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Sample records for yeast cell separation

The price of independence: cell separation in fission yeast.

PubMed

Martín-García, Rebeca; Santos, Beatriz

2016-04-01

The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.
Continuous-flow separation of live and dead yeasts using reservoir-based dielectrophoresis (rDEP)

NASA Astrophysics Data System (ADS)

Patel, Saurin; Showers, Daniel; Vedantam, Pallavi; Tzeng, Tzuen-Rong; Qian, Shizhi; Xuan, Xiangchun

2012-11-01

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening etc. We develop a novel microfluidic approach to continuous separation of yeast cells by viability inside a reservoir. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeasts and continuously sort them from live ones. We term this approach reservoir-based dielectrophoresis (rDEP). The transporting, focusing, and trapping of live and dead yeast cells at the reservoir-microchannel junction are studied separately by varying the DC-biased AC electric fields. These phenomena can all be reasonably predicted by a 2D numerical model. We find that the AC to DC field ratio for live yeast trapping is higher than that for dead cells because the former experiences a weaker rDEP while having a larger electrokinetic mobility. It is this difference in the AC to DC field ratio that enables the viability-based yeast cell separation. The rDEP approach has unique advantages over existing DEP-based techniques such as the occupation of zero channel space and the elimination of in-channel mechanical or electrical parts. NSF
Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

PubMed Central

Li, Ping; Shao, Yize; Jin, Hui

2015-01-01

Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084
Entropy-based separation of yeast cells using a microfluidic system of conjoined spheres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Kai-Jian; Qin, S.-J., E-mail: shuijie.qin@gmail.com; Bai, Zhong-Chen

2013-11-21

A physical model is derived to create a biological cell separator that is based on controlling the entropy in a microfluidic system having conjoined spherical structures. A one-dimensional simplified model of this three-dimensional problem in terms of the corresponding effects of entropy on the Brownian motion of particles is presented. This dynamic mechanism is based on the Langevin equation from statistical thermodynamics and takes advantage of the characteristics of the Fokker-Planck equation. This mechanism can be applied to manipulate biological particles inside a microfluidic system with identical, conjoined, spherical compartments. This theoretical analysis is verified by performing a rapid andmore » a simple technique for separating yeast cells in these conjoined, spherical microfluidic structures. The experimental results basically match with our theoretical model and we further analyze the parameters which can be used to control this separation mechanism. Both numerical simulations and experimental results show that the motion of the particles depends on the geometrical boundary conditions of the microfluidic system and the initial concentration of the diffusing material. This theoretical model can be implemented in future biophysics devices for the optimized design of passive cell sorters.« less
Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis.

PubMed

Li, Ping; Shao, Yize; Jin, Hui; Yu, Hong-Guo

2015-04-27

Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. © 2015 Li et al.
Dielectric modelling of cell division for budding and fission yeast

NASA Astrophysics Data System (ADS)

Asami, Koji; Sekine, Katsuhisa

2007-02-01

The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast.
A Modified MuDPIT Separation Identified 4,488 Proteins in a System Wide Analysis of Quiescence in Yeast

PubMed Central

Webb, Kristofor J.; Xu, Tao; Park, Sung Kyu; Yates, John R.

2013-01-01

A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near complete yeast proteome from a whole cell tryptic digest. This modified on-line two dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4,269 protein identifications were made from 4,189 distinguishable protein families from yeast during log phase growth. The “Micro” MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days’ time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations ‘oxidation reduction’, ‘catabolic processing’ and ‘cellular response to oxidative stress’ was seen in the quiescent cellular fraction, consistent with their long lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of ‘Ribosome’ and ‘Proteasome’, further defining the complex nature of yeast populations present during stationary phase growth. In total 4,488 distinguishable protein families were identified in all cellular conditions tested. PMID:23540446
CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection.

PubMed

Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

2007-07-01

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.
Extraction of the number of peroxisomes in yeast cells by automated image analysis.

PubMed

Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

2006-01-01

An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.
Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

PubMed

Palková, Zdena; Váchová, Libuše

2016-09-01

Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.
Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle

PubMed Central

Chen, Katherine C.; Csikasz-Nagy, Attila; Gyorffy, Bela; Val, John; Novak, Bela; Tyson, John J.

2000-01-01

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID:10637314
Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission

PubMed Central

Sato, Mamiko; Muñoz, Javier; Moreno, M. Belén; Clemente-Ramos, Jose Angel; Ramos, Mariona; Okada, Hitoshi; Osumi, Masako; Durán, Angel; Ribas, Juan Carlos

2012-01-01

Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure. PMID:22891259
Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

PubMed

Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

2017-07-03

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Cell separation technique in dilectrophoretic chip with bulk electrode

NASA Astrophysics Data System (ADS)

Iliescu, Ciprian; Tay, Francis E. H.; Xu, Guolin; Yu, Liming

2006-01-01

This paper presents a new technique for separation of two cell populations in a dielectrophoretic chip with bulk silicon electrode. A characteristic of the dielectrophoretic chip is its "sandwich" structure: glass/silicon/glass that generates a unique definition of the microfluidic channel with conductive walls (silicon) and isolating floor and ceiling (glass). The structure confers the opportunity to use the electrodes not only to generate a gradient of the electric field but also to generate a gradient of velocity of the fluid inside the channel. This interesting combination gives rise to a new solution for dielectrophoretic separation of two cell populations. The separation method consists of four steps. First, the microchannel is field with the cells mixture. Second, the cells are trapped in different locations of the microfluidic channel, the cell population which exhibits positive dielectrophoresis is trapped in the area where the distance between the electrodes is the minimum whilst, the other population that exhibit negative dielectrophoresis is trapped where the distance between electrodes is the maximum. In the next step, increasing the flow in the microchannel will result in an increased hydrodynamic force that sweeps the cells trapped by positive dielectrophoresis out of the chip. In the last step, the electric field is removed and the second population is sweep out and collected at the outlet. The device was tested for separation of dead yeast cells from live yeast cells. The paper presents analytical aspects of the separation method a comparative study between different electrode profiles and experimental results.
Separation of similar yeast strains by IEF techniques.

PubMed

Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

2009-06-01

Rapid and reliable identification of the etiological agents of infectious diseases, especially species that are hardly distinguishable by routinely used laboratory methods, e.g. Candida albicans from C. dubliniensis, is necessary for early administration of an appropriate therapy. Similarly, the differentiation between biofilm-positive and biofilm-negative yeast strains is necessary for the choice of a therapeutic strategy due to higher resistance of the biofilm-positive strains to antifungals. In this study rapid separation and identification of similar strains of Candida, cells and/or their lysates, based on IEF are outlined. The isoelectric points of the monitored "similar pairs" of Candidas, C. albicans and C. dubliniensis and the biofilm-positive C. parapsilosis, C. tropicalis and their biofilm-negative strains were determined by CIEF with UV detection in the acidic pH gradient. The differences between their isoelectric points were up to 0.3 units of pI. Simultaneously, a fast and a simple technique was developed for the lysis of the outer membrane cell and characteristic fingerprints were found in lysate electrophoreograms and in gels from the capillary or the gel IEF, respectively.
Yeast fuel cell: Application for desalination

NASA Astrophysics Data System (ADS)

Mardiana, Ummy; Innocent, Christophe; Cretin, Marc; Buchari, Buchari; Gandasasmita, Suryo

2016-02-01

Yeasts have been implicated in microbial fuel cells as biocatalysts because they are non-pathogenic organisms, easily handled and robust with a good tolerance in different environmental conditions. Here we investigated baker's yeast Saccharomyces cerevisiae through the oxidation of glucose. Yeast was used in the anolyte, to transfer electrons to the anode in the presence of methylene blue as mediator whereas K3Fe(CN)6 was used as an electron acceptor for the reduction reaction in the catholyte. Power production with biofuel cell was coupled with a desalination process. The maximum current density produced by the cell was 88 mA.m-2. In those conditions, it was found that concentration of salt was removed 64% from initial 0.6 M after 1-month operation. This result proves that yeast fuel cells can be used to remove salt through electrically driven membrane processes and demonstrated that could be applied for energy production and desalination. Further developments are in progress to improve power output to make yeast fuel cells applicable for water treatment.
Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast
Characterization of winemaking yeast by cell number-size distribution analysis through flow field-flow fractionation with multi-wavelength turbidimetric detection.

PubMed

Zattoni, Andrea; Melucci, Dora; Reschiglian, Pierluigi; Sanz, Ramsés; Puignou, Lluís; Galceran, Maria Teresa

2004-10-29

Yeasts are widely used in several areas of food industry, e.g. baking, beer brewing, and wine production. Interest in new analytical methods for quality control and characterization of yeast cells is thus increasing. The biophysical properties of yeast cells, among which cell size, are related to yeast cell capabilities to produce primary and secondary metabolites during the fermentation process. Biophysical properties of winemaking yeast strains can be screened by field-flow fractionation (FFF). In this work we present the use of flow FFF (FlFFF) with turbidimetric multi-wavelength detection for the number-size distribution analysis of different commercial winemaking yeast varieties. The use of a diode-array detector allows to apply to dispersed samples like yeast cells the recently developed method for number-size (or mass-size) analysis in flow-assisted separation techniques. Results for six commercial winemaking yeast strains are compared with data obtained by a standard method for cell sizing (Coulter counter). The method here proposed gives, at short analysis time, accurate information on the number of cells of a given size, and information on the total number of cells.
A simplified sheathless cell separation approach using combined gravitational-sedimentation-based prefocusing and dielectrophoretic separation.

PubMed

Luo, Tao; Fan, Lei; Zeng, Yixiao; Liu, Ya; Chen, Shuxun; Tan, Qiulin; Lam, Raymond H W; Sun, Dong

2018-05-04

Prefocusing of the cell mixture is necessary for achieving a high-efficiency and continuous dielectrophoretic (DEP) cell separation. However, prefocusing through sheath flow requires a complex and tedious peripheral system for multi-channel fluid control, hindering the integration of DEP separation systems with other microfluidic functionalities for comprehensive clinical and biological tasks. This paper presented a simplified sheathless cell separation approach that combines gravitational-sedimentation-based sheathless prefocusing and DEP separation methods. Through gravitational sedimentation in a tubing, which was inserted into the inlet of a microfluidic chip with an adjustable steering angle, the cells were focused into a stream at the upstream region of a microchannel prior to separation. Then, a DEP force was applied at the downstream region of the microchannel for the active separation of the cells. Through this combined strategy, the peripheral system for the sheath flow was no longer required, and thus the integration of cell separation system with additional microfluidic functionalities was facilitated. The proposed sheathless scheme focused the mixture of cells with different sizes and dielectric properties into a stream in a wide range of flow rates without changing the design of the microfluidic chip. The DEP method is a label-free approach that can continuously separate cells on the basis of the sizes or dielectric properties of the cells and thus capable of greatly flexible cell separation. The efficiency of the proposed approach was experimentally assessed according to its performance in the separation of human acute monocytic leukemia THP-1 cells from yeast cells with respect to different sizes and THP-1 cells from human acute myelomonocytic leukemia OCI-AML3 cells with respect to different dielectric properties. The experimental results revealed that the separation efficiency of the method can surpass 90% and thus effective in separating cells on
Accumulation and metabolism of selenium by yeast cells.

PubMed

Kieliszek, Marek; Błażejak, Stanisław; Gientka, Iwona; Bzducha-Wróbel, Anna

2015-07-01

This paper examines the process of selenium bioaccumulation and selenium metabolism in yeast cells. Yeast cells can bind elements in ionic from the environment and permanently integrate them into their cellular structure. Up to now, Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica yeasts have been used primarily in biotechnological studies to evaluate binding of minerals. Yeast cells are able to bind selenium in the form of both organic and inorganic compounds. The process of bioaccumulation of selenium by microorganisms occurs through two mechanisms: extracellular binding by ligands of membrane assembly and intracellular accumulation associated with the transport of ions across the cytoplasmic membrane into the cell interior. During intracellular metabolism of selenium, oxidation, reduction, methylation, and selenoprotein synthesis processes are involved, as exemplified by detoxification processes that allow yeasts to survive under culture conditions involving the elevated selenium concentrations which were observed. Selenium yeasts represent probably the best absorbed form of this element. In turn, in terms of wide application, the inclusion of yeast with accumulated selenium may aid in lessening selenium deficiency in a diet.

Guidelines and recommendations on yeast cell death nomenclature.

PubMed

Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J; Breitenbach, Michael; Burhans, William C; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W; Grant, Chris M; Greenwood, Michael T; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D; Outeiro, Tiago F; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F; Sharon, Amir; Sigrist, Stephan J; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B; Tuite, Mick; Vögtle, F-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J; Zhao, Richard Y; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

2018-01-01

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
Guidelines and recommendations on yeast cell death nomenclature

PubMed Central

Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J.; Breitenbach, Michael; Burhans, William C.; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F.; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B.; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W.; Grant, Chris M.; Greenwood, Michael T.; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M.; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P.; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A.; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D.; Outeiro, Tiago F.; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F.; Sharon, Amir; Sigrist, Stephan J.; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M.; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B.; Tuite, Mick; Vögtle, F.-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J.; Zhao, Richard Y.; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

2018-01-01

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research. PMID:29354647
Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

NASA Astrophysics Data System (ADS)

Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.
A multivariate approach using attenuated total reflectance mid-infrared spectroscopy to measure the surface mannoproteins and β-glucans of yeast cell walls during wine fermentations.

PubMed

Moore, John P; Zhang, Song-Lei; Nieuwoudt, Hélène; Divol, Benoit; Trygg, Johan; Bauer, Florian F

2015-11-18

Yeast cells possess a cell wall comprising primarily glycoproteins, mannans, and glucan polymers. Several yeast phenotypes relevant for fermentation, wine processing, and wine quality are correlated with cell wall properties. To investigate the effect of wine fermentation on cell wall composition, a study was performed using mid-infrared (MIR) spectroscopy coupled with multivariate methods (i.e., PCA and OPLS-DA). A total of 40 yeast strains were evaluated, including Saccharomyces strains (laboratory and industrial) and non-Saccharomyces species. Cells were fermented in both synthetic MS300 and Chardonnay grape must to stationery phase, processed, and scanned in the MIR spectrum. PCA of the fingerprint spectral region showed distinct separation of Saccharomyces strains from non-Saccharomyces species; furthermore, industrial wine yeast strains separated from laboratory strains. PCA loading plots and the use of OPLS-DA to the data sets suggested that industrial strains were enriched with cell wall proteins (e.g., mannoproteins), whereas laboratory strains were composed mainly of mannan and glucan polymers.
Oxidative Stress and Programmed Cell Death in Yeast

PubMed Central

Farrugia, Gianluca; Balzan, Rena

2012-01-01

Yeasts, such as Saccharomyces cerevisiae, have long served as useful models for the study of oxidative stress, an event associated with cell death and severe human pathologies. This review will discuss oxidative stress in yeast, in terms of sources of reactive oxygen species (ROS), their molecular targets, and the metabolic responses elicited by cellular ROS accumulation. Responses of yeast to accumulated ROS include upregulation of antioxidants mediated by complex transcriptional changes, activation of pro-survival pathways such as mitophagy, and programmed cell death (PCD) which, apart from apoptosis, includes pathways such as autophagy and necrosis, a form of cell death long considered accidental and uncoordinated. The role of ROS in yeast aging will also be discussed. PMID:22737670
Mitochondrial fission proteins regulate programmed cell death in yeast.

PubMed

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.
Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

PubMed

Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

2015-09-02

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.
Lateral separation of colloids or cells by dielectrophoresis augmented by AC electroosmosis.

PubMed

Zhou, Hao; White, Lee R; Tilton, Robert D

2005-05-01

Colloidal particles and biological cells are patterned and separated laterally adjacent to a micropatterned electrode array by applying AC electric fields that are principally oriented normally to the electrode array. This is demonstrated for yeast cells, red blood cells, and colloidal polystyrene particles of different sizes and zeta-potentials. The separation mechanism is observed experimentally to depend on the applied field frequency and voltage. At high frequencies, particles position themselves in a manner that is consistent with dielectrophoresis, while at low frequencies, the positioning is explained in terms of a strong coupling between gravity, the vertical component of the dielectrophoretic force, and the Stokes drag on particles induced by AC electroosmotic flow. Compared to high frequency dielectrophoretic separations, the low frequency separations are faster and require lower applied voltages. Furthermore, the AC electroosmosis coupling with dielectrophoresis may enable cell separations that are not feasible based on dielectrophoresis alone.
Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

NASA Astrophysics Data System (ADS)

Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

2006-02-01

We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.
Differential Adsorption of Ochratoxin A and Anthocyanins by Inactivated Yeasts and Yeast Cell Walls during Simulation of Wine Aging

PubMed Central

Petruzzi, Leonardo; Baiano, Antonietta; De Gianni, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria; Bevilacqua, Antonio

2015-01-01

The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify the intensity of the phenomenon. The aim of this study was to examine OTA adsorption by two strains of Saccharomyces cerevisiae (the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a yeast cell wall preparation. Experiments were conducted using Nero di Troia red wine contaminated with 2 μg/L OTA and supplemented with yeast biomass (20 g/L). The samples were analyzed periodically to assess mycotoxin concentration, chromatic characteristics, and total anthocyanins over 84 days of aging. Yeast cell walls revealed the highest OTA-adsorption in comparison to thermally-inactivated cells (50% vs. 43% toxin reduction), whilst no significant differences were found for the amount of adsorbed anthocyanins in OTA-contaminated and control wines. OTA and anthocyanins adsorption were not competitive phenomena. Unfortunately, the addition of yeast cells to wine could cause color loss; therefore, yeast selection should also focus on this trait to select the best strain. PMID:26516913
Immobilisation increases yeast cells' resistance to dehydration-rehydration treatment.

PubMed

Borovikova, Diana; Rozenfelde, Linda; Pavlovska, Ilona; Rapoport, Alexander

2014-08-20

This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains. Copyright © 2014 Elsevier B.V. All rights reserved.
Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

PubMed

Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

2015-01-01

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.
Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts.

PubMed

Zhang, Ning; Fan, Yuxuan; Li, Chen; Wang, Qiming; Leksawasdi, Noppol; Li, Fuli; Wang, Shi'an

2018-05-01

Non-model yeasts within basidiomycetes have considerable importance in agriculture, industry, and environment, but they are not as well studied as ascomycetous yeasts. Serving as a basic technique, nuclear DNA staining is widely used in physiology, ecology, cell biology, and genetics. However, it is unclear whether the classical nuclear DNA staining method for ascomycetous yeasts is applicable to basidiomycetous yeasts. In this study, 5 yeasts ineffectively stained by the classical propidium iodide (PI) staining method were identified from 23 representative basidiomycetous yeasts. Pretreatment of cells using dimethyl sulfoxide (DMSO) or snailase markedly improved cell penetration to PI and thus enabled DNA content determination by flow cytometry on the recalcitrant yeasts. The pretreatments are efficient, simple, and fast, avoiding tedious mutagenesis or genetic engineering used in previous reports. The heterogeneity of cell penetration to PI among basidiomycetous yeasts was attributed to the discrepancy in cell wall polysaccharides instead of capsule or plasma membrane. This study also indicated that care must be taken in attributing PI-negative staining as viable cells when studying non-model microorganisms.
Correlating yeast cell stress physiology to changes in the cell surface morphology: atomic force microscopic studies.

PubMed

Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

2006-07-06

Atomic Force Microscopy (AFM) has emerged as a powerful biophysical tool in biotechnology and medicine to investigate the morphological, physical, and mechanical properties of yeasts and other biological systems. However, properties such as, yeasts' response to environmental stresses, metabolic activities of pathogenic yeasts, cell-cell/cell-substrate adhesion, and cell-flocculation have rarely been investigated so far by using biophysical tools. Our recent results obtained by AFM on one strain each of Saccharomyces cerevisiae and Schizosaccharomyces pombe show a clear correlation between the physiology of environmentally stressed yeasts and the changes in their surface morphology. The future directions of the AFM related techniques in relation to yeasts are also discussed.
Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

PubMed Central

Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

2015-01-01

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry. PMID:26295574
Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

NASA Astrophysics Data System (ADS)

Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.
Sake yeast strains have difficulty in entering a quiescent state after cell growth cessation.

PubMed

Urbanczyk, Henryk; Noguchi, Chiemi; Wu, Hong; Watanabe, Daisuke; Akao, Takeshi; Takagi, Hiroshi; Shimoi, Hitoshi

2011-07-01

Sake yeast strains produce a high concentration of ethanol during sake brewing compared to laboratory yeast strains. As ethanol fermentation by yeast cells continues even after cell growth stops, analysis of the physiological state of the stationary phase cells is very important for understanding the mechanism of producing higher concentrations of ethanol. We compared the physiological characteristics of stationary phase cells of both sake and laboratory yeast strains in an aerobic batch culture and under sake brewing conditions. We unexpectedly found that sake yeast cells in the stationary phase had a lower buoyant density and stress tolerance than did the laboratory yeast cells under both experimental conditions. These results suggest that it is difficult for sake yeast cells to enter a quiescent state after cell growth has stopped, which may be one reason for the higher fermentation rate of sake yeast compared to laboratory yeast strains. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Sharing the cell's bounty - organelle inheritance in yeast.

PubMed

Knoblach, Barbara; Rachubinski, Richard A

2015-02-15

Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.
Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

NASA Astrophysics Data System (ADS)

Choi, Jinsu; Lee, Hojae; Choi, Insung S.; Yang, Sung Ho

2017-09-01

Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.
Yeasts in floral nectar: a quantitative survey

PubMed Central

Herrera, Carlos M.; de Vega, Clara; Canto, Azucena; Pozo, María I.

2009-01-01

Background and Aims One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. Methods The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. Key Results Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 105 cells mm−3). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 104 cells mm−3 were commonplace, and densities >105 cells mm−3 were not rare. About one-fifth of species at each site had mean yeast cell densities >104 cells mm−3. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. Conclusions Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

PubMed

Faraghat, Shabnam A; Hoettges, Kai F; Steinbach, Max K; van der Veen, Daan R; Brackenbury, William J; Henslee, Erin A; Labeed, Fatima H; Hughes, Michael P

2017-05-02

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.
High power density yeast catalyzed microbial fuel cells

NASA Astrophysics Data System (ADS)

Ganguli, Rahul

Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density
Preparation of corncob grits as a carrier for immobilizing yeast cells for ethanol production.

PubMed

Lee, Sang-Eun; Lee, Choon Geun; Kang, Do Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

2012-12-01

In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.
Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

PubMed

Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

2016-08-01

The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Yeast cell wall supplementation alters the metabolic responses of crossbred heifers to an endotoxin challenge

USDA-ARS?s Scientific Manuscript database

This study examined the effect of feeding yeast cell wall (YCW) products on the metabolic responses of newly-received heifers to endotoxin challenge. Heifers (n = 24; 219 ± 2.4 kg) were separated into treatment groups receiving a Control diet (n = 8), YCW-A (2.5 grams/heifer/d; n = 8) or YCW-C (2.5 ...
Yeast cell surface display for lipase whole cell catalyst and its applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less
Transcription in Yeast: Separation and Properties of Multiple RNA Polymerases

PubMed Central

Adman, Ray; Schultz, Loren D.; Hall, Benjamin D.

1972-01-01

Four peaks of DNA-directed RNA polymerase activity are resolved by salt gradient elution of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named IA, IB, II, and III in order of elution, all appear to come from cell nuclei. Only enzyme II is sensitive to α-amanitin. All enzymes are more active with Mn++ than with Mg++ as divalent ion. Enzymes IB and II have salt optima in the range 0.05-0.10 M (NH4)2SO4, whereas enzyme III is maximally active at 0.20-0.25 M (NH4)2SO4. With optimal salt concentration and saturating DNA, the template preference ratio, activity on native calfthymus DNA divided by activity on denatured calf-thymus DNA, is 2.2 for IB, 0.4 for II, and 3.5 for III. None of the yeast polymerases was inhibited by rifamycin SV. Rifamycin AF/013 effectively inhibited polymerases IB, II, and III. PMID:4558656
Extracellular electron transfer in yeast-based biofuel cells: A review.

PubMed

Hubenova, Yolina; Mitov, Mario

2015-12-01

This paper reviews the state-of-the art of the yeast-based biofuel cell research and development. The established extracellular electron transfer (EET) mechanisms in the presence and absence of exogenous mediators are summarized and discussed. The approaches applied for improvement of mediator-less yeast-based biofuel cells performance are also presented. The overview of the literature shows that biofuel cells utilizing yeasts as biocatalysts generate power density in the range of 20 to 2440 mW/m(2), which values are comparable with the power achieved when bacteria are used instead. The electrons' origin and the contribution of the glycolysis, fermentation, aerobic respiration, and phosphorylation to the EET are commented. The reported enhanced current generation in aerobic conditions presumes reconsideration of some basic MFC principles. The challenges towards the practical application of the yeast-based biofuel cells are outlined. Copyright © 2015 Elsevier B.V. All rights reserved.
Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

PubMed

Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

2014-03-03

The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.
Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.

PubMed

Méndez, S P; González, E B; Sanz-Medel, A

2001-05-01

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast. Copyright 2001 John Wiley & Sons, Ltd.
Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

PubMed

Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

2011-07-01

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the ﬁrst attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.
[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.
Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi

2011-02-25

Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way towardmore » the individual tracking of proteins of interest inside living yeast cells.« less
[A Modified Procedure to Isolate Synchronous Cells from Yeasts with Continuous Percoll Density Gradient and Their Raman Discrimination].

PubMed

Huang, Shu-shi; Lai, Jun-zhuo; Lu, Ming-qian; Cheng, Qin; Liao, Wei; Chen, Li-mei

2015-08-01

A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains, as well as Raman spectra discrimination of single yeast cells was reported. About 1.75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19,320 g, 20 °C with an angle rotor for 15 min to form continuous densities gradient (1.00~1.31 g · mL(-1)), approximately 100 μL sample was overlaid onto the preformed continuous density gradient carefully, subsequently, centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 °C. Yeast samples could be observed that the suspensions were separated into two cell fractions obviously. Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC), phase contrast microscope and synchronous culture to distinguish their morphological and growth trait. The results showed that the lower fraction cells were unbudded, mostly unicellular, highly refractive, homogeneous and uniform in size, and represented growth characteristic synchronously; Their protoplasm had relatively high density, and contained significant concentrations of glycogen; all of which were accordant with description of quiescent yeast cells and G0 cells in previously published paper. It was shown that lower fraction was quiescent cells, synchronous G0 cells as well. A Raman tweezers setup was used to investigate the differences between two fractions, G0 cells and non G0 cells, at a single cell level. The result showed that both G0 cells and the non G0 cells had the same characteristic peaks corresponding biological macromolecules including proteins, carbohydrates and nucleic acids, but all characteristic peak intensities of G0 cells were higher than that of non G0 cells, implied that the macromolecular substance content of G0 cells was more higher. Principal component
Astaxanthinogenesis in the yeast Phaffia rhodozyma - optimization of low-cost culture media and yeast cell-wall lysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fontana, J.D.; Baron, M.; Guimaraes, M.F.

Astaxanthin is a diketo-dihydroxy-carotenoid produced by Phaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only amore » slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni{sup 2} (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. 13 refs., 6 figs.« less
Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast.

PubMed

Gruhlke, Martin C H; Portz, Daniela; Stitz, Michael; Anwar, Awais; Schneider, Thomas; Jacob, Claus; Schlaich, Nikolaus L; Slusarenko, Alan J

2010-12-15

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes. Copyright © 2010 Elsevier Inc. All rights reserved.
Studying the replicative life span of yeast cells.

PubMed

Sinclair, David A

2013-01-01

The budding yeast Saccharomyces cerevisiae is a useful model for elucidating the pathways that control life span and the influence of environmental factors, such as calorie restriction (CR). For 75 years, CR has been studied for its ability to delay diseases of aging in mammals, from cancer to cardiovascular disease (McCay et al., Nutr Rev 33:241-243, 1975). In many other species, reducing calorie intake extends life span, including unicellular organisms (Jiang et al., FASEB J 14:2135-2137, 2000; Lin et al., Science 289:2126-2128, 2000), invertebrates (Rogina and Helfand, Proc Natl Acad Sci U S A 101:15998-16003, 2004), and rodents (Martín-Montalvo et al., Oncogene 30:505-520, 2011). Here we describe how to calorically restrict yeast cells, the methods used to determine the replicative life span (RLS) of budding yeast cells, how to selectively kill daughter cells using the mother enrichment program (MEP), how to measure recombination frequency at the rDNA locus, how to isolate large quantities of old cells, and how to analyze the circular forms of DNA known as extrachromosomal rDNA circles (ERCs), a cause of aging in S. cerevisiae (Petes, Cell 19:765-774, 1980; Sinclair and Guarente, Cell 91:1033-1042, 1997; Defossez et al., Mol Cell 3:447-455, 1999).
Yeast cell wall supplementation alters the physiological and acute phase responses of crossbred heifers to an endotoxin challenge

USDA-ARS?s Scientific Manuscript database

A study was conducted to determine the effect of feeding yeast cell wall (YCW) products on the physiological and acute phase responses of crossbred newly-received heifers to an endotoxin challenge. Heifers (n = 24; 219 ± 2.4 kg) were separated into treatment groups receiving a Control diet (n = 8), ...
Aroma formation by immobilized yeast cells in fermentation processes.

PubMed

Nedović, V; Gibson, B; Mantzouridou, T F; Bugarski, B; Djordjević, V; Kalušević, A; Paraskevopoulou, A; Sandell, M; Šmogrovičová, D; Yilmaztekin, M

2015-01-01

Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes. Copyright © 2014 John Wiley & Sons, Ltd.
Binding mechanism of patulin to heat-treated yeast cell.

