12 research outputs found
Dermacozines HāJ Isolated from a Deep-Sea Strain of <i>Dermacoccus abyssi</i> from Mariana Trench Sediments
<i>Dermacoccus abyssi</i> sp. nov. strains MT1.1 and
MT1.2 are actinomycetes isolated from a Mariana Trench sediment at
a depth of 10ā898 m. The fermentation process using complex
media led to the production of three new pigmented heteroaromatic
(oxidized and reduced) phenazine compounds, dermacozines HāJ
(<b>1</b>ā<b>3</b>). Extensive use was made of
1D and 2D NMR experiments and high-resolution MS to determine the
structures of the compounds. The new dermacozines showed radical scavenging
activity, and the highest activity was observed for dermacozine H
(<b>1</b>), with an IC<sub>50</sub> value of 18.8 Ī¼M
Fluorine Speciation Analysis Using Reverse Phase Liquid Chromatography Coupled Off-Line to Continuum Source Molecular Absorption Spectrometry (CS-MAS): Identification and Quantification of Novel Fluorinated Organic Compounds in Environmental and Biological Samples
Driven by increasing demand for the monitoring of industrial
perfluorinated
compounds (PFCs), the identification of novel fluorine containing
compounds (FOCs) and the tracking of organofluorine drugs and their
degradation products, there is a clear need for sensitive, fluorine-specific
detection of unknown FOCs. Here we report the first ever direct fluorine-specific
(speciation) method; capable of individually detecting untargeted
FOCs in environmental and biological samples through the application
of continuum source molecular absorption spectrometry (CS-MAS) using
a commercial CS-AAS. Two model FOCs (2,4,6, trifluorobenzoic acid
(TFBA) and 5-fluoroindol-5-carboxylic acid (FICA)) were used, achieving
fluorine-specific detection across a range of 0.1 to 300 ng/mL fluorine,
corresponding to a limit of detection of 4 pg F and 5.26 nM for both
compounds. Both TFBA and FICA showed a similar response to CS-MAS
detection, potentially enabling the quantification of fluorine content
in novel FOCs without having molecular standards available. This paper
also reports the use of reverse-phase high performance liquid chromatography
(RP-HPLC) coupled off-line with CS-MAS for the identification of single
organofluorines in a mixture of FOCs via fraction collection. The
linear range of both FOCs was determined to be from 1 to 500 ng/mL.
The limits of detection of those species were just above 1 ng/mL (100
pg) and can therefore compete with targeted analytical methods such
as ESI-MS. Finally, as a proof of principle the analysis of a fluoride-containing
groundwater sample from Ghana demonstrated that this method can be
used in the detection of novel FOCs, with identification achieved
through parallel ESI-MS. Coupled HPLCāCS-MAS/ESI-MS is the
first analytical methodology capable of selectively detecting and
identifying novel FOCs, making possible the quantification of all
fluorine containing compounds in one sample. This is the necessary
analytical requirement to perform <i>fluoronomics</i>
Isolation and Synthesis of Pulmonarins A and B, Acetylcholinesterase Inhibitors from the Colonial Ascidian <i>Synoicum pulmonaria</i>
Pulmonarins A and B are two new dibrominated
marine acetylcholinesterase
inhibitors that were isolated and characterized from the sub-Arctic
ascidian <i>Synoicum pulmonaria</i> collected off the Norwegian
coast. The structures of natural pulmonarins A and B were tentatively
elucidated by spectroscopic methods and later verified by comparison
with synthetically prepared material. Both pulmonarins A and B displayed
reversible, noncompetitive acetylcholinesterase inhibition comparable
to several known natural acetylcholinesterase inhibitiors. Pulmonarin
B was the strongest inhibitor, with an inhibition constant (<i>K</i><sub>i</sub>) of 20 Ī¼M. In addition to reversible,
noncompetitive acetylcholinesterase inhibition, the compounds displayed
weak antibacterial activity but no cytotoxicity or other investigated
bioactivities
Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A<sub>2</sub> and Azaspirofuran A
In
search for novel antiseizure drugs (ASDs), the European FP7-funded
PharmaSea project used zebrafish embryos and larvae as a drug discovery
platform to screen marine natural products to identify promising antiseizure
hits in vivo for further development. Within the framework of this
project, seven known heterospirocyclic Ī³-lactams, namely, pseurotin
A, pseurotin A<sub>2</sub>, pseurotin F1, 11-<i>O</i>-methylpseurotin
A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated
from the bioactive marine fungus <i>Aspergillus fumigatus</i>, and their antiseizure activity was evaluated in the larval zebrafish
pentylenetetrazole (PTZ) seizure model. Pseurotin A<sub>2</sub> and
azaspirofuran A were identified as antiseizure hits, while their close
chemical analogues were inactive. Besides, electrophysiological analysis
from the zebrafish midbrain demonstrated that pseurotin A<sub>2</sub> and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges.
