Journal articles: 'Yeast fluorescence'

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Karreman, Robert J., and George G. Lindsey. "A Rapid Method to Determine the Stress Status of Saccharomyces cerevisiae by Monitoring the Expression of a Hsp12:Green Fluorescent Protein (GFP) Construct under the Control of the Hsp12 Promoter." Journal of Biomolecular Screening 10, no. 3 (April 2005): 253–59. http://dx.doi.org/10.1177/1087057104273485.

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The gene for the green fluorescent protein (GFP) was fused in-frame to the 3′ end of HSP12. This construct was regulated by the HSP12 promoter in a pYES2 yeast expression vector. No fluorescence was observed in yeast growing exponentially in glucose-containing medium, but fluorescence was observed when the yeast entered the stationary phase. Fluorescence microscopy indicated that the fusion protein was localized to the peripheral regions of the cell as well as to the cytoplasm and the tonoplast. Subjecting the yeast to a variety of stresses known to induce HSP12 transcription, including salt, osmotic, ethanol, and heat stress, resulted in a time-dependent increase in GFP fluorescence. The use of this system as a method to assess the general stress status of yeast growing in an industrial application is proposed.

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Jones, Laura M., Danielle Dunham, Monique Y. Rennie, Jeffrey Kirman, Andrea J. Lopez, Klara C. Keim, William Little, et al. "In vitro detection of porphyrin-producing wound bacteria with real-time fluorescence imaging." Future Microbiology 15, no. 5 (March 2020): 319–32. http://dx.doi.org/10.2217/fmb-2019-0279.

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Aim: Fluorescence imaging can visualize polymicrobial populations in chronic and acute wounds based on porphyrin fluorescence. We investigated the fluorescent properties of specific wound pathogens and the fluorescence detected from bacteria in biofilm. Methods: Utilizing Remel Porphyrin Test Agar, 32 bacterial and four yeast species were examined for red fluorescence under 405 nm violet light illumination. Polymicrobial biofilms, supplemented with δ-aminolevulinic acid, were investigated similarly. Results: A total of 28/32 bacteria, 1/4 yeast species and polymicrobial biofilms produced red fluorescence, in agreement with their known porphyrin production abilities. Conclusion: These results identify common wound pathogens capable of producing porphyrin-specific fluorescence and support clinical observations using fluorescence imaging to detect pathogenic bacteria in chronic wounds.

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Rosseau, S., W. Seeger, H. Pralle, and J. Lohmeyer. "Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification." American Journal of Physiology-Lung Cellular and Molecular Physiology 267, no. 2 (August 1, 1994): L211—L217. http://dx.doi.org/10.1152/ajplung.1994.267.2.l211.

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The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

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Thiebault, F., and J. Coulon. "Influence of carbon source and surface hydrophobicity on the aggregation of the yeastKluyveromyces bulgaricus." Canadian Journal of Microbiology 51, no. 1 (January 1, 2005): 91–94. http://dx.doi.org/10.1139/w04-106.

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Aggregation of the yeast Kluyveromyces bulgaricus is mediated by the galactose-specific lectin KbCWL1. This lectin contains hydrophobic amino acids and its activity is calcium dependent. A specific fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid in the free acid form (ANS; Sigma Chemical Co., St. Louis, Missouri), was used to study the hydrophobic areas on the cellular surface of K. bulgaricus. Changes in surface hydrophobicity during the growth and aggregation of yeast cells were studied. Surface hydrophobicity increased during growth and depended on the amount of yeast cells in the culture medium. During growth, the size of the hydrophobic areas on the cell surface was measured using ANS and was found to increase with the percentage of flocculating yeasts. Our results strongly suggest that the hydrophobic areas of the cell walls of yeast cells are involved in the aggregation of K. bulgaricus.Key words: aggregation, carbon source, fluorescence probe, hydrophobicity, yeast.

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Skruzny, Pohl, and Abella. "FRET Microscopy in Yeast." Biosensors 9, no. 4 (October 11, 2019): 122. http://dx.doi.org/10.3390/bios9040122.

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Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

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Audus, K. L., M. R. Tavakoli-Saberi, H. Zheng, and E. N. Boyce. "Chlorhexidine Effects on Membrane Lipid Domains of Human Buccal Epithelial Cells." Journal of Dental Research 71, no. 6 (June 1992): 1298–303. http://dx.doi.org/10.1177/00220345920710060601.

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The effect of chlorhexidine gluconate on the adherence of Candida albicans to human buccal epithelial cells (BEC) and drug-induced alterations in BEC membrane-lipid packing order were examined. Treatment of BEC with attached yeasts with 0.1 and 0.2% chlorhexidine resulted in significant yeast detachment after 90 and 60 min, respectively. Following pre-treatment of BEC with > 0.1% chlorhexidine, yeast adherence was inhibited by > 80%. In parallel experiments, the fluorescence anisotropy of BEC labeled with fluorescent membrane probes-diphenylhexatriene (DPH) and trimethylammonium DPH-was assessed following exposure to chlorhexidine. The fluorescence anisotropy decreased with increasing concentrations of chlorhexidine, which indicated that the drug decreased epithelial-cell membrane-lipid packing order. Chlorhexidine concentrations that altered epithelial-cell membrane-lipid packing order, particularly in superficial regions, were similar to those drug concentrations required for detachment of adherent yeasts. Similar results were obtained with a second antifungal, nystatin A. While the effects of chlorhexidine on the buccal-cell membrane-lipid packing order were not reversed by multiple washings, the opposite situation occurred with nystatin A. The results suggest that chlorhexidine-induced alterations ofBEC membrane-lipid order may be involved in the antifungal actions of the drug.

