Structural characterization of N-acyl-homoserine lactones from bacterial quorum sensing using LC-MS/MS analyses after Paternò-Büchi derivatization in solution.

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs.

Chemical Investigation of the Calcareous Marine Sponge Pericharax heteroraphis, Clathridine-A Related Derivatives Isolation, Synthesis and Osteogenic Activity.

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3-6 and the homodimeric complex (clathridine A)2 Zn2+ (9), together with the new unstable heterodimeric complex (clathridine A-clathridimine)Zn2+ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)2CH2 (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model.

Conventional Epithelial-Off Corneal Crosslinking in Patients With Progressive Keratoconus: 10-Year Outcomes.

PURPOSE: Corneal crosslinking (CXL) is the standard treatment of progressive keratoconus (KC). We evaluated the safety and 10-year outcomes of conventional "epithelial-off" CXL for progressive KC for the first time in a cohort in France. METHODS: We conducted a retrospective review of patients undergoing conventional CXL (Dresden protocol) in our tertiary ophthalmology department from 2006 to 2011 with 10-year follow-up. The primary outcome was change in preoperative versus postoperative keratometry measured by maximum keratometry (Kmax), steep keratometry (K2), flat keratometry (K1), mean keratometry (Km), and topographic cylinder. Secondary outcomes were changes in visual and refractive outcomes. We report postoperative complications and adverse events. RESULTS: Eighty-nine eyes from 76 patients (67% male patients, mean age 22.7 ± 7.6 years) were included. Mean Kmax (-2.31 ± 2.98 diopters (D); P < 0.00001), K2 (-2.07 ± 3.15 D; P < 0.00001), K1 (-1.00 ± 2.29 D; P = 0.00008), Km (-1.53 ± 2.47 D; P < 0.00001), and topographic cylinder (-1.15 ± 2.53 D; P = 0.00004) significantly decreased 10 years after CXL compared with preoperative baseline. Significant decreases were still observed between 5 and 10 years after for mean Kmax, mean K2, mean K1, and mean Km. Mean distance best spectacle-corrected visual acuity and mean manifest refraction spherical equivalent were significantly improved after 10 years versus before CXL. The 10-year rate of repeat CXL was n = 3/76 patients (4%) (all younger than 18 years at first CXL) and of loss of >3 lines in best spectacle-corrected visual acuity was n = 1/76 patients (1%). CONCLUSIONS: Progressive KC was effectively stabilized with a prolonged flattening and maintenance of functional vision improvements after 10 years. Repeat CXL was rare and only required among younger patients.

Multiscale organisation of lead carboxylates in artistic oil binders.

The supramolecular and mesoscopic architectures of lead-saponified linseed oil, used by painters since the Renaissance, have been characterised and linked to their rheological properties. The multi-scale organization of saponified oils has been demonstrated by SAXS (Small Angle X-ray Scattering), FF-TEM (Freeze-Fracture Transmission Electron Microscopy) and DIC (Differential Interference Contrast): some of the lead soaps (formed when the oil is heated in the presence of PbO) are organized into microscopic lamellar domains, distributed in a continuous matrix made up of unorganized species (partially saponified triglycerides, glycerol, remaining soaps, etc.). The concentration of lead soaps in the oil controls the average size and interaction between the lamellar domains. Linseed oil + PbO 17 mol% is viscous and consists of aggregates of lamellar domains isolated within the continuous unorganized matrix. In contrast, in linseed oil + PbO 50 mol%, the domains are homogeneously dispersed and form what can be described as a three-dimensional network, giving the system viscoelastic properties.

Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.

MALDI mass spectrometry imaging has gained major interest in the field of chemical imaging. This technique makes it possible to locate tens to hundreds of ionic signals on the sample surface without any a priori. One of the current challenges is still the limited ability to annotate signals in order to convert m/z values into probable chemical structures. At the same time, data obtained by LC-MS/MS have benefited from the development of numerous chemoinformatics tools, in particular molecular networks, for their efficient annotation. For the first time, we present here the combination of MALDI-FT-ICR imaging with molecular networks from MALDI-MS/MS data directly acquired on plant tissue sections. Annotation improvements are demonstrated, paving the way for new annotation pipelines for MALDI imaging.

Oxygen Exposure and Tolerance Shapes the Cell Wall-Associated Lipids of the Skin Commensal Cutibacterium acnes.

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m/z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes.

N 2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing.

Modified nucleotides in non-coding RNAs, such as tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last 'orphan' modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing.

Wood Degradation by Fomitiporia mediterranea M. Fischer: Exploring Fungal Adaptation Using Metabolomic Networking.