Next, to determine whether these findings translate to mammalians,
both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor
seizure model. They lowered the seizure duration dose-dependently,
thereby confirming their antiseizure properties and suggesting activity
against drug-resistant seizures. Finally, in a thorough ADMET assessment,
pseurotin A<sub>2</sub> and azaspirofuran A were found to be drug-like.
Based on the prominent antiseizure activity in both species and the
drug-likeness, we propose pseurotin A<sub>2</sub> and azaspirofuran
A as lead compounds that are worth further investigation for the treatment
of epileptic seizures. This study not only provides the first evidence
of antiseizure activity of pseurotins and azaspirofurans, but also
demonstrates the value of the zebrafish model in (marine) natural
product drug discovery in general, and for ASD discovery in particular
Chaxapeptin, a Lasso Peptide from Extremotolerant <i>Streptomyces leeuwenhoekii</i> Strain C58 from the Hyperarid Atacama Desert
Lasso
peptides are ribosomally synthesized and post-translationally
modified peptides (RiPPs) that possess a unique ālariat knotā
structural motif. Genome mining-targeted discovery of new natural
products from microbes obtained from extreme environments has led
to the identification of a gene cluster directing the biosynthesis
of a new lasso peptide, designated as chaxapeptin <b>1</b>,
in the genome of <i>Streptomyces leeuwenhoekii</i> strain
C58 isolated from the Atacama Desert. Subsequently, <b>1</b> was isolated and characterized using high-resolution electrospray
ionization mass spectrometry and nuclear magnetic resonance methods.
The lasso nature of <b>1</b> was confirmed by calculating its
nuclear Overhauser effect restraint-based solution structure. Chaxapeptin <b>1</b> displayed a significant inhibitory activity in a cell invasion
assay with human lung cancer cell line A549
Titan cells can be induced <i>in vitro</i> and have all the properties of <i>in vivo</i> induced cells.
<p>H99 haploid cells pre-grown in YNB and incubated in 10%HI-FCS at 37Ā°C, 5%CO<sub>2</sub> for 3 days. Resulting colonies were A) counterstained with India ink to reveal capsule and B) fixed and stained for DNA content relative to haploid and diploid controls. Left panel shows YNB grown haploid control. Middle panel shows total induced population (blue). In addition, induced cells were passaged through an 11 Ī¼m filter to enrich for large cells (red). Right panel shows all three populations. C) H99 haploid cells were pre-grown in YNB and incubated in 10%HI-FCS at the indicated OD<sub>600</sub> at 37Ā°C, 5%CO<sub>2</sub> for 3 days. Scale bar = 10 Ī¼m. D,E) H99 haploid and KN994B7#16 diploid cells pre-grown in either YNB or YPD were inoculated into 10%HI-FCS at OD<sub>600</sub> = 0.001, (37Ā°C, 5%CO<sub>2</sub>, 3 days) and then counterstained with India ink and analysed. Representative micrographs (D) and diameters for >200 cells excluding or including capsule (E) are shown. Significance was assessed using Mann Whitney U (***p<0.0001).</p
Titan cells are primed by YNB and can be maintained in 10Ć¼S.