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Guenther, Margarita, Falko Altenkirch, Kai Ostermann, Gerhard Rödel, Ingo Tobehn-Steinhäuser, Steffen Herbst, Stefan Görlandt, and Gerald Gerlach. "Optical and impedimetric study of genetically modified cells for diclofenac sensing." Journal of Sensors and Sensor Systems 8, no. 1 (May 21, 2019): 215–22. http://dx.doi.org/10.5194/jsss-8-215-2019.

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Abstract. Whole-cell biosensors, based on genetically modified yeast cells, were employed to detect anthropogenic micropollutants (e.g. drugs). Specific stimuli, e.g. traces of drugs, lead to the induction of fluorescence in the respective cells. Receptors of the cells detect specific signal molecules and induce the formation of fluorescent proteins. In this work, genetically modified cells of the yeast Saccharomyces cerevisiae BY4741 were confined in a four-chamber microfluidic cell, providing an optical monitoring of the cell behaviour and their supply with the nutrients. The measurements of the time-dependent fluorescence intensity were performed with different concentrations of the drug diclofenac, and the sensitivity of yeast cells to diclofenac was demonstrated. Cell viability was monitored by simultaneous impedance recording.

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Vanek, Martin, Filip Mravec, Martin Szotkowski, Dana Byrtusova, Andrea Haronikova, Milan Certik, Volha Shapaval, and Ivana Marova. "Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth." EuroBiotech Journal 2, no. 2 (April 1, 2018): 114–20. http://dx.doi.org/10.2478/ebtj-2018-0015.

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AbstractRed yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.

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Stasyuk, Natalia Ye, Galina Z. Gayda, Roman Ja Serkiz, and Mykhailo V. Gonchar. "Cell Imaging with Fluorescent Bi-Metallic Nanoparticles." JOURNAL OF ADVANCES IN CHEMISTRY 11, no. 4 (March 9, 2015): 3499–511. http://dx.doi.org/10.24297/jac.v11i4.6694.

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Last decades various imaging techniques have been applied in biological and biomedical research, such as magnetic resonance imaging, different types of tomography, fluorescence/bioluminescence, ultrasound, as well as multimodality approaches. Fluorescence imaging, especially in combination with nanoscale materials, is a very prospective tool for experiments in vivo and clinical applications due to its high temporal and spatial resolutions. Fluorescent nanoparticles (NPs), having ability to interact with biomolecules both on the surface of and inside the cells, may revolutionize the cell imaging approaches for diagnostics and therapy. In our investigation we report about new method of cell imaging with fluorescent bi-metallic NPs synthesized by chemical reduction of the relevant ions. As the model of living organism, the cells of yeast Hansenula polymorpha were used. All NPs in minimal concentration (up to 0.05 mM) was proved to be non-toxic for yeast cells. The NPs and NPs-modified cells were characterized with the methods of UV-VIS spectroscopy, scanning electron microscopy, atom force microscopy, transmission electron microscopy and fluorescence microscopy. The bimetallic NPs, possessing the stable fluorescence in solution and inside the cells, allow to observe the phenomenon of NPs transferring from parental to daughter cells through at least three generations followed by releasing from the modified cells. The fluorescent NPs synthesized being small, non-toxic and fluorescent was shown to be perspective tool for cell imaging.

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10

Monosov, E. Z., T. J. Wenzel, G. H. Lüers, J. A. Heyman, and S. Subramani. "Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells." Journal of Histochemistry & Cytochemistry 44, no. 6 (June 1996): 581–89. http://dx.doi.org/10.1177/44.6.8666743.

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We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

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Ramirez-Castrillon, Mauricio, Victoria Jaramillo-Garcia, Helio Barros, João Henriques, Valter Stefani, and Patricia Valente. "Dataset of Nile Red Fluorescence Readings with Different Yeast Strains, Solvents, and Incubation Times." Data 5, no. 3 (September 1, 2020): 77. http://dx.doi.org/10.3390/data5030077.