Fomitiporia mediterranea M. Fischer (Fmed) is a white-rot wood-decaying fungus associated with one of the most important and challenging diseases in vineyards: Esca. To relieve microbial degradation, woody plants, including Vitis vinifera, use structural and chemical weapons. Lignin is the most recalcitrant of the wood cell wall structural compounds and contributes to wood durability. Extractives are constitutive or de novo synthesized specialized metabolites that are not covalently bound to wood cell walls and are often associated with antimicrobial properties. Fmed is able to mineralize lignin and detoxify toxic wood extractives, thanks to enzymes such as laccases and peroxidases. Grapevine wood's chemical composition could be involved in Fmed's adaptation to its substrate. This study aimed at deciphering if Fmed uses specific mechanisms to degrade grapevine wood structure and extractives. Three different wood species, grapevine, beech, and oak. were exposed to fungal degradation by two Fmed strains. The well-studied white-rot fungus Trametes versicolor (Tver) was used as a comparison model. A simultaneous degradation pattern was shown for Fmed in the three degraded wood species. Wood mass loss after 7 months for the two fungal species was the highest with low-density oak wood. For the latter wood species, radical differences in initial wood density were observed. No differences between grapevine or beech wood degradation rates were observed after degradation by Fmed or by Tver. Contrary to the Tver secretome, one manganese peroxidase isoform (MnP2l, jgi protein ID 145801) was the most abundant in the Fmed secretome on grapevine wood only. Non-targeted metabolomic analysis was conducted on wood and mycelium samples, using metabolomic networking and public databases (GNPS, MS-DIAL) for metabolite annotations. Chemical differences between non-degraded and degraded woods, and between mycelia grown on different wood species, are discussed. This study highlights Fmed physiological, proteomic and metabolomic traits during wood degradation and thus contributes to a better understanding of its wood degradation mechanisms.

A new series of bioactive Mo(V)2O2S2-based thiosemicarbazone complexes: Solution and DFT studies, and antifungal and antioxidant activities.

This paper deals with the synthesis, characterization, and studies of biological properties of a series of 5 coordination compounds based on binuclear core [Mo(V)2O2S2]2+ with thiosemicarbazones ligands bearing different substituents on the R1 position of the ligand. The complexes are first studied using MALDI-TOF mass spectrometry and NMR spectroscopy to determine their structures in solution in relation to single-crystal X-Ray diffraction data. In a second part, the antifungal and antioxidative activities are explored and the high potential of these coordination compounds compared to the uncoordinated ligands is demonstrated for these properties. Finally, DFT calculation provides important support to the solution studies by identifying the most stable isomers in each [Mo2O2S2]2+/Ligand system, while the determination of HUMO and LUMO levels is performed to explain the antioxidative properties of these systems.

Keratoconus and the Impact of Treatment on Patients' Quality of Life: A Qualitative Study.

INTRODUCTION: Keratoconus has a significant impact on patients' quality of life (QoL), from diagnosis to the advanced stages of the disease. The aim of this research was to identify domains of QoL affected by this disease and its treatment. METHODS: Phone interviews were conducted using a semi-structured interview guide, with patients with keratoconus stratified according to their current treatment. A board of keratoconus experts helped identify the guide's main themes. RESULTS: Thirty-five patients (rigid contact lenses, n = 9; cross-linking, n = 9; corneal ring implants, n = 8; and corneal transplantation, n = 9) were interviewed by qualitative researchers. Phone interviews revealed several QoL domains affected by the disease and its treatments: "psychological", "social life", "professional life", "financial costs" and "student life". All domains were impacted, independently of the treatment history. Few differences were found between treatment regimens and keratoconus stages. Qualitative analysis enabled the development of a conceptual framework based on Wilson and Cleary's model for patient outcomes common to all patients. This conceptual model describes the relationship between patients' characteristics, their symptoms, their environment, their functional visual impairment and the impact on their QoL. CONCLUSIONS: These qualitative findings supported the generation of a questionnaire to evaluate the impact of keratoconus and its treatment on patients' QoL. Cognitive debriefings confirmed its content validity. The questionnaire is applicable for all stages of keratoconus and treatments and may help tracking change over time in regular clinical settings. Psychometric validation is yet to be performed before its use in research and clinical practices.

Ocular sequelae of epidermal necrolysis: French national audit of practices, literature review and proposed management.

Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) are serious and rare diseases, most often drug-induced, and their incidence has been estimated at 6 cases/million/year in France. SJS and TEN belong to the same spectrum of disease known as epidermal necrolysis (EN). They are characterized by more or less extensive epidermal detachment, associated with mucous membrane involvement, and may be complicated during the acute phase by fatal multiorgan failure. SJS and TEN can lead to severe ophthalmologic sequelae. There are no recommendations for ocular management during the chronic phase. We conducted a national audit of current practice in the 11 sites of the French reference center for toxic bullous dermatoses and a review of the literature to establish therapeutic consensus guidelines. Ophthalmologists and dermatologists from the French reference center for epidermal necrolysis were asked to complete a questionnaire on management practices in the chronic phase of SJS/TEN. The survey focused on the presence of a referent ophthalmologist at the center, the use of local treatments (artificial tears, corticosteroid eye drops, antibiotic-corticosteroids, antiseptics, vitamin A ointment (VA), cyclosporine, tacrolimus), the management of trichiatic eyelashes, meibomian dysfunction, symblepharons, and corneal neovascularization, as well as the contactologic solutions implemented. Eleven ophthalmologists and 9 dermatologists from 9 of the 11 centers responded to the questionnaire. Based on questionnaire results, 10/11 ophthalmologists systematically prescribed preservative-free artificial tears, and 11/11 administered VA. Antiseptic or antibiotic eye drops or antibiotic-corticosteroid eye drops were recommended as needed by 8/11 and 7/11 ophthalmologists, respectively. In case of chronic inflammation, topical cyclosporine was consistently proposed by 11/11 ophthalmologists. The removal of trichiatic eyelashes was mainly performed by 10/11 ophthalmologists. Patients were referred to a reference center for fitting of scleral lenses (10/10,100%). Based on this practice audit and literature review, we propose an evaluation form to facilitate ophthalmic data collection in the chronic phase of EN and we also propose an algorithm for the ophthalmologic management of ocular sequelae.

Tuning the nuclearity of [Mo2O2S2]2+-based assemblies by playing with the degree of flexibility of bis-thiosemicarbazone ligands.

[MoV2O2S2]2+-based thiosemicarbazone complexes appear as very promising molecules for biological applications due to the intrinsic properties of their components. This paper deals with the synthesis and characterization of six coordination complexes obtained by the reaction of [MoV2O2S2]2+ clusters with bis-thiosemicarbazone ligands that contain flexible or rigid spacers between the two thiosemicarbazone units. Interestingly, structural characterization by single-crystal X-ray diffraction, MALDI-TOF MS technique and NMR spectroscopy revealed that the nuclearity of the complex is controlled by the nature of the spacer between the thiosemicarbazone units. Binuclear complexes, namely [MoV2O2S2(L1-3)], are isolated with flexible spacers while tetranuclear complexes [(MoV2O2S2)2(L4-6)2] are formed when the bis-thiosemicarbazone ligands are built on rigid spacers.

Nitrogen Enriched Solid-State Cultivation for the Overproduction of Azaphilone Red Pigments by Penicillium sclerotiorum SNB-CN111.

Azaphilones are microbial specialized metabolites employed as yellow, orange, red or purple pigments. In particular, yellow azaphilones react spontaneously with functionalized nitrogen groups, leading to red azaphilones. In this study, a new two-step solid-state cultivation process to produce specific red azaphilones pigments was implemented, and their chemical diversity was explored based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and a molecular network. This two-step procedure first implies a cellophane membrane allowing accumulating yellow and orange azaphilones from a Penicillium sclerotiorum SNB-CN111 strain, and second involves the incorporation of the desired functionalized nitrogen by shifting the culture medium. The potential of this solid-state cultivation method was finally demonstrated by overproducing an azaphilone with a propargylamine side chain, representing 16% of the metabolic crude extract mass.

Grafting of Anionic Decahydro-Closo-Decaborate Clusters on Keggin and Dawson-Type Polyoxometalates: Syntheses, Studies in Solution, DFT Calculations and Electrochemical Properties.

Herein we report the synthesis of a new class of compounds associating Keggin and Dawson-type Polyoxometalates (POMs) with a derivative of the anionic decahydro-closo-decaborate cluster [B10H10]2- through aminopropylsilyl ligand (APTES) acting as both a linker and a spacer between the two negatively charged species. Three new adducts were isolated and fully characterized by various NMR techniques and MALDI-TOF mass spectrometry, notably revealing the isolation of an unprecedented monofunctionalized SiW10 derivative stabilized through intramolecular H-H dihydrogen contacts. DFT as well as electrochemical studies allowed studying the electronic effect of grafting the decaborate cluster on the POM moiety and its consequences on the hydrogen evolution reaction (HER) properties.

Combining OSMAC, metabolomic and genomic methods for the production and annotation of halogenated azaphilones and ilicicolins in termite symbiotic fungi.

We gathered a collection of termite mutualistic strains from French Guiana to explore the metabolites of symbiotic microorganisms. Molecular networks reconstructed from a metabolomic analysis using LC-ESI-MS/MS methodology led us to identify two families of chlorinated polyketides, i.e., azaphilones from Penicillium sclerotiorum and ilicicolins from Neonectria discophora. To define the biosynthetic pathways related to these two types of scaffolds, we used a whole genome sequencing approach followed by hybrid assembly from short and long reads. We found two biosynthetic gene clusters, including two FAD-dependent halogenases. To exploit the enzymatic promiscuity of the two identified FAD halogenases, we sought to biosynthesize novel halogenated metabolites. An OSMAC strategy was used and resulted in the production of brominated analogs of ilicicolins and azaphilones as well as iodinated analogs of azaphilones.