PubMed

Guo, C; Yuan, Y; Yue, T; Hatab, S; Wang, Z

2012-12-01

This study aims to assess the removal mechanism of patulin using heat-treated Saccharomyces cerevisiae cells and identify the role of different cell wall components in the binding process. In order to understand the binding mechanism, viable cells, heat-treated cells, cell wall and intracellular extract were performed to assess their ability to remove patulin. Additionally, the effects of chemical and enzymatic treatments of yeast on the binding ability were tested. The results showed that there was no significant difference between viable (53·28%) and heat-treated yeast cells (51·71%) in patulin binding. In addition, the cell wall fraction decreased patulin by 35·05%, and the cell extract nearly failed to bind patulin. Treatments with protease E, methanol, formaldehyde, periodate or urea significantly decreased (P < 0·05) the ability of heat-treated cells to remove patulin. Fourier transform infrared (FTIR) analysis indicated that more functional groups were involved in the binding process of heat-treated cells. Polysaccharides and protein are important components of yeast cell wall involved in patulin removal. In addition, hydrophobic interactions play a major role in binding processes. Heat-treated S. cerevisiae cells could be used to control patulin contamination in the apple juice industry. Also, our results proof that the patulin removal process is based mainly on the adsorption not degradation. © 2012 The Society for Applied Microbiology.

Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

ERIC Educational Resources Information Center

Deutch, Charles E.; Marshall, Pamela A.

2008-01-01

In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…
Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

PubMed

Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.
Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

PubMed Central

Smith, Ida M.; Christensen, Jeffrey E.; Arneborg, Nils; Jespersen, Lene

2014-01-01

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current
Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

PubMed Central

Pais, Thiago M.; Foulquié-Moreno, María R.; Hubmann, Georg; Duitama, Jorge; Swinnen, Steve; Goovaerts, Annelies; Yang, Yudi; Dumortier, Françoise; Thevelein, Johan M.

2013-01-01

The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance. PMID:23754966
Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.
Charge-based separation of particles and cells with similar sizes via the wall-induced electrical lift.

PubMed

Thomas, Cory; Lu, Xinyu; Todd, Andrew; Raval, Yash; Tzeng, Tzuen-Rong; Song, Yongxin; Wang, Junsheng; Li, Dongqing; Xuan, Xiangchun

2017-01-01

The separation of particles and cells in a uniform mixture has been extensively studied as a necessity in many chemical and biomedical engineering and research fields. This work demonstrates a continuous charge-based separation of fluorescent and plain spherical polystyrene particles with comparable sizes in a ψ-shaped microchannel via the wall-induced electrical lift. The effects of both the direct current electric field in the main-branch and the electric field ratio in between the inlet branches for sheath fluid and particle mixture are investigated on this electrokinetic particle separation. A Lagrangian tracking method based theoretical model is also developed to understand the particle transport in the microchannel and simulate the parametric effects on particle separation. Moreover, the demonstrated charge-based separation is applied to a mixture of yeast cells and polystyrene particles with similar sizes. Good separation efficiency and purity are achieved for both the cells and the particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Effect of whole yeast cell product supplementation (CitriStim®) on immune responses and cecal microflora species in pullet and layer chickens during an experimental coccidial challenge.

PubMed

Markazi, Ashley D; Perez, Victor; Sifri, Mamduh; Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2017-07-01

Three separate experiments were conducted to study the effects of whole yeast cell product supplementation in pullets and layer hens. Body weight gain, fecal and intestinal coccidial oocyst counts, cecal microflora species, cytokine mRNA amounts, and CD4+ and CD8+ T-cell populations in the cecal tonsils were analyzed following an experimental coccidial infection. In Experiment I, day-old Leghorn layer chicks were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product (CitriStim®, ADM, Decatur, IL). At 21 d of age, birds were challenged with 1 × 105 live coccidial oocysts. Supplementation with whole yeast cell product decreased the fecal coccidial oocyst count at 7 (P = 0.05) and 8 (P < 0.01) d post-challenge. In Experiment II, 27-week old Leghorn layer hens were fed 3 experimental diets with 0, 0.05 or 0.1% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 25 of whole yeast cell product feeding. Supplementation with whole yeast cell product decreased the coccidial oocyst count in the intestinal content (P < 0.01) at 5, 13, and 38 d post-coccidial challenge. Supplementation with whole yeast cell product increased relative proportion of Lactobacillus (P < 0.01) in the cecal tonsils 13 d post-coccidial challenge. Supplementation with whole yeast cell product decreased CD8+ T cell percentages (P < 0.05) in the cecal tonsils at 5 d post-coccidial challenge. In Experiment III, 32-week-old Leghorn layer hens were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 66 of whole yeast cell product feeding. At 5 d post-coccidial challenge, whole yeast cell product supplementation down-regulated (P = 0.01) IL-10 mRNA amount. It could be concluded that supplementing whole yeast cell product can help minimize coccidial infection in both growing pullets and layer chickens. © 2017 Poultry Science Association Inc.
Downsides and benefits of unicellularity in budding yeast

NASA Astrophysics Data System (ADS)

Balazsi, Gabor; Chen, Lin; Kuzdzal-Fick, Jennie

Yeast cells that do not separate after cell division form clumps. Clumping was shown to aid utilization of certain sugars, but its effects in stressful conditions are unknown. Generally speaking, what are the costs and benefits of unicellularity versus clumping multicellularity in normal and stressful conditions? To address this question, we evolved clumping yeast towards unicellularity by continuously propagating only those cells that remain suspended in liquid culture after settling. Whole-genome sequencing indicated that mutations in the AMN1 (antagonist of mitotic exit network) gene underlie the changes from clumping to unicellular phenotypes in these evolved yeast cells. Simple models predict that clumping should hinder growth in normal conditions while being protective in stress. Accordingly, we find experimentally that yeast clumps are more resistant to freeze/thaw, hydrogen peroxide, and ethanol stressors than their unicellular counterparts. On the other hand, unicellularity seems to be advantageous in normal conditions. Overall, these results reveal the downsides and benefits of unicellularity in different environmental conditions and uncover its genetic bases in yeast. This research was supported by the NIH Director's New Innovator Award Program (1DP2 OD006481-01), by NSF/IOS 1021675 and the Laufer Center for Physical & Quantitative Biology.
Human Cpr (Cell Cycle Progression Restoration) Genes Impart a Far(-) Phenotype on Yeast Cells

PubMed Central

Edwards, M. C.; Liegeois, N.; Horecka, J.; DePinho, R. A.; Sprague-Jr., G. F.; Tyers, M.; Elledge, S. J.

1997-01-01

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G(1) arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G(1) arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles. PMID:9383053
Assessment of yeast Saccharomyces cerevisiae component binding to Mycobacterium avium subspecies paratuberculosis using bovine epithelial cells.

PubMed

Li, Ziwei; You, Qiumei; Ossa, Faisury; Mead, Philip; Quinton, Margaret; Karrow, Niel A

2016-03-01

Since yeast Saccharomyces cerevisiae and its components are being used for the prevention and treatment of enteric diseases in different species, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium spp. paratuberculosis (MAP). This study aimed to identify potential yeast derivatives that may be used to help prevent MAP infection. The adherence of mCherry-labeled MAP to bovine mammary epithelial cell line (MAC-T cells) and bovine primary epithelial cells (BECs) co-cultured with yeast cell wall components (CWCs) from four different yeast strains (A, B, C and D) and two forms of dead yeast from strain A was investigated. The CWCs from all four yeast strains and the other two forms of dead yeast from strain A reduced MAP adhesion to MAC-T cells and BECs in a concentration-dependent manner after 6-h of exposure, with the dead yeast having the greatest effect. The following in vitro binding studies suggest that dead yeast and its' CWCs may be useful for reducing risk of MAP infection.
Performance study of sugar-yeast-ethanol bio-hybrid fuel cells

NASA Astrophysics Data System (ADS)

Jahnke, Justin P.; Mackie, David M.; Benyamin, Marcus; Ganguli, Rahul; Sumner, James J.

2015-05-01

Renewable alternatives to fossil hydrocarbons for energy generation are of general interest for a variety of political, economic, environmental, and practical reasons. In particular, energy from biomass has many advantages, including safety, sustainability, and the ability to be scavenged from native ecosystems or from waste streams. Microbial fuel cells (MFCs) can take advantage of microorganism metabolism to efficiently use sugar and other biomolecules as fuel, but are limited by low power densities. In contrast, direct alcohol fuel cells (DAFCs) take advantage of proton exchange membranes (PEMs) to generate electricity from alcohols at much higher power densities. Here, we investigate a novel bio-hybrid fuel cell design prepared using commercial off-the-shelf DAFCs. In the bio-hybrid fuel cells, biomass such as sugar is fermented by yeast to ethanol, which can be used to fuel a DAFC. A separation membrane between the fermentation and the DAFC is used to purify the fermentate while avoiding any parasitic power losses. However, shifting the DAFCs from pure alcohol-water solutions to filtered fermented media introduces complications related to how the starting materials, fermentation byproducts, and DAFC waste products affect both the fermentation and the long-term DAFC performance. This study examines the impact of separation membrane pore size, fermentation/fuel cell byproducts, alcohol and salt concentrations, and load resistance on fuel cell performance. Under optimized conditions, the performance obtained is comparable to that of a similar DAFC run with a pure alcohol-water mixture. Additionally, the modified DAFC can provide useable amounts of power for weeks.
One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE PAGES

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; ...

2016-11-16

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less
One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less
Modeling Yeast Cell Polarization Induced by Pheromone Gradients

NASA Astrophysics Data System (ADS)

Yi, Tau-Mu; Chen, Shanqin; Chou, Ching-Shan; Nie, Qing

2007-07-01

Yeast cells respond to spatial gradients of mating pheromones by polarizing and projecting up the gradient toward the source. It is thought that they employ a spatial sensing mechanism in which the cell compares the concentration of pheromone at different points on the cell surface and determines the maximum point, where the projection forms. Here we constructed the first spatial mathematical model of the yeast pheromone response that describes the dynamics of the heterotrimeric and Cdc42p G-protein cycles, which are linked in a cascade. Two key performance objectives of this system are (1) amplification—converting a shallow external gradient of ligand to a steep internal gradient of protein components and (2) tracking—following changes in gradient direction. We used simulations to investigate amplification mechanisms that allow tracking. We identified specific strategies for regulating the spatial dynamics of the protein components (i.e. their changing location in the cell) that would enable the cell to achieve both objectives.
Isolation of total RNA from yeast cell cultures.

PubMed

Ares, Manuel

2012-10-01

This article describes two procedures for isolating total RNA from yeast cell cultures. The first allows the convenient isolation of total RNA from early log-phase cultures (vegetative cells). RNA isolated in this way is intact and sufficiently pure for use in microarray experiments, primer extension, and RNase protection mapping. With additional treatment to remove contaminating genomic DNA, the preparation is suitable for reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), cDNA library construction, high-throughput sequencing of RNA, or other manipulations. However, compared to vegetative cells, the isolation of RNA from cells late in meiosis (asci and ascospores) requires additional effort. This is because a tough cell wall composed of heavily cross-linked polysaccharides and proteins is built around the four spores during meiosis and ascospore development. Therefore, an alternative protocol is presented for extracting RNA from cells late in meiosis. This alternative may also be preferable for cells from stationary cultures or from yeast strains and other fungal species isolated from the environment.
Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

PubMed

Chen, Xianzhong

2017-03-04

The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.
Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance

PubMed Central

Chen, Xianzhong

2017-01-01

ABSTRACT The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed. PMID:27459271
Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

PubMed Central

Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

2016-01-01

The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527
Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.
Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast.

PubMed

Goshima, G; Yanagida, M

2000-03-17

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.

Modifying infrared scattering effects of single yeast cells with plasmonic metal mesh

NASA Astrophysics Data System (ADS)

Malone, Marvin A.; Prakash, Suraj; Heer, Joseph M.; Corwin, Lloyd D.; Cilwa, Katherine E.; Coe, James V.

2010-11-01

The scattering effects in the infrared (IR) spectra of single, isolated bread yeast cells (Saccharomyces cerevisiae) on a ZnSe substrate and in metal microchannels have been probed by Fourier transform infrared imaging microspectroscopy. Absolute extinction [(3.4±0.6)×10-7 cm2 at 3178 cm-1], scattering, and absorption cross sections for a single yeast cell and a vibrational absorption spectrum have been determined by comparing it to the scattering properties of single, isolated, latex microspheres (polystyrene, 5.0 μm in diameter) on ZnSe, which are well modeled by the Mie scattering theory. Single yeast cells were then placed into the holes of the IR plasmonic mesh, i.e., metal films with arrays of subwavelength holes, yielding "scatter-free" IR absorption spectra, which have undistorted vibrational lineshapes and a rising generic IR absorption baseline. Absolute extinction, scattering, and absorption spectral profiles were determined for a single, ellipsoidal yeast cell to characterize the interplay of these effects.
Stochastic Petri Net extension of a yeast cell cycle model.

PubMed

Mura, Ivan; Csikász-Nagy, Attila

2008-10-21

This paper presents the definition, solution and validation of a stochastic model of the budding yeast cell cycle, based on Stochastic Petri Nets (SPN). A specific family of SPNs is selected for building a stochastic version of a well-established deterministic model. We describe the procedure followed in defining the SPN model from the deterministic ODE model, a procedure that can be largely automated. The validation of the SPN model is conducted with respect to both the results provided by the deterministic one and the experimental results available from literature. The SPN model catches the behavior of the wild type budding yeast cells and a variety of mutants. We show that the stochastic model matches some characteristics of budding yeast cells that cannot be found with the deterministic model. The SPN model fine-tunes the simulation results, enriching the breadth and the quality of its outcome.
Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

PubMed Central

Peng, Xu; Karuturi, R. Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

2005-01-01

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC. PMID:15616197
Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.
Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

PubMed Central

Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

2006-01-01

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994
Yeast-assisted synthesis of polypyrrole: Quantification and influence on the mechanical properties of the cell wall.

PubMed

Andriukonis, Eivydas; Stirke, Arunas; Garbaras, Andrius; Mikoliunaite, Lina; Ramanaviciene, Almira; Remeikis, Vidmantas; Thornton, Barry; Ramanavicius, Arunas

2018-04-01

In this study, the metabolism of yeast cells (Saccharomyces cerevisiae) was utilized for the synthesis of the conducting polymer - polypyrrole (Ppy).Yeast cells were modified in situ by synthesized Ppy. The Ppy was formed in the cell wall by redox-cycling of [Fe(CN) 6 ] 3-/4- , performed by the yeast cells. Fluorescence microscopy, enzymatic digestions, atomic force microscopy and isotope ratio mass spectroscopy were applied to determine both the polymerization reaction itself and the polymer location in yeast cells. Ppy formation resulted in enhanced resistance to lytic enzymes, significant increase of elasticity and alteration of other mechanical cell wall properties evaluated by atomic force microscopy (AFM). The suggested method of polymer synthesis allows the introduction of polypyrrole structures within the cell wall, which is build up from polymers consisting of carbohydrates. This cell wall modification strategy could increase the usefulness of yeast as an alternative energy source in biofuel cells, and in cell based biosensors. Copyright © 2018 Elsevier B.V. All rights reserved.
Stable current outputs and phytate degradation by yeast-based biofuel cell.

PubMed

Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

2014-09-01

In this paper, we report for the first time that Candida melibiosica 2491 yeast strain expresses enhanced phytase activity when used as a biocatalyst in biofuel cells. The polarization also results in an increase of the yeast biomass. Higher steady-state electrical outputs, assigned to earlier production of an endogenous mediator, were achieved at continuous polarization under constant load. The obtained results prove that the C. melibiosica yeast-based biofuel cell could be used for simultaneous electricity generation and phytate bioremediation. In addition, the higher phytase activity obtained by interruptive polarization suggests a new method for increasing the phytase yield from microorganisms. Copyright © 2014 John Wiley & Sons, Ltd.
Reproductive potential and instability of the rDNA region of the Saccharomyces cerevisiae yeast: Common or separate mechanisms of regulation?

PubMed

Zadrag-Tecza, Renata; Skoneczna, Adrianna

2016-11-01

The yeast Saccharomyces cerevisiae is a unicellular organism commonly used as a model to explain mechanisms of aging in multicellular organisms. It is used as a model organism for both replicative and chronological aging. Replicative aging is defined as the number of daughter cells produced by an individual cell during its life. A widely accepted hypothesis assumes that replicative aging of yeast is related to the existence of a so called "senescence factor" that gradually accumulates in the mother cell, which consequently leads to its death. One of the earliest proposed "senescence factors" were extrachromosomal rDNA circles (ERCs). However, their role in the regulation of the replicative lifespan is somewhat controversial and subject to discussion. In this paper, we propose a more comprehensive approach to this problem by analysing the length of life and the correlation between the cell size and the replicative lifespan of yeast cells with different level of ERCs, i.e. Δrad52 and Δsgs1 mutants. This analysis shows that it is not the accumulation of ERCs but genomic instability and hypertrophy that play an important role in the regulation of reproductive potential and total lifespan of the S. cerevisiae yeast. However, these two factors have a different impact on various phases of the yeast cell life, i.e. reproductive and post-reproductive phases. Copyright © 2016 Elsevier Inc. All rights reserved.
Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis

PubMed Central

Koch, Bailey A.; Han, Xuemei

2017-01-01

Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery. PMID:28609436
Cell wall of pathogenic yeasts and implications for antimycotic therapy.

PubMed

Cassone, A

1986-01-01

Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).
The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors.

PubMed

Christiaens, Joaquin F; Franco, Luis M; Cools, Tanne L; De Meester, Luc; Michiels, Jan; Wenseleers, Tom; Hassan, Bassem A; Yaksi, Emre; Verstrepen, Kevin J

2014-10-23

Yeast cells produce various volatile metabolites that are key contributors to the pleasing fruity and flowery aroma of fermented beverages. Several of these fruity metabolites, including isoamyl acetate and ethyl acetate, are produced by a dedicated enzyme, the alcohol acetyl transferase Atf1. However, despite much research, the physiological role of acetate ester formation in yeast remains unknown. Using a combination of molecular biology, neurobiology, and behavioral tests, we demonstrate that deletion of ATF1 alters the olfactory response in the antennal lobe of fruit flies that feed on yeast cells. The flies are much less attracted to the mutant yeast cells, and this in turn results in reduced dispersal of the mutant yeast cells by the flies. Together, our results uncover the molecular details of an intriguing aroma-based communication and mutualism between microbes and their insect vectors. Similar mechanisms may exist in other microbes, including microbes on flowering plants and pathogens. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Fabrication of perforated isoporous membranes via a transfer-free strategy: enabling high-resolution separation of cells.

PubMed

Ou, Yang; Lv, Chang-Jiang; Yu, Wei; Mao, Zheng-Wei; Wan, Ling-Shu; Xu, Zhi-Kang

2014-12-24

Thin perforated membranes with ordered pores are ideal barriers for high-resolution and high-efficiency selective transport and separation of biological species. However, for self-assembled thin membranes with a thickness less than several micrometers, an additional step of transferring the membranes onto porous supports is generally required. In this article, we present a facile transfer-free strategy for fabrication of robust perforated composite membranes via the breath figure process, and for the first time, demonstrate the application of the membranes in high-resolution cell separation of yeasts and lactobacilli without external pressure, achieving almost 100% rejection of yeasts and more than 70% recovery of lactobacilli with excellent viability. The avoidance of the transfer step simplifies the fabrication procedure of composite membranes and greatly improves the membrane homogeneity. Moreover, the introduction of an elastic triblock copolymer increases the interfacial strength between the membrane and the support, and allows the preservation of composite membranes in a dry state. Such perforated ordered membranes can also be applied in other size-based separation systems, enabling new opportunities in bioseparation and biosensors.
Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

PubMed Central

Mauzeroll, Janine; Bard, Allen J.

2004-01-01

The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV–visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-μm-diameter electrode situated 10 μm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster. PMID:15148374
Quantitative description of ion transport via plasma membrane of yeast and small cells.

PubMed

Volkov, Vadim

2015-01-01

Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions.
Quantitative description of ion transport via plasma membrane of yeast and small cells

PubMed Central

Volkov, Vadim

2015-01-01

Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporary resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions. PMID:26113853
Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

PubMed Central

Stagge, Franziska; Mitronova, Gyuzel Y.; Belov, Vladimir N.; Wurm, Christian A.; Jakobs, Stefan

2013-01-01

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae. PMID:24205303
Mitochondrial origin of extracelullar transferred electrons in yeast-based biofuel cells.

PubMed

Hubenova, Yolina; Mitov, Mario

2015-12-01

The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7±1.3%, respectively 38.8±1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.
Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230
Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

PubMed

Takahashi, Keisuke G; Izumi-Nakajima, Nakako; Mori, Katsuyoshi

2017-11-01

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and
Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

PubMed

Chen, Ke-Quan; Li, Jian; Ma, Jiang-Feng; Jiang, Min; Wei, Ping; Liu, Zhong-Min; Ying, Han-Jie

2011-01-01

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources. Crown Copyright Â© 2010. Published by Elsevier Ltd. All rights reserved.
Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.
Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

ERIC Educational Resources Information Center

Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

2005-01-01

In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…
Study the oxidative injury of yeast cells by NADH autofluorescence

NASA Astrophysics Data System (ADS)

Liang, Ju; Wu, Wen-Lan; Liu, Zhi-Hong; Mei, Yun-Jun; Cai, Ru-Xiu; Shen, Ping

2007-06-01

Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H 2O 2 and ONOO - have been recorded in detail in this work. In the presence of different amounts of H 2O 2 and ONOO -, necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.
Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

PubMed

Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

2009-05-01

Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.
Fundamentals of affinity cell separations.

PubMed

Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

2018-03-01

Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

NASA Astrophysics Data System (ADS)

Procházka, Václav; Cifra, Michal; Kulha, Pavel; Ižák, Tibor; Rezek, Bohuslav; Kromka, Alexander

2017-02-01

Diamond thin films provide unique features as substrates for cell cultures and as bio-electronic sensors. Here we employ solution-gated field effect transistors (SGFET) based on nanocrystalline diamond thin films with H-terminated surface which exhibits the sub-surface p-type conductive channel. We study an influence of yeast cells (Saccharomyces cerevisiae) on electrical characteristics of the diamond SGFETs. Two different cell culture solutions (sucrose and yeast peptone dextrose-YPD) are used, with and without the cells. We have found that transfer characteristics of the SGFETs exhibit a negative shift of the gate voltage by -26 mV and -42 mV for sucrose and YPD with cells in comparison to blank solutions without the cells. This effect is attributed to a local pH change in close vicinity of the H-terminated diamond surface due to metabolic processes of the yeast cells. The pH sensitivity of the diamond-based SGFETs, the role of cell and protein adhesion on the gate surface and the role of negative surface charge of yeast cells on the SGFETs electrical characteristics are discussed as well.
Analysis of ribosomal RNA stability in dead cells of wine yeast by quantitative PCR.

PubMed

Sunyer-Figueres, Merce; Wang, Chunxiao; Mas, Albert

2018-04-02

During wine production, some yeasts enter a Viable But Not Culturable (VBNC) state, which may influence the quality and stability of the final wine through remnant metabolic activity or by resuscitation. Culture-independent techniques are used for obtaining an accurate estimation of the number of live cells, and quantitative PCR could be the most accurate technique. As a marker of cell viability, rRNA was evaluated by analyzing its stability in dead cells. The species-specific stability of rRNA was tested in Saccharomyces cerevisiae, as well as in three species of non-Saccharomyces yeast (Hanseniaspora uvarum, Torulaspora delbrueckii and Starmerella bacillaris). High temperature and antimicrobial dimethyl dicarbonate (DMDC) treatments were efficient in lysing the yeast cells. rRNA gene and rRNA (as cDNA) were analyzed over 48 h after cell lysis by quantitative PCR. The results confirmed the stability of rRNA for 48 h after the cell lysis treatments. To sum up, rRNA may not be a good marker of cell viability in the wine yeasts that were tested. Copyright © 2018 Elsevier B.V. All rights reserved.
Mechanics and morphogenesis of fission yeast cells.

PubMed

Davì, Valeria; Minc, Nicolas

2015-12-01

The integration of biochemical and biomechanical elements is at the heart of morphogenesis. While animal cells are relatively soft objects which shape and mechanics is mostly regulated by cytoskeletal networks, walled cells including those of plants, fungi and bacteria are encased in a rigid cell wall which resist high internal turgor pressure. How these particular mechanical properties may influence basic cellular processes, such as growth, shape and division remains poorly understood. Recent work using the model fungal cell fission yeast, Schizosaccharomyces pombe, highlights important contribution of cell mechanics to various morphogenesis processes. We envision this genetically tractable system to serve as a novel standard for the mechanobiology of walled cell. Copyright © 2015 Elsevier Ltd. All rights reserved.
Evaluation of a recombinant yeast cell estrogen screening assay.

PubMed Central

Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

1997-01-01

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720
Fuel-Cell Water Separator

NASA Technical Reports Server (NTRS)

Burke, Kenneth Alan; Fisher, Caleb; Newman, Paul

2010-01-01

The main product of a typical fuel cell is water, and many fuel-cell configurations use the flow of excess gases (i.e., gases not consumed by the reaction) to drive the resultant water out of the cell. This two-phase mixture then exits through an exhaust port where the two fluids must again be separated to prevent the fuel cell from flooding and to facilitate the reutilization of both fluids. The Glenn Research Center (GRC) has designed, built, and tested an innovative fuel-cell water separator that not only removes liquid water from a fuel cell s exhaust ports, but does so with no moving parts or other power-consuming components. Instead it employs the potential and kinetic energies already present in the moving exhaust flow. In addition, the geometry of the separator is explicitly intended to be integrated into a fuel-cell stack, providing a direct mate with the fuel cell s existing flow ports. The separator is also fully scalable, allowing it to accommodate a wide range of water removal requirements. Multiple separators can simply be "stacked" in series or parallel to adapt to the water production/removal rate. GRC s separator accomplishes the task of water removal by coupling a high aspect- ratio flow chamber with a highly hydrophilic, polyethersulfone membrane. The hydrophilic membrane readily absorbs and transports the liquid water away from the mixture while simultaneously resisting gas penetration. The expansive flow path maximizes the interaction of the water particles with the membrane while minimizing the overall gas flow restriction. In essence, each fluid takes its corresponding path of least resistance, and the two fluids are effectively separated. The GRC fuel-cell water separator has a broad range of applications, including commercial hydrogen-air fuel cells currently being considered for power generation in automobiles.
Drying enhances immunoactivity of spent brewer's yeast cell wall β-D-glucans.

PubMed

Liepins, Janis; Kovačova, Elena; Shvirksts, Karlis; Grube, Mara; Rapoport, Alexander; Kogan, Grigorij

2015-07-20

Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast β-D-glucans as BRM, and drying as an efficient pretreatment to increase β-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified β-D-glucan. However, drying increased purified β-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted β-D-glucan. This is corroborated with FT-IR analyses of the β-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality β-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast β-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran. Copyright © 2015 Elsevier B.V. All rights reserved.
Glycerol production by fermenting yeast cells is essential for optimal bread dough fermentation.

PubMed

Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M; Verstrepen, Kevin J

2015-01-01

Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.
Cell population modelling of yeast glycolytic oscillations.

PubMed Central

Henson, Michael A; Müller, Dirk; Reuss, Matthias

2002-01-01

We investigated a cell-population modelling technique in which the population is constructed from an ensemble of individual cell models. The average value or the number distribution of any intracellular property captured by the individual cell model can be calculated by simulation of a sufficient number of individual cells. The proposed method is applied to a simple model of yeast glycolytic oscillations where synchronization of the cell population is mediated by the action of an excreted metabolite. We show that smooth one-dimensional distributions can be obtained with ensembles comprising 1000 individual cells. Random variations in the state and/or structure of individual cells are shown to produce complex dynamic behaviours which cannot be adequately captured by small ensembles. PMID:12206713
Visualization and Image Analysis of Yeast Cells.

PubMed

Bagley, Steve

2016-01-01

When converting real-life data via visualization to numbers and then onto statistics the whole system needs to be considered so that conversion from the analogue to the digital is accurate and repeatable. Here we describe the points to consider when approaching yeast cell analysis visualization, processing, and analysis of a population by screening techniques.
Forces in yeast flocculation

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

2015-01-01

In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.
[Export of an invertase by yeast cells (Candida utilis)].

PubMed

Alekseeva, O V; Sabirzianova, T A; Celiakh, I O; Kalebina, T S; Kulaev, I S

2014-01-01

Export and accumulation of various forms of invertase (EC 3.2.1.26) in the cell wall and culture liquid of the yeast Candida utilis was investigated. It was found that the high-molecular-weight CW-form of invertase is present in the cell wall. This form is not exported into the culture liquid, and it is by a third more glycosylated than the previously described exported S-form. It was shown that one of the two liquid forms of invertase exported into the culture-the glycosylated S-form--is retained in the cell wall, while the other one--the nonglycosylated F-form--was not detected in the cell wall. Based on these results, as well as data on the distribution dynamics of the enzyme in the culture liquid and in the cell wall during different growth stages of a yeast culture, we suggested that the nonglycosylated form was exported into the culture liquid via the zone of abnormal cell wall permeability and the glycosylated forms of this enzyme (both exported and nonexported) did not use this pathway (the degree of N-glycosylation is an important factor determining the final localization of the enzyme).
The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi, E-mail: hiro@bs.naist.jp

Highlights: Black-Right-Pointing-Pointer NO is produced from L-arginine in response to elevated temperature in yeast. Black-Right-Pointing-Pointer Tah18 was first identified as the yeast protein involved in NO synthesis. Black-Right-Pointing-Pointer Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiologicalmore » role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.« less
Engineering tolerance to industrially relevant stress factors in yeast cell factories.

PubMed

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R; Thevelein, Johan M

2017-06-01

The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. © FEMS 2017.
Engineering tolerance to industrially relevant stress factors in yeast cell factories

PubMed Central

Deparis, Quinten; Claes, Arne; Foulquié-Moreno, Maria R.

2017-01-01

Abstract The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way. PMID:28586408

Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

PubMed

Tipper, Donald J; Szomolanyi-Tsuda, Eva

2016-01-01

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.
Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

PubMed

Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

2014-02-01

The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.
A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

ERIC Educational Resources Information Center

Stambuk, Boris U.