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We used Nile red to estimate lipid content in oleaginous yeasts using a high-throughput approach. We measured the fluorescence intensity of Nile red using different solvents, yeast strains, and incubation times in optimized excitation/emission wavelengths. The data show the relative fluorescence units (RFU) for Nile red excitation, using 1× PBS, 1× PBS and 5% v/v isopropyl alcohol, 50% v/v glycerol, culture medium A-gly broth, and A-gly broth supplemented with 5% v/v DMSO. In addition, we showed the RFU for the Nile red dye for different oleaginous and non-oleaginous yeast strains, such as Meyerozyma guilliermondii BI281A, Yarrowia lipolytica QU21 and Saccharomyces cerevisiae MRC164. Other measurements of lipid accumulation kinetics were shown for the above and additional yeast strains. These datasets provide the guidelines to obtain the optimal solvent system and the minimal interaction time for the Nile red dye to enter in the cells and obtain a stable readout.

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12

Bradley, Michael E., and Susan W. Liebman. "Destabilizing Interactions Among [PSI +] and [PIN +] Yeast Prion Variants." Genetics 165, no. 4 (December 1, 2003): 1675–85. http://dx.doi.org/10.1093/genetics/165.4.1675.

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AbstractThe yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi–]or[pin–], respectively, or the multiple infectious amyloid-like forms called [PSI+]or[PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.

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13

Rubel, Aleksander A., Viktoria V. Korzhova, Alsu F. Saifitdinova, Kirill S. Antonez, Sergey G. Inge-Vechtomov, and Alexey P. Galkin. "PRION PROTEIN AND AMYLOID BETA PEPTIDE INTERACT IN THE YEAST SACCHAROMYCES CEREVISIAE." Ecological genetics 10, no. 1 (March 28, 2012): 74–80. http://dx.doi.org/10.17816/ecogen10174-80.

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SUMMARY: The possibility of interaction between Prion Protein and amyloid beta peptide in living cells of yeast Saccharomyces cerevisiae have been investigated by fluorescence 3D microscopy. Using the FR ET technique, it was shown that amyloid beta peptide and PrP interact in yeast cells. In the future, the yeast model can be used for investigation of the fine mechanisms of this interaction by fluorescence microscopy.

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Eiden-Plach, Antje, Tatjana Zagorc, Tanja Heintel, Yvonne Carius, Frank Breinig, and Manfred J. Schmitt. "Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe." Applied and Environmental Microbiology 70, no. 2 (February 2004): 961–66. http://dx.doi.org/10.1128/aem.70.2.961-966.2004.

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ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

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Bordonné, Rémy. "Functional Characterization of Nuclear Localization Signals in Yeast Sm Proteins." Molecular and Cellular Biology 20, no. 21 (November 1, 2000): 7943–54. http://dx.doi.org/10.1128/mcb.20.21.7943-7954.2000.

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ABSTRACT In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.

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Kim, Hye-Lim, Hyun-Chul Ryu, and Young Shik Park. "Quantitative analysis by flow cytometry of green fluorescent protein-tagged human phenylalanine hydroxylase expressed in Dictyostelium." Pteridines 28, no. 3-4 (December 20, 2017): 153–56. http://dx.doi.org/10.1515/pterid-2017-0015.

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AbstractWe have developed a fluorescence assay system to monitor the protein levels of human phenylalanine hydroxylase (hPAH). Wild-type (WT) and three mutant hPAHs (I65T, L255V, and S349L) were expressed as green fluorescent protein (GFP)-tagged forms in a PAH knockout mutant (pah−) of Dictyostelium discoideum Ax2. The fluorescence-activated cell sorting (FACS) analysis showed that the GFP positive cells were the most frequent in WT but were rare in pah−, demonstrating the successful expression of GFP-tagged hPAHs in Dictyostelium. The fluorescence levels of mutants relative to WT were higher than expected from the protein amounts determined from the non-tagged forms, probably due to the presence of the N-terminal GFP. However, treatment of the cells with cumene hydroperoxide, which is known to accelerate protein degradation, decreased fluorescence levels, suggesting that protein stability changes in individual mutations can be monitored by FACS analysis. For an evaluation study, a putative pharmacological chaperone effect of yeast extract on S349L was examined by Western blot and FACS analysis. Both the protein amount and the fluorescence levels were increased by yeast extract, supporting that the FACS analysis could replace the time- and labor-consuming procedures such as the Western blot and cell culture. The fluorescence-based cell assay system may be valuable for the high-throughput screening of pharmacological chaperones for phenylketonuria mutations.

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17

TESTET, Eric, Jeanny LAROCHE-TRAINEAU, Abdelmajid NOUBHANI, Denis COULON, Odile BUNOUST, Nadine CAMOUGRAND, Stephen MANON, René LESSIRE, and Jean-Jacques BESSOULE. "Ypr140wp, ‘the yeast tafazzin’, displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis." Biochemical Journal 387, no. 3 (April 26, 2005): 617–26. http://dx.doi.org/10.1042/bj20041491.

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When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w–green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wΔ cells is discussed.

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Kawai-Noma, Shigeko, Chan-Gi Pack, Tomoko Kojidani, Haruhiko Asakawa, Yasushi Hiraoka, Masataka Kinjo, Tokuko Haraguchi, Hideki Taguchi, and Aiko Hirata. "In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells." Journal of Cell Biology 190, no. 2 (July 19, 2010): 223–31. http://dx.doi.org/10.1083/jcb.201002149.

