Straightforward N-Acyl Homoserine Lactone Discovery and Annotation by LC-MS/MS-based Molecular Networking.

N-Acyl-l-homoserine lactones (AHLs) are a large family of signaling molecules in "quorum sensing" communication. This mechanism is present in a number of bacterial physiological phenomena, including pathogenic phenomena. In this study, we described a simple and accessible way to detect, annotate, and quantify these compounds from bacterial culture media. Analytical standards and ethyl acetate bacterial extracts containing AHLs were analyzed by an ultra-high-performance liquid chromatography system coupled to a mass spectrometer using a nontargeted FullMS data-dependent MS2 method. The results were processed in MZmine2 and then analyzed by a Feature-Based Molecular Networking (FBMN) workflow in the Global Natural Products Social Networking (GNPS) platform for the discovery and annotation of known and unknown AHLs. Our group analyzed 31 AHL standards and included the MS2 spectra in the spectral library of the GNPS platform. We also provide the 31 standard AHL spectrum list for inclusion in molecular networking analyses. FBMN analysis annotated 30 out of 31 standards correctly. Then, as an example, a set of five bacterial extracts was prepared for AHL annotation. Following the method described in this Article, 5 known and 11 unknown AHLs were properly annotated using the FBMN-based molecular network approach. This study offers the possibility for the automatic annotation of known AHLs and the search for nonreferenced AHLs in bacterial extracts in a somewhat straightforward approach even without acquiring analytical standards. The method also provides relative quantification information.

A Comparative Metabolomics Approach Demonstrates That Octocrylene Accumulates in Stylophora pistillata Tissues as Derivatives and That Octocrylene Exposure Induces Mitochondrial Dysfunction and Cell Senescence.

The objective of this work was to study the effect of octocrylene on Stylophora pistillata and to compare the metabolomic response of this coral species to that obtained with Pocillopora damicornis. When S. pistillata coral was exposed to octocrylene, it accumulated octocrylene derivatives similar to P. damicornis. Octocrylene-fatty acid conjugates were found, as well as octocrylene heterosides. Furthermore, the tissue concentrations of various acylcarnitines and three sphingoid bases increased significantly. This phenomenon was indicative of mitochondrial dysfunction and the induction of cellular senescence processes in exposed corals. Overall, the responses of the two corals to octocrylene pollution were consistent. The proven impact of octocrylene on a second coral species suggests that potential environmental octocrylene pollution could impact many reef-building species. Furthermore, this work demonstrates that octocrylene may be modified in vivo by many organisms and that levels of octocrylene contamination in the food chain have probably been underestimated until now.

Antiparasitic Ovalicin Derivatives from Pseudallescheria boydii, a Mutualistic Fungus of French Guiana Termites.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

Benzophenone Accumulates over Time from the Degradation of Octocrylene in Commercial Sunscreen Products.

Benzophenone is a mutagen, carcinogen, and endocrine disruptor. Its presence in food products or food packaging is banned in the United States. Under California Proposition 65, there is no safe harbor for benzophenone in any personal care products, including sunscreens, anti-aging creams, and moisturizers. The purpose of this study was to determine (1) if benzophenone was present in a wide variety of commercial sun protection factor (SPF)/sunscreen products, (2) whether benzophenone concentration in the product increased over time, and (3) if the degradation of octocrylene was the likely source for benzophenone contamination. Benzophenone concentration was assayed in nine commercial sunscreen products from the European Union and eight from the United States (in triplicate), including two single ingredient sources of octocrylene. These same SPF items were subjected to the United States Food and Drug Administration (U.S. FDA)-accelerated stability aging protocol for 6 weeks. Benzophenone was measured in the accelerated-aged products. Sixteen octocrylene-containing product lines that were recently purchased had an average concentration of 39 mg/kg benzophenone, ranging from 6 mg/kg to 186 mg/kg. Benzophenone was not detectable in the product that did not contain octocrylene. After subjecting the 17 products to the U.S. FDA-accelerated stability method, the 16 octocrylene-containing products had an average concentration of 75 mg/kg, ranging from 9.8 mg/kg to 435 mg/kg. Benzophenone was not detectable in the product that did not contain octocrylene. Benzophenone was detected in the pure octocrylene manufactured ingredient. Octocrylene generates benzophenone through a retro-aldol condensation. In vivo, up to 70% of the benzophenone in these sunscreen products may be absorbed through the skin. U.S. FDA has established a zero tolerance for benzophenone as a food additive. In the United States, there were 2999 SPF products containing octocrylene in 2019. The safety of octocrylene as a benzophenone generator in SPF or any consumer products should be expeditiously reviewed by regulatory agencies.

Optimization method for quantification of sunscreen organic ultraviolet filters in coastal sands.

Most organic ultraviolet filters are very lipophilic and some of them are difficult to quantify in the environment. This article describes an optimization method for the quantification of these compounds in sand samples with diverse compositions. The standard additions method was used. The search for a unique high-performance liquid chromatography method to analyze all these filters along with the search for optimal detection conditions are presented in detail. The best extraction solvent was methanol, and the best conditions for analysis and detection involved the use of a high-performance liquid chromatography system equipped with a biphenyl column (2.6 µm, 150 × 4.6 mm), and an Orbitrap MS detector. We also demonstrated that sample freeze-drying can induce significant loss of some of the ultraviolet filters.