2000-01-01

Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)
The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast

PubMed Central

Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José M.; Arroyo, Javier

2017-01-01

Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI) pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies. PMID:29371494
Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

PubMed Central

Aslankoohi, Elham; Rezaei, Mohammad Naser; Vervoort, Yannick; Courtin, Christophe M.; Verstrepen, Kevin J.

2015-01-01

Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO2 production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts. PMID:25764309
Reconstruction of a yeast cell from x-ray diffraction data

DOE PAGES

Thibault, Pierre; Elser, Veit; Jacobsen, Chris; ...

2006-06-21

We provide details of the algorithm used for the reconstruction of yeast cell images in the recent demonstration of diffraction microscopy by Shapiro, Thibault, Beetz, Elser, Howells, Jacobsen, Kirz, Lima, Miao, Nieman & Sayre. Two refinements of the iterative constraint-based scheme are developed to address the current experimental realities of this imaging technique, which include missing central data and noise. A constrained power operator is defined whose eigenmodes allow the identification of a small number of degrees of freedom in the reconstruction that are negligibly constrained as a result of the missing data. To achieve reproducibility in the algorithm's output,more » a special intervention is required for these modes. Weak incompatibility of the constraints caused by noise in both direct and Fourier space leads to residual phase fluctuations. This problem is addressed by supplementing the algorithm with an averaging method. The effect of averaging may be interpreted in terms of an effective modulation transfer function, as used in optics, to quantify the resolution. The reconstruction details are prefaced with simulations of wave propagation through a model yeast cell. These show that the yeast cell is a strong-phase-contrast object for the conditions in the experiment.« less
Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

PubMed Central

Kretschmer, M; Schellenberger, W; Otto, A; Kessler, R; Hofmann, E

1987-01-01

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A. PMID:2825652
Separating biological cells

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1979-01-01

Technique utilizing electric field to promote biological cell separation from suspending medium in zero gravity increases speed, reduces sedimentation, and improves efficiency of separation in normal gravity.
Reduction of Nucleic Acid Content in Candida Yeast Cells by Bovine Pancreatic Ribonuclease A Treatment

PubMed Central

Castro, A. C.; Sinskey, A. J.; Tannenbaum, S. R.

1971-01-01

Yeast as a source of protein for human consumption is limited by its relatively high nucleic acid content. In this study, we developed an enzymatic method of decreasing the nucleic acid content. Candida utilis cells, heat-shocked at 80 C for 30 sec, were treated with bovine pancreatic ribonuclease A. Maximum leakage of nucleic acid was observed when the incubation temperature was between 55 and 65 C, the pH of the system from 6.75 to 8.0, and the enzyme-to-cell ratio 1:10,000 on a weight-by-weight basis. Other factors, such as yeast strain, age of cells, and method of propagation, did not influence the susceptibility of the yeast cells to the action of ribonuclease. Buffers and monovalent cations had no inhibiting effects. Magnesium and calcium ions at concentrations greater than 0.001 m showed marked inhibition on the rate of nucleic acid leakage. This enzymatic method reduced the nucleic acid content of yeast cells from 7.5 to 9.0% to 1.5 to 2.0% with no significant concomitant loss of protein. PMID:5165838
Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

PubMed Central

Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Qin, Jiufu; Siewers, Verena; Nielsen, Jens

2016-01-01

Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value. PMID:27222209
A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

PubMed

Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

2016-12-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.
A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

PubMed Central

Ball, David A.

2016-01-01

The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947
Novel platform for minimizing cell loss on separation process: Droplet-based magnetically activated cell separator

NASA Astrophysics Data System (ADS)

Kim, Youngho; Hong, Su; Lee, Sang Ho; Lee, Kangsun; Yun, Seok; Kang, Yuri; Paek, Kyeong-Kap; Ju, Byeong-Kwon; Kim, Byungkyu

2007-07-01

To reduce the problem of cell loss due to adhesion, one of the basic phenomena in microchannel, we proposed the droplet-based magnetically activated cell separator (DMACS). Based on the platform of the DMACS—which consists of permanent magnets, a coverslip with a circle-shaped boundary, and an injection tube—we could collect magnetically (CD45)-labeled (positive) cells with high purity and minimize cell loss due to adhesion. To compare separation efficiency between the MACS and the DMACS, the total number of cells before and after separation with both the separators was counted by flow cytometry. We could find that the number (3241/59940) of cells lost in the DMACS is much less than that (22360/59940) in the MACS while the efficiency of cell separation in the DMACS (96.07%) is almost the same as that in the MACS (96.72%). Practically, with fluorescent images, it was visually confirmed that the statistical data are reliable. From the viability test by using Hoechst 33 342, it was also demonstrated that there was no cell damage on a gas-liquid interface. Conclusively, DMACS will be a powerful tool to separate rare cells and applicable as a separator, key component of lab-on-a-chip.
Trichoderma sp. spores and Kluyveromyces marxianus cells magnetic separation: Immobilization on chitosan-coated magnetic nanoparticles.

PubMed

Palacios-Ponce, Sócrates; Ramos-González, Rodolfo; Ruiz, Héctor A; Aguilar, Miguel A; Martínez-Hernández, José L; Segura-Ceniceros, Elda P; Aguilar, Cristóbal N; Michelena, Georgina; Ilyina, Anna

2017-07-03

In the present study, the interactions between chitosan-coated magnetic nanoparticles (C-MNP) and Trichoderma sp. spores as well as Kluyveromyces marxianus cells were studied. By Plackett-Burman design, it was demonstrated that factors which directly influenced on yeast cell immobilization and magnetic separation were inoculum and C-MNP quantity, stirring speed, interaction time, and volume of medium, while in the case of fungal spores, the temperature also was disclosed as an influencing factor. Langmuir and Freundlich models were applied for the mathematical analysis of adsorption isotherms at 30°C. For Trichoderma sp. spore adsorption isotherm, the highest correlation coefficient was observed for lineal function of Langmuir model with a maximum adsorption capacity at 5.00E + 09 spores (C-MNP g -1 ). Adsorption isotherm of K. marxianus cells was better adjusted to Freundlich model with a constant (K f ) estimated as 2.05E + 08 cells (C-MNP g -1 ). Both systems may have a novel application in fermentation processes assisted with magnetic separation of biomass.
Neural network analysis of electrodynamic activity of yeast cells around 1 kHz

NASA Astrophysics Data System (ADS)

Janca, R.

2011-12-01

This paper deals with data analysis of electrodynamic activity of two mutants of yeast cells, cell cycle of which is synchronized and non-synchronized, respectively. We used data already published by Jelinek et al. and treat them with data mining method based on the multilayer neural network. Intersection of data mining and statistical distribution of the noise shows significant difference between synchronized and non-synchronized yeasts not only in total power, but also discrete frequencies.
Biostimulation effects of low-energy laser radiation on yeast cell suspensions

NASA Astrophysics Data System (ADS)

Anghel, Sorin; Stanescu, Constantin S.; Giosanu, Dana; Neagu, Ionica; Savulescu, Geta; Iorga-Siman, Ion

2000-02-01

This paper presents work to determine the effects produced by low energy laser radiation on the metabolism and growth of a yeast cell suspension. As experimental material, we used young yeast culture in liquid medium, then distributed on a solid medium, to obtain isolated colonies. As laser source, we used a He-Ne laser, and the irradiation was made with different exposure times. Form each irradiated material, a sample of white grape sterile must was sowed, that has fermented at 18 divided by 20 degrees C for 10 divided by 15 days, after that some properties was tested. Some microscopic studies were also made. The results prove some influence of low energy laser irradiation, which can induce mutations, with new properties of the irradiated material. These mutations can be obtained in a positive sense, with new and important perspectives in wine industry. Also, we observed an inhibitory effect of the laser radiation on the yeast cell growth, due, probably to the too high values of the exposure.
Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

PubMed

Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

2018-02-08

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.
A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation

PubMed Central

Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine

2017-01-01

Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation. PMID:28922393
Effects of metal salt catalysts on yeast cell growth in ethanol conversion

Treesearch

Chung-Yun Hse; Yin Lin

2009-01-01

The effects of the addition of metal salts and metal salt-catalyzed hydrolyzates on yeast cell growth in ethanol fermentation were investigated. Four yeast strains (Saccharomyces cerevisiae WT1, Saccharomyces cerevisiae MT81, Candida sp. 1779, and Klumaromyces fragilis), four metal salts (CuCl2, FeCl3, AgNO3, and I2), two metal salt-catalyzed hydrolyzates (...
Molten carbonate fuel cell separator

DOEpatents

Nickols, Richard C.

1986-09-02

In a stacked array of molten carbonate fuel cells, a fuel cell separator is positioned between adjacent fuel cells to provide isolation as well as a conductive path therebetween. The center portion of the fuel cell separator includes a generally rectangular, flat, electrical conductor. Around the periphery of the flat portion of the separator are positioned a plurality of elongated resilient flanges which form a gas-tight seal around the edges of the fuel cell. With one elongated flange resiliently engaging a respective edge of the center portion of the separator, the sealing flanges, which are preferably comprised of a noncorrosive material such as an alloy of yttrium, iron, aluminum or chromium, form a tight-fitting wet seal for confining the corrosive elements of the fuel cell therein. This arrangement permits a good conductive material which may be highly subject to corrosion and dissolution to be used in combination with a corrosion-resistant material in the fuel cell separator of a molten carbonate fuel cell for improved fuel cell conductivity and a gas-tight wet seal.

Molten carbonate fuel cell separator

DOEpatents

Nickols, R.C.

1984-10-17

In a stacked array of molten carbonate fuel cells, a fuel cell separator is positioned between adjacent fuel cells to provide isolation as well as a conductive path therebetween. The center portion of the fuel cell separator includes a generally rectangular, flat, electrical conductor. Around the periphery of the flat portion of the separator are positioned a plurality of elongated resilient flanges which form a gas-tight seal around the edges of the fuel cell. With one elongated flange resiliently engaging a respective edge of the center portion of the separator, the sealing flanges, which are preferably comprised of a noncorrosive material such as an alloy of yttrium, iron, aluminum or chromium, form a tight-fitting wet seal for confining the corrosive elements of the fuel cell therein. This arrangement permits a good conductive material which may be highly subject to corrosion and dissolution to be used in combination with a corrosion-resistant material in the fuel cell separator of a molten carbonate fuel cell for improved fuel cell conductivity and a gas-tight wet seal.
Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells

PubMed Central

Koyama, Sumihiro; Tsubouchi, Taishi; Usui, Keiko; Uematsu, Katsuyuki; Tame, Akihiro; Nogi, Yuichi; Ohta, Yukari; Hatada, Yuji; Kato, Chiaki; Miwa, Tetsuya; Toyofuku, Takashi; Nagahama, Takehiko; Konishi, Masaaki; Nagano, Yuriko; Abe, Fumiyoshi

2015-01-01

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications. PMID:26187908
Physiological analysis of yeast cells by flow cytometry during serial-repitching of low-malt beer fermentation.

PubMed

Kobayashi, Michiko; Shimizu, Hiroshi; Shioya, Suteaki

2007-05-01

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.
Stacking Oxygen-Separation Cells

NASA Technical Reports Server (NTRS)

Schroeder, James E.

1991-01-01

Simplified configuration and procedure developed for assembly of stacks of solid-electrolyte cells separating oxygen from air electrochemically. Reduces number of components and thus reduces probability of such failures as gas leaks, breakdown of sensitive parts, and electrical open or short circuits. Previous, more complicated version of cell described in "Improved Zirconia Oxygen-Separation Cell" (NPO-16161).
Classification of yeast cells from image features to evaluate pathogen conditions

NASA Astrophysics Data System (ADS)

van der Putten, Peter; Bertens, Laura; Liu, Jinshuo; Hagen, Ferry; Boekhout, Teun; Verbeek, Fons J.

2007-01-01

Morphometrics from images, image analysis, may reveal differences between classes of objects present in the images. We have performed an image-features-based classification for the pathogenic yeast Cryptococcus neoformans. Building and analyzing image collections from the yeast under different environmental or genetic conditions may help to diagnose a new "unseen" situation. Diagnosis here means that retrieval of the relevant information from the image collection is at hand each time a new "sample" is presented. The basidiomycetous yeast Cryptococcus neoformans can cause infections such as meningitis or pneumonia. The presence of an extra-cellular capsule is known to be related to virulence. This paper reports on the approach towards developing classifiers for detecting potentially more or less virulent cells in a sample, i.e. an image, by using a range of features derived from the shape or density distribution. The classifier can henceforth be used for automating screening and annotating existing image collections. In addition we will present our methods for creating samples, collecting images, image preprocessing, identifying "yeast cells" and creating feature extraction from the images. We compare various expertise based and fully automated methods of feature selection and benchmark a range of classification algorithms and illustrate successful application to this particular domain.
From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Okazaki, Fumiyoshi; Djohan, Apridah Cameliawati; Hara, Kiyotaka Y; Asai-Nakashima, Nanami; Teramura, Hiroshi; Andriani, Ade; Tominaga, Masahiro; Wakai, Satoshi; Kahar, Prihardi; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2016-01-01

Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. We successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.
Synchronization of glycolytic oscillations in a yeast cell population.

PubMed

Danø, S; Hynne, F; De Monte, S; d'Ovidio, F; Sørensen, P G; Westerhoff, H

2001-01-01

The mechanism of active phase synchronization in a suspension of oscillatory yeast cells has remained a puzzle for almost half a century. The difficulty of the problem stems from the fact that the synchronization phenomenon involves the entire metabolic network of glycolysis and fermentation, and consequently it cannot be addressed at the level of a single enzyme or a single chemical species. In this paper it is shown how this system in a CSTR (continuous flow stirred tank reactor) can be modelled quantitatively as a population of Stuart-Landau oscillators interacting by exchange of metabolites through the extracellular medium, thus reducing the complexity of the problem without sacrificing the biochemical realism. The parameters of the model can be derived by a systematic expansion from any full-scale model of the yeast cell kinetics with a supercritical Hopf bifurcation. Some parameter values can also be obtained directly from analysis of perturbation experiments. In the mean-field limit, equations for the study of populations having a distribution of frequencies are used to simulate the effect of the inherent variations between cells.
Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

PubMed

Wang, Hongwei; Lang, Qiaolin; Li, Liang; Liang, Bo; Tang, Xiangjiang; Kong, Lingrang; Mascini, Marco; Liu, Aihua

2013-06-18

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.
Candida ecuadorensis sp. nov., an ascomycetous yeast species found in two separate regions of Ecuador.

PubMed

James, Stephen A; Carvajal Barriga, Enrique Javier; Barahona, Patricia Portero; Cross, Kathryn; Bond, Christopher J; Roberts, Ian N

2013-01-01

In the course of an on-going study aimed at cataloguing the natural yeast biodiversity found in Ecuador, two strains (CLQCA 13-025 and CLQCA 20-004(T)) were isolated from samples of cow manure and rotten wood collected in two separate provinces of the country (Orellana and Bolívar). These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to belong to the Metschnikowia clade and to be most closely related to Candida suratensis, a species recently discovered in a mangrove forest in Thailand. The species name of Candida ecuadorensis sp. nov. is proposed to accommodate these strains, with strain CLQCA 20-004(T) (=CBS 12653(T) = NCYC 3782(T)) designated as the type strain.
Nanoscopic morphological changes in yeast cell surfaces caused by oxidative stress: an atomic force microscopic study.

PubMed

Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

2009-06-01

Nanoscopic changes in the cell surface morphology of the yeasts Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354), due to their exposure to varying concentrations of hydrogen peroxide (oxidative stress), were investigated using an atomic force microscope (AFM). Increasing hydrogen peroxide concentration led to a decrease in cell viabilities and mean cell volumes, and an increase in the surface roughness of the yeasts. In addition, AFM studies revealed that oxidative stress caused cell compression in both S. cerevisiae and Schiz. pombe cells and an increase in the number of aged yeasts. These results confirmed the importance and usefulness of AFM in investigating the morphology of stressed microbial cells at the nanoscale. The results also provided novel information on the relative oxidative stress tolerance of S. cerevisiae and Schiz. pombe.
Yeast Based Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.
Synthesis of polypyrrole within the cell wall of yeast by redox-cycling of [Fe(CN)6](3-)/[Fe(CN)6](4-).

PubMed

Ramanavicius, Arunas; Andriukonis, Eivydas; Stirke, Arunas; Mikoliunaite, Lina; Balevicius, Zigmas; Ramanaviciene, Almira

2016-02-01

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)6](3-)ions, which were generated from K4[Fe(CN)6], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)6](4-). After 24h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated. Copyright © 2015 Elsevier Inc. All rights reserved.
Study of budding yeast colony formation and its characterizations by using circular granular cell

NASA Astrophysics Data System (ADS)

Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

2016-03-01

Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.
S-Adenosyl-L-methionine protects the probiotic yeast, Saccharomyces boulardii, from acid-induced cell death.

PubMed

Cascio, Vincent; Gittings, Daniel; Merloni, Kristen; Hurton, Matthew; Laprade, David; Austriaco, Nicanor

2013-02-13

Saccharomyces boulardii is a probiotic yeast routinely used to prevent and to treat gastrointestinal disorders, including the antibiotic-associated diarrhea caused by Clostridium difficile infections. However, only 1-3% of the yeast administered orally is recovered alive in the feces suggesting that this yeast is unable to survive the acidic environment of the gastrointestinal tract. We provide evidence that suggests that S. boulardii undergoes programmed cell death (PCD) in acidic environments, which is accompanied by the generation of reactive oxygen species and the appearance of caspase-like activity. To better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. Significantly, functional annotation revealed that the up-regulated genes were significantly over-represented in cell death pathways Finally, we show that S-adenosyl-L-methionine (AdoMet), a commercially available, FDA-approved dietary supplement, enhances the viability of S. boulardii in acidic environments, most likely by preventing programmed cell death. In toto, given the observation that many of the proven health benefits of S. boulardii are dependent on cell viability, our data suggests that taking S. boulardii and AdoMet together may be a more effective treatment for gastrointestinal disorders than taking the probiotic yeast alone.
A systems-level approach for metabolic engineering of yeast cell factories.

PubMed

Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

2012-03-01

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Heavy ion induced DNA-DSB in yeast and mammalian cells

NASA Technical Reports Server (NTRS)

Loebrich, M.; Ikpeme, S.; Kiefer, J.

1994-01-01

Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.
Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

PubMed

Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

2015-05-01

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.
Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

PubMed Central

Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

2012-01-01

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti
Immobilization of yeast cells with ionic hydrogel carriers by adhesion-multiplication.

PubMed

Zhaoxin, L; Fujimura, T

2000-12-01

The mixture of an ionic monomer, 2-acrylamido 2-methylpropanesulfonic acid (TBAS), and a series of poly(ethylene glycol) dimethacrylate (nG) monomers were copolymerized with 60Co gamma-rays, and the produced ionic hydrogel polymers were used for immobilization of yeast cells. The cells were adhered onto the surface of the hydrogel polymers and intruded into the interior of the polymers with growing. The immobilized yeast cells with these hydrogel polymers had higher ethanol productivity than that of free cells. The yield of ethanol with poly(TBAS-14G) carrier was the highest and increased by 3.5 times compared to the free cells. It was found that the ethanol yield increased with the increase of glycol number in poly(ethylene glycol) dimethacrylate. The state of the immobilized cells was observed with microscope, and it was also found that the difference in the ethanol productivity is mainly due to the difference in the internal structure and properties of polymer carrier, such as surface charge, hydrophilicity, and swelling ability of polymer carrier.
Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation.

PubMed Central

Peña, A; Ramírez, J; Rosas, G; Calahorra, M

1995-01-01

The internal pH of yeast cells was determined by measuring the fluorescence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), which was introduced into the cells by electroporation. This may be a suitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm and to avoid the vacuole, and therefore it probably measured actual cytoplasmic pH. (v) Correction factors to obtain a more precise estimation of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the introduction of other substances into cells. Values for the cytoplasmic pHs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole. PMID:7860582

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

PubMed

Attfield, P V; Kletsas, S; Veal, D A; van Rooijen, R; Bell, P J

2000-08-01

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.
Interaction Between Yeasts and Zinc

NASA Astrophysics Data System (ADS)

Nicola, Raffaele De; Walker, Graeme

Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses
Building Better Biosensors for Exploration into Deep-Space, Using Humanized Yeast

NASA Technical Reports Server (NTRS)

Liddell, Lauren; Santa Maria, Sergio; Tieze, Sofia; Bhattacharya, Sharmila

2017-01-01

1.BioSentinel is 1 of 13 secondary payloads hitching a ride beyond Low Earth Orbit on Exploration Mission 1 (EM-1), set to launch from NASAs Space Launch System in 2019. EM-1 is our first opportunity to investigate the effects of the deep space environment on a eukaryotic biological system, the budding yeast S. cerevisiae. Though separated by a billion years of evolution we share hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast is an ideal biosensor for detecting typesextent of damage induced by deep-space radiation.We will fly desiccated cells, then rehydrate to wake them up when the automated payload is ready to initiate the experiment. Rehydration solution contains SC (Synthetic Complete) media and alamarBlue, an indicator for changes in growth and metabolism. Telemetry of LED readings will then allow us to detect how cells respond throughout the mission. The desiccation-rehydration process can be extremely damaging to cells, and can severely diminish our ability to accurately measure and model cellular responses to deep-space radiation. The aim of this study is to develop a better biosensor: yeast strains that are more resistant to desiccation stress. We will over-express known cellular protectants, including hydrophilin Sip18, the protein disaggregase Hsp104, and thioredoxin Trx2, a responder to oxidative stress, then measure cell viability after desiccation to determine which factors improve stress tolerance. Over-expression of SIP18 in wine yeast starter cultures was previously reported to increase viability following desiccation stress by up to 70. Thus, we expect similar improvements in our space-yeast strains. By designing better yeast biosensors we can better prepare for and mitigate the potential dangers of deep-space radiation for future missions.This work is funded by NASAs AES program.
Recent advances in yeast cell-surface display technologies for waste biorefineries.

PubMed

Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

2016-09-01

Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.
The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

PubMed Central

Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

2002-01-01

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast. PMID:11916725
Harmonic generation by yeast cells in response to low-frequency electric fields

NASA Astrophysics Data System (ADS)

Nawarathna, D.; Claycomb, J. R.; Cardenas, G.; Gardner, J.; Warmflash, D.; Miller, J. H., Jr.; Widger, W. R.

2006-05-01

We report on harmonic generation by budding yeast cells (Saccharomyces cerevisiae, 108cells/ml ) in response to sinusoidal electric fields with amplitudes ranging from zero to 5V/cm in the frequency range 10-300Hz . The cell-generated harmonics are found to exhibit strong amplitude and frequency dependence. Sodium metavanadate, an inhibitor of the proton pump known as H+ -ATPase, and glucose, a substrate of H+ -ATPase, are found to increase harmonic production at low amplitudes while reducing it at large amplitudes. This P-type proton pump can be driven by an oscillatory transmembrane potential, and its nonlinear response is believed to be largely responsible for harmonic production at low frequencies in yeast cells. We find that the observed harmonics show dramatic changes with time and in their field and frequency dependence after perturbing the system by adding an inhibitor, substrate, or membrane depolarizer to the cell suspension.
Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

PubMed Central

Bohnert, K. Adam; Gould, Kathleen L.

2012-01-01

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state. PMID:23093943
Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

DOEpatents

Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

2012-03-20

Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.
S-Adenosyl-L-Methionine protects the probiotic yeast, Saccharomyces boulardii, from acid-induced cell death

PubMed Central

2013-01-01

Background Saccharomyces boulardii is a probiotic yeast routinely used to prevent and to treat gastrointestinal disorders, including the antibiotic-associated diarrhea caused by Clostridium difficile infections. However, only 1-3% of the yeast administered orally is recovered alive in the feces suggesting that this yeast is unable to survive the acidic environment of the gastrointestinal tract. Results We provide evidence that suggests that S. boulardii undergoes programmed cell death (PCD) in acidic environments, which is accompanied by the generation of reactive oxygen species and the appearance of caspase-like activity. To better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. Significantly, functional annotation revealed that the up-regulated genes were significantly over-represented in cell death pathways Finally, we show that S-adenosyl-L-methionine (AdoMet), a commercially available, FDA-approved dietary supplement, enhances the viability of S. boulardii in acidic environments, most likely by preventing programmed cell death. Conclusions In toto, given the observation that many of the proven health benefits of S. boulardii are dependent on cell viability, our data suggests that taking S. boulardii and AdoMet together may be a more effective treatment for gastrointestinal disorders than taking the probiotic yeast alone. PMID:23402325
A multiplex culture system for the long-term growth of fission yeast cells.

PubMed

Callens, Céline; Coelho, Nelson C; Miller, Aaron W; Sananes, Maria Rosa Domingo; Dunham, Maitreya J; Denoual, Matthieu; Coudreuse, Damien

2017-08-01

Maintenance of long-term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin-encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo-pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long-term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
Yeast proteome map (last update).

PubMed

Perrot, Michel; Moes, Suzette; Massoni, Aurélie; Jenoe, Paul; Boucherie, Hélian

2009-10-01

The identification of proteins separated on 2-D gels is essential to exploit the full potential of 2-D gel electrophoresis for proteomic investigations. For this purpose we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification by mass spectrometry of 100 novel yeast protein spots that have so far not been tackled due to their scarcity on our standard 2-D gels. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 716. They correspond to 485 unique proteins. Among these, 154 were resolved into several isoforms. The present data set can now be expanded to report for the first time a map of 363 protein isoforms that significantly deepens our knowledge of the yeast proteome. The reference map and a list of all identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).
Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

NASA Astrophysics Data System (ADS)

Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

2008-09-01

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.
T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072
Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

PubMed Central

2014-01-01

Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production
Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.

PubMed

Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

2016-01-01

Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.
Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leão, Mariana; Gomes, Sara; Bessa, Cláudia

In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either permore » se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.« less
X-ray irradiation of yeast cells

NASA Astrophysics Data System (ADS)

Masini, Alessandra; Batani, Dimitri; Previdi, Fabio; Conti, Aldo; Pisani, Francesca; Botto, Cesare; Bortolotto, Fulvia; Torsiello, Flavia; Turcu, I. C. Edmond; Allott, Ric M.; Lisi, Nicola; Milani, Marziale; Costato, Michele; Pozzi, Achille; Koenig, Michel

1997-10-01

Saccharomyces Cerevisiae yeast cells were irradiated using the soft X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterized by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency.
Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

PubMed

Linke, Bettina; Schröder, Kersten; Arter, Juliane; Gasperazzo, Tatiana; Woehlecke, Holger; Ehwald, Rudolf

2010-09-01

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.
Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

PubMed

Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

2016-10-24

Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.
The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

PubMed

Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

2014-10-16

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Yeast flocculation: New story in fuel ethanol production.

PubMed

Zhao, X Q; Bai, F W

2009-01-01

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.
Separation of CHO cells using hydrocyclones.

PubMed

Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

2008-01-01

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.
Study of amyloids using yeast

PubMed Central

Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

2012-01-01

Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100
Form and function of topologically associating genomic domains in budding yeast.

PubMed

Eser, Umut; Chandler-Brown, Devon; Ay, Ferhat; Straight, Aaron F; Duan, Zhijun; Noble, William Stafford; Skotheim, Jan M

2017-04-11

The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.
How do fission yeast cells grow and connect growth to the mitotic cycle?

PubMed

Sveiczer, Ákos; Horváth, Anna

2017-05-01

To maintain size homeostasis in a unicellular culture, cells should coordinate growth to the division cycle. This is achieved via size control mechanisms (also known as size checkpoints), i.e. some events during the mitotic cycle supervene only if the cell has reached a critical size. Rod-shaped cells like those of fission yeast are ideal model organisms to study these checkpoints via time-lapse microphotography. By applying this method, once we can analyse the growth process between two consecutive divisions at a single (or even at an 'average') cellular level, moreover, we can also position the size checkpoint(s) at the population level. Finally, any of these controls can be abolished in appropriate cell cycle mutants, either in steady-state or in induction synchronised cultures. In the latter case, we produce abnormally oversized cells, and microscopic experiments with them clearly show the existence of a critical size above which the size checkpoint ceases (becomes cryptic). In this review, we delineate the development of our knowledge both on the growth mode of fission yeast and on the operating size control(s) during its mitotic cycle. We finish these historical stories with our recent findings, arguing that three different size checkpoints exist in the fission yeast cell cycle, namely in late G1, in mid G2 and in late G2, which has been concluded by analysing these controls in several cell cycle mutants.
Nitrile Metabolizing Yeasts

NASA Astrophysics Data System (ADS)

Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing
Nutrient depletion modifies cell wall adsorption activity of wine yeast.

PubMed

Sidari, R; Caridi, A

2016-06-01

Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions-early, mid, 'adjusted' for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in 'adjusted' for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters.
Development of a novel microbial sensor with baker's yeast cells for monitoring temperature control during cold food chain.

PubMed

Kogure, H; Kawasaki, S; Nakajima, K; Sakai, N; Futase, K; Inatsu, Y; Bari, M L; Isshiki, K; Kawamoto, S

2005-01-01

A novel microbial sensor containing a commercial baker's yeast with a high freeze tolerance was developed for visibly detecting inappropriate temperature control of food. When the yeast cells fermented glucose, the resulting gas production triggered the microbial sensor. The biosensor was a simple, small bag containing a solution of yeast cells, yeast extract, glucose, and glycerol sealed up with multilayer transparent film with barriers against oxygen and humidity. Fine adjustment of gas productivity in the biosensor at low temperatures was achieved by changing either or both concentrations of glucose and yeast cells. Moreover, the amount of time that food was exposed to inappropriate temperatures could be deduced by the amount of gas produced in the biosensor. The biosensor was stable without any functional loss for up to 1 week in frozen storage. The biosensor could offer a useful tool for securing food safety by maintaining low-temperature control in every stage from farm to fork, including during transportation, in the store, and at home.
Dynamic acoustic field activated cell separation (DAFACS).

