Complementary Strategies to Unlock Biosynthesis Gene Clusters Encoding Secondary Metabolites in the Filamentous Fungus Podospora anserina.

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

Online acetylcholinesterase inhibition evaluation by high-performance liquid chromatography-mass spectrometry hyphenated with an immobilized enzyme reactor.

A high-performance liquid chromatography-mass spectrometry technique hyphenated on-line with an immobilized enzyme reactor (IMER) was developed by the use of 3 known acetylcholinesterase (AChE) inhibitors (galanthamine, huperzine A and tacrine). This bioanalytical device allows qualitative comparison of the inhibitory strengths of AChE inhibitors. The AChE inhibitory strengths were evaluated and compared by the corresponding acetylcholine peak areas (mass signal) obtained after a chromatographic separation and the elution through the IMER. Only one injection of the analytes is needed to get this comparative analysis. This bioanalytical device was then applied to the extract of a natural plant, Lycoris radiata, which is known to contain AChE inhibitors such as galanthamine and lycoramine. Aside from the demonstration of the inhibitory activity of the two known AChE inhibitors, the AChE inhibitory activity of another compound (dihydro-latifaliumin C) was revealed. This is the first report describing the AChE inhibitory activity of this compound.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Severity of manchineel fruit (Hippomane mancinella) poisoning: A retrospective case series of 97 patients from French Poison Control Centers.

In this retrospective series of 97 cases of manchineel fruit ingestion reported to French Poison Control Centers between 2009 and 2017, we investigated cases of poisoning due to manchineel fruit (from the Hippomane mancinella tree). This fruit is known to be responsible for oropharyngeal and gastrointestinal tract lesions and possibly hypotension and bradycardia (previously attributed to the presence of physostigmine). The most commonly observed clinical signs were oropharyngeal pain, abdominal pain, diarrhea and oropharyngeal irritation. No major gastrointestinal tract lesions were observed in the five cases in which upper gastrointestinal (GI) endoscopy was performed. One case of laryngeal edema and one case of bradycardia were observed, but analysis of the harvested fruits did not confirm the presence of physostigmine. Ingestion of manchineel fruit can cause mild abdominal pain and digestive irritation, requiring medical attention. Rarely, when several fruits have been ingested, severe oropharyngeal injury or hemodynamic disorders may require otorhinolaryngological consultation or cardiac monitoring for several hours, respectively.

Clerodane furanoditerpenoids as the probable cause of toxic hepatitis induced by Tinospora crispa.

Tinospora crispa is a popular traditional herbal plant commonly used throughout the world for treatment of various diseases, in particular type 2 diabetes mellitus. We report here a new case of toxic hepatitis in a 57-year old male patient in the French West Indies following the consumption of two aqueous extracts of fresh Tinospora crispa stems. It thus differs from two previously reported cases that concerned the chronic intake of powdered dry stems delivered in solid oral dosage forms (i.e. pellets and tablets). Liquid Chromatography-Diode Array Detection-Mass Spectrometry (LC/DAD/MS) analyses were performed on an aqueous extract of the offending sample that mimics the swallowed preparation. They revealed the presence of species-specific molecular marker borapetoside C (1) and thus enabled an unambiguous phytochemical identification. The exploration of tandem MS/MS data obtained by ultra-high performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-HRMS) allowed the identification of 17 additional cis-clerodane-type furanoditerpenoid lactones, analogues of 1. These results support the hypothesis that the mechanisms underlying hepatotoxicity of Tinospora crispa are the same as those encountered with furanoditerpenoids-containing plants such as Teucrium chamaedrys or Dioscorea bulbifera. In the context of type 2 diabetes treatment, we recommend that Tinospora crispa intake should be more closely monitored for signs of hepatotoxicity.

Molecular Fingerprint Comparison of Closely Related Rose Varieties based on UHPLC-HRMS Analysis and Chemometrics.

INTRODUCTION: The "Jardin de Granville" modern rose variety not only combines the morphological properties of its two parental cultivars, but also possesses better agronomic characteristics (abundant blooms, strong growth and vitality, high resistance to common rose diseases). In addition, it shows remarkable biological properties such as a high ability to decrease inflammatory and oxidative stress on skin cells. That is why Parfums Christian Dior selected this rose variety to be an active ingredient in luxury cosmetics. OBJECTIVES: To identify the characteristic molecular signature of "Jardin de Granville" compared with its parents "Annapurna" and "John Clare", by the mean of a non-targeted metabolomic comparison. MATERIAL AND METHODS: Wood, flower and leaf hydro-alcoholic extracts were analysed by UHPLC-ESI-HRMS. The fingerprints were then submitted to unsupervised multivariate analyses involving principal component analysis (PCA) and hierarchical ascendant classification (HAC). Analysis of variance (ANOVA) was finally performed to highlight the significant differences in each group of organs. RESULTS: The extracts were composed of phenolic compounds such as hydrolysable and condensed tannins and flavonol derivatives. Three groups of extracts were clustered as a function of the variety. The compounds overexpressed in "Jardin de Granville" variety were highlighted thanks to ANOVA test. Flower was the most discriminative organ with 15 overexpressed molecules. Auto MS/MS analyses led to their tentative identifications. CONCLUSION: The non-targeted metabolomic approach revealed the importance of tannins to discriminate close rose varieties. The overexpressed hydrolysable tannins characteristic of "Jardin de Granville" can be responsible for the antioxidant and anti-inflammatory properties of the rose cosmetic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.