0000000000221298
AUTHOR
Pascale Winckler
Using CRISPR/Cas platform for Genetic Modification of Commercial Saccharomyces cerevisiae strains
International audience; Traditional wine fermentation is a complex microbial process initiated by various yeast species classified as Saccharomyces and non-Saccharomyces species.To better understand the different interactions occurring within wine fermentations and track a specific yeast population, we wish to obtain GFP-tagged yeast cells that stably expres fluorescence signal without compromising the fermentative capability of the strain.To this end, the CRISPR/Cas system was investigated to genetically modify the commercial Saccharomyces Saccharomyces cerevisiae diploid strain Lalvin EC 1118
Harnessing medically relevant metals onto water-soluble subphthalocyanines: towards bimodal imaging and theranostics
International audience; Subphthalocyanine (SubPc), a putative fluorophore for optical imaging (OI), was conjugated to chelating ligands (DOTA, DTPA) affording water-soluble conjugates complexed with (non-radioactive) metals relevant to the following medical imaging techniques/therapies: MRI (Gd), PET (Cu, Ga), SPECT (In, Ga, Lu), RIT (Cu, Lu, Y), and NCT (Gd). Magneto-optical properties of ditopic gadolinium species (and optical properties of other metal containing species) were examined (brightness (ε × Φ F) and relaxivity R 1) and fluorescence confocal/biphoton microscopy studies were conducted. † Electronic supplementary information (ESI) available: Experimental characterization, NMR and…
Surviving the heat: heterogeneity of response inSaccharomyces cerevisiaeprovides insight into thermal damage to the membrane
Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for…
Multispectral fluorescence sensitivity to acidic and polyphenolic changes in Chardonnay wines – The case study of malolactic fermentation
International audience; In this study, stationary and time-resolvedfluorescence signatures, were statistically and chemometrically analyzed among three typologies of Chardonnay wines (A, B and C) with the objectives to evaluate their sensitivity to acidic and polyphenolic changes. For that purpose, a dataset was built using Excitation Emission Matrices of fluorescence (N = 103) decomposed by a Parallel Factor Analysis (PARAFAC), andfluorescence decays (N = 22), mathematically fitted, using the conventional exponential modeling and the phasor plot representation. Wine PARAFAC component C4 coupledwith its phasor plot g and s values enable the description of malolactic fermentation (MLF) occur…
Effect of high hydrostatic pressure on extraction of B-phycoerythrin from Porphyridium cruentum: Use of confocal microscopy and image processing
International audience; The aim of the study was to extract B-phycoerythrin from Porphyridium cruentum while preserving its structure. The high hydrostatic pressure treatments were chosen as extraction technology. Different methods have been used to observe the effects of the treatment: spectrophotometry and confocal laser scanning microscopy followed by image processing analysis. Image processing led to the generation of masks used for the identification of three clusters: intra, extra and intercellular. All methods showed that high hydrostatic pressure treatments between 50 and 500 MPa failed to extract B-phycoerythrin from Porphyridium cruentum cells. The fluorescence emission was negati…
5èmes Journées Scientifiques et Techniques du Réseau des Microscopistes INRA. « Imagerie cellulaire en science du vivant : Cryo-microscopies, Dynamiques in vivo et Interactions moléculaires »
National audience; L’imagerie cellulaire, dans le domaine des sciences du vivant, est essentielle à la compréhension des phénomènes cellulaires subcellulaires régulant le fonctionnement des cellules et tissus. En microscopie électronique, la mise en oeuvre de cryo-méthodes permet de préserver l’intégrité moléculaire et spatiale des protéines et lipides intracellulaires ou membranaires (de faible poids moléculaire, présentes en faible quantité ou très labiles) dont la localisation in situ est recherchée afin de caractériser leur organisation, participer à la détermination de leur fonction au sein de la cellule et d’analyser leur distribution. Ces techniques, en pleine évolution, seront abord…
Subphthalocyanines: addressing water-solubility, nano-encapsulation, and activation for optical imaging of B16 melanoma cells
Water-soluble disulfonato-subphthalocyanines (SubPcs) or hydrophobic nano-encapsulated SubPcs are efficient probes for the fluorescence imaging of cells. 20 nm large liposomes (TEM and DLS) incorporated about 13% SubPc. Moreover, some of these fluorophores were found to be pH activatable.
Visualization of RNA-Quadruplexes in Live Cells
Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.
Physico-chemical state influences in vitro release profile of curcumin from pectin beads
International audience; Curcumin is a polyphenolic compound with diverse effects interesting to develop health benefit products but its formulation in functional foods or in food supplement is hampered by its poor water solubility and susceptibility to alkaline conditions, light, oxidation and heat. Encapsulation of curcumin could be a mean to overcome these difficulties. In this paper, curcumin was encapsulated by ionotropic gelation method in low methoxyl pectin beads associated with different surfactants: Solutol®, Transcutol® and sodium caseinate. After encapsulation, physico-chemical properties of encapsulated curcumin such as its solubility, physical state, tautomeric forms and encaps…
Resveratrol-induced xenophagy promotes intracellular bacteria clearance in intestinal epithelial cells and macrophages
International audience; Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be bene ficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebra f…
Cellular imaging using BODIPY-, pyrene- and phthalocyanine-based conjugates
International audience; Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in the green/red have been synthesized (as the form of dyads-pentads), studied by spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanine-pyrene 1 was achieved by cyclotetramerization of pyrenyldicyanobenzene, whereas phthalocyanine-BODIPY 2c was synthesized by Sonogashira coupling between tetraiodophthalocyanine and meso-alkynylBODIPY. The standard four-steps BODIPY synthesis was applied to the BODIPY-pyrene dyad 3 starting from pyrenecarbaldehyde and dimethylpyrrole. H-1, C-13, F-19, (BNMR)-B-11, ICP, MS, and UV/Vis spectroscopic analyses demonstra…
Shedding Light on the Formation and Structure of Kombucha Biofilm Using Two-Photon Fluorescence Microscopy
Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in thede novoformation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected t…
The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.
The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analyzed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the amount of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ub…
CCDC 1014064: Experimental Crystal Structure Determination
Related Article: Yann Bernhard, Pascale Winckler, Remi Chassagnon, Philippe Richard, Élodie Gigot, Jean-Marie Perrier-Cornet, Richard A. Decréau|2014|Chem.Commun.|50|13975|doi:10.1039/C4CC05503A