PubMed

Skotis, G D; Cumming, D R S; Roberts, J N; Riehle, M O; Bernassau, A L

2015-02-07

Advances in diagnostics, cell and stem cell technologies drive the development of application-specific tools for cell and particle separation. Acoustic micro-particle separation offers a promising avenue for high-throughput, label-free, high recovery, cell and particle separation and isolation in regenerative medicine. Here, we demonstrate a novel approach utilizing a dynamic acoustic field that is capable of separating an arbitrary size range of cells. We first demonstrate the method for the separation of particles with different diameters between 6 and 45 μm and secondly particles of different densities in a heterogeneous medium. The dynamic acoustic field is then used to separate dorsal root ganglion cells. The shearless, label-free and low damage characteristics make this method of manipulation particularly suited for biological applications. Advantages of using a dynamic acoustic field for the separation of cells include its inherent safety and biocompatibility, the possibility to operate over large distances (centimetres), high purity (ratio of particle population, up to 100%), and high efficiency (ratio of separated particles over total number of particles to separate, up to 100%).
Yeast Droplets

NASA Astrophysics Data System (ADS)

Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

2002-11-01

It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.
Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

PubMed

Niki, Hironori

2014-03-01

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.
Tributyltin induces Yca1p-dependent cell death of yeast Saccharomyces cerevisiae.

PubMed

Chahomchuen, Thippayarat; Akiyama, Koichi; Sekito, Takayuki; Sugimoto, Naoko; Okabe, Masaaki; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 microM for the parent strain BY4741 and 3 microM for the pdr5Delta mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the yca1 gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.
Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

PubMed

Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

2012-01-01

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.
Quality testing of an innovative cascade separation system for multiple cell separation

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Moszczynska, Aleksandra; Albrecht, Bernd; Heinrich, Jan-Michael; Tarnok, Attila

2012-03-01

Isolation of different cell types from mixed samples in one separation step by FACS is feasible but expensive and slow. It is cheaper and faster but still challenging by magnetic separation. An innovative bead-based cascade-system (pluriSelect GmbH, Leipzig, Germany) relies on simultaneous physical separation of different cell types. It is based on antibody-mediated binding of cells to beads of different size and isolation with sieves of different mesh-size. We validated pluriSelect system for single parameter (CD3) and simultaneous separation of CD3 and CD15 cells from EDTA blood-samples. Results were compared with those obtained by MACS (Miltenyi-Biotech) magnetic separation (CD3 separation). pluriSelect separation was done in whole blood, MACS on Ficoll gradient isolated leukocytes, according to the manufacturer's protocols. Isolated and residual cells were immunophenotyped (7-color 8-antibody panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLADR) on a CyFlowML flow cytometer (Partec GmbH). Cell count (Coulter), purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (92-98%), yield (50-60%) and viability (92-98%) of isolated cells. PluriSelect separation was slightly faster than MACS (1.15 h versus 1.5h). Moreover, no preenrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two cell subpopulation directly from whole blood and can provide a simple alternative to FACS. The isolated cells can be used for further research applications.
Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13.

PubMed

Hellström, A; Qvirist, L; Svanberg, U; Veide Vilg, J; Andlid, T

2015-05-01

Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190 mU ml(-1) depending on the media. The Km was 0.28 mmol l(-1) and the specific phytase activity 0.32 U mg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase. © 2015 The Society for Applied Microbiology.
Cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for high yield ethanol production in consolidated bioprocessing.

PubMed

Matano, Yuki; Hasunuma, Tomohisa; Kondo, Akihiko

2013-05-01

The aim of this study is to develop a scheme of cell recycle batch fermentation (CRBF) of high-solid lignocellulosic materials. Two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid-liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. Five consecutive batch fermentation of 200 g/L rice straw hydrothermally pretreated led to an average ethanol titer of 34.5 g/L. Moreover, the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2 g/L. After, five-cycle fermentation, only 3.3 g/L sugar was retained in the fermentation medium, because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. Fermentation ability of the recombinant strain was successfully kept during a five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.
Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

PubMed

Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

2017-08-01

Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.
The yeast actin cytoskeleton.

PubMed

Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

2014-03-01

The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
A multiplex culture system for the long‐term growth of fission yeast cells

PubMed Central

Callens, Céline; Coelho, Nelson C.; Miller, Aaron W.; Sananes, Maria Rosa Domingo; Dunham, Maitreya J.; Denoual, Matthieu

2017-01-01

Abstract Maintenance of long‐term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin‐encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo‐pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long‐term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:28426144
Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae.

PubMed

Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I

2014-05-27

A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae . Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific "master regulator" proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast.

Separation technologies for stem cell bioprocessing.

PubMed

Diogo, Maria Margarida; da Silva, Cláudia Lobato; Cabral, Joaquim M S

2012-11-01

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell-derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato-oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost-effective. Important examples are the need for high-resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity-based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical-based methods requiring no cell labeling and integrated with microscale technologies. Copyright © 2012 Wiley Periodicals, Inc.
Improved Separators For Rechargeable Lithium Cells

NASA Technical Reports Server (NTRS)

Shen, David; Surampudi, Subbarao; Huang, Chen-Kuo; Halpert, Gerald

1994-01-01

Improved pairs of separators proposed for use in rechargeable lithium cells operating at ambient temperature. Block growth of lithium dendrites and help prevent short circuits. Each cell contains one separator made of microporous polypropylene placed next to anode, and one separator made of microporous polytetrafluoroethylene (PTFE) next to cathode. Separators increase cycle lives of secondary lithium cells. Cells to which concept applicable those of Li/TiS(2), Li/NbSe(3), Li/CoO(2), Li/MoS(2), Li/VO(x), and Li/MnO(2) chemical systems. Advantageous in spacecraft, military, communications, automotive, and other applications in which high energy density and rechargeability needed.
Cell separation: Terminology and practical considerations

PubMed Central

Tomlinson, Sophie; Yang, Xuebin B; Kirkham, Jennifer

2013-01-01

Cell separation is a powerful tool in biological research. Increasing usage, particularly within the tissue engineering and regenerative medicine communities, means that researchers from a diverse range of backgrounds are utilising cell separation technologies. This review aims to offer potential solutions to cell sorting problems and to clarify common ambiguities in terminology and experimental design. The frequently used cell separation terms of ‘purity’, ‘recovery’ and ‘viability’ are discussed, and attempts are made to reach a consensus view of their sometimes ambiguous meanings. The importance of appropriate experimental design is considered, with aspects such as marker expression, tissue isolation and original cell population analysis discussed. Finally, specific technical issues such as cell clustering, dead cell removal and non-specific antibody binding are considered and potential solutions offered. The solutions offered may provide a starting point to improve the quality of cell separations achieved by both the novice and experienced researcher alike. PMID:23440031
Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

PubMed

Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

2018-01-01

Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.
Facile and quantitative electrochemical detection of yeast cell apoptosis

NASA Astrophysics Data System (ADS)

Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

2014-03-01

An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.
Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level.

PubMed

Ganeva, V; Galutzov, B; Teissié, J

1995-12-13

The mechanism of electric field mediated macromolecule transfer inside an intact yeast cell was investigated by observing, under a microscope, the fluorescence associated to cells after pulsation in a buffer containing two different hydrophilic fluorescent dyes. In the case of a small probe such as propidium iodide, a long lived permeabilized state was induced by the field as classically observed on wall free systems. Penetration of a 70 kDa FITC dextran was obtained only by using drastic conditions and only a very limited number of yeast cells which took up macromolecules remained viable. Most dextrans were trapped in the wall. A dramatic improvement in transfer of dextrans was observed when the cells were treated by dithiothreitol before pulsation. A cytoplasmic protein leakage was detected after the electric treatment suggesting that an irreversible damage took place in the walls of many pulsed cells. Electroloading of macromolecules in intact yeast cells appears to be controlled by a field induced short lived alteration of the envelope organization.
Surface enhanced Raman scattering analyses of individual silver nanoaggregates on living single yeast cell wall

NASA Astrophysics Data System (ADS)

Sujith, Athiyanathil; Itoh, Tamitake; Abe, Hiroko; Anas, Abdul Aziz; Yoshida, Kenichi; Biju, Vasudevanpillai; Ishikawa, Mitsuru

2008-03-01

We labeled the living yeast cell surface (Saccharomyces cerevisiae strain W303-1A) by silver nanoparticles which can form nanoaggregates and found to show surface enhanced Raman scattering (SERS) activity. Blinking of SERS and its polarization dependence reveal that SERS signals are from amplified electromagnetic field at nanometric Ag nanoparticles gaps with single or a few molecules sensitivity. We tentatively assigned SERS spectra from a yeast cell wall to mannoproteins. Nanoaggregate-by-nanoaggregate variations and temporal fluctuations of SERS spectra are discussed in terms of inhomogeneous mannoprotein distribution on a cell wall and possible ways of Ag nanoaggregate adsorption, respectively.
Antibody-immobilized column for quick cell separation based on cell rolling.

PubMed

Mahara, Atsushi; Yamaoka, Tetsuji

2010-01-01

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column.
Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must.

PubMed

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.
Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must

PubMed Central

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations. PMID:23658613
Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

PubMed

Martínez-Esparza, M; Sarazin, A; Jouy, N; Poulain, D; Jouault, T

2006-07-31

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.
Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

NASA Astrophysics Data System (ADS)

Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

2012-10-01

In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.
Application of anhydrobiosis and dehydration of yeasts for non-conventional biotechnological goals.

PubMed

Rapoport, Alexander; Turchetti, Benedetta; Buzzini, Pietro

2016-06-01

Dehydration of yeast cells causes them to enter a state of anhydrobiosis in which their metabolism is temporarily and reversibly suspended. This unique state among organisms is currently used in the production of active dry yeasts, mainly used in baking and winemaking. In recent decades non-conventional applications of yeast dehydration have been proposed for various modern biotechnologies. This mini-review briefly summarises current information on the application of dry yeasts in traditional and innovative fields. It has been shown that dry yeast preparations can be used for the efficient protection, purification and bioremediation of the environment from heavy metals. The high sorption activity of dehydrated yeasts can be used as an interesting tool in winemaking due to their effects on quality and taste. Dry yeasts are also used in agricultural animal feed. Another interesting application of yeast dehydration is as an additional stage in new methods for the stable immobilisation of microorganisms, especially in cases when biotechnologically important strains have no affinity with the carrier. Such immobilisation methods also provide a new approach for the successful conservation of yeast strains that are very sensitive to dehydration. In addition, the application of dehydration procedures opens up new possibilities for the use of yeast as a model system. Separate sections of this review also discuss possible uses of dry yeasts in biocontrol, bioprotection and biotransformations, in analytical methods as well as in some other areas.
A genome-wide resource of cell cycle and cell shape genes of fission yeast

PubMed Central

Hayles, Jacqueline; Wood, Valerie; Jeffery, Linda; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Salas-Pino, Silvia; Heichinger, Christian; Nurse, Paul

2013-01-01

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape. PMID:23697806
Soft x-ray-controlled dose deposition in yeast cells: techniques, model, and biological assessment

NASA Astrophysics Data System (ADS)

Milani, Marziale; Batani, Dimitri; Conti, Aldo; Masini, Alessandra; Costato, Michele; Pozzi, Achille; Turcu, I. C. Edmond

1996-12-01

A procedure is presented to release soft x-rays onto yeast cell membrane allegedly damaging the resident enzymatic processes connected with fermentation. The damage is expected to be restricted to regulating fermentation processes without interference with respiration. By this technique fermentation is followed leading to CO2 production, and respiration resulting in global pressure measurements. A solid state pressure sensor system has been developed linked to a data acquisition system. Yeast cells cultures have been investigated at different concentrations and with different nutrients. A non-monotone response in CO2 production as a function of the delivered x-ray dose is observed.
The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast

PubMed Central

2017-01-01

The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast. PMID:28939614
Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

PubMed

Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

2017-07-01

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our
Fresenius AS.TEC204 blood cell separator.

PubMed

Sugai, Mikiya

2003-02-01

Fresenius AS.TEC204 is a third-generation blood cell separator that incorporates the continuous centrifugal separation method and automatic control of the cell separation process. Continuous centrifugation separates cell components according to their specific gravity, and different cell components are either harvested or eliminated as needed. The interface between the red blood cell and plasma is optically detected, and the Interface Control (IFC) cooperates with different pumps, monitors and detectors to harvest required components automatically. The system is composed of three major sections; the Front Panel Unit; the Pump Unit, and the Centrifuge Unit. This unit can be used for a wide variety of clinical applications including collection of platelets, peripheral blood stem cells, bone marrow stem cells, granulocytes, mononuclear cells, and exchange of plasma or red cells, and for plasma treatment.
Tim18, a component of the mitochondrial translocator, mediates yeast cell death induced by arsenic.

PubMed

Du, Li; Yu, Yong; Li, Zidong; Chen, Jingsi; Liu, Yan; Xia, Yongjing; Liu, Xiangjun

2007-08-01

Evidence is presented that Tim18, a mitochondria translocase, plays a role in the previously described apoptosis induced by arsenite in Saccharomyces cerevisiae. Tim18 deletion mutant exhibited resistance to arsenite. After arsenite treatment, both the wild type and Tim18-deficient cells showed reactive oxygen species (ROS) production. Arsenite induced the higher expression of tim18 in wild type yeast cells. We found that the tim18 deletion mutant also exhibited resistance to other apoptotic stresses such as acetic acid, H2O2, and hyperosmotic stress. These results suggest that Tim18 is important for yeast cell death induced by arsenic, and it may act downstream of ROS production.
Effect of Selenium on Lipid and Amino Acid Metabolism in Yeast Cells.

PubMed

Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna; Kot, Anna M

2018-04-19

This article discusses the effect of selenium in aqueous solutions on aspects of lipid and amino acid metabolism in the cell biomass of Saccharomyces cerevisiae MYA-2200 and Candida utilis ATCC 9950 yeasts. The yeast biomass was obtained by using waste products (potato wastewater and glycerol). Selenium, at a dose of 20 mg/L of aqueous solution, affected the differentiation of cellular morphology. Yeast enriched with selenium was characterized by a large functional diversity in terms of protein and amino acid content. The protein content in the biomass of S. cerevisiae enriched with selenium (42.6%) decreased slightly as compared to that in the control sample without additional selenium supplementation (48.4%). Moreover, yeasts of both strains enriched with selenium contained a large amount of glutamic acid, aspartic acid, lysine, and leucine. Analysis of fatty acid profiles in S. cerevisiae yeast supplemented with selenium showed an increase in the unsaturated fatty acid content (e.g., C18:1). The presence of margaric acid (C17:0) and hexadecanoic acid (C17:1) was found in the C. utilis biomass enriched with selenium, in contrast to that of S. cerevisiae. These results indicate that selenium may induce lipid peroxidation, which consequently affects the loss of integrity of the cytoplasmic membrane. Yeast enriched with selenium with optimal amino acid and lipid composition can be used to prepare a novel formula of dietary supplements, which can be applied directly to various diets for both humans and animals.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

PubMed Central

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

2016-01-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771
In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE PAGES

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

2016-03-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.
In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.
Label-free ferrohydrodynamic cell separation of circulating tumor cells.

PubMed

Zhao, Wujun; Cheng, Rui; Jenkins, Brittany D; Zhu, Taotao; Okonkwo, Nneoma E; Jones, Courtney E; Davis, Melissa B; Kavuri, Sravan K; Hao, Zhonglin; Schroeder, Carsten; Mao, Leidong

2017-09-12

Circulating tumor cells (CTCs) have significant implications in both basic cancer research and clinical applications. To address the limited availability of viable CTCs for fundamental and clinical investigations, effective separation of extremely rare CTCs from blood is critical. Ferrohydrodynamic cell separation (FCS), a label-free method that conducted cell sorting based on cell size difference in biocompatible ferrofluids, has thus far not been able to enrich low-concentration CTCs from cancer patients' blood because of technical challenges associated with processing clinical samples. In this study, we demonstrated the development of a laminar-flow microfluidic FCS device that was capable of enriching rare CTCs from patients' blood in a biocompatible manner with a high throughput (6 mL h -1 ) and a high rate of recovery (92.9%). Systematic optimization of the FCS devices through a validated analytical model was performed to determine optimal magnetic field and its gradient, ferrofluid properties, and cell throughput that could process clinically relevant amount of blood. We first validated the capability of the FCS devices by successfully separating low-concentration (∼100 cells per mL) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour. Specifically, at ∼100 cancer cells per mL spike ratio, the recovery rates of cancer cells were 92.3 ± 3.6% (H1299 lung cancer), 88.3 ± 5.5% (A549 lung cancer), 93.7 ± 5.5% (H3122 lung cancer), 95.3 ± 6.0% (PC-3 prostate cancer), 94.7 ± 4.0% (MCF-7 breast cancer), and 93.0 ± 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11.1 ± 1.2% (H1299 lung cancer), 10.1 ± 1.7% (A549 lung cancer), 12.1 ± 2.1% (H3122 lung cancer), 12.8 ± 1.6% (PC-3 prostate cancer), 11.9 ± 1.8% (MCF-7 breast cancer), and 12.2 ± 1
Mechanisms of electron transfer between a styrylquinolinium dye and yeast in biofuel cell.

PubMed

Hubenova, Yolina; Bakalska, Rumyana; Hubenova, Eleonora; Mitov, Mario

2016-12-01

In the present study, the influence of the recently synthesized styrylquinolinium dye 4-{(E)-2-[4-(dimethylamino)naphthalen-1-yl]ethenyl}-1-methylquinolinium iodide (DANSQI) on the intracellular processes as well as the electrical outputs of Candida melibiosica 2491 yeast-based biofuel cell was investigated. The addition of nanomolar quantities of DANSQI to the yeast suspension results in an increase of the current outputs right after the startup of the biofuel cells, associated with an electrooxidation of the dye on the anode. After that, the formed cation radical of the dye penetrates the yeast cells, provoking a set of intracellular changes. Studies of the subcellular anolyte fractions show that 1μM dye increased the peroxisomal catalase activity 30-times (1.15±0.06Unit/mg protein) and over twice the mitochondrial cytochrome c oxidase activity (92±5Unit/mg protein). The results obtained by electrochemical and spectrophotometric analyses let to the supposition that the dye acts as subcellular shuttle, on account of its specific intramolecular charge transfer properties. The transition between its benzoid, quinolyl radical and ion forms and their putative role for the extracellular and intracellular charge transfer mechanisms are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.
Thinner, More-Efficient Oxygen-Separation Cells

NASA Technical Reports Server (NTRS)

Clark, Douglas J.; Galica, Leo M.; Losey, Robert W.

1992-01-01

Better gas-distribution plates fabricated more easily. Oxygen-separation cell redesigned to make it more efficient, smaller, lighter, and easier to manufacture. Potential applications include use as gas separators, filters, and fuel cells.
The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast.

PubMed

Leitao, Ricardo M; Kellogg, Douglas R

2017-11-06

The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast. © 2017 Leitao and Kellogg.
Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform.

PubMed

Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt; Lhoussaine, Cédric; Hersen, Pascal; Batt, Gregory

2017-02-01

With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar , a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. © 2017 The Authors.
Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform

PubMed Central

Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; Llamosi, Artémis; Mroz, Filip; Kaczmarek, Adam; Deyell, Matt

2017-01-01

With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar, a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others. PMID:28179544
Rapid cell separation with minimal manipulation for autologous cell therapies

NASA Astrophysics Data System (ADS)

Smith, Alban J.; O'Rorke, Richard D.; Kale, Akshay; Rimsa, Roberts; Tomlinson, Matthew J.; Kirkham, Jennifer; Davies, A. Giles; Wälti, Christoph; Wood, Christopher D.

2017-02-01

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.
Stress-tolerance of baker's-yeast (Saccharomyces cerevisiae) cells: stress-protective molecules and genes involved in stress tolerance.

PubMed

Shima, Jun; Takagi, Hiroshi

2009-05-29

During the fermentation of dough and the production of baker's yeast (Saccharomyces cerevisiae), cells are exposed to numerous environmental stresses (baking-associated stresses) such as freeze-thaw, high sugar concentrations, air-drying and oxidative stresses. Cellular macromolecules, including proteins, nucleic acids and membranes, are seriously damaged under stress conditions, leading to the inhibition of cell growth, cell viability and fermentation. To avoid lethal damage, yeast cells need to acquire a variety of stress-tolerant mechanisms, for example the induction of stress proteins, the accumulation of stress protectants, changes in membrane composition and repression of translation, and by regulating the corresponding gene expression via stress-triggered signal-transduction pathways. Trehalose and proline are considered to be critical stress protectants, as is glycerol. It is known that these molecules are effective for providing protection against various types of environmental stresses. Modifications of the metabolic pathways of trehalose and proline by self-cloning methods have significantly increased tolerance to baking-associated stresses. To clarify which genes are required for stress tolerance, both a comprehensive phenomics analysis and a functional genomics analysis were carried out under stress conditions that simulated those occurring during the commercial baking process. These analyses indicated that many genes are involved in stress tolerance in yeast. In particular, it was suggested that vacuolar H+-ATPase plays important roles in yeast cells under stress conditions.
Characterization and oxidative stability of purslane seed oil microencapsulated in yeast cells biocapsules.

PubMed

Kavosi, Maryam; Mohammadi, Abdorreza; Shojaee-Aliabadi, Saeedeh; Khaksar, Ramin; Hosseini, Seyede Marzieh

2018-05-01

Purslane seed oil, as a potential nutritious source of omega-3 fatty acid, is susceptible to oxidation. Encapsulation in yeast cells is a possible approach for overcoming this problem. In the present study, purslane seed oil was encapsulated in non-plasmolysed, plasmolysed and plasmolysed carboxy methyl cellulose (CMC)-coated Saccharomyces cerevisiae cells and measurements of oil loading capacity (LC), encapsulation efficiency (EE), oxidative stability and the fatty acid composition of oil-loaded microcapsules were made. Furthermore, investigations of morphology and thermal behavior, as well as a Fourier transform-infrared (FTIR) analyses of microcapsules, were performed. The values of EE, LC were approximately 53-65% and 187-231 g kg -1 , respectively. Studies found that the plasmolysis treatment increased EE and LC and decreased the mean peroxide value (PV) of microencapsulated oil. The presence of purslane seed oil in yeast microcapsules was confirmed by FTIR spectroscopy and differential scanning calorimetry analyses. The lowest rate of oxidation belonged to the oil-loaded plasmolysed CMC-coated microcapsules (16.73 meqvO 2 kg -1 ), whereas the highest amount of oxidation regardless of native oil referred to the oil-loaded in non-plasmolysed cells (28.15 meqvO 2 kg -1 ). The encapsulation of purslane seed oil in the yeast cells of S. cerevisiae can be considered as an efficient approach for extending the oxidative stability of this nutritious oil and facilitating its application in food products. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
Separation of cancer cells from white blood cells by pinched flow fractionation.

PubMed

Pødenphant, Marie; Ashley, Neil; Koprowska, Kamila; Mir, Kalim U; Zalkovskij, Maksim; Bilenberg, Brian; Bodmer, Walter; Kristensen, Anders; Marie, Rodolphe

2015-12-21

In this paper, the microfluidic size-separation technique pinched flow fractionation (PFF) is used to separate cancer cells from white blood cells (WBCs). The cells are separated at efficiencies above 90% for both cell types. Circulating tumor cells (CTCs) are found in the blood of cancer patients and can form new tumors. CTCs are rare cells in blood, but they are important for the understanding of metastasis. There is therefore a high interest in developing a method for the enrichment of CTCs from blood samples, which also enables further analysis of the separated cells. The separation is challenged by the size overlap between cancer cells and the 10(6) times more abundant WBCs. The size overlap prevents high efficiency separation, however we demonstrate that cell deformability can be exploited in PFF devices to gain higher efficiencies than expected from the size distribution of the cells.
The glyoxylate pathway contributes to enhanced extracellular electron transfer in yeast-based biofuel cell.

PubMed

Hubenova, Yolina; Hubenova, Eleonora; Slavcheva, Evelina; Mitov, Mario

2017-08-01

This study provides a new insight into our understanding of yeast response to starvation conditions (sole acetate as carbon source) and applied polarization and offers important information about the role of the glyoxylate cycle in the carbohydrate synthesis and extracellular charge transfer processes in biofuel cells. The biosynthetic capabilities of yeast C. melibiosica 2491 and the up/down-regulation of the glyoxylate cycle are evaluated by modifying the cellular metabolism by feedback inhibition or carbohydrate presence and establishing the malate dehydrogenase activity and carbohydrate content together with the electric charge passed through bioelectrochemical system. 10mM malate leads to a decrease of the produced quantity of electricity with ca. 55%. At the same time, 24-times lower intracellular malate dehydrogenase activity is established. At polarization conditions the glyoxylate pathway is up-regulated and huge amount of malate is intra-converted into oxaloacetate. The yeasts are able to synthesize carbohydrates from acetate and a part of them is used for the electricity generation. It is recognized that the enhanced charge transfer in acetate fed yeast-based biofuel cell is implemented by secreted endogenous mediator and changes in the cellular surface redox activity depending on the addition of carbohydrate in the medium. Copyright © 2017 Elsevier B.V. All rights reserved.
Differential electrophoretic separation of cells and its effect on cell viability

NASA Technical Reports Server (NTRS)

Leise, E. M.; Lesane, F.

1974-01-01

An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.
Behavior of yeast cells in aqueous suspension affected by pulsed electric field.

PubMed

El Zakhem, H; Lanoisellé, J-L; Lebovka, N I; Nonus, M; Vorobiev, E

2006-08-15

This work discusses pulsed electric fields (PEF) induced effects in treatment of aqueous suspensions of concentrated yeast cells (S. cerevisiae). The PEF treatment was done using pulses of near-rectangular shape, electric field strength was within E=2-5 kV/cm and the total time of treatment was t(PEF)=10(-4)-0.1 s. The concentration of aqueous yeast suspensions was in the interval of C(Y)=0-22 (wt%), where 1% concentration corresponds to the cellular density of 2x10(8) cells/mL. Triton X-100 was used for studying non-ionic surfactant additive effects. The electric current peak value I was measured during each pulse application, and from these data the electrical conductivity sigma was estimated. The PEF-induced damage results in increase of sigma with t(PEF) increasing and attains its saturation level sigma approximately sigma(max) at long time of PEF treatment. The value of sigma(max) reflects the efficiency of damage. The reduced efficiency of damage at suspension volume concentration higher than phi(Y) approximately 32 vol% is explained by the percolation phenomenon in the randomly packed suspension of near-spherical cells. The higher cytoplasmic ions leakage was observed in presence of surfactant. Experiments were carried out in the static and continuous flow treatment chambers in order to reveal the effects of mixing in PEF-treatment efficiency. A noticeable aggregation of the yeast cells was observed in the static flow chamber during the PEF treatment, while aggregation was not so pronounced in the continuous flow chamber. The nature of the enhanced aggregation under the PEF treatment was revealed by the zeta-potential measurements: these data demonstrate different zeta-potential signs for alive and dead cells. The effect of the electric field strength on the PEF-induced extraction of the intracellular components of S. cerevisiae is discussed.
[Thermoresistance in Saccharomyces cerevisiae yeasts].

PubMed

Kaliuzhin, V A

2011-01-01

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.
Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

PubMed

Yoshikawa, Yuki; Nasuno, Ryo; Kawahara, Nobuhiro; Nishimura, Akira; Watanabe, Daisuke; Takagi, Hiroshi

2016-07-01

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes. Copyright © 2016 Elsevier Inc. All rights reserved.
21 CFR 172.898 - Bakers yeast glycan.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and dried cell walls of the yeast...
Structure of gels layers with cells

NASA Astrophysics Data System (ADS)

Pokusaev, B. G.; Karlov, S. P.; Vyazmin, A. V.; Nekrasov, D. A.; Zakharov, N. S.; Khramtsov, D. P.; Skladnev, D. A.; Tyupa, D. V.

2017-11-01

The structure of two-layer agarose gels containing yeast cells is investigated experimentally by spectrometry, to shed a light on the theoretical foundations for the development of bioreactors by the method of 3D bioprinting. Due to division, cells overcome the layer of the dispersion phase separating successively applied layers of the agarose gel. However a gel layer of 100 μm thick with a high concentration of silver nanoparticles completely excludes the infiltration of yeast cells through it. A special sort of agarose is suggested where the concentration of silver nanoparticles formed by cells from salt of silver can serve as an indicator of the state of the yeast cells in the volume of the gel.

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...
21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...
21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...
21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...
21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...
Yeast Genetics for Delineating Bax/Bcl Pathway of Cell Death Regulation.

DTIC Science & Technology

1998-07-01

differences in tosol. The cytosol also became electron dense ("cyto- the copy number of the episomal plasmid from which solic condensation"), similar to...Cell Death & Differ . 3, 229-236. (1993). The C. eheans cell death gene ccd-3 encodes a protein similar ¶Xhitc. K., Tahaoglu, E., and Steller, H. (1996...components may be used in different functional contexts. Similar modules might exist in metazoan apoptotic pathways. Even though yeast does not contain
Increase of ethanol productivity by cell-recycle fermentation of flocculating yeast.

PubMed

Wang, F Z; Xie, T; Hui, M

2011-01-01

Using the recombinant flocculating Angel yeast F6, long-term repeated batch fermentation for ethanol production was performed and a high volumetric productivity resulted from half cells not washed and the optimum opportunity of residual glucose 20 g l(-1) of last medium. The obtained highest productivity was 2.07 g l-(1) h(-1), which was improved by 75.4% compared with that of 1.18 g l(-1) h(-1) in the first batch fermentation. The ethanol concentration reached 8.4% corresponding to the yield of 0.46 g g(-1). These results will contribute greatly to the industrial production of fuel ethanol using the commercial method with the flocculating yeast.
ODE, RDE and SDE models of cell cycle dynamics and clustering in yeast.