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Yeast prion [PSI+] is caused by aggregated structures of the Sup35 protein. Although Sup35 forms typical amyloid fibrils in vitro, there is no direct evidence for the fibrillar structures of Sup35 in vivo. We analyzed [PSI+] cells in which Sup35 fused with green fluorescent protein (GFP) formed aggregates visible by fluorescence microscopy using thin-section electron microscopy (EM). Rapid-freeze EM combined with an immunogold-labeling technique as well as correlative light EM, which allows high-resolution imaging by EM of the same structure observed by light (fluorescence) microscopy, shows that the aggregates contain bundled fibrillar structures of Sup35-GFP. Additional biochemical and fluorescent correlation spectroscopy results suggest that the Sup35 oligomers diffused in the [PSI+] lysates adopt fibril-like shapes. Our findings demonstrate that [PSI+] cells contain Sup35 fibrillar structures closely related to those formed in vitro and provide insight into the molecular mechanism by which Sup35 aggregates are assembled and remodeled in [PSI+] cells.

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Zheng, Hu-Zhi, Hui-Hui Liu, Shao-Xing Chen, Zhe-Xue Lu, Zhi-Ling Zhang, Dai-Wen Pang, Zhi-Xiong Xie, and Ping Shen. "Yeast Transformation Process Studied by Fluorescence Labeling Technique." Bioconjugate Chemistry 16, no. 2 (March 2005): 250–54. http://dx.doi.org/10.1021/bc049833v.

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Riechers, Sean-Patrick, Ulf Stahl, and Christine Lang. "Endocytic uptake of fluorescence labelled DNA in yeast." Journal of Basic Microbiology 50, no. 1 (February 2010): 83–89. http://dx.doi.org/10.1002/jobm.200900279.

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Kukulski, Wanda, Martin Schorb, Sonja Welsch, Andrea Picco, Marko Kaksonen, and John A. G. Briggs. "Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision." Journal of Cell Biology 192, no. 1 (January 3, 2011): 111–19. http://dx.doi.org/10.1083/jcb.201009037.

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Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.

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Barnard, Emma, Neil V. McFerran, Alan Trudgett, John Nelson, and David J. Timson. "Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 479–82. http://dx.doi.org/10.1042/bst0360479.

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BiFC (bimolecular fluorescence complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein–protein interactions in this model organism.

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Bell, P. J. L., D. Deere, J. Shen, B. Chapman, P. H. Bissinger, P. V. Attfield, and D. A. Veal. "A Flow Cytometric Method for Rapid Selection of Novel Industrial Yeast Hybrids." Applied and Environmental Microbiology 64, no. 5 (May 1, 1998): 1669–72. http://dx.doi.org/10.1128/aem.64.5.1669-1672.1998.

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ABSTRACT We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 Ã¯Â¿Â½ 106 cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.

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Mazo-Vargas, Anyimilehidi, Heungwon Park, Mert Aydin, and Nicolas E. Buchler. "Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy." Molecular Biology of the Cell 25, no. 22 (November 5, 2014): 3699–708. http://dx.doi.org/10.1091/mbc.e14-07-1187.

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Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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Bouix, Marielle, Agnès Grabowski, Monique Charpentier, Jean-Yves Leveau, and Bruno Duteurtre. "Rapid detection of microbial contamination in grape juice by flow cytometry." OENO One 33, no. 1 (March 31, 1999): 31. http://dx.doi.org/10.20870/oeno-one.1999.33.1.1038.

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This study presents an application of flow cytometry to evaluate rapidly the viable micro-organisms in grape juice. In this method, viable cells are firstly specitically labelled with a fluorescent reagent. The sample is then injected into the flow cytometer where the labelled micro-organisms are individually illuminated by a laser beam. The emission of fluorescence is measured. The system counts the number of fluorescent events and prints out a histogram of the fluorescence intensity which is characteristic of the micro-organism being analysed. In laboratory conditions, preliminary trials have been undertaken with an artificially inoculated grape juice with pure yeast and bacteria cultures. This method succeeded in counting simultaneously yeasts and bacteria within 15 minutes, with a high degree of sensitivity, 5.103 yeasts perml and 5.104 bacteria per ml. This technique can also be applied to the detection of mould contamination and the test has been done with Botrytis spores. The method makes direct cell counts possible and is capable of analysing 30 samples per hour. It can be automatised and easily used in industrial laboratory. During the last harvest, more than a thousand of must samples were controled using this technique. The results let to determine the yeast contamination level of a grape juice tank even before unloading. The results obtained by flow cytometry were compared to the plate count reference method. The correlation between cytometry and count by plate culture was 99 p. cent for the threshold of 5.1 04 yeasts/ml which seemed to point out a high contamination. By using this flow cytometry method during the harvest period, the results were supplied in real time. This allowed a rapid selection of the musts, depending upon the scale of their contamination and improved the quality of the wine by corrective actions.

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Bae, Jungu, Kouichi Kuroda, and Mitsuyoshi Ueda. "Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface." Applied and Environmental Microbiology 81, no. 1 (October 10, 2014): 59–66. http://dx.doi.org/10.1128/aem.02864-14.

