Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

Novel phenoxazine dyes : laccase-mediated synthesis, physico-chemical properties and applications

Laccases are members of the multicopper oxidase family which are involved in numerous biosynthetic pathways in fungi. Among these, one laccase has been shown to produce in vitro a natural pigment, namely the cinnabarinic acid, which is related to the actinomycin chromophore. The core structure of this chromophore, i.e 2-amino-3H-phenoxazin-3-one, was found to possess a marked potential for the fluorescent labelling in the field of molecular biology. As water-solubility is one of the critical features for biological application, efficient and original synthetic routes are needed to provide new hydrophilic fluorophores inspired from natural pigments. In the frame of the European research project SOPHIED, we have investigated the biomimetic synthesis of novel aminophenoxazinone dyes featuring tuneable water-solubility. Our synthetic route involves a key step of biocatalysed oxidative cascade with a total of 6e- oxidation mediated by a laccase. Sulfonylated and phosphorylated aminophenol building-blocks were synthesised with a total regioselectivity control and further processed as water-soluble substrates by our biocatalyst to afford novel fluorophores. The investigation of the scope and limitations of our biocatalytic process led to a new mechanistic proposal for the key step of oxidative cyclisation initiated by the laccase. Fluorescent properties of these heterocyclic scaffolds were evaluated and further assayed in a live-cell imaging application. This results in the discovery of a promising fluorophore, presumably a bulk endocytic tracer with targeting by default to lysosomes. Lastly the serendipitous discovery of an intramolecular cyclisation due to a neighbouring substituent effect of the acidic group (SO3H and PO(OH)2) in our aminophenol derivatives led to the development of an alternative synthetic route towards novel benzoxazole derivatives of potential interest in medicinal chemistry.Les Laccases font partie de la famille des oxydases à cuivre, enzymes principalement impliquées dans de nombreuses voies de biosynthèse chez les champignons. Parmi les isozymes répertoriés, une laccase a permis de réaliser avec succès la formation in vitro d’un pigment d’origine naturelle dénommé « acide cinnabarinique ». Ce composé comprend un motif hétérocyclique identique à celui du groupement chromophore de l’Actinomycin D qui s’avère être un puissant agent antitumoral d’origine fongique. Le motif hétérocyclique commun dénommé 2-amino-3H-phenoxazin-3-one, possède un potentiel avéré pour une application telle que le marquage par fluorescence dans le domaine de la biologie cellulaire et/ou moléculaire. Une excellente solubilité en milieux aqueux représente une caractéristique incontournable pour de telles applications biologiques. Dés lors, de nouvelles voies de synthèses présentant simultanément originalité et efficacité doivent être continuellement élaborées afin de produire de nouveaux marqueurs fluorescents hydrophiles inspirés de pigments naturels. Dans le cadre d’un projet de recherche européen nommé SOPHIED, nous avons élaboré la synthèse biomimétique de nouveaux colorants de type aminophenoxazinone possédant une solubilité en milieux aqueux modulable grâce aux groupements fonctionnels présents sur ceux-ci. Notre voie de synthèse implique une étape clé de cascade oxydante pour un total de six électrons initiée par un bio-catalyseur, une laccase. Nos blocs de constructions, des molécules de type aminophénols présentant des groupements fonctionnels dérivés d’acides sulfoniques et phosphoniques, ont été synthétisés avec un contrôle total de la régio-sélectivité. Ces substrats hydrophiles ont été transformés par une laccase en nouveaux colorants fluorescents avec une complète sélectivité et d’excellents rendements. Une investigation des potentialités et limitations de notre synthèse bio-catalytique a permis de suggérer une proposition mécanistique pour la cascade oxydante, principalement une variante de celle communément acceptée pour la production de pigments naturels. Néanmoins, bien que cette proposition ait le mérite d’expliquer certains de nos résultats plutôt surprenant, elle reste une suggestion et en aucun cas une quelconque affirmation. Les propriétés de fluorescence de ces composés hétérocycliques ont été évaluées et testés comme marqueurs fluorescents de structures cellulaires vivantes. Grâce à cette application, un de ces hétérocycles s’est révélé prometteur en tant que traceur pour endocytose avec une localisation intracellulaire finale dans les lysosomes. Finalement, la découverte accidentelle d’une cyclisation intramoléculaire due à un effet activant des groupes fonctionnels acides, type sulfonique et phosphorique, dans nos substrats aromatiques à mené à une brève investigation d’une voie alternative de synthèse de benzoxazoles car ce motif structural est largement représenté dans diverses molécules bioactives d’origine naturelle ou synthétique.(CHIM 3) -- UCL, 201

Novel 4- and 7-Sulfonylated 2-Substituted Benzoxazoles

The efficient synthesis of sulfonylated benzoxazoles at positions C4 or C7 is reported. The condensation reactions involve original anilide acetal reagents which, upon acid catalysis, allow an easy cyclization reaction with the sulfonylated o-aminophenol partners. This method circumvents the classical use of orthoesters which drawback is the limited access to aromatic reagents

Extrapulmonary small cell carcinoma following augmentation enterocystoplasty

Tumours developing after urinary tract or bladder reconstruction with intestinal tissue have well been described. Adenocarcinomas and transitional cell carcinomas are the most frequent histological features. We report here a case of small cell carcinoma developing after augmentation enterocystoplasty. This rare observation is an opportunity to review the literature about extrapulmonary small cell carcinoma and tumours developing after uro-intestinal reconstruction surgery.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Non-symmetrically substituted phenoxazinones from laccase-mediated oxidative cross-coupling of aminophenols: an experimental and theoretical insight

Oxidative cross-coupling reactions of substituted o-aminophenols were catalyzed by a commercial laccase to produce non-symmetrically substituted phenoxazinones for the first time. Identification by 1H-, 13C- and 31P-NMR, and by HPLC-PDA and HPLC-MS/MS of exclusively two kinds of substituted phenoxazinones out of four potential heterocyclic frameworks was confirmed by a DFT study. The redox-properties of the substrates, their relative rates of conversion and the rigid docking of selected substrates led to a revisited mechanistic pathway for phenoxazinones biosynthesis. Our suggestions concern both the first formal two-electron oxidation by laccase and the first intermolecular 1,4-conjugated addition which secures the observed regioselectivity

The Double-Trunk-Mask: A Simple System to Save Oxygen Supplies

Dear editor, Since March 2020, after China, Europe and the rest of the world are facing the COVID-19 pandemic, caused by the SARS-COV-2 virus [1]. This health crisis has led to oxygen shortages in developing countries but also in nursing homes because of the limited capacity of oxy-concentrators, when present, to provide an adequate oxygen flow [2-4]. [...