PubMed

Boczko, Erik M; Gedeon, Tomas; Stowers, Chris C; Young, Todd R

2010-07-01

Biologists have long observed periodic-like oxygen consumption oscillations in yeast populations under certain conditions, and several unsatisfactory explanations for this phenomenon have been proposed. These ‘autonomous oscillations’ have often appeared with periods that are nearly integer divisors of the calculated doubling time of the culture. We hypothesize that these oscillations could be caused by a form of cell cycle synchronization that we call clustering. We develop some novel ordinary differential equation models of the cell cycle. For these models, and for random and stochastic perturbations, we give both rigorous proofs and simulations showing that both positive and negative growth rate feedback within the cell cycle are possible agents that can cause clustering of populations within the cell cycle. It occurs for a variety of models and for a broad selection of parameter values. These results suggest that the clustering phenomenon is robust and is likely to be observed in nature. Since there are necessarily an integer number of clusters, clustering would lead to periodic-like behaviour with periods that are nearly integer divisors of the period of the cell cycle. Related experiments have shown conclusively that cell cycle clustering occurs in some oscillating yeast cultures.
Proteomics analysis for asymmetric inheritance of preexisting proteins between mother and daughter cells in budding yeast.

PubMed

Okada, Mitsuhiro; Kusunoki, Shunta; Ishibashi, Yuko; Kito, Keiji

2017-06-01

In budding yeast, a mother cell can produce a finite number of daughter cells over its life. The accumulation of a variety of types of damaged components has an impact on the aging process. Asymmetrical inheritance during cell division causes these aberrant intracellular constituents to be retained in mother cells and prevents them from segregating to daughter cells. However, the understanding of asymmetrical inheritance of individual proteins that are damaged or old age, and their relevance to the aging process, has been limited. The aim of this study is to propose a proteomics strategy for asymmetrical inheritance of preexisting proteins between mother and daughter cells. During synchronous culture for one generation, newly synthesized proteins were labeled with stable isotope amino acids to discriminate preexisting proteins originally expressed in mother cells, followed by separation of mother and daughter cells using a conventional method based on biotin labeling. Isotope incorporation ratios for individual proteins were quantified using mass spectrometry. We successfully identified 21 proteins whose preexisting versions were asymmetrically inherited in mother cells, including plasma membrane transporter involved in the aging process and organelle-anchoring proteins related to the stress response to misfolded proteins. Thus, our approach would be useful for making catalog of asymmetrically inherited proteins. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

PubMed Central

2013-01-01

Background Numerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance. Results The cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L). Conclusions The PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose. PMID:23835302
Creutzfeldt-Jakob disease and mad cows: lessons learnt from yeast cells.

PubMed

Hofmann, J; Wolf, H; Grassmann, A; Arndt, V; Graham, J; Vorberg, I

2012-01-24

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including humans. The proteinaceous nature of the infectious agent, the prion, and its propagation, challenge established dogmas in biology. It is now widely accepted that prion diseases are caused by unconventional agents principally composed of a misfolded host-encoded protein, PrP. Surprisingly, major break-throughs in prion research came from studies on functionally unrelated proteins in yeast and filamentous fungi. Aggregates composed of these proteins act as epigenetic elements of inheritance that can propagate their alternative states by a conformational switch into an ordered ß-sheet rich polymer just like mammalian prions. Since their discovery prions of lower eukaryotes have provided invaluable insights into all aspects of prion biogenesis. Importantly, yeast prions provide proof-of-principle that distinct protein conformers can be infectious and can serve as genetic elements that have the capacity to encipher strain specific information. As a powerful and tractable model system, yeast prions will continue to increase our understanding of prion-host cell interaction and potential mechanisms of protein-based epigenetic inheritance.
Microfluidic immunomagnetic cell separation from whole blood.

PubMed

Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

2016-02-01

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. Copyright © 2015 Elsevier B.V. All rights reserved.
Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

PubMed

Ramanavicius, A; Morkvenaite-Vilkonciene, I; Kisieliute, A; Petroniene, J; Ramanaviciene, A

2017-01-01

In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals. Copyright © 2016 Elsevier B.V. All rights reserved.
The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our datamore » strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.« less
Septin Organization and Functions in Budding Yeast

PubMed Central

Glomb, Oliver; Gronemeyer, Thomas

2016-01-01

The septins are a conserved family of GTP-binding proteins present in all eukaryotic cells except plants. They were originally discovered in the baker's yeast Saccharomyces cerevisiae that serves until today as an important model organism for septin research. In yeast, the septins assemble into a highly ordered array of filaments at the mother bud neck. The septins are regulators of spatial compartmentalization in yeast and act as key players in cytokinesis. This minireview summarizes the recent findings about structural features and cell biology of the yeast septins. PMID:27857941
Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

PubMed

Bodenberger, Nicholas; Kubiczek, Dennis; Paul, Patrick; Preising, Nico; Weber, Lukas; Bosch, Ramona; Hausmann, Rudolf; Gottschalk, Kay-Eberhard; Rosenau, Frank

2017-03-01

Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 μm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 μM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.
A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

PubMed Central

Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

2014-01-01

In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009
Measuring mitotic spindle dynamics in budding yeast

NASA Astrophysics Data System (ADS)

Plumb, Kemp

In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.
Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Effects of different yeast cell wall supplements added to maize- or wheat-based diets for broiler chickens.

PubMed

Morales-López, R; Auclair, E; Van Immerseel, F; Ducatelle, R; García, F; Brufau, J

2010-06-01

1. Three experiments were carried out to study the effects of two experimental yeast cell wall (YCW) supplements, one from the yeast extract industry and the other from the brewery industry, added to maize or wheat based-diets, on performance and intestinal parameters of broiler chickens (Ross 308). 2. In the first and second experiments, a completely randomised block design with 4 experimental treatments was used: T-1) Negative control, no additives T-2) Positive control, avilamycin group (10 mg/kg feed), T-3) Yeast extract-YCW (500 mg/kg), and T-4) Brewery-YCW (500 mg/kg feed). There were 6 replicates of 20 (experiment 1) and 22 (experiment 2) chicks per treatment. 3. In experiment 1 (wheat based diets), yeast extract-YCW increased BW and daily feed intake (42 d). The effects were comparable to those of avilamycin. In experiment 2 (maize based diet), avilamycin, yeast extract-YCW and brewery-YCW treatments improved the feed conversion ratio with respect to the negative control group (0 to 14 d). 4. At 24 d, in both experiments, the ileal nutrient digestibility and ileal bacterial counts were not affected by any experimental treatment. In maize diets, lower intestinal viscosity was obtained with avilamycin, yeast extract-YCW and brewery-YCW than with the negative control. In wheat diets, yeast extract-YCW and brewery-YCW reduced intestinal viscosity. 5. A third experiment was conducted to study the effect of yeast extract-YCW on animal performance, intestinal mucosa morphology and intestinal viscosity. A 2 x 2 factorial arrangement of treatments was used; one factor was the dietary yeast extract-YCW supplementation (0 or 500 mg/kg feed) and the other the cereal in the diet (maize or wheat). 6. At 43 d, the heaviest BW was in chickens fed on yeast extract-YCW compared to those given the negative control. At 22 d, yeast extract-YCW increased villus height, mucus thickness and number of goblet cells with respect to negative control. 7. Results of these experiments

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

PubMed Central

Menacho-Márquez, Mauricio; Rodríguez-Hernández, Carlos J; Villaronga, M Ángeles; Pérez-Valle, Jorge; Gadea, José; Belandia, Borja; Murguía, José R

2015-01-01

β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses. PMID:25590579
Isolation and characterization of ethanol tolerant yeast strains

PubMed Central

Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

2013-01-01

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092
21 CFR 184.1983 - Bakers yeast extract.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting from concentration of the solubles of mechanically ruptured cells of a selected strain of yeast, Saccharomyces...
Origins of multicellular evolvability in snowflake yeast

PubMed Central

Ratcliff, William C.; Fankhauser, Johnathon D.; Rogers, David W.; Greig, Duncan; Travisano, Michael

2015-01-01

Complex life has arisen through a series of ‘major transitions’ in which collectives of formerly autonomous individuals evolve into a single, integrated organism. A key step in this process is the origin of higher-level evolvability, but little is known about how higher-level entities originate and gain the capacity to evolve as an individual. Here we report a single mutation that not only creates a new level of biological organization, but also potentiates higher-level evolvability. Disrupting the transcription factor ACE2 in Saccharomyces cerevisiae prevents mother–daughter cell separation, generating multicellular ‘snowflake’ yeast. Snowflake yeast develop through deterministic rules that produce geometrically defined clusters that preclude genetic conflict and display a high broad-sense heritability for multicellular traits; as a result they are preadapted to multicellular adaptation. This work demonstrates that simple microevolutionary changes can have profound macroevolutionary consequences, and suggests that the formation of clonally developing clusters may often be the first step to multicellularity. PMID:25600558
Yeast and Mammalian Metallothioneins Functionally Substitute for Yeast Copper-Zinc Superoxide Dismutase

NASA Astrophysics Data System (ADS)

Tamai, Katherine T.; Gralla, Edith B.; Ellerby, Lisa M.; Valentine, Joan S.; Thiele, Dennis J.

1993-09-01

Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.
Toxicology of the aqueous extract from the flowers of Butea monosperma Lam. and it's metabolomics in yeast cells.

PubMed

Khan, Washim; Gupta, Shreesh; Ahmad, Sayeed

2017-10-01

Due to lack of scientific evidence for the safety of Butea monosperma (Fabaceae), our study aimed to carry out its toxicological profile and to identify its metabolic pattern in yeast cell. The effect of aqueous extract of B. monosperma flower on glucose uptake in yeast cell was evaluated through optimizing pH, temperature, incubation time, substrate concentration and kinetic parameters. Further, the metabolic pattern of extract as such and in yeast cell were analyzed by gas chromatography-mass spectrometry. Mice were administered aqueous extract up to 6000 and 4000 mg/kg for acute oral and intraperitoneal toxicity, respectively, while up to 4500 mg/kg for sub-acute oral toxicity (30 days). Elongation in the lag and log phase was observed in yeast cells supplemented with extract as compared to control. A maximum of 184.9% glucose uptake was observed whereas kinetic parameters (K m and V max ) were 1.38 and 41.91 mol/s, respectively. Out of 75 metabolites found in the extract, 14 and 18 metabolites were utilized by yeast cell after 15 and 30 min of incubation, respectively. The LD 50 of extract administered through intraperitoneal route was estimated to be 3500 mg/kg. The extract did not elicit any significant difference (P ≥ 0.05) in weight gain, food consumption, water intake, hematological, biochemical parameters and histological changes as compared to the normal control. Results ascertained the safety of B. monosperma flower extract which can be explored as potential candidates for the development of anti-diabetic phytopharmaceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.
Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast

PubMed Central

Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M.; Rosebrock, Adam P.; Futcher, Bruce; Cross, Frederick R.

2009-01-01

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. PMID:19841732
Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

PubMed

Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas; Klipp, Edda

2016-09-01

The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. © 2016 The Authors.
Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis

PubMed Central

Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas

2016-01-01

The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377
Electrochemical cell and separator plate thereof

DOEpatents

Baker, Bernard S.; Dharia, Dilip J.

1979-10-02

A fuel cell includes a separator plate having first and second flow channels extending there through contiguously with an electrode and respectively in flow communication with the cell electrolyte and in flow isolation with respect to such electrolyte. In fuel cell system arrangement, the diverse type channels are supplied in common with process gas for thermal control purposes. The separator plate is readily formed by corrugation of integral sheet material. 10 figs.
Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

DOE PAGES

Huang, Xiaojing; Nelson, Johanna; Kirz, Janos; ...

2009-11-01

We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below -170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.
Extraction of brewer's yeasts using different methods of cell disruption for practical biodiesel production.

PubMed

Řezanka, Tomáš; Matoulková, Dagmar; Kolouchová, Irena; Masák, Jan; Viden, Ivan; Sigler, Karel

2015-05-01

The methods of preparation of fatty acids from brewer's yeast and its use in production of biofuels and in different branches of industry are described. Isolation of fatty acids from cell lipids includes cell disintegration (e.g., with liquid nitrogen, KOH, NaOH, petroleum ether, nitrogenous basic compounds, etc.) and subsequent processing of extracted lipids, including analysis of fatty acid and computing of biodiesel properties such as viscosity, density, cloud point, and cetane number. Methyl esters obtained from brewer's waste yeast are well suited for the production of biodiesel. All 49 samples (7 breweries and 7 methods) meet the requirements for biodiesel quality in both the composition of fatty acids and the properties of the biofuel required by the US and EU standards.
Separator material for electrochemical cells

DOEpatents

Cieslak, W.R.; Storz, L.J.

1991-03-26

An electrochemical cell is characterized as utilizing an aramid fiber as a separator material. The aramid fibers are especially suited for lithium/thionyl chloride battery systems. The battery separator made of aramid fibers possesses superior mechanical strength, chemical resistance, and is flame retardant.
Separator material for electrochemical cells

DOEpatents

Cieslak, Wendy R.; Storz, Leonard J.

1991-01-01

An electrochemical cell characterized as utilizing an aramid fiber as a separator material. The aramid fibers are especially suited for lithium/thionyl chloride battery systems. The battery separator made of aramid fibers possesses superior mechanical strength, chemical resistance, and is flame retardant.
Cell separation using tilted-angle standing surface acoustic waves

PubMed Central

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-01-01

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles–cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological–biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150
Cell separation using tilted-angle standing surface acoustic waves.

PubMed

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-09-09

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.
Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093
Electrophoretic cell separation using microspheres. [purification of lymphocytes

NASA Technical Reports Server (NTRS)

Smolka, A.; Sachs, G.

1980-01-01

Methods of cell separation based on the electrokinetic properties of the cell membrane offer a degree of discrimination among cell populations which is not available with methods based on cell size or density alone. Studies aimed at extending red cell separations using microspheres to purification of lymphocytes.
Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin.

PubMed

Zhu, Hanyu; Liu, Dongmei; Wang, Yuanyuan; Ren, Danfeng; Zheng, Liesheng; Chen, Liguo; Ma, Aimin

2017-08-01

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens. The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate. Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.
Yeast as a model for Ras signalling.

PubMed

Tisi, Renata; Belotti, Fiorella; Martegani, Enzo

2014-01-01

For centuries yeast species have been popular hosts for classical biotechnology processes, such as baking, brewing, and wine making, and more recently for recombinant proteins production, thanks to the advantages of unicellular organisms (i.e., ease of genetic manipulation and rapid growth) together with the ability to perform eukaryotic posttranslational modifications. Moreover, yeast cells have been used for few decades as a tool for identifying the genes and pathways involved in basic cellular processes such as the cell cycle, aging, and stress response. In the budding yeast S. cerevisiae the Ras/cAMP/PKA pathway is directly involved in the regulation of metabolism, cell growth, stress resistance, and proliferation in response to the availability of nutrients and in the adaptation to glucose, controlling cytosolic cAMP levels and consequently the cAMP-dependent protein kinase (PKA) activity. Moreover, Ras signalling has been identified in several pathogenic yeasts as a key controller for virulence, due to its involvement in yeast morphogenesis. Nowadays, yeasts are still useful for Ras-like proteins investigation, both as model organisms and as a test tube to study variants of heterologous Ras-like proteins.

Arming Technology in Yeast-Novel Strategy for Whole-cell Biocatalyst and Protein Engineering.

PubMed

Kuroda, Kouichi; Ueda, Mitsuyoshi

2013-09-09

Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.
Tolerant industrial yeast Saccharomyces cerevisiae posses a more robust cell wall integrity signaling pathway against 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde

USDA-ARS?s Scientific Manuscript database

Cell wall integrity signaling pathway in Saccharomyces cerevisiae is a conserved function for detecting and responding to cell stress conditions but less understood for industrial yeast. We dissected gene expression dynamics for a tolerant industrial yeast strain NRRL Y-50049 in response to challeng...
Microbially induced separation of quartz from calcite using Saccharomyces cerevisiae.

PubMed

Padukone, S Usha; Natarajan, K A

2011-11-01

Cells of Saccharomyces cerevisiae and their metabolites were successfully utilized to achieve selective separation of quartz and calcite through microbially induced flotation and flocculation. S. cerevisiae was adapted to calcite and quartz minerals. Adsorption studies and electrokinetic investigations were carried out to understand the changes in the surface chemistry of yeast cells and the minerals after mutual interaction. Possible mechanisms in microbially induced flotation and flocculation are outlined. Copyright © 2011 Elsevier B.V. All rights reserved.
Cell separation and electrofusion in space

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Hofmann, G. A.

1990-01-01

In microgravity, free-fluid electrophoretic methods for separating living cells and proteins are improved significantly by the absence of gravity-driven phenomena. Cell fusion, culture, and other bioprocessing steps are being investigated to understand the limits of earth-based processing. A multistep space bioprocess is described that includes electrophoretic separation of human target cells, single-cell manipulations using receptor-specific antibodies, electrofusion to produce immortal hybridomas, gentle suspension culture, and monoclonal antibody recovery using continuous-flow electrophoresis or recirculating isoelectric focusing. Improvements in several key steps already have been demonstrated by space experiments, and others will be studied on Space Station Freedom.
Separator development and testing of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Gonzalez-Sanabria, O. D.; Manzo, M. A.

1984-01-01

The components, design, and operating characteristics of Ni-H2 cells batteries were improved. A separator development program was designed to develop a separator that is resistant to penetration by oxygen and loose active material from then nickel electrode, while retraining the required chemical and thermal stability, reservoir capability, and high ionic conductivity. The performance of the separators in terms of cell operating voltage was to at least match that of state-of-the-art separators while eliminating the separator problems. The separators were submitted to initial screening tests and those which successfully completed the tests were built into Ni-H2 cells for short term testing. The separators with the best performance are tested for long term performance and life.
Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

PubMed

Elsutohy, Mohamed M; Chauhan, Veeren M; Markus, Robert; Kyyaly, Mohammed Aref; Tendler, Saul J B; Aylott, Jonathan W

2017-05-11

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.
Separation of Corn Fiber and Conversion to Fuels and Chemicals Phase II: Pilot-scale Operation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbas, Charles; Beery, Kyle; Orth, Rick

2007-09-28

The purpose of the Department of Energy (DOE)-supported corn fiber conversion project, “Separation of Corn Fiber and Conversion to Fuels and Chemicals Phase II: Pilot-scale Operation” is to develop and demonstrate an integrated, economical process for the separation of corn fiber into its principal components to produce higher value-added fuel (ethanol and biodiesel), nutraceuticals (phytosterols), chemicals (polyols), and animal feed (corn fiber molasses). This project has successfully demonstrated the corn fiber conversion process on the pilot scale, and ensured that the process will integrate well into existing ADM corn wet-mills. This process involves hydrolyzing the corn fiber to solubilize 50%more » of the corn fiber as oligosaccharides and soluble protein. The solubilized fiber is removed and the remaining fiber residue is solvent extracted to remove the corn fiber oil, which contains valuable phytosterols. The extracted oil is refined to separate the phytosterols and the remaining oil is converted to biodiesel. The de-oiled fiber is enzymatically hydrolyzed and remixed with the soluble oligosaccharides in a fermentation vessel where it is fermented by a recombinant yeast, which is capable of fermenting the glucose and xylose to produce ethanol. The fermentation broth is distilled to remove the ethanol. The stillage is centrifuged to separate the yeast cell mass from the soluble components. The yeast cell mass is sold as a high-protein yeast cream and the remaining sugars in the stillage can be purified to produce a feedstock for catalytic conversion of the sugars to polyols (mainly ethylene glycol and propylene glycol) if desirable. The remaining materials from the purification step and any materials remaining after catalytic conversion are concentrated and sold as a corn fiber molasses. Additional high-value products are being investigated for the use of the corn fiber as a dietary fiber sources.« less
Big data mining powers fungal research: recent advances in fission yeast systems biology approaches.

PubMed

Wang, Zhe

2017-06-01

Biology research has entered into big data era. Systems biology approaches therefore become the powerful tools to obtain the whole landscape of how cell separate, grow, and resist the stresses. Fission yeast Schizosaccharomyces pombe is wonderful unicellular eukaryote model, especially studying its division and metabolism can facilitate to understanding the molecular mechanism of cancer and discovering anticancer agents. In this perspective, we discuss the recent advanced fission yeast systems biology tools, mainly focus on metabolomics profiling and metabolic modeling, protein-protein interactome and genetic interaction network, DNA sequencing and applications, and high-throughput phenotypic screening. We therefore hope this review can be useful for interested fungal researchers as well as bioformaticians.
Electrophoretic cell separation by means of immunomicrospheres

NASA Technical Reports Server (NTRS)

Rembaum, A.; Smolka, A. J. K.

1980-01-01

The electrophoretic mobility of fixed human red blood cells immunologically labeled with polymeric (4-vinyl)pyridine or polyglutaraldehyde microspheres was altered to a considerable extent. This observation was utilized in the preparative scale electrophoretic separation of human and turkey fixed red blood cells, whose mobilities under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. It is suggested that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immuno-specific labeling of target populations using microspheres.
High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

1995-11-14

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.
High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

1995-01-01

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.
Accelerating Yeast Prion Biology using Droplet Microfluidics

NASA Astrophysics Data System (ADS)

Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

2012-02-01

Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.
Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

PubMed

Kochan, Kamila; Peng, Huadong; Wood, Bayden R; Haritos, Victoria S

2018-01-01

Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. Firstly, a strong correlation ( r 2 > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. Vibrational spectroscopy analysis allowed the simultaneous
Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

PubMed

Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

2014-01-01

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.
Yeast Genetics and Biotechnological Applications

NASA Astrophysics Data System (ADS)

Mishra, Saroj; Baranwal, Richa

Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast Saccharomyces cerevisiae has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 μm plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.
Stripe-patterned thermo-responsive cell culture dish for cell separation without cell labeling.

PubMed

Kumashiro, Yoshikazu; Ishihara, Jun; Umemoto, Terumasa; Itoga, Kazuyoshi; Kobayashi, Jun; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-02-11

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 μm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast.

PubMed

Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

2013-08-01

Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. © 2013 The Authors. Aging Cell published by John Wiley & Sons Ltd and the Anatomical Society.
Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, Wesley C.; Hatfield, J. Michael

1983-01-01

Various techniques for separating the hormone-producing cell types from the rat anterior pituitary gland are examined. The purity, viability, and responsiveness of the separated cells depend on the physiological state of the donor, the tissue dissociation procedures, the staining technique used for identification of cell type, and the cell separation technique. The chamber-gradient setup and operation, the characteristics of the gradient materials, and the separated cell analysis of velocity sedimentation techniques (in particular Staput and Celsep) are described. Consideration is given to the various types of materials used in density gradient centrifugation and the operation of a gradient generating device. The use of electrophoresis to separate rat pituitary cells is discussed.
Microspore Separation in the quartet 3 Mutants of Arabidopsis Is Impaired by a Defect in a Developmentally Regulated Polygalacturonase Required for Pollen Mother Cell Wall Degradation1

PubMed Central

Rhee, Seung Y.; Osborne, Erin; Poindexter, Patricia D.; Somerville, Chris R.

2003-01-01

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development. PMID:14551328
Enhanced phytate dephosphorylation by using Candida melibiosica yeast-based biofuel cell.

PubMed

Hubenova, Yolina; Georgiev, Danail; Mitov, Mario

2014-10-01

We report for the first time that Candida melibiosica expresses enhanced phytase activity when grown under biofuel cell polarization in a nutrient-poor medium, containing only fructose as a carbohydrate source. Phytase activity during the cultivation under polarization reached up to 25 U per g dry biomass, exceeding with 20 ± 3 % those of the control. A participation of the enzyme in the adaptation processes to the stress conditions is proposed. In addition, steady-state electrical outputs were achieved during biofuel cell operation at continuous polarization under constant load. The obtained results show that C. melibiosica yeast-based biofuel cell could be used for simultaneous electricity generation and phytate bioremediation.

The rotating spectrometer: Biotechnology for cell separations

NASA Technical Reports Server (NTRS)

Noever, David A.

1991-01-01

An instrument for biochemical studies, called the rotating spectrometer, separates previously inseparable cell cultures. The rotating spectrometer is intended for use in pharmacological studies which require fractional splitting of heterogeneous cell cultures based on cell morphology and swimming behavior. As a method to separate and concentrate cells in free solution, the rotating method requires active organism participation and can effectively split the large class of organisms known to form spontaneous patterns. Examples include the biochemical star, an organism called Tetrahymena pyriformis. Following focusing in a rotating frame, the separation is accomplished using different radial dependencies of concentrated algal and protozoan species. The focusing itself appears as concentric rings and arises from the coupling between swimming direction and Coriolis forces. A dense cut is taken at varying radii, and extraction is replenished at an inlet. Unlike standard separation and concentrating techniques such as filtration or centrifugation, the instrument is able to separate motile from immotile fractions. For a single pass, typical split efficiencies can reach 200 to 300 percent compared to the inlet concentration.
Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

PubMed

Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

2017-11-01

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.
Characteristics of an immobilized yeast cell system using very high gravity for the fermentation of ethanol.

PubMed

Ji, Hairui; Yu, Jianliang; Zhang, Xu; Tan, Tianwei

2012-09-01

The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280 g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65 h with an average ethanol concentration and ethanol yield of 130.12 g/L and 0.477 g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28 days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75 L. The bioreactor was operated for 26 days at a dilution rate of 0.015 h(-1). The ethanol concentration of the effluent reached 130.77 g/L ethanol while an average 8.18 g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.
Cell separation using electric fields

NASA Technical Reports Server (NTRS)

Eppich, Henry M. (Inventor); Mangano, Joseph A. (Inventor)

2003-01-01

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.
Cell separation using electric fields

NASA Technical Reports Server (NTRS)

Mangano, Joseph (Inventor); Eppich, Henry (Inventor)

2009-01-01

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.
Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

PubMed

Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

2017-07-25

We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected
Separator plate for a fuel cell

DOEpatents

Petri, Randy J.; Meek, John; Bachta, Robert P.; Marianowski, Leonard G.

1996-01-01

A separator plate for a fuel cell comprising an anode current collector, a cathode current collector and a main plate, the main plate disposed between the anode current collector and the cathode current collector. The anode current collector forms a flattened peripheral wet seal structure and manifold wet seal structure on the anode side of the separator plate and the cathode current collector forms a flattened peripheral wet seal structure and manifold wet seal structure on the cathode side of the separator plate. In this manner, the number of components required to manufacture and assemble a fuel cell stack is reduced.
Wine yeasts for the future.

PubMed

Fleet, Graham H

2008-11-01

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.
sup 31 P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brindle, K.; Braddock, P.; Fulton, S.

1990-04-03

Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creating kinase activities similar to those found in mammalian heart muscle. {sup 31}P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06more » and 0.1 measured directly in cell extracts.« less
A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.
The yeast replicative aging model.

PubMed

He, Chong; Zhou, Chuankai; Kennedy, Brian K

2018-03-08

It has been nearly three decades since the budding yeast Saccharomyces cerevisiae became a significant model organism for aging research and it has emerged as both simple and powerful. The replicative aging assay, which interrogates the number of times a "mother" cell can divide and produce "daughters", has been a stalwart in these studies, and genetic approaches have led to the identification of hundreds of genes impacting lifespan. More recently, cell biological and biochemical approaches have been developed to determine how cellular processes become altered with age. Together, the tools are in place to develop a holistic view of aging in this single-celled organism. Here, we summarize the current state of understanding of yeast replicative aging with a focus on the recent studies that shed new light on how aging pathways interact to modulate lifespan in yeast. Copyright © 2018. Published by Elsevier B.V.
UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi

Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in amore » buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.« less
Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

2013-05-01

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.
Unravel lipid accumulation mechanism in oleaginous yeast through single cell systems biology study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiaoliang; Ding, Shiyou

Searching for alternative and clean energy is one of the most important tasks today. Our research aimed at finding the best living condition for certain types of oleaginous yeasts for efficient lipid production. We found that R. glutinis yeast cells has great variability in lipid production among cells while Y. lipolytica cells has similar oil production ability. We found some individual cells shows much higher level of oil production. In order to further study these cases, we employed a label-free chemical sensitive microscopy method call stimulated Raman scattering (SRS). With SRS, we could measure the lipid content in each cell.more » We combined SRS microscopy with microfluidic device so that we can isolate cells with high fat content. We also developed SRS imaging technique that has higher imaging speed, which is highly desirable for high throughput cell screening and sorting. Since these cells has similar genome, it must be the transcriptome caused their difference in oil production. We developed a single cell transcriptome sequencing method to study which genes are responsible for elevated oil production. These methods that are developed for this project can easily be applied for many other areas of research. For example, the single transcriptome can be used to study the transcriptomes of other cell types. The high-speed SRS microscopy techniques can be used to speed up chemical imaging for lablefree histology or imaging distribution of chemicals in tissues of live mice or in humans. The developed microfluidic platform can be used to sort other type of cells, e.g., white blood cells for diagnosis of cancer or other blood diseases.« less
Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11.

PubMed

Hirayama, Satoru; Shimizu, Masashi; Tsuchiya, Noriko; Furukawa, Soichi; Watanabe, Daisuke; Shimoi, Hitoshi; Takagi, Hiroshi; Ogihara, Hirokazu; Morinaga, Yasushi

2015-05-01

We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

PubMed

Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

2016-01-01

"pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.
Separator plate for a fuel cell

DOEpatents

Petri, R.J.; Meek, J.; Bachta, R.P.; Marianowski, L.G.

1996-04-02

A separator plate is described for a fuel cell comprising an anode current collector, a cathode current collector and a main plate, the main plate disposed between the anode current collector and the cathode current collector. The anode current collector forms a flattened peripheral wet seal structure and manifold wet seal structure on the anode side of the separator plate and the cathode current collector forms a flattened peripheral wet seal structure and manifold wet seal structure on the cathode side of the separator plate. In this manner, the number of components required to manufacture and assemble a fuel cell stack is reduced. 9 figs.
The structure of a β-(1→3)-d-glucan from yeast cell walls

PubMed Central

Manners, David J.; Masson, Alan J.; Patterson, James C.

1973-01-01

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched β-(1→3)-glucan of high molecular weight (about 240000) containing 3% of β-(1→6)-glucosidic interchain linkages. The minor component is a branched β-(1→6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major β-(1→3)-glucan component may vary considerably in degree of branching. PMID:4359920
Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

PubMed

Recouvreux, Pierre; Sokolowski, Thomas R; Grammoustianou, Aristea; ten Wolde, Pieter Rein; Dogterom, Marileen

2016-02-16

Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns.
Simple method for the extraction and reversed-phase high-performance liquid chromatographic analysis of carotenoid pigments from red yeasts (Basidiomycota, Fungi).