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ABSTRACTProximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on theSaccharomyces cerevisiaecell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.

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Yvanoff, Charlotte, Stefania Torino, and Ronnie G. Willaert. "Robotic Cell Printing for Constructing Living Yeast Cell Microarrays in Microfluidic Chips." Fermentation 6, no. 1 (February 14, 2020): 26. http://dx.doi.org/10.3390/fermentation6010026.

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Living cell microarrays in microfluidic chips allow the non-invasive multiplexed molecular analysis of single cells. Here, we developed a simple and affordable perfusion microfluidic chip containing a living yeast cell array composed of a population of cell variants (green fluorescent protein (GFP)-tagged Saccharomyces cerevisiae clones). We combined mechanical patterning in 102 microwells and robotic piezoelectric cell dispensing in the microwells to construct the cell arrays. Robotic yeast cell dispensing of a yeast collection from a multiwell plate to the microfluidic chip microwells was optimized. The developed microfluidic chip and procedure were validated by observing the growth of GFP-tagged yeast clones that are linked to the cell cycle by time-lapse fluorescence microscopy over a few generations. The developed microfluidic technology has the potential to be easily upscaled to a high-density cell array allowing us to perform dynamic proteomics and localizomics experiments.

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Plamont, Marie-Aude, Emmanuelle Billon-Denis, Sylvie Maurin, Carole Gauron, Frederico M. Pimenta, Christian G. Specht, Jian Shi, et al. "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo." Proceedings of the National Academy of Sciences 113, no. 3 (December 28, 2015): 497–502. http://dx.doi.org/10.1073/pnas.1513094113.

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This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

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Siavoshi, Farideh, Marzieh Sahraee, Samira Heydari, Abdolfattah Sarrafnejad, Parastoo Saniee, Atefeh Tavakolian, and Sheida Heidarian. "Sugar-Rich Foods Carry Osmotolerant Yeasts with Intracellular Helicobacter Pylori and Staphylococcus spp." Middle East Journal of Digestive Diseases 12, no. 3 (July 19, 2020): 182–93. http://dx.doi.org/10.34172/mejdd.2020.181.

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BACKGROUND Sugar-rich foods are of the main components of daily human meals. These foods with high sugar and low water content kill bacteria. However, osmotolerant yeasts survive and multiply. The aim of this study was to examine the occurrence of intracellular Helicobacter pylori(H. pylori) and Staphylococcus spp. in yeast isolates from sugar-rich foods. METHODS Thirty-two yeast isolates from fresh fruits, dried fruits, commercial foods, and miscellaneous foods were identified by the sequencing of amplified products of 26S rDNA. Fluorescence microscopy and LIVE/DEAD bacterial viability kit were used to examine the occurrence of live bacteria inside the yeast’s vacuole. Immunofluorescence assay was used to confirm the identity of intracellular bacteria as H. pylori and Staphylococcus. Polymerase chain reaction (PCR) was used for the detection of 16S rDNA of H. pylori and Staphylococcus in the total DNA of yeasts. RESULTS Yeasts were identified as members of seven genera; Candida, Saccharomyces, Zygosaccharomyces, Pichia, Meyerozyma, Metschnikowia, and Wickerhamomyces. Intravacuolar bacteria were stained green with a bacterial viability kit, revealing that they were alive. Immunofluorescence assay confirmed the identity of intracellular H. pylori and Staphylococcus spp. PCR results revealed that among the 32 isolated yeasts, 53% were H. pylori-positive, 6% were Staphylococcus-positive, 18.7% were positive for both, and 21.8% were negative for both. CONCLUSION Detection of H. pylori- and Staphylococcus-16S rDNA in yeast isolates from dried fruits, and commercial foods showed the occurrence of more than one kind of endosymbiotic bacterium in yeasts’ vacuoles. While the establishment of H. pylori and Staphylococcus in yeast is a sophisticated survival strategy, yeast serves as a potent bacterial reservoir.

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Doyle, Timothy C., James E. Hansen, and Emil Reisler. "Tryptophan Fluorescence of Yeast Actin Resolved via Conserved Mutations." Biophysical Journal 80, no. 1 (January 2001): 427–34. http://dx.doi.org/10.1016/s0006-3495(01)76025-0.

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Bhatta, Hemant, and Ewa M. Goldys. "Characterization of yeast strains by fluorescence lifetime imaging microscopy." FEMS Yeast Research 8, no. 1 (February 2008): 81–87. http://dx.doi.org/10.1111/j.1567-1364.2007.00340.x.

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Hahne, Katja, Gerhard Rödel, and Kai Ostermann. "A fluorescence‐based yeast sensor for monitoring acetic acid." Engineering in Life Sciences 21, no. 5 (January 18, 2021): 303–13. http://dx.doi.org/10.1002/elsc.202000006.

