) <i>A. nitens</i> H154, (d) <i>A.</i> aff. <i>nitens</i> H099, (e) <i>Annulohypoxylon</i> sp. H213, (f) <i>Annulohypoxylon</i> sp. H255, (g) <i>Hypoxylon monticulosum</i> H188, (h) <i>H</i>. <i>lenormandii</i> H212, (i) <i>H. investiens</i> H259, (j) <i>H. perforatum</i> SUT218, (k) <i>H. duranii</i> H250, (l) <i>H. haematostroma</i> H114, (m) <i>H. crocopeplum</i> H119, (n) <i>H. pelliculosum</i> H227, (o) <i>H. diatrypeoides</i> H226, (p) <i>H. rubiginosum</i> SUT082, (q) <i>H. fendleri</i> SUT061, (r) <i>H. investiens</i> H259, (s) <i>H. haematostroma</i> H114, (t) <i>H. haematostroma</i> SUT293, (u) <i>A. stygium</i> SUT010, (v) <i>H. duranii</i> SUT284, (w) <i>H. investiens</i> SUT041, (x) <i>A. nitens</i> SUT249, (y) <i>H. monticulosum</i> SUT185 and (z) <i>A. nitens</i> SUT025.</p

DataSheet_1_Metabarcoding of soil environmental DNA to estimate plant diversity globally.pdf

IntroductionTraditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods.MethodsWe sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data.ResultsLarge-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region.DiscussioneDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region.</p