PubMed

Weber, Roland W S; Anke, Heidrun; Davoli, Paolo

2007-03-23

A simple method for the extraction of carotenoid pigments from frozen wet cells of red yeasts (Basidiomycota) and their analysis by reversed-phase HPLC using a C(18) column and a water/acetone solvent system is described. Typical red yeast carotenoids belonging to an oxidative series from the monocyclic gamma-carotene to 2-hydroxytorularhodin and from the bicyclic beta-carotene to astaxanthin were separated. Pigment identity was confirmed by LC-atmospheric pressure chemical ionisation (APCI) mass spectrometry using similar chromatographic conditions.

Influence of Zero-Shear on Yeast Development

NASA Technical Reports Server (NTRS)

McGinnis, Michael R.

1997-01-01

The objective of the research was to begin evaluating the effect of zero-shear on the development of the cell wall of Saccharomyces cerevisiae employing the High Aspect Rotating-Wall Vessel (HARV) NASA bioreactor. This particular yeast has enormous potential for research as a model eukaryotic system on the International Space Station, as well as the production of food stuffs' at the future lunar colony. Because the cell wall is the barrier between the cell and the environment, its form and function as influenced by microgravity is of great importance. Morphologic studies revealed that the circularity and total area of the individual yeast cells were essentially the same in both the control and test HARV's. The growth rates were also essentially the same. In zero-shear, the yeast grew in clumps consisting of rudimentary pseudohyphae in contrast to solitary budding cells in the control. Based upon mechanical and sonic shear applied to the yeast cells, those grown in zero-shear had stronger cell walls and septa. This suggests that there are structural differences, most likely related to the chitin skeleton of the cell wall. From this research further NASA support was obtained to continue the work. Investigations will deal with gene expression and ultrastructure. These will lead to a clearer assessment of the value of S. cerevisiae eukaryotic as a model for space station research.
Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

PubMed Central

Roy, Adhiraj; Kim, Jeong-Ho

2014-01-01

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370
Role of intracellular freezing in the death of cells cooled at supraoptimal rates. [Preservation of erythrocytes, bone marrow cells, and yeasts by freezing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mazur, P.

1976-01-01

Cooling velocity is one of the major factors that determines whether viable cells can be frozen to temperatures that permit indefinite storage. Cooling either too slowly or too rapidly tends to be damaging. Optimum cooling rates are reported for mouse marrow stem cells, yeast, and human red cells.
Electrolytic cell. [For separating anolyte and catholyte

DOEpatents

Bullock, J.S.; Hale, B.D.

1984-09-14

An apparatus is described for the separation of the anolyte and the catholyte during electrolysis. The electrolyte flows through an electrolytic cell between the oppositely charged electrodes. The cell is equipped with a wedge-shaped device, the tapered end being located between the electrodes on the effluent side of the cell. The wedge diverts the flow of the electrolyte to either side of the wedge, substantially separating the anolyte and the catholyte.
The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

PubMed

Meseroll, Rebecca A; Cohen-Fix, Orna

2016-11-01

In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
Growth control of the eukaryote cell: a systems biology study in yeast.

PubMed

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David Cj; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom Pj; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the
Growth control of the eukaryote cell: a systems biology study in yeast

PubMed Central

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David CJ; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom PJ; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome
Biocompatible and label-free separation of cancer cells from cell culture lines from white blood cells in ferrofluids.

PubMed

Zhao, Wujun; Cheng, Rui; Lim, So Hyun; Miller, Joshua R; Zhang, Weizhong; Tang, Wei; Xie, Jin; Mao, Leidong

2017-06-27

This paper reports a biocompatible and label-free cell separation method using ferrofluids that can separate a variety of low-concentration cancer cells from cell culture lines (∼100 cancer cells per mL) from undiluted white blood cells, with a throughput of 1.2 mL h -1 and an average separation efficiency of 82.2%. The separation is based on the size difference of the cancer cells and white blood cells, and is conducted in a custom-made biocompatible ferrofluid that retains not only excellent short-term viabilities but also normal proliferations of 7 commonly used cancer cell lines. A microfluidic device is designed and optimized specifically to shorten the time of live cells' exposure to ferrofluids from hours to seconds, by eliminating time-consuming off-chip sample preparation and extraction steps and integrating them on-chip to achieve a one-step process. As a proof-of-concept demonstration, a ferrofluid with 0.26% volume fraction was used in this microfluidic device to separate spiked cancer cells from cell lines at a concentration of ∼100 cells per mL from white blood cells with a throughput of 1.2 mL h -1 . The separation efficiencies were 80 ± 3%, 81 ± 5%, 82 ± 5%, 82 ± 4%, and 86 ± 6% for A549 lung cancer, H1299 lung cancer, MCF-7 breast cancer, MDA-MB-231 breast cancer, and PC-3 prostate cancer cell lines, respectively. The separated cancer cells' purity was between 25.3% and 28.8%. In addition, the separated cancer cells from this strategy showed an average short-term viability of 94.4 ± 1.3%, and these separated cells were cultured and demonstrated normal proliferation to confluence even after the separation process. Owing to its excellent biocompatibility and label-free operation and its ability to recover low concentrations of cancer cells from white blood cells, this method could lead to a promising tool for rare cell separation.
Beer and bread to brains and beyond: can yeast cells teach us about neurodegenerative disease?

PubMed

Gitler, Aaron D

2008-01-01

For millennia, humans have harnessed the astonishing power of yeast, producing such culinary masterpieces as bread, beer and wine. Therefore, in this new millennium, is it very farfetched to ask if we can also use yeast to unlock some of the modern day mysteries of human disease? Remarkably, these seemingly simple cells possess most of the same basic cellular machinery as the neurons in the brain. We and others have been using the baker's yeast, Saccharomyces cerevisiae, as a model system to study the mechanisms of devastating neurodegenerative diseases such as Parkinson's, Huntington's, Alzheimer's and amyotrophic lateral sclerosis. While very different in their pathophysiology, they are collectively referred to as protein-misfolding disorders because of the presence of misfolded and aggregated forms of various proteins in the brains of affected individuals. Using yeast genetics and the latest high-throughput screening technologies, we have identified some of the potential causes underpinning these disorders and discovered conserved genes that have proven effective in preventing neuron loss in animal models. Thus, these genes represent new potential drug targets. In this review, I highlight recent work investigating mechanisms of cellular toxicity in a yeast Parkinson's disease model and discuss how similar approaches are being applied to additional neurodegenerative diseases.
Separator for electrochemical cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffin, R.A.

1988-12-27

An electrochemical cell is described comprising a sealed casing; an anode, a cathode, a separator positioned between the anode and the cathode, and a non-aqueous electrolyte sealed in the casing; a pair of electrical terminals on the casing; means for electrically isolating the electrical terminals from each other; and means for electrically connecting the anode to one terminal and the cathode to the other terminal; wherein the anode is comprised of lithium foil, the cathode is comprised of manganese dioxide, and the separator consists essentially of a microporous polypropylene film having a thickness of about 1.5 mils and internal voidsmore » of about 60% by volume; wherein the anode, cathode, and separator are spirally wound together in a jelly roll configuration.« less
Application of cell-surface engineering for visualization of yeast in bread dough: development of a fluorescent bio-imaging technique in the mixing process of dough.

PubMed

Maeda, Tatsuro; Shiraga, Seizaburo; Araki, Tetsuya; Ueda, Mitsuyoshi; Yamada, Masaharu; Takeya, Koji; Sagara, Yasuyuki

2009-07-01

Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at -30 degrees C and sliced at 10 microm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.
Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans

PubMed Central

Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G.

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions. PMID:29107946
Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

PubMed

Jung, Sook-In; Rodriguez, Natalie; Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G; Park, Hyunsook

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.
Yeast surface display of dehydrogenases in microbial fuel-cells.

PubMed

Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

2016-12-01

Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. Copyright © 2016 Elsevier B.V. All rights reserved.
In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast.

PubMed

Bermejo, Clara; Ewald, Jennifer C; Lanquar, Viviane; Jones, Alexander M; Frommer, Wolf B

2011-08-15

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation. © The Authors Journal compilation © 2011 Biochemical Society
Two Pathways of Sphingolipid Biosynthesis Are Separated in the Yeast Pichia pastoris*

PubMed Central

Ternes, Philipp; Wobbe, Tobias; Schwarz, Marnie; Albrecht, Sandra; Feussner, Kirstin; Riezman, Isabelle; Cregg, James M.; Heinz, Ernst; Riezman, Howard; Feussner, Ivo; Warnecke, Dirk

2011-01-01

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis. PMID:21303904
Continuous beer fermentation using immobilized yeast cell bioreactor systems.

PubMed

Brányik, Tomás; Vicente, António A; Dostálek, Pavel; Teixeira, José A

2005-01-01

Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO(2) removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.
Quasi-programmed aging of budding yeast: a trade-off between programmed processes of cell proliferation, differentiation, stress response, survival and death defines yeast lifespan

PubMed Central

Arlia-Ciommo, Anthony; Piano, Amanda; Leonov, Anna; Svistkova, Veronika; Titorenko, Vladimir I

2014-01-01

Recent findings suggest that evolutionarily distant organisms share the key features of the aging process and exhibit similar mechanisms of its modulation by certain genetic, dietary and pharmacological interventions. The scope of this review is to analyze mechanisms that in the yeast Saccharomyces cerevisiae underlie: (1) the replicative and chronological modes of aging; (2) the convergence of these 2 modes of aging into a single aging process; (3) a programmed differentiation of aging cell communities in liquid media and on solid surfaces; and (4) longevity-defining responses of cells to some chemical compounds released to an ecosystem by other organisms populating it. Based on such analysis, we conclude that all these mechanisms are programs for upholding the long-term survival of the entire yeast population inhabiting an ecological niche; however, none of these mechanisms is a ʺprogram of agingʺ - i.e., a program for progressing through consecutive steps of the aging process. PMID:25485579
Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sifontes, Ángela B., E-mail: asifonte@ivic.gob.ve; González, Gema; Tovar, Leidy M.

2013-02-15

Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m{sup 2} g{sup −1}. -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N{sub 2} adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity,more » respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m{sup 2} g{sup −1}.« less
Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature.

PubMed

Zakhartsev, Maksim; Reuss, Matthias

2018-04-26

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μ_max), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μ_max, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (SSC-index) negatively depends on μ_max. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μ_max. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Conditions of activation of yeast plasma membrane ATPase.

PubMed

Sychrová, H; Kotyk, A

1985-04-08

The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose. The activated but not the control ATPase was sensitive to oligomycin. No activation was possible in a cell-free extract with added glucose. The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.
Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

PubMed

Hager, Natalie A; Krasowski, Collin J; Mackie, Timothy D; Kolb, Alexander R; Needham, Patrick G; Augustine, Andrew A; Dempsey, Alison; Szent-Gyorgyi, Christopher; Bruchez, Marcel P; Bain, Daniel J; Kwiatkowski, Adam V; O'Donnell, Allyson F; Brodsky, Jeffrey L

2018-05-21

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inwardly rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorescence-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation induction in haploid yeast after split-dose radiation-exposure. I. Fractionated UV-irradiation.

PubMed

Schenk, K; Zölzer, F; Kiefer, J

1989-01-01

Mutation induction was investigated in wild-type haploid yeast Saccharomyces cerevisiae after split-dose UV-irradiation. Cells were exposed to fractionated 254 nm-UV-doses separated by intervals from 0 to 6 h with incubation either on non-nutrient or nutrient agar between. The test parameter was resistance to canavanine. If modifications of sensitivity due to incubation are appropriately taken into account there is no change of mutation frequency.
Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

PubMed Central

Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

2011-01-01

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950
Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

PubMed

Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

2011-01-31

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.
How peroxisomes partition between cells. A story of yeast, mammals and filamentous fungi.

PubMed

Knoblach, Barbara; Rachubinski, Richard A

2016-08-01

Eukaryotic cells are subcompartmentalized into discrete, membrane-enclosed organelles. These organelles must be preserved in cells over many generations to maintain the selective advantages afforded by compartmentalization. Cells use complex molecular mechanisms of organelle inheritance to achieve high accuracy in the sharing of organelles between daughter cells. Here we focus on how a multi-copy organelle, the peroxisome, is partitioned in yeast, mammalian cells, and filamentous fungi, which differ in their mode of cell division. Cells achieve equidistribution of their peroxisomes through organelle transport and retention processes that act coordinately, although the strategies employed vary considerably by organism. Nevertheless, we propose that mechanisms common across species apply to the partitioning of all membrane-enclosed organelles. Copyright © 2016 Elsevier Ltd. All rights reserved.
The rotating spectrometer: New biotechnology for cell separations

NASA Technical Reports Server (NTRS)

Noever, David A.; Matsos, Helen C.

1990-01-01

An instrument for biochemical studies, called the rotating spectrometer, separates previously inseparable cell cultures. The rotating spectrometer is intended for use in pharmacological studies which require fractional splitting of heterogeneous cell cultures based on cell morphology and swimming behavior. As a method to separate and concentrate cells in free solution, the rotating method requires active organism participation and can effectively split the large class of organisms known to form spontaneous patterns. Examples include the biochemical star, an organism called Tetrahymena pyriformis. Following focusing in a rotated frame, the separation is accomplished using different radial dependencies of concentrated algal and protozoan species. The focusing itself appears as concentric rings and arises from the coupling between swimming direction and Coriolis forces. A dense cut is taken at varying radii and extraction is replenished at an inlet. Unlike standard separation and concentrating techniques such as filtration or centrifugation, the instrument is able to separate motile from immotile fractions. For a single pass, typical split efficiencies can reach 200 to 300 percent compared to the inlet concentration.
[Continuous ethanol fermentation coupled with recycling of yeast flocs].

PubMed

Wang, Bo; Ge, Xu-Meng; Li, Ning; Bai, Feng-Wu

2006-09-01

A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220g/L of reducing sugar, supplemented with 1.5g/L (NH4)2HPO4 and 2.5g/L KH2PO4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057h(-1). The yeast flocs separated by sedimentation were recycled into the first fermentor as two different models: activation-recycle and direct recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at the high dilution rates. The broth containing more than 101g/L ethanol, 3.2g/L residual reducing sugar and 7.7g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77g/(L x h), which increased by more than 70% compared with that achieved in the same tank in series system without recycling of yeast cells.
Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2014-01-07

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.
Eighteen new oleaginous yeast species.

PubMed

Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

2016-07-01

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.
Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

PubMed

Farbo, Maria Grazia; Urgeghe, Pietro Paolo; Fiori, Stefano; Marceddu, Salvatore; Jaoua, Samir; Migheli, Quirico

2016-01-18

Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20 μg/kg OTA and after 48 h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72-120 h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.
Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

PubMed

Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.
Fuel cell system with separating structure bonded to electrolyte

DOEpatents

Bourgeois, Richard Scott; Gudlavalleti, Sauri; Quek, Shu Ching; Hasz, Wayne Charles; Powers, James Daniel

2010-09-28

A fuel cell assembly comprises a separating structure configured for separating a first reactant and a second reactant wherein the separating structure has an opening therein. The fuel cell assembly further comprises a fuel cell comprising a first electrode, a second electrode, and an electrolyte interposed between the first and second electrodes, and a passage configured to introduce the second reactant to the second electrode. The electrolyte is bonded to the separating structure with the first electrode being situated within the opening, and the second electrode being situated within the passage.
Acoustic separation of circulating tumor cells

PubMed Central

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I.; Drabick, Joseph J.; El-Deiry, Wafik S.; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-01-01

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state. PMID:25848039
Acoustic separation of circulating tumor cells.

PubMed

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I; Drabick, Joseph J; El-Deiry, Wafik S; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-04-21

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.
Separation and sorting of cells in microsystems using physical principles

NASA Astrophysics Data System (ADS)

Lee, Gi-Hun; Kim, Sung-Hwan; Ahn, Kihoon; Lee, Sang-Hoon; Park, Joong Yull

2016-01-01

In the last decade, microfabrication techniques have been combined with microfluidics and applied to cell biology. Utilizing such new techniques, various cell studies have been performed for the research of stem cells, immune cells, cancer, neurons, etc. Among the various biological applications of microtechnology-based platforms, cell separation technology has been highly regarded in biological and clinical fields for sorting different types of cells, finding circulating tumor cells (CTCs), and blood cell separation, amongst other things. Many cell separation methods have been created using various physical principles. Representatively, these include hydrodynamic, acoustic, dielectrophoretic, magnetic, optical, and filtering methods. In this review, each of these methods will be introduced, and their physical principles and sample applications described. Each physical principle has its own advantages and disadvantages. The engineers who design the systems and the biologists who use them should understand the pros and cons of each method or principle, to broaden the use of microsystems for cell separation. Continuous development of microsystems for cell separation will lead to new opportunities for diagnosing CTCs and cancer metastasis, as well as other elements in the bloodstream.
Effect of Shock Waves Generated by Pulsed Electric Discharges in Water on Yeast Cells and Virus Particles

NASA Astrophysics Data System (ADS)

Girdyuk, A. E.; Gorshkov, A. N.; Egorov, V. V.; Kolikov, V. A.; Snetov, V. N.; Shneerson, G. A.

2018-02-01

The aim of this study is to determine the optimal parameters of the electric pulses and shock waves generated by them for the soft destruction of the virus and yeast envelopes with no changes in the structure of antigenic surface albumin and in the cell morphology in order to use them to produce antivirus vaccines and in biotechnology. The pulse electric discharges in water have been studied for different values of amplitude, pulse duration and the rate of the rise in the current. A mathematical model has been developed to estimate the optimal parameters of pulsed electric charges and shock waves for the complete destruction of the yeast cell envelopes and virus particles at a minimum of pulses.
The secretory pathway: exploring yeast diversity.

PubMed

Delic, Marizela; Valli, Minoska; Graf, Alexandra B; Pfeffer, Martin; Mattanovich, Diethard; Gasser, Brigitte

2013-11-01

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Co-fermentation of glucose, xylose and/or cellobiose by yeast

DOEpatents

Jeffries, Thomas W.; Willis, Laura B.; Long, Tanya M.; Su, Yi-Kai

2013-09-10

Provided herein are methods of using yeast cells to produce ethanol by contacting a mixture comprising xylose with a Spathaspora yeast cell under conditions suitable to allow the yeast to ferment at least a portion of the xylose to ethanol. The methods allow for efficient ethanol production from hydrolysates derived from lignocellulosic material and sugar mixtures including at least xylose and glucose or xylose, glucose and cellobiose.
Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Hatfield, J. Michael

1984-01-01

Data concerned with analyzing the cellular organization of the rat anterior pituitary gland are examined. The preparation of the cell suspensions and the methods used to separate pituitary cell types are described. Particular emphasis is given to velocity sedimentation at unit gravity, density gradient centrifugation, affinity methods, fluorescence activated cell sorting, and density gradient and continuous-flow electrophoresis. The difficulties encountered when attempting to compare data from different pituitary cell separation studies are discussed, and results from various experiments are presented. The functional capabilities of the separated cell populations can be tested in various culture systems.

The evaluation of layered separators for nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Gahn, Randall F.

1991-01-01

The concept of using layered separators to achieve the required electrolyte retention and bubble pressure fo nickel-hydrogen cells was evaluated in a boilerplate cell test. Zircar cloth, polyethylene paper and polypropylene felt were combined with a layer of radiation-grafted polyethylene film to achieve the required properties. Three cells of each layered separator were built and tested by characterization cycling and by low earth orbit cycling for 5000 cycles at 80 percent DOD. Three cells containing asbestos separators were used as the reference.
A minimal mathematical model combining several regulatory cycles from the budding yeast cell cycle.

PubMed

Sriram, K; Bernot, G; Képès, F

2007-11-01

A novel topology of regulatory networks abstracted from the budding yeast cell cycle is studied by constructing a simple nonlinear model. A ternary positive feedback loop with only positive regulations is constructed with elements that activates the subsequent element in a clockwise fashion. A ternary negative feedback loop with only negative regulations is constructed with the elements that inhibit the subsequent element in an anticlockwise fashion. Positive feedback loop exhibits bistability, whereas the negative feedback loop exhibits limit cycle oscillations. The novelty of the topology is that the corresponding elements in these two homogeneous feedback loops are linked by the binary positive feedback loops with only positive regulations. This results in the emergence of mixed feedback loops in the network that displays complex behaviour like the coexistence of multiple steady states, relaxation oscillations and chaos. Importantly, the arrangement of the feedback loops brings in the notion of checkpoint in the model. The model also exhibits domino-like behaviour, where the limit cycle oscillations take place in a stepwise fashion. As the aforementioned topology is abstracted from the budding yeast cell cycle, the events that govern the cell cycle are considered for the present study. In budding yeast, the sequential activation of the transcription factors, cyclins and their inhibitors form mixed feedback loops. The transcription factors that involve in the positive regulation in a clockwise orientation generates ternary positive feedback loop, while the cyclins and their inhibitors that involve in the negative regulation in an anticlockwise orientation generates ternary negative feedback loop. The mutual regulation between the corresponding elements in the transcription factors and the cyclins and their inhibitors generates binary positive feedback loops. The bifurcation diagram constructed for the whole system can be related to the different events of the
Nutrient supplements boost yeast transformation efficiency

PubMed Central

Yu, Sheng-Chun; Dawson, Alexander; Henderson, Alyssa C.; Lockyer, Eloise J.; Read, Emily; Sritharan, Gayathri; Ryan, Marjah; Sgroi, Mara; Ngou, Pok M.; Woodruff, Rosie; Zhang, Ruifeng; Ren Teen Chia, Travis; Liu, Yu; Xiang, Yiyu; Spanu, Pietro D.

2016-01-01

Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening. PMID:27760994
Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2013-05-14

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.
Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2017-09-12

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.
Genetically modified yeast species and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2011-05-17

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.
Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2016-08-09

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.
Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine

PubMed Central

Jales, Alessandra; Huang, Bruce; Fernando, Romaine I.; Hodge, James W.; Ardiani, Andressa; Apelian, David

2013-01-01

The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and metastasis in vivo, and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the development and characterization of an immunological platform for the efficient targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury protein. We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and expand brachyury-specific CD4+ and CD8+ T cells in vitro that, in turn, can effectively lyse human tumor cells expressing the brachyury protein. Vaccination of mice with recombinant yeast-brachyury is also shown here to elicit brachyury-specific CD4+ and CD8+ T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in patients with advanced tumors; to our knowledge, this is the first vaccine platform aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage. PMID:24125763
The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less
Pseudozyma and other non-Candida opportunistic yeast bloodstream infections in a large stem cell transplant center.

PubMed

Pande, Anupam; Non, Lemuel R; Romee, Rizwan; Santos, Carlos A Q

2017-04-01

Non-Candida opportunistic yeasts are emerging causes of bloodstream infection (BSI) in immunocompromised hosts. However, their clinical presentation, management, and outcomes in stem cell transplant (SCT) recipients are not well described. We report the first case to our knowledge of Pseudozyma BSI in a SCT recipient. He had evidence of cutaneous involvement, which has not been previously described in the literature. He became infected while neutropenic and receiving empiric micafungin, which is notable because Pseudozyma is reported to be resistant to echinocandins. He was successfully treated with the sequential use of liposomal amphotericin B and voriconazole. A review of the literature revealed nine reported instances of Pseudozyma fungemia. We performed a retrospective review of 3557 SCT recipients at our institution from January 2000 to June 2015 and identified four additional cases of non-Candida yeast BSIs. These include two with Cryptococcus, one with Trichosporon, and one with Saccharomyces. Pseudozyma and other non-Candida yeasts are emerging pathogens that can cause severe and disseminated infections in SCT recipients and other immunocompromised hosts. Clinicians should have a high degree of suspicion for echinocandin-resistant yeasts, if patients develop breakthrough yeast BSIs while receiving echinocandin therapy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Effect of temperature on replicative aging of the budding yeast Saccharomyces cerevisiae.

PubMed

Molon, Mateusz; Zadrag-Tecza, Renata

2016-04-01

The use of the budding yeast Saccharomyces cerevisiae in gerontological studies was based on the assumption that the reproduction limit of a single cell (replicative aging) is a consequence of accumulation of a hypothetical universal "senescence factor" within the mother cell. However, some evidence suggests that molecules or structures proposed as the "aging factor", such as rDNA circles, oxidatively damaged proteins (with carbonyl groups) or mitochondria, have little effect on replicative lifespan of yeast cells. Our results also suggest that protein aggregates associated with Hsp104, treated as a marker of yeast aging, do not seem to affect the numeric value of replicative lifespan of yeast. What these results indicate, however, is the need for finding a different way of expressing age and longevity of yeast cells instead of the commonly used number of daughters produced over units of time, as in the case of other organisms. In this paper, we show that the temperature has a stronger influence on the time of life (the total lifespan) than on the reproductive potential of yeast cells.
Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression*

PubMed Central

Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.

2014-01-01

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692
Beta-glucan-depleted, glycopeptide-rich extracts from Brewer's and Baker's yeast (Saccharomyces cerevisiae) lower interferon-gamma production by stimulated human blood cells in vitro.

PubMed

Williams, Roderick; Dias, Daniel A; Jayasinghe, Nirupama; Roessner, Ute; Bennett, Louise E

2016-04-15

Regulation of the human immune system requires controlled pro- and anti-inflammatory responses for host defence against infection and disease states. Yeasts (Saccharomyces cerevisiae), as used in brewing and baking, are mostly known for ability to stimulate the human immune-system predominantly reflecting the pro-inflammatory cell wall β-glucans. However, in this study, using food-compatible processing methods, glycopeptide-enriched and β-glucan-depleted products were each prepared from Brewer's and Baker's yeasts, which suppressed production of interferon-γ (IFN-γ) in human whole blood cell assay, signifying that anti-inflammatory factors are also present in yeast. Anti-inflammatory bioactivities of products prepared from Brewer's and Baker's yeast were compared with the commercial yeast product, Epicor®. While unfractionated Epicor was inactive, the C18 resin-binding fractions of Brewer's and Baker's yeast products and Epicor dose-dependently lowered IFN-γ, demonstrating that Epicor also contained both pro-inflammatory (β-glucans) and anti-inflammatory components. Anti-inflammatory activity was attributed to C18 resin-binding species glyco-peptides in Epicor and experimental yeast products. This study demonstrated that pro- and anti-inflammatory factors could be resolved and enriched in yeasts by suitable processing, with potential to improve specific activities. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.
The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization.

PubMed

Qi, Aisha; Yeo, Leslie; Friend, James; Ho, Jenny

2010-02-21

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.
High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

PubMed Central

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; Shapiro, David; Kirz, Janos; Marchesini, Stefano; Neiman, Aaron M.; Turner, Joshua J.; Jacobsen, Chris

2010-01-01

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11–13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy. PMID:20368463
High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

DOE PAGES

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; ...

2010-04-20

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane andmore » freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.« less
Uptake of yeast (Saccharomyces boulardii) in normal and rotavirus treated intestine.

PubMed Central

Cartwright-Shamoon, J; Dickson, G R; Dodge, J; Carr, K E

1996-01-01

BACKGROUND: There has recently been a growing interest in the use of the non-pathogenic yeast Saccharomyces boulardii, in the treatment of gastrointestinal disorders, including diarrhoea. The full effects of administration of the yeast are not fully understood. AIMS: To investigate the morphological effects of inoculated S boulardii on mouse intestinal villi, both in control animals and those treated with rotavirus. METHODS: Seven day old BALB/c seronegative mice were intubated with either rotavirus (30 microliters orally) or S boulardii (1.5 g/kg) or both rotavirus and S boulardii administered together. Control animals were given saline only. Animals were killed by decapitation 48 hours post-treatment. The middle region of the small intestine was studied using light microscopy and transmission and scanning electron microscopy, including backscattered electron imaging. RESULTS: Animals treated with rotavirus with or without S boulardii developed severe diarrhoea and showed morphological villous changes such as stromal separation and increased epithelial vacuolation. Specimens treated with S boulardii contained yeast particles within the mucosal tissues. CONCLUSION: The administration of S boulardii did not influence the changes produced by rotavirus, but yeast particles appeared to be taken up by the villous mucosa, with the predominant route apparently being uptake between adjacent epithelial cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8991857
Multiple Origins of the Pathogenic Yeast Candida orthopsilosis by Separate Hybridizations between Two Parental Species.

PubMed

Schröder, Markus S; Martinez de San Vicente, Kontxi; Prandini, Tâmara H R; Hammel, Stephen; Higgins, Desmond G; Bagagli, Eduardo; Wolfe, Kenneth H; Butler, Geraldine

2016-11-01

Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.
Border cell release: Cell separation without cell wall degradation?

PubMed

Mravec, Jozef

2017-07-03

Plant border cells are specialized cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localized cell separation which is essential for their release to the environment is little understood. Here I present in situ analysis of Brachypodium distachyon, a model organism for grasses which possess type II primary cell walls poor in pectin content. Results suggest similarity in spatial dynamics of pectic homogalacturonan during dicot and monocot border cell release. Integration of observations from different species leads to the hypothesis that this process most likely does not involve degradation of cell wall material but rather uses unique cell wall structural and compositional means enabling both the rigidity of the root cap as well as detachability of given cells on its surface.
Wall effects in continuous microfluidic magneto-affinity cell separation.

PubMed

Wu, Liqun; Zhang, Yong; Palaniapan, Moorthi; Roy, Partha

2010-05-01

Continuous microfluidic magneto-affinity cell separator combines unique microscale flow phenomenon with advantageous nanobead properties, to isolate cells with high specificity. Owing to the comparable size of the cell-bead complexes and the microchannels, the walls of the microchannel exert a strong influence on the separation of cells by this method. We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid shear forces. Separation of a human colon carcinoma cell line from a mixture of red blood cells, with folic acid conjugated 1 microm and 200 nm beads, is reported.

Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

NASA Astrophysics Data System (ADS)

Sakharkar, Kishore R.; Sakharkar, Meena K.

The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.
[Treatment of oil-manufacturing wastewater by yeast-SBR system].

PubMed

Lü, Wen-zhou; Liu, Ying; Huang, Yi-zhen

2008-04-01

Eight yeast strains were applied to a sequencing batch reactor (SBR) to treat high-strength oil-containing wastewater. The removal performance, yeast cultivation method and key factors affecting the stability of system were discussed. The results show yeast sludge with MLSS of 19 g/L and SVI of 35 mL/g can be obtained in 6 d in an open system without any molds and bacteria inhibitor addition; In 30 d continuous wastewater treatment, COD and oil removal rate achieve 86.8%-96.9% and above 99.5% respectively under the influent conditions of the COD of 9000-23000 mg/L and oil of 4500-16000 mg/L; Short period of pH impact brings reversible effects on the system and the sludge retention time can affect the SVI of the yeast; Absence of nitrogen induces morphology conversion of some yeast cells from single cell to filamentous one and impairs the settling capability of the yeast.
Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in
Mechanisms of Contact-Mediated Killing of Yeast Cells on Dry Metallic Copper Surfaces▿

PubMed Central

Quaranta, Davide; Krans, Travis; Santo, Christophe Espírito; Elowsky, Christian G.; Domaille, Dylan W.; Chang, Christopher J.; Grass, Gregor

2011-01-01

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC2(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated. PMID:21097600
Atomic force microscopic study of the effects of ethanol on yeast cell surface morphology.

PubMed

Canetta, Elisabetta; Adya, Ashok K; Walker, Graeme M

2006-02-01

The detrimental effects of ethanol toxicity on the cell surface morphology of Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354) were investigated using an atomic force microscope (AFM). In combination with culture viability and mean cell volume measurements AFM studies allowed us to relate the cell surface morphological changes, observed on nanometer lateral resolution, with the cellular stress physiology. Exposing yeasts to increasing stressful concentrations of ethanol led to decreased cell viabilities and mean cell volumes. Together with the roughness and bearing volume analyses of the AFM images, the results provided novel insight into the relative ethanol tolerance of S. cerevisiae and Sc. pombe.
Crossflow microfiltration of yeast suspensions in tubular filters.

PubMed

Redkar, S G; Davis, R H

1993-01-01

Crossflow microfiltration experiments were performed on yeast suspensions through 0.2-microns pore size ceramic and polypropylene tubes at various operating conditions. The initial transient flux decline follows dead-end filtration theory, with the membrane resistance determined from the initial flux and the specific cake resistance determined from the rate of flux decline due to cake buildup. For long times, the observed fluxes reach steady or nearly steady values, presumably as a result of the cake growth being arrested by the shear exerted at its surface. The steady-state fluxes increase with increasing shear rate and decreasing feed concentration, and they are nearly independent of transmembrane pressure. The steady-state fluxes for unwashed yeast in deionized water or fermentation media are typically 2-4 times lower than those predicted by a model based on the properties of nonadhesive, rigid spheres undergoing shear-induced back-diffusion. In contrast, the steady-state fluxes observed for washed yeast cells in deionized water are only 10-30% below the predicted values. The washed yeast cells also exhibited specific cake resistances that are an order of magnitude lower than those for the unwashed yeast. The differences are due to the presence of extracellular proteins and other macromolecules in the unwashed yeast suspensions. These biopolymers cause higher cell adhesion and resistance in the cake layer, so that the cells at the top edge are not free to diffuse away. This is manifested as a concentration jump from the edge of the cake layer to the sheared suspension adjacent to it.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Surface Interference with Plasma Membrane and Transport Processes in Yeasts.

PubMed

Francois, Jean Marie

2016-01-01

The wall of the yeast Saccharomyces cerevisiae is a shell of about 120 nm thick, made of two distinct layers, which surrounds the cell. The outer layer is constituted of highly glycosylated proteins and the inner layer is composed of β-glucan and chitin. These two layers are interconnected through covalent linkages leading to a supramolecular architecture that is characterized by physical and chemical properties including rigidity, porosity and biosorption. The later property results from the presence of highly negative charged phosphate and carboxylic groups of the cell wall proteins, allowing the cell wall to act as an efficient barrier to metals ions, toxins and organic compounds. An intimate connection between cell wall and plasma membrane is indicated by the fact that changes in membrane fluidity results in change in cell wall nanomechanical properties. Finally, cell wall contributes to transport processes through the use of dedicated cell wall mannoproteins, as it is the case for Fit proteins implicated in the siderophore-iron bound transport and the Tir/Dan proteins family in the uptake of sterols.
Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…
Between science and industry-applied yeast research.

PubMed

Korhola, Matti

2018-03-01

I was fortunate to enter yeast research at the Alko Research Laboratories with a strong tradition in yeast biochemistry and physiology studies. At the same time in the 1980s there was a fundamental or paradigm change in molecular biology research with discoveries in DNA sequencing and other analytical and physical techniques for studying macromolecules and cells. Since that time biotechnological research has expanded the traditional fermentation industries to efficient production of industrial and other enzymes and specialty chemicals. Our efforts were directed towards improving the industrial production organisms: minerals enriched yeasts (Se, Cr, Zn) and high glutathione content yeast, baker´s, distiller´s, sour dough and wine yeasts, and the fungal Trichoderma reesei platform for enzyme production. I am grateful for the trust of my colleagues in several leadership positions at the Alko Research Laboratories, Yeast Industry Platform and at the international yeast community.
A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks.

PubMed

Laomettachit, Teeraphan; Chen, Katherine C; Baumann, William T; Tyson, John J

2016-01-01

To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a "standard component" modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with "standard components" can capture in quantitative detail many essential properties of cell cycle control in budding yeast.
A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks

PubMed Central

Laomettachit, Teeraphan; Chen, Katherine C.; Baumann, William T.

2016-01-01

To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a “standard component” modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with “standard components” can capture in quantitative detail many essential properties of cell cycle control in budding yeast. PMID:27187804
Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

PubMed Central

Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

2015-01-01

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876
The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

PubMed

Padman, Benjamin S; Bach, Markus; Lucarelli, Giuseppe; Prescott, Mark; Ramm, Georg

2013-11-01

Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.
Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae.

PubMed

Morimoto, Yuji; Tani, Motohiro

2015-02-01

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen-activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan-type N-glycans. © 2014 John Wiley & Sons Ltd.
Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

PubMed

Aung, Hsu Mon; Huangteerakul, Chananya; Panvongsa, Wittaya; Jensen, Amornrat N; Chairoungdua, Arthit; Sukrong, Suchada; Jensen, Laran T

2018-09-15

Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF). Copyright © 2018 Elsevier B.V. All rights reserved.
Aging and differentiation in yeast populations: elders with different properties and functions.

PubMed

Palková, Zdena; Wilkinson, Derek; Váchová, Libuše

2014-02-01

Over the past decade, it has become evident that similarly to cells forming metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified both in yeast liquid cultures and within multicellular structures occupying solid surfaces. Most current knowledge on different cell types comes from studies of the spatiotemporal internal architecture of colonies developing on various media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS production, and others. These differences can affect longevity of particular subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the gradients of certain compounds and the position of cells within the structure affect cellular differentiation. In this review, we summarize and compare the properties of diverse types of differentiated chronologically aging yeast cells that have been identified in colonies growing on different media, as well as of those found in liquid cultures. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
A new methodology to obtain wine yeast strains overproducing mannoproteins.

PubMed

Quirós, Manuel; Gonzalez-Ramos, Daniel; Tabera, Laura; Gonzalez, Ramon

2010-04-30

Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the beta-1,3-glucan present in the yeast cell wall. Among their outstanding enological properties, yeast mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid bacteria. The development of a non-recombinant method to obtain enological yeast strains overproducing mannoproteins would therefore be very useful. Our previous experience on the genetic determinants of the release of these molecules by Saccharomyces cerevisiae has allowed us to propose a new methodology to isolate and characterize wine yeast that overproduce mannoproteins. The described methodology is based on the resistance of the killer 9 toxin produced by Williopsis saturnus, a feature linked to an altered biogenesis of the yeast cell wall. Copyright 2010 Elsevier B.V. All rights reserved.
Biosorption of nickel by yeasts in an osmotically unsuitable environment.

PubMed

Breierová, Emilia; Certík, Milan; Kovárová, Annamaria; Gregor, Tomas

2008-01-01

The tolerance, sorption of nickel(II) ions, and changes in the production and composition of exopolymers of eight yeast strains grown under nickel presence with/without NaCl were studied. Strains of Pichia anomala and Candida maltosa known as the most resistant yeasts against nickel tolerated up to 3 mM Ni2+. NaCl addition decreased both the resistance of the yeast strains toward nickel ions and the sorption of metal ions into cells. All yeasts absorbed nickel predominantly into exopolymers (glycoproteins) and on the surface of cells. However, while the amount of polysaccharide moieties of exoglycoproteins of most of the resistant yeasts was induced by stress conditions, the ratio polysaccharide/protein in the exopolymers remained unchanged in the sensitive species Cystofilobasidium. The exopolymer composition might play a key role in yeast adaptation to stress conditions caused by heavy metal ions.
Rare Cell Separation and Analysis by Magnetic Sorting

PubMed Central

Zborowski, Maciej; Chalmers, Jeffrey J.

2011-01-01

Summary The separation and or isolation of rare cells using magnetic forces is commonly used and growing in use ranging from simple sample prep for further studies to a FDA approved, clinical diagnostic test. This grown is the result of both the demand to obtain homogeneous rare cells for molecular analysis and the dramatic increases in the power of permanent magnets that even allow the separation of some unlabeled cells based on intrinsic magnetic moments, such as malaria parasite-infected red blood cells. PMID:21812408
Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

PubMed Central

2014-01-01

Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase. PMID:24949272

Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts.

PubMed

Barbosa, Catarina; Lage, Patrícia; Vilela, Alice; Mendes-Faia, Arlete; Mendes-Ferreira, Ana

2014-01-01

Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase.
Fed-batch hydrolysate addition and cell separation by settling in high cell density lignocellulosic ethanol fermentations on AFEX™ corn stover in the Rapid Bioconversion with Integrated recycling Technology process.

PubMed

Sarks, Cory; Jin, Mingjie; Balan, Venkatesh; Dale, Bruce E

2017-09-01

The Rapid Bioconversion with Integrated recycling Technology (RaBIT) process uses enzyme and yeast recycling to improve cellulosic ethanol production economics. The previous versions of the RaBIT process exhibited decreased xylose consumption using cell recycle for a variety of different micro-organisms. Process changes were tested in an attempt to eliminate the xylose consumption decrease. Three different RaBIT process changes were evaluated in this work including (1) shortening the fermentation time, (2) fed-batch hydrolysate addition, and (3) selective cell recycling using a settling method. Shorting the RaBIT fermentation process to 11 h and introducing fed-batch hydrolysate addition eliminated any xylose consumption decrease over ten fermentation cycles; otherwise, decreased xylose consumption was apparent by the third cell recycle event. However, partial removal of yeast cells during recycle was not economical when compared to recycling all yeast cells.
Cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, Steven T.; Feikert, John H.; Kachmitter, James L.; Pekala, Richard W.

1995-01-01

An improved multi-cell electrochemical energy storage device, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack.
Redox potential driven aeration during very-high-gravity ethanol fermentation by using flocculating yeast.

PubMed

Liu, Chen-Guang; Hao, Xue-Mi; Lin, Yen-Han; Bai, Feng-Wu

2016-05-10

Ethanol fermentation requires oxygen to maintain high biomass and cell viability, especially under very-high-gravity (VHG) condition. In this work, fermentation redox potential (ORP) was applied to drive the aeration process at low dissolved oxygen (DO) levels, which is infeasible to be regulated by a DO sensor. The performance and characteristics of flocculating yeast grown under 300 and 260 g glucose/L conditions were subjected to various aeration strategies including: no aeration; controlled aeration at -150, -100 and -50 mV levels; and constant aeration at 0.05 and 0.2 vvm. The results showed that anaerobic fermentation produced the least ethanol and had the highest residual glucose after 72 h of fermentation. Controlled aerations, depending on the real-time oxygen demand, led to higher cell viability than the no-aeration counterpart. Constant aeration triggered a quick biomass formation, and fast glucose utilization. However, over aeration at 0.2 vvm caused a reduction of final ethanol concentration. The controlled aeration driven by ORP under VHG conditions resulted in the best fermentation performance. Moreover, the controlled aeration could enhance yeast flocculating activity, promote an increase of flocs size, and accelerate yeast separation near the end of fermentation.
Redox potential driven aeration during very-high-gravity ethanol fermentation by using flocculating yeast

PubMed Central

Liu, Chen-Guang; Hao, Xue-Mi; Lin, Yen-Han; Bai, Feng-Wu

2016-01-01

Ethanol fermentation requires oxygen to maintain high biomass and cell viability, especially under very-high-gravity (VHG) condition. In this work, fermentation redox potential (ORP) was applied to drive the aeration process at low dissolved oxygen (DO) levels, which is infeasible to be regulated by a DO sensor. The performance and characteristics of flocculating yeast grown under 300 and 260 g glucose/L conditions were subjected to various aeration strategies including: no aeration; controlled aeration at −150, −100 and −50 mV levels; and constant aeration at 0.05 and 0.2 vvm. The results showed that anaerobic fermentation produced the least ethanol and had the highest residual glucose after 72 h of fermentation. Controlled aerations, depending on the real-time oxygen demand, led to higher cell viability than the no-aeration counterpart. Constant aeration triggered a quick biomass formation, and fast glucose utilization. However, over aeration at 0.2 vvm caused a reduction of final ethanol concentration. The controlled aeration driven by ORP under VHG conditions resulted in the best fermentation performance. Moreover, the controlled aeration could enhance yeast flocculating activity, promote an increase of flocs size, and accelerate yeast separation near the end of fermentation. PMID:27161047
Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

PubMed

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.
Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability

PubMed Central

Fehrmann, Steffen; Bottin-Duplus, Hélène; Leonidou, Andri; Mollereau, Esther; Barthelaix, Audrey; Wei, Wu; Steinmetz, Lars M; Yvert, Gaël

2013-01-01

Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent Pmet17-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced Pmet17-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation. PMID:24104478
Effects of Amphotericin B and Three Azole Derivatives on the Lipids of Yeast Cells of Paracoccidioides brasiliensis

PubMed Central

Hahn, Rosane Christine; Hamdan, Júnia Soares

2000-01-01

Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC. PMID:10858371
Bioethanol production from uncooked raw starch by immobilized surface-engineered yeast cells.

PubMed

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

2008-03-01

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.
Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

NASA Astrophysics Data System (ADS)

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.
Optical spectral analysis of ultra-weak photon emission from tissue culture and yeast cells

NASA Astrophysics Data System (ADS)

Nerudová, Michaela; Červinková, Kateřina; Hašek, Jiří; Cifra, Michal

2015-01-01

Optical spectral analysis of the ultra-weak photon emission (UPE) could be utilized for non-invasive diagnostic of state of biological systems and for elucidation of underlying mechanisms of UPE generation. Optical spectra of UPE from differentiated HL-60 cells and yeast cells (Saccharomyces cerevisiae) were investigated. Induced photon emission of neutrophil-like cells and spontaneous photon emission of yeast cells were measured using highly sensitive photomultiplier module Hamamatsu H7360-01 in a thermally regulated light-tight chamber. The respiratory burst of neutrophil-like HL-60 cells was induced with the PMA (phorbol 12-myristate, 13-acetate). PMA activates an assembly of NADPH oxidase, which induces a rapid formation of reactive oxygen species (ROS). Long-pass edge filters (wavelength 350, from 400 to 600 with 25 nm resolution and 650 nm) were used for optical spectral analysis. Propagation of error of indirect measurements and standard deviation were used to assess reliability of the measured spectra. Results indicate that the photon emission from both cell cultures is detectable in the six from eight examined wavelength ranges with different percentage distribution of cell suspensions, particularly 450-475, 475-500, 500-525, 525-550, 550-575 and 575-600 nm. The wavelength range of spectra from 450 to 550 nm coincides with the range of photon emission from triplet excited carbonyls (350-550 nm). The both cells cultures emitted photons in wavelength range from 550 to 600 nm but this range does not correspond with any known emitter. To summarize, we have demonstrated a clear difference in the UPE spectra between two organisms using rigorous methodology and error analysis.
Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform.

PubMed

Shibuta, Mayu; Tamura, Masato; Kanie, Kei; Yanagisawa, Masumi; Matsui, Hirofumi; Satoh, Taku; Takagi, Toshiyuki; Kanamori, Toshiyuki; Sugiura, Shinji; Kato, Ryuji

2018-06-09

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells.

PubMed

Stöckl, Petra; Zankl, Christina; Hütter, Eveline; Unterluggauer, Hermann; Laun, Peter; Heeren, Gino; Bogengruber, Edith; Herndler-Brandstetter, Dietmar; Breitenbach, Michael; Jansen-Dürr, Pidder

2007-09-15

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.
Cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, S.T.; Feikert, J.H.; Kachmitter, J.L.; Pekala, R.W.

1995-02-28

An improved multi-cell electrochemical energy storage device is described, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack. 2 figs.
Digital Image Analysis of Yeast Single Cells Growing in Two Different Oxygen Concentrations to Analyze the Population Growth and to Assist Individual-Based Modeling.

PubMed

Ginovart, Marta; Carbó, Rosa; Blanco, Mónica; Portell, Xavier

2017-01-01

Nowadays control of the growth of Saccharomyces to obtain biomass or cellular wall components is crucial for specific industrial applications. The general aim of this contribution is to deal with experimental data obtained from yeast cells and from yeast cultures to attempt the integration of the two levels of information, individual and population, to progress in the control of yeast biotechnological processes by means of the overall analysis of this set of experimental data, and to assist in the improvement of an individual-based model, namely, INDISIM- Saccha . Populations of S. cerevisiae growing in liquid batch culture, in aerobic and microaerophilic conditions, were studied. A set of digital images was taken during the population growth, and a protocol for the treatment and analyses of the images obtained was established. The piecewise linear model of Buchanan was adjusted to the temporal evolutions of the yeast populations to determine the kinetic parameters and changes of growth phases. In parallel, for all the yeast cells analyzed, values of direct morphological parameters, such as area, perimeter, major diameter, minor diameter, and derived ones, such as circularity and elongation, were obtained. Graphical and numerical methods from descriptive statistics were applied to these data to characterize the growth phases and the budding state of the yeast cells in both experimental conditions, and inferential statistical methods were used to compare the diverse groups of data achieved. Oxidative metabolism of yeast in a medium with oxygen available and low initial sugar concentration can be taken into account in order to obtain a greater number of cells or larger cells. Morphological parameters were analyzed statistically to identify which were the most useful for the discrimination of the different states, according to budding and/or growth phase, in aerobic and microaerophilic conditions. The use of the experimental data for subsequent modeling work was then
Digital Image Analysis of Yeast Single Cells Growing in Two Different Oxygen Concentrations to Analyze the Population Growth and to Assist Individual-Based Modeling

PubMed Central

Ginovart, Marta; Carbó, Rosa; Blanco, Mónica; Portell, Xavier

2018-01-01

Nowadays control of the growth of Saccharomyces to obtain biomass or cellular wall components is crucial for specific industrial applications. The general aim of this contribution is to deal with experimental data obtained from yeast cells and from yeast cultures to attempt the integration of the two levels of information, individual and population, to progress in the control of yeast biotechnological processes by means of the overall analysis of this set of experimental data, and to assist in the improvement of an individual-based model, namely, INDISIM-Saccha. Populations of S. cerevisiae growing in liquid batch culture, in aerobic and microaerophilic conditions, were studied. A set of digital images was taken during the population growth, and a protocol for the treatment and analyses of the images obtained was established. The piecewise linear model of Buchanan was adjusted to the temporal evolutions of the yeast populations to determine the kinetic parameters and changes of growth phases. In parallel, for all the yeast cells analyzed, values of direct morphological parameters, such as area, perimeter, major diameter, minor diameter, and derived ones, such as circularity and elongation, were obtained. Graphical and numerical methods from descriptive statistics were applied to these data to characterize the growth phases and the budding state of the yeast cells in both experimental conditions, and inferential statistical methods were used to compare the diverse groups of data achieved. Oxidative metabolism of yeast in a medium with oxygen available and low initial sugar concentration can be taken into account in order to obtain a greater number of cells or larger cells. Morphological parameters were analyzed statistically to identify which were the most useful for the discrimination of the different states, according to budding and/or growth phase, in aerobic and microaerophilic conditions. The use of the experimental data for subsequent modeling work was then
High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317
Design of polymeric immunomicrospheres for cell labelling and cell separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Margel, S.

1978-01-01

Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.
Influence of aeration during propagation of pitching yeast on fermentation and beer flavor.

PubMed

Cheong, Chul; Wackerbauer, Karl; Kang, Soon Ah

2007-02-01

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.
Direct cytotoxicity evaluation of 63S bioactive glass and bone-derived hydroxyapatite particles using yeast model and human chondrocyte cells by microcalorimetry.

PubMed

Doostmohammadi, A; Monshi, A; Fathi, M H; Karbasi, S; Braissant, O; Daniels, A U

2011-10-01

In this study, the cytotoxicity evaluation of prepared 63S bioactive glass and bone-derived hydroxyapatite particles with yeast and human chondrocyte cells was carried out using isothermal micro-nano calorimetry (IMNC), which is a new method for studying cell/biomaterial interactions. Bioactive glass particles were made via sol-gel method and hydroxyapatite was obtained from bovine bone. Elemental analysis was carried out by XRF and EDXRF. Amorphous structure of the glass and completely crystalline structure of HA were detected by XRD analysis. Finally, the cytotoxicity of bioactive glass and bone-derived HA particles with yeast and cultured human chondrocyte cells was evaluated using IMNC. The results confirmed the viability, growth and proliferation of human chondrocyte cells in contact with 63S bioactive glass, and bone-derived HA particles. Also the results indicated that yeast model which is much easier to handle, can be considered as a good proxy and can provide a rapid primary estimate of the ranges to be used in assays involving human cells. All of these results confirmed that IMNC is a convenient method which caters to measuring the cell-biomaterial interactions alongside the current methods.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

PubMed

Laun, Peter; Bruschi, Carlo V; Dickinson, J Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.
Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing

PubMed Central

Laun, Peter; Bruschi, Carlo V.; Dickinson, J. Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast. PMID:17986449
Yeast selection for fuel ethanol production in Brazil.

PubMed

Basso, Luiz C; de Amorim, Henrique V; de Oliveira, Antonio J; Lopes, Mario L

2008-11-01

Brazil is one of the largest ethanol biofuel producers and exporters in the world and its production has increased steadily during the last three decades. The increasing efficiency of Brazilian ethanol plants has been evident due to the many technological contributions. As far as yeast is concerned, few publications are available regarding the industrial fermentation processes in Brazil. The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high viability during recycling and very high implantation capability into industrial fermenters). Results also suggest that the great yeast biodiversity found in distillery environments could be an important source of strains. This is because during yeast cell recycling, selective pressure (an adaptive evolution) is imposed on cells, leading to strains with higher tolerance to the stressful conditions of the industrial fermentation.
Yeast as a model system for mammalian seven-transmembrane segment receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeansonne, N.E.

1994-05-01

Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomalmore » location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.« less
Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamei, Yuka; Tai, Akiko; Dakeyama, Shota

Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be themore » potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.« less
[Mitochondria inheritance in yeast saccharomyces cerevisiae].

PubMed

Fizikova, A Iu

2011-01-01

The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.
Dielectrophoretic separation of micron and submicron particles: a review.

PubMed

Dash, Swagatika; Mohanty, Swati

2014-09-01

This paper provides an overview on separation of micron and submicron sized biological (cells, yeast, virus, bacteria, etc.) and nonbiological particles (latex, polystyrene, CNTs, metals, etc.) by dielectrophoresis (DEP), which finds wide applications in the field of medical and environmental science. Mathematical models to predict the electric field, flow profile, and concentration profiles of the particles under the influence of DEP force have also been covered in this review. In addition, advancements made primarily in the last decade, in the area of electrode design (shape and arrangement), new materials for electrode (carbon, silicon, polymers), and geometry of the microdevice, for efficient DEP separation of particles have been highlighted. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Separation of cancer cells from a red blood cell suspension using inertial force.

PubMed

Tanaka, Tatsuya; Ishikawa, Takuji; Numayama-Tsuruta, Keiko; Imai, Yohsuke; Ueno, Hironori; Matsuki, Noriaki; Yamaguchi, Takami

2012-11-07

The circulating tumor cell (CTC) test has recently become popular for evaluating prognosis and treatment efficacy in cancer patients. The accuracy of the test is strongly dependent on the precision of the cancer cell separation. In this study, we developed a multistage microfluidic device to separate cancer cells from a red blood cell (RBC) suspension using inertial migration forces. The device was able to effectively remove RBCs up to the 1% hematocrit (Hct) condition with a throughput of 565 μL min(-1). The collection efficiency of cancer cells from a RBC suspension was about 85%, and the enrichment of cancer cells was about 120-fold. Further improvements can be easily achieved by parallelizing the device. These results illustrate that the separation of cancer cells from RBCs is possible using only inertial migration forces, thus paving the way for the development of a novel microfluidic device for future CTC tests.
Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells.

PubMed

Gustavsson, Anna-Karin; van Niekerk, David D; Adiels, Caroline B; Kooi, Bob; Goksör, Mattias; Snoep, Jacky L

2014-06-01

Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP. The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/webMathematica/UItester.jsp?modelName=gustavsson5. [Database section added 14 May 2014 after original online publication]. © 2014 FEBS.
An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.

PubMed

van Brabant, A J; Buchanan, C D; Charboneau, E; Fangman, W L; Brewer, B J

2001-04-01

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.
Model of Fission Yeast Cell Shape Driven by Membrane-Bound Growth Factors and the Cytoskeleton

PubMed Central

Drake, Tyler; Vavylonis, Dimitrios

2013-01-01

Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism) with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular level in the future
A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.
Whole-Genome Analysis of Three Yeast Strains Used for Production of Sherry-Like Wines Revealed Genetic Traits Specific to Flor Yeasts

PubMed Central

Eldarov, Mikhail A.; Beletsky, Alexey V.; Tanashchuk, Tatiana N.; Kishkovskaya, Svetlana A.; Ravin, Nikolai V.; Mardanov, Andrey V.

2018-01-01

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for biological wine aging. We have sequenced the genomes of three flor strains originated from different geographic regions and used for production of sherry-like wines in Russia. According to the obtained phylogeny of 118 yeast strains, flor strains form very tight cluster adjacent to the main wine clade. SNP analysis versus available genomes of wine and flor strains revealed 2,270 genetic variants in 1,337 loci specific to flor strains. Gene ontology analysis in combination with gene content evaluation revealed a complex landscape of possibly adaptive genetic changes in flor yeast, related to genes associated with cell morphology, mitotic cell cycle, ion homeostasis, DNA repair, carbohydrate metabolism, lipid metabolism, and cell wall biogenesis. Pangenomic analysis discovered the presence of several well-known “non-reference” loci of potential industrial importance. Events of gene loss included deletions of asparaginase genes, maltose utilization locus, and FRE-FIT locus involved in iron transport. The latter in combination with a flor-yeast-specific mutation in the Aft1 transcription factor gene is likely to be responsible for the discovered phenotype of increased iron sensitivity and improved iron uptake of analyzed strains. Expansion of the coding region of the FLO11 flocullin gene and alteration of the balance between members of the FLO gene family are likely to positively affect the well-known propensity of flor strains for velum formation. Our study provides new insights in the nature of genetic variation in flor yeast strains and demonstrates that different adaptive properties of flor yeast strains could have evolved through different mechanisms of genetic variation. PMID:29867869
Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

PubMed

Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

2015-01-01

The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd.
DNA double strand break induction in yeast.

PubMed

Kiefer, J; Egenolf, R; Ikpeme, S E

2002-01-01

The induction of DNA double strand breaks (DSBs) by accelerated heavy ions was systematically measured in diploid yeast cells. Particles were provided by the accelerators at GSI, Darmstadt, and HMI, Berlin. DNA was separated using pulsed field gel electrophoresis and the intensity of the largest bands used to determine the loss of molecular weight. Since the DNA content of each chromosome is exactly known absolute values for DSB induction can be measured without calibration procedures. Ions used range from protons to uranium with LET values between 2 and about 15,000 keV.micron-1. Induction cross sections increase in the lower LET region approaching a plateau around 200 keV.micron-1. With higher LET values the dependence can no longer be described by a common curve with each ion showing a specific behaviour. With very heavy particles the influence of the penumbra becomes obvious: cross sections decrease with LET because of the reduced penumbra extensions. Classical target theory would predict cross sections to follow a simple saturation function which is not substantiated by the data. Track structure analysis as introduced by Butts and Katz in 1967 is also not able to predict the experimental results. A semi-empirical fit indicates a linear-quadratic dependence of induction cross sections on LET up to about 1000 keV.micron-1. RBE for DSB induction rises above unity reaching a maximum of about 2.5 around 200 keV.micron-1. This is different from many experiments in mammalian cells and is presumably due to differences in chromatin structure since yeast cells seem to lack a functional III histone.
Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization

PubMed Central

Witte, Kristen; Strickland, Devin; Glotzer, Michael

2017-01-01

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment. DOI: http://dx.doi.org/10.7554/eLife.26722.001 PMID:28682236
Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells.

PubMed

Zhang, Weijia; Kai, Kazuharu; Choi, Dong Soon; Iwamoto, Takayuki; Nguyen, Yen H; Wong, Helen; Landis, Melissa D; Ueno, Naoto T; Chang, Jenny; Qin, Lidong

2012-11-13

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.
Three-dimensional spatiotemporal tracking of fluorine-18 radiolabeled yeast cells via positron emission particle tracking

DOE PAGES

Langford, Seth T.; Wiggins, Cody S.; Santos, Roque; ...