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LASORSA, Francesco M., Pasquale SCARCIA, Ralf ERDMANN, Ferdinando PALMIERI, Hanspeter ROTTENSTEINER, and Luigi PALMIERI. "The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes." Biochemical Journal 381, no. 3 (July 27, 2004): 581–85. http://dx.doi.org/10.1042/bj20040856.

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The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a ΔpH across the peroxisomal membrane, acidic inside.

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Marutyan, Seda V., Liparit H. Navasardyan, Hamlet G. Badalyan, and Mariam A. Shahinyan. "INFLUENCE OF X-RADIATION ON STRUCTURE OF DNA OF YEASTS C. guilliermondii NP-4." IZVESTIYA VYSSHIKH UCHEBNYKH ZAVEDENIY KHIMIYA KHIMICHESKAYA TEKHNOLOGIYA 59, no. 3 (July 12, 2018): 90. http://dx.doi.org/10.6060/tcct.20165903.5319.

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Investigation of fluorescence, electrophoretic and melting parameters of DNA of yeasts Candida guilliermondii NP-4 after X-radiation and posradiation repair was carried out. It was shown that under influence of X-radiation the rate of saturation of DNA by ethidium bromide and the melting temperature of yeast DNA was increased, and the melting interval was decreased. After repair period the rate of saturation of DNA by ethidium bromide and the melting temperature of yeast DNA were the highest, and the melting interval was the lowest, and in the electrophoregramms or repaired DNA there were additional low molecular fractions which testify about possible changes in DNA primary and secondary structures under influence of X-radiation and postradiation repair.

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Marley, Elaine, Phyllis Brown, Dave Leeman, and Carol Donnelly. "Analysis of Citrinin in Cereals, Red Yeast Rice Dietary Supplement, and Animal Feed by Immunoaffinity Column Cleanup and LC with Fluorescence Detection." Journal of AOAC INTERNATIONAL 99, no. 4 (July 1, 2016): 1025–31. http://dx.doi.org/10.5740/jaoacint.16-0060.

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Abstract The analysis of citrinin in various cereals (wheat, oats, maize, rice, and rye and multigrain breakfast cereal), red yeast rice (dietary supplement and traditional medicine), distillers dried grain with solubles, and barley (animal feed) was carried out using a citrinin immunoaffinity column (IAC) for sample cleanup before LC analysis with fluorescence detection (LC-fluorescence). To establish method performance characteristics, wheat was spiked with citrinin at levels of 10–200 μg/kg, whereas red yeast rice was spiked at levels of 100–3000 μg/kg. Methanol–water (75 + 25, v/v) was used for the extraction of cereals and animal feed, and extraction was with 100% methanol for red yeast rice. Cleanup used a commercial citrinin IAC, followed by LC-fluorescence (λex, 330 nm; λem, 500 nm). Recoveries ranged from 80 to 110%, with r from 0.7 to 4.3%. The LOQ for citrinin in both wheat and red yeast rice was 10 μg/kg, with an LOD of 3 μg/kg. Satisfactory performance was demonstrated in a proficiency testing exercise for a sample of maize contaminated with both citrinin and ochratoxin A. It was concluded that the commercial citrinin IAC was capable of providing an efficient and effective cleanup of complex food and feed matrixes to enable citrinin to be reliably determined with the specific LC-fluorescence system used.

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Pollard, Thomas D. "Progress towards understanding the mechanism of cytokinesis in fission yeast." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 425–30. http://dx.doi.org/10.1042/bst0360425.

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We use fission yeast to study the molecular mechanism of cytokinesis. We benefit from a long history in genetic analysis of the cell cycle in fission yeast, which provided the most complete inventory of cytokinesis proteins. We used fluorescence microscopy of proteins tagged with fluorescent proteins to establish the temporal and spatial pathway for the assembly and constriction of the contractile ring. We combined biochemical analysis of purified proteins (myosin-II, profilin, formin Cdc12p and cofilin), observations of fluorescent fusion proteins in live cells and mathematical modelling to formulate and test a simple hypothesis for the assembly of the contractile ring. This model involves the formation of 65 nodes containing myosin-II and formin Cdc12p around the equator of the cell. As a cell enters anaphase, actin filaments grow from formin Cdc12p in these nodes. Myosin captures actin filaments from adjacent nodes and pulls intermittently to condense the nodes into a contractile ring.

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Prick, Tanja, Michael Thumm, Karl Köhrer, Dieter Häussinger, and Stephan Vom Dahl. "In yeast, loss of Hog1 leads to osmosensitivity of autophagy." Biochemical Journal 394, no. 1 (January 27, 2006): 153–61. http://dx.doi.org/10.1042/bj20051243.