Antimicrobial Activity and Synergy Investigation of Hypericum scabrum Essential Oil with Antifungal Drugs.

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32-64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, ß-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.

Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry.

In recent years, use of supercritical-fluid chromatography (SFC) with CO2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC-HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

Carneic Acids from an Endophytic Phomopsis sp. as Dengue Virus Polymerase Inhibitors.

Thirteen carneic acids were isolated from the fungal endophyte Phomopsis sp. SNB-LAP1-7-32. Their structures were identified by mass spectrometry and extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Compounds 1-13 were investigated for their antipolymerase activities against DENV polymerase and Zika NS5. Five of them exhibited significant inhibition of dengue polymerase with IC50 values in the 10 to 20 µM range without cytotoxicity. None inhibited Zika virus NS5 protein.

Paecilosetin Derivatives as Potent Antimicrobial Agents from Isaria farinosa.

Fifty-seven entomopathogenic microorganisms were screened against human pathogens and subjected to mass spectrometry molecular networking based dereplication. Isaria farinosa BSNB-1250, shown to produce potentially novel biologically active metabolites, was grown on a large scale on potato dextrose agar, and paecilosetin (1) and five new analogues (2-6) were subsequently isolated. The structures of the new compounds were elucidated using 1D and 2D NMR. The absolute configurations of compounds 1-6 were determined using Mosher ester derivatives (1, 3, 4), comparison of experimental and calculated ECD spectra (2-4 and 6), and single-crystal X-ray diffraction analysis (5). Compounds 1 and 5 exhibited strong antibacterial activity against MSSA and MRSA with MIC values of 1-2 µg/mL.

Marine Microbial Diversity as a Source of Bioactive Natural Products.

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Metabolomic Insights into Marine Phytoplankton Diversity.

The democratization of sequencing technologies fostered a leap in our knowledge of the diversity of marine phytoplanktonic microalgae, revealing many previously unknown species and lineages. The evolutionary history of the diversification of microalgae can be inferred from the analysis of their genome sequences. However, the link between the DNA sequence and the associated phenotype is notoriously difficult to assess, all the more so for marine phytoplanktonic microalgae for which the lab culture and, thus, biological experimentation is very tedious. Here, we explore the potential of a high-throughput untargeted metabolomic approach to explore the phenotypic-genotypic gap in 12 marine microalgae encompassing 1.2 billion years of evolution. We identified species- and lineage-specific metabolites. We also provide evidence of a very good correlation between the molecular divergence, inferred from the DNA sequences, and the metabolomic divergence, inferred from the complete metabolomic profiles. These results provide novel insights into the potential of chemotaxonomy in marine phytoplankton and support the hypothesis of a metabolomic clock, suggesting that DNA and metabolomic profiles co-evolve.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Potent and Non-Cytotoxic Antibacterial Compounds Against Methicillin-Resistant Staphylococcus aureus Isolated from Psiloxylon mauritianum, A Medicinal Plant from Reunion Island.

With the occurrence of antibiotic-resistant Staphylococcus aureus strains, identification of new anti-staphylococcal drugs has become a necessity. It has long been demonstrated that plants are a large and diverse source of antibacterial compounds. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, was chemically investigated for its reported biological activity against S. aureus. Aspidin VB, a phloroglucinol derivative never before described, together with Aspidin BB, were first isolated from the ethyl acetate extract of P. mauritianum leaves. Their structures were elucidated from spectroscopic data. Aspidin VB exhibited strong antibacterial activity against standard and methicillin-resistant S. aureus strains, with a minimal inhibition concentration (MIC) of 0.25 µg/mL, and no cytotoxicity was observed at 10-5 M in MRC5 cells. Due to its biological activities, Aspidin VB appears to be a good natural lead in the fight against S. aureus.

Metabolomics Reveal That Octocrylene Accumulates in Pocillopora damicornis Tissues as Fatty Acid Conjugates and Triggers Coral Cell Mitochondrial Dysfunction.

Octocrylene (OC) is an ingredient used in many sunscreens and cosmetics worldwide. Our group evaluated the toxicity of OC in corals. Adult Pocillopora damicornis coral was treated with OC at concentrations of 5, 50, 300, and 1000 µg/L. Most polyps were closed at concentrations of 300 µg/L and higher. Further, metabolomic profiling provided crucial information regarding OC accumulation in coral tissues and OC toxicity. First, we demonstrated that OC was transformed into fatty acid conjugates via oxidation of the ethylhexyl chain, yielding very lipophilic OC analogues that accumulate in coral tissues. Second, the differential analysis of coral profiles revealed higher levels of 15 acylcarnitines, suggesting abnormal fatty acid metabolism related to mitochondrial dysfunction. The formation of OC analogues suggests that OC concentrations measured in the environment, and organisms may have been largely underestimated. Overall, these results call for an in-depth evaluation of OC toxicity and the reevaluation of the actual OC accumulation rate in the ocean's food chain, including OC-fatty acid conjugates.

