Dermacozines H–J 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 10 898 m. The fermentation process using complex media led to the production of three new pigmented heteroaromatic (oxidized and reduced) phenazine compounds, dermacozines H–J (<b>1</b>–<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 IC₅₀ value of 18.8 μM

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 HPLC–CS-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>_i) of 20 μM. 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 A₂ 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 γ-lactams, namely, pseurotin A, pseurotin A₂, 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 A₂ 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 A₂ 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 A₂ 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 A₂ 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 “lariat knot” 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 37°C, 5%CO₂ 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 μm 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 OD₆₀₀ at 37°C, 5%CO₂ for 3 days. Scale bar = 10 μm. D,E) H99 haploid and KN994B7#16 diploid cells pre-grown in either YNB or YPD were inoculated into 10%HI-FCS at OD₆₀₀ = 0.001, (37°C, 5%CO₂, 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 10üS.

<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, 37°C, 5%CO₂). Representative micrographs and cell size quantification are shown. B) Titan cells were induced for 24 hr, enriched for cells >11μm, and stained with CFW (10 μg/ml), then returned to inducing conditions for 48 hr. Representative micrographs show CFW (chitin) and mAb 18B7-labeled capsule. Scale bar = 10 μm.</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⁺ CD11b⁺ populations and then analysed for Ly6G and Ly6C markers. Indicated populations of CD45⁺ CD11b⁺ cells in individual mice were analysed for the two conditions (* p = 0.0299; **p = 0.0079; ***p<0.0002). B) Indicated populations of CD45⁺ CD11b⁺ Ly6G- cells in individual mice were further analysed for SigLecF and Ly6C markers (** p = 0.0085). C) Indicated populations of CD45⁺ CD11b⁺ 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 μM or 20 μM N-Acetylmuramyl-L-alanyl-D-isoglutamine (NMAiGn) as indicated. Representative micrographs are shown for live cells (CFW, SytoxGreen). Scale bar = 10 μm. 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 30°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 μm) 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 ¹H NMR and ¹H-¹³C 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 OD₆₀₀ = 0.001 in 10% FCS (3 days, 37°C, 5%CO₂). 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 μm. 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