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In mammalian liver, proteolysis is regulated by the cellular hydration state in a microtubule- and p38MAPK (p38 mitogen-activated protein kinase)-dependent fashion. Osmosensing in liver cells towards proteolysis is achieved by activation of integrin receptors. The yeast orthologue of p38MAPK is Hog1 (high-osmolarity glycerol 1), which is involved in the hyperosmotic-response pathway. Since it is not known whether starvation-induced autophagy in yeast is osmosensitive and whether Hog1 is involved in this process, we performed fluorescence microscopy experiments. The hog1Δ cells exhibited a visible decrease of autophagy in hypo-osmotic and hyperosmotic nitrogen-starvation medium as compared with normo-osmolarity, as determined by GFP (green fluorescent protein)–Atg8 (autophagy-related 8) fluorescence. Western blot analysis of GFP–Atg8 degradation showed that WT (wild-type) cells maintained a stable autophagic activity over a broad osmolarity range, whereas hog1Δ cells showed an impaired autophagic actitivity during hypo- and hyper-osmotic stress. In [3H]leucine-pre-labelled yeast cells, the proteolysis rate was osmodependent only in hog1Δ cells. Neither maturation of pro-aminopeptidase I nor vitality was affected by osmotic stress in either yeast strain. In contrast, rapamycin-dependent autophagy, as measured by degradation of GFP–Atg8, did not significantly respond to hypo-osmotic or hyperosmotic stress in hog1Δ or WT cells. We conclude that Hog1 plays a role in the stabilization machinery of nitrogen-deprivation-induced autophagy in yeast cells during ambient osmolarity changes. This could be an analogy to the p38MAPK pathway in mammalian liver, where osmosensing towards p38MAPK is required for autophagy regulation by hypo-osmotic or amino-acid-induced cell swelling. A phenotypic difference is observed in rapamycin-induced autophagy, which does not seem to respond to extracellular osmolarity changes in hog1Δ cells.

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Bento-Oliveira, Andreia, Filipa C. Santos, Joaquim Trigo Marquês, Pedro M. R. Paulo, Thomas Korte, Andreas Herrmann, H. Susana Marinho, and Rodrigo F. M. de Almeida. "Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide." Biomolecules 10, no. 6 (June 6, 2020): 871. http://dx.doi.org/10.3390/biom10060871.

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The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterol-enriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

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Matamala-Valdés, Lillian, Kimberly Sánchez-Alonzo, Cristian Parra, Katia Sáez, Alejandro Aguayo-Reyes, and Apolinaria García. "Detection of intracellular Helicobacter pylori in Candida. SPP from neonate oral swabs." Revista da Associação Médica Brasileira 64, no. 10 (October 2018): 928–35. http://dx.doi.org/10.1590/1806-9282.64.10.928.

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SUMMARY BACKGROUND: There is evidence of detection of Helicobacter pylori (H. pylori) in the stool of newborns and in the yeast that colonizes the oral cavity of this age group. However, there is a lack of research to confirm it. This study proposes to determine the existence of the bacteria at an early age, specifically in newborns. OBJECTIVE: To identify intracellular H. pylori in oral yeasts and to detect antigens of the bacteria in newborn stools. METHODOLOGY: Cross-sectional and descriptive study. Samples were obtained from infants (oral swab and meconium). Identification of yeast species was performed using the following techniques: CHROMagar Candida, Germinal Tube Test and API Candida Identification System, then the yeasts were observed by light microscopy and fluorescence. Detection of H. pylori antigen in meconium and PCR were performed to amplify specific genes of the bacterium (rRNA16S, cagA, vacA s1a, vacA s1b, vacA s2, vacA m1, vacA m2 and dupA). RESULTS: Intracellular H. pylori was detected in yeast of the species Candida glabrata (C. glabrata) isolated from an oral swab of a newborn. CONCLUSION: The results of this study evidenced the existence of intracellular H. pylori in newborns.

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Colling, Lisa, Richard N. Carter, Michael Essmann, and Bryan Larsen. "Evaluation of Relative Yeast Cell Surface Hydrophobicity Measured by Flow Cytometry." Infectious Diseases in Obstetrics and Gynecology 13, no. 1 (2005): 43–48. http://dx.doi.org/10.1155/2005/739101.

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Objective:To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties.Methods:Yeast isolates were suspended in phosphate-buffered saline and mixed with deep blue-dyed polystyrene microspheres. Flow cytometry was used to detect the degree of microsphere binding to yeast cells. Different strains of yeast were compared for intrinsic microsphere binding activity and changes in growth conditions were invoked to modify the relative surface hydrophobicity.Results:Commercially available blue-dyed polystyrene microspheres showed strong fluorescence in the FL3 channel, whereas yeast cells did not show appreciable FL3 fluorescence. Microspheres and yeast were generally distinguishable on the basis of size revealed by forward light scatter. This method showed a wide variation in intrinsic cell surface hydrophobicity amongCandida albicansstrains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25°C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37°C, or 50 mg/dl glucose, respectively. Growth in1×10−9M estradiol had no appreciable effect on hydrophobicity.Conclusions:Stained latex microspheres fluoresced in the FL3 channel of the flow cytometer and bound to yeast cells to an extent related to the surface hydrophobicity of the yeast. Binding detected by flow cytometry showed that clinical yeast isolates varied in intrinsic binding capacity and this binding ability was altered by different growth conditions. The implications for virulence regulation among yeast isolates are discussed.