Antibacterial, anti-adherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4.

The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL-1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL-1 and 1.17 mg mL-1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.

Structural Identification of Antibacterial Lipids from Amazonian Palm Tree Endophytes through the Molecular Network Approach.

A library of 197 endophytic fungi and bacteria isolated from the Amazonian palm tree Astrocaryum sciophilum was extracted and screened for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Four out of five antibacterial ethyl acetate extracts were also cytotoxic for the MRC-5 cells line. Liquid chromatography coupled to tandem mass spectrometry (UPHLC-HRMS/MS) analyses combined with molecular networking data processing were carried out to allow the identification of depsipeptides and cyclopeptides responsible for the cytotoxicity in the dataset. Specific ion clusters from the active Luteibacter sp. extract were also highlighted using an MRSA activity filter. A chemical study of Luteibacter sp. was conducted leading to the structural characterization of eight fatty acid exhibiting antimicrobial activity against MRSA in the tens of µg/mL range.

Characterization, Diversity, and Structure-Activity Relationship Study of Lipoamino Acids from Pantoea sp. and Synthetic Analogues.

A biological evaluation of a library of extracts from entomopathogen strains showed that Pantoea sp. extract has significant antimicrobial and insecticidal activities. Three hydroxyacyl-phenylalanine derivatives were isolated from this strain. Their structures were elucidated by a comprehensive analysis of their NMR and MS spectroscopic data. The antimicrobial and insecticidal potencies of these compounds were evaluated, and compound 3 showed 67% mortality against Aedes aegypti larvae at a concentration of 100 ppm, and a minimum inhibitory concentration (MIC) of 16 µg/mL against methicillin-resistant Staphylococcus aureus. Subsequently, hydroxyacyl-phenylalanine analogues were synthesized to better understand the structure-activity relationships within this class of compounds. Bioassays highlighted the antimicrobial potential of analogues containing saturated medium-chain fatty acids (12 or 14 carbons), whereas an unsaturated long-chain fatty acid (16 carbons) imparted larvicidal activity. Finally, using a molecular networking-based approach, several close analogues of the isolated and newly synthesized lipoamino acids were discovered in the Pantoea sp. extract.

Chemical Variability of the Essential Oil of Origanum ehrenbergii Boiss. from Lebanon, Assessed by Independent Component Analysis (ICA) and Common Component and Specific Weight Analysis (CCSWA).

Origanum ehrenbergii Boiss., an endemic plant to Lebanon, is widely acknowledged in Lebanese traditional medicine. The aim of the present study was to evaluate the influence of the drying method, region, and time of harvest on yield and chemical composition of O. ehrenbergii essential oils (EOs). Plants were harvested monthly throughout 2013 and 2014, from two different regions, Aabadiye and Qartaba, then dried using two drying methods: lyophilization and shade-drying at 4 °C. EO was extracted by hydrodistillation and analyzed by GC/MS. GC-MS data, combined with independent component analysis (ICA) and common component and specific weight analysis (CCSWA), showed that drying techniques, region of harvest, and soil composition have no effect on the chemical composition of O. ehrenbergii EOs. Of the factors analyzed, only harvesting time affected the EO composition of this species. High and stable amounts of carvacrol, associated with reliable antimicrobial activities, were detected in material harvested between March and October. EOs obtained from plants harvested in Aabadiye in January and February showed high amounts of thymoquinone, related to anti-inflammatory and cytotoxic effects. The use of ICA and CCSWA was proven to be efficient, and allowed the development of a discriminant model for the classification of O. ehrenbergii chemotype and the determination of the best harvesting time.

Aedes aegypti Larvicidal Sesquiterpene Alkaloids from Maytenus oblongata.

Four new sesquiterpene alkaloids (1-4) with a ß-dihydroagrofuran skeleton and a new triterpenoid (5) were isolated from an ethyl acetate extract of Maytenus oblongata stems. Their structures were elucidated using 1D and 2D NMR spectroscopy as well as MS and ECD experiments. The M. oblongata stem EtOAc extract and the pure compounds isolated were tested for larvicidal activity against Aedes aegypti under laboratory conditions, and compounds 2 and 3 were found to be active.

Betulinic Acid, The First Lupane-Type Triterpenoid Isolated from Both a Phomopsis sp. and Its Host Plant Diospyros carbonaria Benoist.

Following a biological screening using a dengue replicon virus-cell-based assay, Diospyros carbonaria AcOEt extract was investigated, affording six known lupane-type triterpenoids endowed with anti-DENV-2 NS5 polymerase activity. The study of the associated microbial community of this species permitted us to identify 38 endophytes belonging to five different orders. Nine out of these 38 strains showed significant activity on the dengue replicon assay. The chemical investigation of the most active one, Phomopsis sp. SNB-LAP1-7-32, led to the isolation of betulinic acid, an anti-viral secondary metabolite isolated previously from the host plant. This result is the first example of a lupane-type triterpenoid isolated from both an endophyte and its host plant. Its presence in the Phomopsis strain may result from gene transfer and/or specific niche selection.