12 research outputs found
Discovery, Isolation, and Structure Elucidation of Dretamycin
The Candida albicans fitness test
is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous
deletion of a particular gene. In the presence of an antifungal inhibitor,
a subset of these mutants exhibits a growth phenotype of hypersensitivity
or hyposensitivity. Collectively these mutants reflect aspects of
the mechanism of action of the compound in question. In the course
of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which
yielded a fitness profile exhibiting significant hypersensitivity
of the <i>DRE2</i> heterozygote and hyposensitivity of the <i>DIP5</i> heterozygote. Herein we report the production, isolation,
and structure elucidation of dretamycin
The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.
<p>ND = Not determined</p><p>Pfu/ml = plaque forming units/ml</p><p>ffu/ml = focus forming units/ml.</p><p><sup>a</sup> PCR/LDR was performed on cloned RNA fragments for all viruses except EBOV and DENV while</p><p><sup>b</sup>dilutions of culture supernatants were used for the latter two viruses. <i>Zaire ebolavirus</i>’95 was used for determination of LOD.</p><p>The limit of detection of the PCR/LDR/Universal Array assay using <i>in vitro</i> transcribed RNA or whole virus.</p
Details of viral cultures and nucleic acids used in the study: RNA viruses.
<p>* The exact sequence of the stock used is not known. The GenBank sequence of the parent strain is indicated.</p><p>Details of viral cultures and nucleic acids used in the study: RNA viruses.</p
Comparison of universal array profile of viral RNA/DNA tested for the corresponding zip-codes.
<p>Normalized average signal intensity for the zip-codes assigned to each virus are presented. The color bars are the signals obtained with the indicated virus (positives). The black bars are the signals produced by the other ten viruses. A signal was considered positive if the intensity of the zip-code spot was at least 10-fold higher than the uniform background level of fluorescence of the array slide. Although a few other viruses produced low-level positive signals for zip18, this did not result in any false positive results since positive signals from at least two addresses was required for a positive identification. In the future, this issue would be rectified by switching to a different zip-code. The average signal intensity for the positives ranged from 31.2 to 123.4, depending on the virus. The average signal intensity for the negatives ranged from 0.3 to 6.2, thus they were not considered positive signals.</p
Schematic of the PCR/LDR assay for detection of VHF viruses.
<p>For each virus (ebolavirus is shown as a representative virus), 1–2 different regions are amplified by RT-PCR using forward and reverse primers, each with minimal degeneracy and all containing universal tails to prevent the formation of primer dimers. Cy-3 labeled downstream LDR primers and single base-discriminating upstream primers with unique zip-code complements (20-30-mers) are targeted to specific sequences/SNPs within the PCR amplicons. Ligation of two adjacent oligonucleotides annealed to a complementary DNA target occurs in the presence of thermostable ligase only if the nucleotides are perfectly matched at the junction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref055" target="_blank">55</a>]. The zip-code complements on the 5’ end of fluorescently labeled LDR products anneal to specific complementary zip-code addresses on a universal array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref056" target="_blank">56</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref057" target="_blank">57</a>]. A positive signal on the universal array is detected as a fluorescent spot. Primers for the ligation reaction were designed targeting 2 or 3 areas within each PCR amplicon. Each virus could produce a maximum of six ligation products, except for VAR and VACC, for which there were a maximum of 5 each. The detection of 2 or more ligation products was required for the detection and identification of a virus. Representative arrays that detect and identify <i>Ebola Zaire</i>, Lassa and Yellow fever viruses are shown.</p
Hematoxylin-eosin stain of liver tissue.
<p>The arrow head indicates an example of cell necrosis (hepatocytes with homogeneously eosinophilic, or pink, cytoplasm and pyknotic or karyolytic nuclei). Hepatocytes occasionally have small basophilic intracytoplasmic inclusions observed in dark blue (arrow).</p
Immunohistochemical test results showing abundant volepox antigen.
<p>The antigen stained in red (arrow heads) within epithelial cells of a stomach specimen.</p
Electron microscopic examination of the inclusion bodies.
<p>Three types of ATIs were observed; inclusions containing virions throughout (Fig. 5A), inclusions without virions (Fig. 5B), and inclusions with virions at the periphery (Fig. 5C). The ATIs examined had varying morphologies that included both non-condensed and mature virions inside and/or around the periphery of the inclusions (Fig. 5D, E). B-type inclusions (BTIs) were also observed (Fig. 5F). The arrow head in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043881#pone-0043881-g005" target="_blank">figure 5A, and 5D</a>, shows a mature volepox virion; the arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043881#pone-0043881-g005" target="_blank">figure 5D, and 5E</a>, show immature or non-condensed virions.</p
Viable virus content per specimen.
<p>Volepox virus loads (PFU/mL) per specimen taken during the necropsy of deceased and euthanized mice on days 6, 7 and 8 pi. <b>*</b>Swabs from the survivors were taken on day 7 pi.</p
Characteristic lesions observed on <i>P.californicus</i> infected with Volepox virus.
<p>Epidermal ulcers with crusting on paw (A), vulvae and perineum (B), tongue and nares (C). Serosal petechiae and focal gastrointestinal tract necrosis (D).</p