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Guo, Wenjun, Sunil Kumar, Frederik Görlitz, Edwin Garcia, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, et al. "Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells." SLAS TECHNOLOGY: Translating Life Sciences Innovation 24, no. 3 (January 10, 2019): 308–20. http://dx.doi.org/10.1177/2472630318819240.

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We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

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Pearson, Chad G., Paul S. Maddox, E. D. Salmon, and Kerry Bloom. "Budding Yeast Chromosome Structure and Dynamics during Mitosis." Journal of Cell Biology 152, no. 6 (March 19, 2001): 1255–66. http://dx.doi.org/10.1083/jcb.152.6.1255.

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Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

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Snyman, Carla, Julie Mekoue Nguela, Nathalie Sieczkowski, Matteo Marangon, and Benoit Divol. "Optimised Extraction and Preliminary Characterisation of Mannoproteins from Non-Saccharomyces Wine Yeasts." Foods 10, no. 5 (April 22, 2021): 924. http://dx.doi.org/10.3390/foods10050924.

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The exogenous application of yeast-derived mannoproteins presents many opportunities for the improvement of wine technological and oenological properties. Their isolation from the cell wall of Saccharomycescerevisiae has been well studied. However, investigations into the efficiency of extraction methods from non-Saccharomyces yeasts are necessary to explore the heterogeneity in structure and composition that varies between yeast species, which may influence wine properties such as clarity and mouthfeel. In this study, nine yeast strains were screened for cell wall mannoprotein content using fluorescence microscopy techniques. Four species were subsequently exposed to a combination of mechanical and enzymatic extraction methods to optimize mannoprotein yield. Yeast cells subjected to 4 min of ultrasound treatment applied at 80% of the maximum possible amplitude with a 50% duty cycle, followed by an enzymatic treatment of 4000 U lyticase per g dry cells weight, showed the highest mannoprotein-rich yield from all species. Furthermore, preliminary evaluation of the obtained extracts revealed differences in carbohydrate/protein ratios between species and with increased enzyme incubation time. The results obtained in this study form an important step towards further characterization of extraction treatment impact and yeast species effect on the isolated mannoproteins, and their subsequent influence on wine properties.

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Vrabioiu, Alina M., and Timothy J. Mitchison. "Structural insights into yeast septin organization from polarized fluorescence microscopy." Nature 443, no. 7110 (September 2006): 466–69. http://dx.doi.org/10.1038/nature05109.

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Ye, Bao-Fen, Zhi-Jie Zhang, and Huang-Xian Ju. "Fluorescence Study on the Interaction between Naproxen and Yeast DNA." Chinese Journal of Chemistry 23, no. 1 (January 2005): 58–62. http://dx.doi.org/10.1002/cjoc.200590013.

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Bhatta, Hemant, Ewa M. Goldys, and Robert P. Learmonth. "Use of fluorescence spectroscopy to differentiate yeast and bacterial cells." Applied Microbiology and Biotechnology 71, no. 1 (June 2006): 121–26. http://dx.doi.org/10.1007/s00253-005-0309-y.

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Wadsworth, Gable M., and Harold Kim. "Single-Probe Fluorescence in Situ Hybridization (FISH) in Budding Yeast." Biophysical Journal 110, no. 3 (February 2016): 21a. http://dx.doi.org/10.1016/j.bpj.2015.11.171.

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BAYLOR, KEN J., CLAIRE M. SHINE, JAMES J. A. HEFFRON, and JAMES MCINTOSH. "Isocyanate binding to yeast glutathione reductase measured by fluorescence spectroscopy." Biochemical Society Transactions 25, no. 1 (February 1, 1997): 47S. http://dx.doi.org/10.1042/bst025047s.

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Wang, Ye, Wen Qian Li, Xun Li, Hao Shi, and Fei Wang. "Constructon of Yeast Surface Display of ProROL by Using Flo1p Anchor System." Advanced Materials Research 512-515 (May 2012): 356–60. http://dx.doi.org/10.4028/www.scientific.net/amr.512-515.356.

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A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system containing N-terminal flocculation functional domain (874 residues, FS), derived from Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) and green fluorescent protein (GFP) gene were successfully cloned and genetically fused to the anchor system with their C-terminus free. Fluorescence microscope was used to detect the GFP displayed on the recombinant P. pastoris cell surface. The results showed that the yeast surface display system using Flo1p as the anchor protein was successfully constructed, and the activity of ProROL displayed on KM71H reached to 217.15 U/g, much higher than 61.30 U/g reported by Matsumoto. Besides, the yeast surface display system could effectively shorten the fermentation time compared with traditional fermentation.

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James, Joel, Nikita Fiji, Debasish Roy, Daniel Andrew MG, Mohamed Sham Shihabudeen, Debarati Chattopadhyay, and Kavitha Thirumurugan. "A rapid method to assess reactive oxygen species in yeast using H2DCF-DA." Analytical Methods 7, no. 20 (2015): 8572–75. http://dx.doi.org/10.1039/c5ay02278a.

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Journal articles on the topic 'Yeast fluorescence'

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