<p>A) H99 cells pre-cultured in either YPD or YNB were washed 6 times in 1xPBS and then inoculated at the indicated OD in 10% FCS (3 days, 37Ā°C, 5%CO<sub>2</sub>). Representative micrographs and cell size quantification are shown. B) Titan cells were induced for 24 hr, enriched for cells >11Ī¼m, and stained with CFW (10 Ī¼g/ml), then returned to inducing conditions for 48 hr. Representative micrographs show CFW (chitin) and mAb 18B7-labeled capsule. Scale bar = 10 Ī¼m.</p
Zc1 and H99 infections differentially impact host immune response.
<p>FACS analyses for immune cell recruitment to the lungs for mice infected with H99 or Zc1 (day 7 p.i., n = 5 per group). A) Concatenated analysis for 5 mice under each condition were gated for CD45<sup>+</sup> CD11b<sup>+</sup> populations and then analysed for Ly6G and Ly6C markers. Indicated populations of CD45<sup>+</sup> CD11b<sup>+</sup> cells in individual mice were analysed for the two conditions (* p = 0.0299; **p = 0.0079; ***p<0.0002). B) Indicated populations of CD45<sup>+</sup> CD11b<sup>+</sup> Ly6G- cells in individual mice were further analysed for SigLecF and Ly6C markers (** p = 0.0085). C) Indicated populations of CD45<sup>+</sup> CD11b<sup>+</sup> cells in individual mice were further analysed for MHCII markers (** p = 0.0027).</p
Titan cell induction is mediated by host relevant ligands and pre-culture condition.
<p>A) H99 cells pre-cultured in YNB were induced for Titans following 3 days growth in 10% FCS, 10% BAL, or 10 Ī¼M or 20 Ī¼M N-Acetylmuramyl-L-alanyl-D-isoglutamine (NMAiGn) as indicated. Representative micrographs are shown for live cells (CFW, SytoxGreen). Scale bar = 10 Ī¼m. B) Daughters arising from Titan mothers induced using BAL (left, T7) or NMAiGn (right, T1, T5, T6) were isolated and allowed to proliferate for 72 hr at 30Ā°C on YPD agar prior to fixation and staining for DNA content (DAPI). Populations were analysed by flow cytometry. C) Quantification of cells induced by various conditions (FCS, BAL, MNAiGn, <i>E</i>. <i>coli</i>, <i>S</i>. <i>pneumonia</i>) (n>300). D) Lung homogenates from mice unexposed or exposed to Pen/Strep for 7 days prior to <i>C</i>. <i>neoformans</i> inhalation infection. Representative micrographs (CFW, scale bar = 20 Ī¼m) and quantification are shown. n>500 for each condition. E,F) FCS was fractionated by size exclusion chromatography (E) and the resulting fractions were tested for capacity to induce Titan cells. F) Analysis by <sup>1</sup>H NMR and <sup>1</sup>H-<sup>13</sup>C HSQC revealed peaks consistent with sugar and amino acid structures.</p
<i>In vitro</i> Titanisation predicts <i>in vivo</i> outcome.
<p>A,B) H99, Zc1, Zc8, and Zc12 clinical isolates were pre-cultured in either YPD or YNB and then inoculated OD<sub>600</sub> = 0.001 in 10% FCS (3 days, 37Ā°C, 5%CO<sub>2</sub>). A) Representative micrographs of live cells stained with CFW and SytoxGreen or counterstained with India ink and cell size quantification are shown. B) Representative lung histology from mice infected under the indicated conditions and sacrificed on day 7. Cell size was measured for each condition (n>500 cells for each condition). C,D) Quantification of cell size (n>300; p = 0.001) and histology from mice infected with H99 or Zc1 for 7 days prior to <i>C</i>. <i>neoformans</i> inhalation infection. Scale bar = 20 Ī¼m. E,F,G) Balb/C mice (n = 10 per group) were infected with either Zc1 or H99 intra-nasally and followed for 28 days. E) Kaplan-Meyer survival curve (humane endpoint) (p = 0.007). CFUs for lung (F, p = 0.907) and brain (G, p<0.0001) were measured on day of cull.</p