New Keratoconus Risk Factors: A Cross-Sectional Case-Control Study.

Purpose: To evaluate risk factors associated with keratoconus in a monocentric cross-sectional case-control study. Methods: This observational study occurred from June 2019 to February 2021 in a university hospital (France). The case group consisted of 195 patients with keratoconus in at least one eye who were followed up by a corneal specialist. The control group consisted of 195 patients without any evidence of keratoconus on slit-lamp examination and corneal topography, who were matched 1 : 1 to controls by age and sex. Data were collected by a self-completed paper questionnaire before the consultation, and a multivariate logistic regression was performed. Results: Multivariate analysis revealed significant associations of keratoconus with family history (odds ratio [OR] = 22.2, p < 0.001), rubbing eyes (OR = 10.9, p < 0.001), allergy (any kind) (OR = 3.80, p < 0.001), smoking exposure (OR = 2.08, p=0.017), and dry eyes (OR = 1.77, p=0.045f). The worst eye was associated with the more rubbed eye (p < 0.001) and the more pressed eye during the night according to sleeping position (p < 0.041). Conclusion: This study confirmed the association between keratoconus and eye rubbing, family history, and allergy. It highlighted the role of pressure on the eyes during sleep. Other less known risk factors such as dry eyes and smoking exposure should be explored in future studies.

Synthesis, Structures, and Solution Studies of a New Class of [Mo2O2S2]-Based Thiosemicarbazone Coordination Complexes.

This paper deals with the synthesis, structural studies, and behavior in solution of unprecedented coordination complexes built by the association of a panel of 14 representative thiosemicarbazone ligands with the cluster [Mo2O2S2]2+. These complexes have been thoroughly characterized both in the solid state and in solution by XRD and by NMR, respectively. In particular, HMBC 1H{15N} and 1H DOSY NMR experiments bring important elements for understanding the complexes' behavior in solution. These studies demonstrate that playing on the nature and the position of various substituents on the ligands strongly influences the coordination modes of the ligands as well as the numbers of isomers in solution, mainly 2 products for the majority of complexes and up to 5 for some of them.

Classification of Environmental Strains from Order to Genus Levels Using Lipid and Protein MALDI-ToF Fingerprintings and Chemotaxonomic Network Analysis.

During the last two decades, MALDI-ToF mass spectrometry has become an efficient and widely-used tool for identifying clinical isolates. However, its use for classification and identification of environmental microorganisms remains limited by the lack of reference spectra in current databases. In addition, the interpretation of the classical dendrogram-based data representation is more difficult when the quantity of taxa or chemotaxa is larger, which implies problems of reproducibility between users. Here, we propose a workflow including a concurrent standardized protein and lipid extraction protocol as well as an analysis methodology using the reliable spectra comparison algorithm available in MetGem software. We first validated our method by comparing protein fingerprints of highly pathogenic bacteria from the Robert Koch Institute (RKI) open database and then implemented protein fingerprints of environmental isolates from French Guiana. We then applied our workflow for the classification of a set of protein and lipid fingerprints from environmental microorganisms and compared our results to classical genetic identifications using 16S and ITS region sequencing for bacteria and fungi, respectively. We demonstrated that our protocol allowed general classification at the order and genus level for bacteria whereas only the Botryosphaeriales order can be finely classified for fungi.

Dereplication of Acetogenins from Annona muricata by Combining Tandem Mass Spectrometry after Lithium and Copper Postcolumn Cationization and Molecular Networks.

Annonaceous acetogenins are natural products held responsible for atypical Parkinsonism due to chronic consumption in traditional medicine or as food, leading to the development of analytical strategies for their complete chemical characterization in complex mixtures. Characterization by tandem mass spectrometry (MS/MS) of acetogenins using collision-induced dissociation from lithium adducts provides additional structural information compared to protonated or sodiated species such as ketone location on the acetogenin backbone. However, very low intensity diagnostic ions together with the lack of extensive structural information regarding position of OH and THF substituents limit this approach. Copper adducts led to diagnostic fragment ions that allow us to identify the position of oxygen rings and hydroxyl substituents. Fragmentation rules were established on the basis of acetogenin standards allowing the identification of 45 over the 77 analogues observed in an extract of Annona muricata by LC-MS/MS using postcolumn infusion of copper sulfate (CuSO4) solution. Molecular networks that were generated thanks to specific fragmentations obtained with copper led to the distinction of THF ring position or to the identification of hydroxylated lactone, for instance.

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.