2017-07-06

A method for Positron Emission Particle Tracking (PEPT) based on optical feature point identification techniques is demonstrated for use in low activity tracking experiments. Furthermore, a population of yeast cells of approximately 125,000 members is activated to roughly 55 Bq/cell by 18F uptake. An in vitro particle tracking experiment is performed with nearly 20 of these cells after decay to 32 Bq/cell. These cells are successfully identified and tracked simultaneously in this experiment. Our work extends the applicability of PEPT as a cell tracking method by allowing a number of cells to be tracked together, and demonstrating tracking for verymore » low activity tracers.« less
Three-dimensional spatiotemporal tracking of fluorine-18 radiolabeled yeast cells via positron emission particle tracking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langford, Seth T.; Wiggins, Cody S.; Santos, Roque

A method for Positron Emission Particle Tracking (PEPT) based on optical feature point identification techniques is demonstrated for use in low activity tracking experiments. Furthermore, a population of yeast cells of approximately 125,000 members is activated to roughly 55 Bq/cell by 18F uptake. An in vitro particle tracking experiment is performed with nearly 20 of these cells after decay to 32 Bq/cell. These cells are successfully identified and tracked simultaneously in this experiment. Our work extends the applicability of PEPT as a cell tracking method by allowing a number of cells to be tracked together, and demonstrating tracking for verymore » low activity tracers.« less
High gradient magnetic field microstructures for magnetophoretic cell separation.

PubMed

Abdel Fattah, Abdel Rahman; Ghosh, Suvojit; Puri, Ishwar K

2016-08-01

Microfluidics has advanced magnetic blood fractionation by making integrated miniature devices possible. A ferromagnetic microstructure array that is integrated with a microfluidic channel rearranges an applied magnetic field to create a high gradient magnetic field (HGMF). By leveraging the differential magnetic susceptibilities of cell types contained in a host medium, such as paramagnetic red blood cells (RBCs) and diamagnetic white blood cells (WBCs), the resulting HGMF can be used to continuously separate them without attaching additional labels, such as magnetic beads, to them. We describe the effect of these ferromagnetic microstructure geometries have on the blood separation efficacy by numerically simulating the influence of microstructure height and pitch on the HGMF characteristics and resulting RBC separation. Visualizations of RBC trajectories provide insight into how arrays can be optimized to best separate these cells from a host fluid. Periodic microstructures are shown to moderate the applied field due to magnetic interference between the adjacent teeth of an array. Since continuous microstructures do not similarly weaken the resultant HGMF, they facilitate significantly higher RBC separation. Nevertheless, periodic arrays are more appropriate for relatively deep microchannels since, unlike continuous microstructures, their separation effectiveness is independent of depth. The results are relevant to the design of microfluidic devices that leverage HGMFs to fractionate blood by separating RBCs and WBCs. Copyright © 2016 Elsevier B.V. All rights reserved.

A Slowed Cell Cycle Stabilizes the Budding Yeast Genome.

PubMed

Vinton, Peter J; Weinert, Ted

2017-06-01

During cell division, aberrant DNA structures are detected by regulators called checkpoints that slow division to allow error correction. In addition to checkpoint-induced delay, it is widely assumed, though rarely shown, that merely slowing the cell cycle might allow more time for error detection and correction, thus resulting in a more stable genome. Fidelity by a slowed cell cycle might be independent of checkpoints. Here we tested the hypothesis that a slowed cell cycle stabilizes the genome, independent of checkpoints, in the budding yeast Saccharomyces cerevisiae We were led to this hypothesis when we identified a gene ( ERV14 , an ER cargo membrane protein) that when mutated, unexpectedly stabilized the genome, as measured by three different chromosome assays. After extensive studies of pathways rendered dysfunctional in erv14 mutant cells, we are led to the inference that no particular pathway is involved in stabilization, but rather the slowed cell cycle induced by erv14 stabilized the genome. We then demonstrated that, in genetic mutations and chemical treatments unrelated to ERV14 , a slowed cell cycle indeed correlates with a more stable genome, even in checkpoint-proficient cells. Data suggest a delay in G2/M may commonly stabilize the genome. We conclude that chromosome errors are more rarely made or are more readily corrected when the cell cycle is slowed (even ∼15 min longer in an ∼100-min cell cycle). And, some chromosome errors may not signal checkpoint-mediated responses, or do not sufficiently signal to allow correction, and their correction benefits from this "time checkpoint." Copyright © 2017 by the Genetics Society of America.
Live cell imaging of mitochondrial movement along actin cables in budding yeast.

PubMed

Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A

2004-11-23

Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.
In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1.

PubMed

Bovo, Fernanda; Franco, Larissa Tuanny; Rosim, Roice Eliana; Barbalho, Ricardo; de Oliveira, Carlos Augusto Fernandes

2015-06-01

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.
In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1

PubMed Central

Bovo, Fernanda; Franco, Larissa Tuanny; Rosim, Roice Eliana; Barbalho, Ricardo; de Oliveira, Carlos Augusto Fernandes

2015-01-01

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins. PMID:26273277
Design and simulation of a microfluidic device for acoustic cell separation.

PubMed

Shamloo, Amir; Boodaghi, Miad

2018-03-01

Experimental acoustic cell separation methods have been widely used to perform separation for different types of blood cells. However, numerical simulation of acoustic cell separation has not gained enough attention and needs further investigation since by using numerical methods, it is possible to optimize different parameters involved in the design of an acoustic device and calculate particle trajectories in a simple and low cost manner before spending time and effort for fabricating these devices. In this study, we present a comprehensive finite element-based simulation of acoustic separation of platelets, red blood cells and white blood cells, using standing surface acoustic waves (SSAWs). A microfluidic channel with three inlets, including the middle inlet for sheath flow and two symmetrical tilted angle inlets for the cells were used to drive the cells through the channel. Two interdigital transducers were also considered in this device and by implementing an alternating voltage to the transducers, an acoustic field was created which can exert the acoustic radiation force to the cells. Since this force is dependent to the size of the cells, the cells are pushed towards the midline of the channel with different path lines. Particle trajectories for different cells were obtained and compared with a theoretical equation. Two types of separations were observed as a result of varying the amplitude of the acoustic field. In the first mode of separation, white blood cells were sorted out through the middle outlet and in the second mode of separation, platelets were sorted out through the side outlets. Depending on the clinical needs and by using the studied microfluidic device, each of these modes can be applied to separate the desired cells. Copyright © 2017 Elsevier B.V. All rights reserved.
Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations.

PubMed

Han, Ki-Ho; Frazier, A Bruno

2006-02-01

This paper presents the characterization of continuous single-stage and three-stage cascade paramagnetic capture (PMC) mode magnetophoretic microseparators for high efficiency separation of red and white blood cells from diluted whole blood based on their native magnetic properties. The separation mechanism for both PMC microseparators is based on a high gradient magnetic separation (HGMS) method. This approach enables separation of blood cells without the use of additives such as magnetic beads. Experimental results for the single-stage PMC microseparator show that 91.1% of red blood cells were continuously separated from the sample at a volumetric flow rate of 5 microl h-1. In addition, the three-stage cascade PMC microseparator continuously separated 93.5% of red blood cells and 97.4% of white blood cells from whole blood at a volumetric flow rate of 5 microl h-1.
Revealing of Saccharomyces cerevisiae yeast cell wall proteins capable of binding thioflavin T, a fluorescent dye specifically interacting with amyloid fibrils.

PubMed

Gorkovskii, A A; Bezsonov, E E; Plotnikova, T A; Kalebina, T S; Kulaev, I S

2009-11-01

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.
HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

PubMed

Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

2016-01-01

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.
Impact of osmotic stress and ethanol inhibition in yeast cells on process oscillation associated with continuous very-high-gravity ethanol fermentation

PubMed Central

2013-01-01

Background VHG fermentation is a promising process engineering strategy aiming at improving ethanol titer, and thus saving energy consumption for ethanol distillation and distillage treatment. However, sustained process oscillation was observed during continuous VHG ethanol fermentation, which significantly affected ethanol fermentation performance of the system. Results Sustained process oscillation was investigated in continuous VHG ethanol fermentation, and stresses exerted on yeast cells by osmotic pressure from unfermented sugars and ethanol inhibition developed within the fermentation system were postulated to be major factors triggering this phenomenon. In this article, steady state was established for continuous ethanol fermentation with LG medium containing 120 g/L glucose, and then 160 g/L non-fermentable xylose was supplemented into the LG medium to simulate the osmotic stress on yeast cells under the VHG fermentation condition, but the fermentation process was still at steady state, indicating that the impact of osmotic stress on yeast cells was not the main reason for the process oscillation. However, when 30 g/L ethanol was supplemented into the LG medium to simulate the ethanol inhibition in yeast cells under the VHG fermentation condition, process oscillation was triggered, which was augmented with extended oscillation period and exaggerated oscillation amplitude as ethanol supplementation was increased to 50 g/L, but the process oscillation was gradually attenuated when the ethanol supplementations were stopped, and the steady state was restored. Furthermore, gas stripping was incorporated into the continuous VHG fermentation system to in situ remove ethanol produced by Saccharomyces cerevisiae, and the process oscillation was also attenuated, but restored after the gas stripping was interrupted. Conclusions Experimental results indicated that ethanol inhibition rather than osmotic stress on yeast cells is one of the main factors triggering the
Uncoupling reproduction from metabolism extends chronological lifespan in yeast

PubMed Central

Nagarajan, Saisubramanian; Kruckeberg, Arthur L.; Schmidt, Karen H.; Kroll, Evgueny; Hamilton, Morgan; McInnerney, Kate; Summers, Ryan; Taylor, Timothy; Rosenzweig, Frank

2014-01-01

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions. PMID:24706810
Laboratory evolution of copper tolerant yeast strains

PubMed Central

2012-01-01

Background Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts. Results We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts. Conclusions Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper
Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

PubMed

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2016-11-01

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm 2 . This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10 5 to 1.4 × 10 6 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.
Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

PubMed

Yukawa, Masashi; Yamauchi, Tomoaki; Kurisawa, Naoaki; Ahmed, Shakil; Kimura, Ken-Ichi; Toda, Takashi

2018-07-01

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest. Copyright © 2018 Elsevier Inc. All rights reserved.
Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

PubMed Central

Ohnuki, Shinsuke; Okada, Hiroki; Friedrich, Anne; Kanno, Yoichiro; Goshima, Tetsuya; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Tamura, Hiroyasu; Nakamura, Ryo; Hirata, Dai; Fukuda, Hisashi; Shimoi, Hitoshi; Kitamoto, Katsuhiko; Watanabe, Daisuke; Schacherer, Joseph; Akao, Takeshi; Ohya, Yoshikazu

2017-01-01

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies. PMID:28642365
Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.
Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

PubMed

Parry, J M; Sharp, D; Tippins, R S; Parry, E M

1979-06-01

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.
Granulocyte-macrophage colony-stimulating factor responses of oral epithelial cells to Candida albicans.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-06-01

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM-CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM-CSF by ELISA. Fixed organisms, germination-deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM-CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM-CSF secretion. GM-CSF responses were contact-dependent, strain-dependent, required yeast viability and were optimal when the yeast germinated into hyphae.
Outward electron transfer by Saccharomyces cerevisiae monitored with a bi-cathodic microbial fuel cell-type activity sensor.

PubMed

Ducommun, Raphaël; Favre, Marie-France; Carrard, Delphine; Fischer, Fabian

2010-03-01

A Janus head-like bi-cathodic microbial fuel cell was constructed to monitor the electron transfer from Saccharomyces cerevisiae to a woven carbon anode. The experiments were conducted during an ethanol cultivation of 170 g/l glucose in the presence and absence of yeast-peptone medium. First, using a basic fuel-cell type activity sensor, it was shown that yeast-peptone medium contains electroactive compounds. For this purpose, 1% solutions of soy peptone and yeast extract were subjected to oxidative conditions, using a microbial fuel cell set-up corresponding to a typical galvanic cell, consisting of culture medium in the anodic half-cell and 0.5 M K(3)Fe(CN)(6) in the cathodic half-cell. Second, using a bi-cathodic microbial fuel cell, it was shown that electrons were transferred from yeast cells to the carbon anode. The participation of electroactive compounds in the electron transport was separated as background current. This result was verified by applying medium-free conditions, where only glucose was fed, confirming that electrons are transferred from yeast cells to the woven carbon anode. Knowledge about the electron transfer through the cell membrane is of importance in amperometric online monitoring of yeast fermentations and for electricity production with microbial fuel cells. Copyright (c) 2009 John Wiley & Sons, Ltd.
Yeast Immobilization Systems for Alcoholic Wine Fermentations: Actual Trends and Future Perspectives

PubMed Central

Moreno-García, Jaime; García-Martínez, Teresa; Mauricio, Juan C.; Moreno, Juan

2018-01-01

Yeast immobilization is defined as the physical confinement of intact cells to a region of space with conservation of biological activity. The use of these methodologies for alcoholic fermentation (AF) offers many advantages over the use of the conventional free yeast cell method and different immobilization systems have been proposed so far for different applications, like winemaking. The most studied methods for yeast immobilization include the use of natural supports (e.g., fruit pieces), organic supports (e.g., alginate), inorganic (e.g., porous ceramics), membrane systems, and multi-functional agents. Some advantages of the yeast-immobilization systems include: high cell densities, product yield improvement, lowered risk of microbial contamination, better control and reproducibility of the processes, as well as reuse of the immobilization system for batch fermentations and continuous fermentation technologies. However, these methods have some consequences on the behavior of the yeasts, affecting the final products of the fermentative metabolism. This review compiles current information about cell immobilizer requirements for winemaking purposes, the immobilization methods applied to the production of fermented beverages to date, and yeast physiological consequences of immobilization strategies. Finally, a recent inter-species immobilization methodology has been revised, where yeast cells are attached to the hyphae of a Generally Recognized As Safe fungus and remain adhered following loss of viability of the fungus. The bio-capsules formed with this method open new and promising strategies for alcoholic beverage production (wine and low ethanol content beverages). PMID:29497415
The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

PubMed

Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M

2016-05-19

Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

PubMed Central

2018-01-01

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368
Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

PubMed Central

Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

2013-01-01

The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133
Apple Can Act as Anti-Aging on Yeast Cells

PubMed Central

Palermo, Vanessa; Mattivi, Fulvio; Silvestri, Romano; La Regina, Giuseppe; Falcone, Claudio; Mazzoni, Cristina

2012-01-01

In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrence of cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such as ascorbic acid (vitamin C) and polyphenols. The substances that hinder the presence of free radicals are also able to protect cells from aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples and their components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate that all the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all apple components. PMID:22970337
Solving the puzzle of yeast survival in ephemeral nectar systems: exponential growth is not enough.

PubMed

Hausmann, Sebastian L; Tietjen, Britta; Rillig, Matthias C

2017-12-01

Flower nectar is a sugar-rich ephemeral habitat for microorganisms. Nectar-borne yeasts are part of the microbial community and can affect pollination by changing nectar chemistry, attractiveness to pollinators or flower temperature if yeast population densities are high. Pollinators act as dispersal agents in this system; however, pollination events lead potentially to shrinking nectar yeast populations. We here examine how sufficiently high cell densities of nectar yeast can develop in a flower. In laboratory experiments, we determined the remaining fraction of nectar yeast cells after nectar removal, and used honeybees to determine the number of transmitted yeast cells from one flower to the next. The results of these experiments directly fed into a simulation model providing an insight into movement and colonization ecology of nectar yeasts. We found that cell densities only reached an ecologically relevant size for an intermediate pollination probability. Too few pollination events reduce yeast inoculation rate and too many reduce yeast population size strongly. In addition, nectar yeasts need a trait combination of at least an intermediate growth rate and an intermediate remaining fraction to compensate for highly frequent decimations. Our results can be used to predict nectar yeast dispersal, growth and consequently their ecological effects. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis.

PubMed

Alazzam, Anas; Mathew, Bobby; Alhammadi, Falah

2017-03-01

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein-labeled MDA-MB-231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA-MB-231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose-dextrose isotonic medium. The variation in response between MDA-MB-231 cancer cells and normal cells to a certain band of alternating-current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Heterologous expression of anti-apoptotic human 14-3-3β/α enhances iron-mediated programmed cell death in yeast

PubMed Central

Eid, Rawan; Zhou, David R.; Arab, Nagla T. T.; Boucher, Eric; Young, Paul G.; Mandato, Craig A.

2017-01-01

The induction of Programmed Cell Death (PCD) requires the activation of complex responses involving the interplay of a variety of different cellular proteins, pathways, and processes. Uncovering the mechanisms regulating PCD requires an understanding of the different processes that both positively and negatively regulate cell death. Here we have examined the response of normal as well as PCD resistant yeast cells to different PCD inducing stresses. As expected cells expressing the pro-survival human 14-3-3β/α sequence show increased resistance to numerous stresses including copper and rapamycin. In contrast, other stresses including iron were more lethal in PCD resistant 14-3-3β/α expressing cells. The increased sensitivity to PCD was not iron and 14-3-3β/α specific since it was also observed with other stresses (hydroxyurea and zinc) and other pro-survival sequences (human TC-1 and H-ferritin). Although microscopical examination revealed little differences in morphology with iron or copper stresses, cells undergoing PCD in response to high levels of prolonged copper treatment were reduced in size. This supports the interaction some forms of PCD have with the mechanisms regulating cell growth. Analysis of iron-mediated effects in yeast mutant strains lacking key regulators suggests that a functional vacuole is required to mediate the synergistic effects of iron and 14-3-3β/α on yeast PCD. Finally, mild sub-lethal levels of copper were found to attenuate the observed inhibitory effects of iron. Taken together, we propose a model in which a subset of stresses like iron induces a complex process that requires the cross-talk of two different PCD inducing pathways. PMID:28854230
Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
[Study on mechanism of inactivated cider yeast adsorbing patulin by Fourier transform infrared spectroscopy].

PubMed

Guo, Cai-Xia; Yue, Tian-Li; Yuan, Ya-Hong; Wang, Zhou-Li; Wang, Ling; Cai, Rui

2013-03-01

The mechanism of patulin adsorption by inactivated cider yeast was studied by chemical modification and FTIR The results of patulin removal by various modified yeast biomass showed that the ability of patulin biosorption by acetone-treated yeast and NaOH-treated yeast increased siginificantly, while the methylation of amino group and esterification of carboxylate functionalities of yeast cell surface caused a decrease in patulin binding, which indicated that amino group and carboxyl group presented in the cell walls of yeast might be involved in the binding of patulin to the yeast. The FTIR analysis indicated that the main functional groups were amino group, carboxyl group and hydroxy group which are associated with protein and polysaccharides.
How do yeast cells become tolerant to high ethanol concentrations?

PubMed

Snoek, Tim; Verstrepen, Kevin J; Voordeckers, Karin

2016-08-01

The brewer's yeast Saccharomyces cerevisiae displays a much higher ethanol tolerance compared to most other organisms, and it is therefore commonly used for the industrial production of bioethanol and alcoholic beverages. However, the genetic determinants underlying this yeast's exceptional ethanol tolerance have proven difficult to elucidate. In this perspective, we discuss how different types of experiments have contributed to our understanding of the toxic effects of ethanol and the mechanisms and complex genetics underlying ethanol tolerance. In a second part, we summarize the different routes and challenges involved in obtaining superior industrial yeasts with improved ethanol tolerance.
Divergent branches of mitochondrial signaling regulate specific genes and the viability of specialized cell types of differentiated yeast colonies.

PubMed

Podholová, Kristýna; Plocek, Vítězslav; Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena

2016-03-29

Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation.
How does yeast respond to pressure?

PubMed

Fernandes, P M B

2005-08-01

The brewing and baking yeast Saccharomyces cerevisiae has been used as a model for stress response studies of eukaryotic cells. In this review we focus on the effect of high hydrostatic pressure (HHP) on S. cerevisiae. HHP exerts a broad effect on yeast cells characteristic of common stresses, mainly associated with protein alteration and lipid bilayer phase transition. Like most stresses, pressure induces cell cycle arrest. Below 50 MPa (500 atm) yeast cell morphology is unaffected whereas above 220 MPa wild-type cells are killed. S. cerevisiae cells can acquire barotolerance if they are pretreated with a sublethal stress due to temperature, ethanol, hydrogen peroxide, or pressure. Nevertheless, pressure only leads to protection against severe stress if, after pressure pretreatment, the cells are also re-incubated at room pressure. We attribute this effect to the inhibition of the protein synthesis apparatus under HHP. The global genome expression analysis of S. cerevisiae cells submitted to HHP revealed a stress response profile. The majority of the up-regulated genes are involved in stress defense and carbohydrate metabolism while most repressed genes belong to the cell cycle progression and protein synthesis categories. However, the signaling pathway involved in the pressure response is still to be elucidated. Nitric oxide, a signaling molecule involved in the regulation of a large number of cellular functions, confers baroprotection. Furthermore, S. cerevisiae cells in the early exponential phase submitted to 50-MPa pressure show induction of the expression level of the nitric oxide synthase inducible isoform. As pressure becomes an important biotechnological tool, studies concerning this kind of stress in microorganisms are imperative.
Fast kinetic studies of plasmid DNA transfer in intact yeast cells mediated by electropulsation.

PubMed

Ganeva, V; Galutzov, B; Teissie, J

1995-09-25

Intact yeast cell Electrotransformation process was investigated. It is a two step process. The plasmid must be pre-mixed and present in contact with the cells during the pulse. During the millisecond field pulse, plasmid DNA is associated to the envelope. It therefore crosses the membrane by a process which lasts several seconds as shown by its sensitivity to a post pulse addition of DNase. Electrotransformation is not supported by an electrophoretic transfer due to the external field nor by a free diffusion across the electropermeabilized envelope. DNA is first bound during the field pulse and then is transferred by a still unknown active process due to cell metabolism.
Stochastic simulations of a synthetic bacteria-yeast ecosystem

PubMed Central

2012-01-01

Background The field of synthetic biology has greatly evolved and numerous functions can now be implemented by artificially engineered cells carrying the appropriate genetic information. However, in order for the cells to robustly perform complex or multiple tasks, co-operation between them may be necessary. Therefore, various synthetic biological systems whose functionality requires cell-cell communication are being designed. These systems, microbial consortia, are composed of engineered cells and exhibit a wide range of behaviors. These include yeast cells whose growth is dependent on one another, or bacteria that kill or rescue each other, synchronize, behave as predator-prey ecosystems or invade cancer cells. Results In this paper, we study a synthetic ecosystem comprising of bacteria and yeast that communicate with and benefit from each other using small diffusible molecules. We explore the behavior of this heterogeneous microbial consortium, composed of Saccharomyces cerevisiae and Escherichia coli cells, using stochastic modeling. The stochastic model captures the relevant intra-cellular and inter-cellular interactions taking place in and between the eukaryotic and prokaryotic cells. Integration of well-characterized molecular regulatory elements into these two microbes allows for communication through quorum sensing. A gene controlling growth in yeast is induced by bacteria via chemical signals and vice versa. Interesting dynamics that are common in natural ecosystems, such as obligatory and facultative mutualism, extinction, commensalism and predator-prey like dynamics are observed. We investigate and report on the conditions under which the two species can successfully communicate and rescue each other. Conclusions This study explores the various behaviors exhibited by the cohabitation of engineered yeast and bacterial cells. The way that the model is built allows for studying the dynamics of any system consisting of two species communicating with one
Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations.

PubMed

Souza, Raquel Santos; Diaz-Albiter, Hector Manuel; Dillon, Vivian Maureen; Dillon, Rod J; Genta, Fernando Ariel

2016-01-01

Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2 h, 100% in 48 h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.
Yeast 5 – an expanded reconstruction of the Saccharomyces cerevisiae metabolic network

PubMed Central

2012-01-01

stoichiometrically constrained yeast metabolic model. It differs from previous reconstructions and models by emphasizing the distinction between the yeast metabolic reconstruction and the stoichiometrically constrained model, and makes both available as Additional file 4 and Additional file 5 and at http://yeast.sf.net/ as separate systems biology markup language (SBML) files. Through this separation, we intend to make the modeling process more accessible, explicit, transparent, and reproducible. PMID:22663945
Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed

Yaremko, M L; Kelemen, P R; Kutza, C; Barker, D; Westbrook, C A

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.
Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed Central

Yaremko, M. L.; Kelemen, P. R.; Kutza, C.; Barker, D.; Westbrook, C. A.

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible. Images Figure 1 Figure 2 Figure 3 PMID:8546231
Electrical control of cell polarization in the fission yeast Schizosaccharomyces pombe.

PubMed

Minc, Nicolas; Chang, Fred

2010-04-27

Electric signals surround tissues and cells and have been proposed to participate in directing cell polarity in processes such as development, wound healing, and host invasion [1, 2]. The application of exogenous electric fields (EFs) can direct cell polarization in cell types ranging from bacteria and fungi to neurons and neutrophils [3-7]. The mechanisms by which EFs modulate cell polarity, however, remain poorly understood. Here we introduce the fission yeast Schizosaccharomyces pombe as a model organism to elucidate the mechanisms underlying this process. In these rod-shaped cells, an exogenous EF reorients cell growth in a direction orthogonal to the field, producing cells with a bent morphology. A candidate genetic screen identifies conserved factors involved in this process: an integral membrane proton ATPase pma1p that regulates intracellular pH, the small GTPase cdc42p, and the formin for3p that assembles actin cables. Interestingly, mutants in these genes still respond to the EF but orient in a different direction, toward the anode. In addition, EFs also cause electrophoretic movement of cell wall synthase complex proteins toward the anode. These data suggest molecular models for how the EF reorients cell polarization by modulating intracellular pH and steering cell polarity factors in multiple directions. Copyright © 2010 Elsevier Ltd. All rights reserved.
An Evolutionary Perspective on Yeast Mating-Type Switching

PubMed Central

Hanson, Sara J.; Wolfe, Kenneth H.

2017-01-01

Cell differentiation in yeast species is controlled by a reversible, programmed DNA-rearrangement process called mating-type switching. Switching is achieved by two functionally similar but structurally distinct processes in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In both species, haploid cells possess one active and two silent copies of the mating-type locus (a three-cassette structure), the active locus is cleaved, and synthesis-dependent strand annealing is used to replace it with a copy of a silent locus encoding the opposite mating-type information. Each species has its own set of components responsible for regulating these processes. In this review, we summarize knowledge about the function and evolution of mating-type switching components in these species, including mechanisms of heterochromatin formation, MAT locus cleavage, donor bias, lineage tracking, and environmental regulation of switching. We compare switching in these well-studied species to others such as Kluyveromyces lactis and the methylotrophic yeasts Ogataea polymorpha and Komagataella phaffii. We focus on some key questions: Which cells switch mating type? What molecular apparatus is required for switching? Where did it come from? And what is the evolutionary purpose of switching? PMID:28476860
Prion-based memory of heat stress in yeast

PubMed Central

Chernova, Tatiana A.; Wilkinson, Keith D.

2017-01-01

ABSTRACT Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices. We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses. PMID:28521568

Prion-based memory of heat stress in yeast.

PubMed

Chernova, Tatiana A; Chernoff, Yury O; Wilkinson, Keith D

2017-05-04

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices. We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.
Method for separating biological cells. [suspended in aqueous polymer systems

NASA Technical Reports Server (NTRS)

Brooks, D. E. (Inventor)

1980-01-01

A method for separating biological cells by suspending a mixed cell population in a two-phase polymer system is described. The polymer system consists of droplet phases with different surface potentials for which the cell populations exhibit different affinities. The system is subjected to an electrostatic field of sufficient intensity to cause migration of the droplets with an attendant separation of cells.
Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals.

PubMed

Borodina, Irina; Nielsen, Jens

2014-05-01

Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals "aging factors" and mechanism of lifespan asymmetry.

PubMed

Yang, Jing; McCormick, Mark A; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K; Li, Hao

2015-09-22

Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.
Preparation of Total RNA from Fission Yeast.

PubMed

Bähler, Jürg; Wise, Jo Ann

2017-04-03

Treatment with hot phenol breaks open fission yeast cells and begins to strip away bound proteins from RNA. Deproteinization is completed by multiple extractions with chloroform/isoamyl alcohol and separation of the aqueous and organic phases using MaXtract gel, an inert material that acts as a physical barrier between the phases. The final step is concentration of the RNA by ethanol precipitation. The protocol can be used to prepare RNA from several cultures grown in parallel, but it is important not to process too many samples at once because delays can be detrimental to RNA quality. A reasonable number of samples to process at once would be three to four for microarray or RNA sequencing analyses and six for preliminary investigations of mutants implicated in RNA metabolism. © 2017 Cold Spring Harbor Laboratory Press.
Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

PubMed Central

Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

2015-01-01

Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575
Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

PubMed

Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A; Holik, John; Grunstein, Michael; Kaufman, Paul D; Friedman, Nir; Rando, Oliver J

2008-11-01

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.
Aerospace nickel-cadmium cell separator qualifications program

NASA Technical Reports Server (NTRS)

Francis, R. W.; Haag, R. L.

1986-01-01

The present space qualified nylon separator, Pellon 2505 ML, is no longer available for aerospace nickel-cadmium (NiCd) cells. As a result of this anticipated unavailability, a joint Government program between the Air Force Space Division and the Naval Research Laboratory was established. Four cell types were procured with both the old qualified and the new unqualified separators. Acceptance, characterization, and life cycling tests are to be performed at the Naval Weapons Support Center, Crane, Ind. (NWSC/Crane). The scheduling and current status of this program are discussed and the progress of testing and available results are projected.
Raman scattering evidence of hydrohalite formation on frozen yeast cells.

PubMed

Okotrub, K A; Surovtsev, N V

2013-02-01

We studied yeast cells in physiological solution during freezing by Raman microspectroscopy technique. The purpose was to find out the origin of a sharp peak near ∼3430cm(-1) in Raman spectrum of frozen mammalian cells, observed earlier (J. Dong et al., Biophys. J. 99 (2010) 2453), which presumably could be used as an indicator of intracellar ice appearance. We have shown that this line (actually doublet of 3408 and 3425cm(-1)) corresponds to Raman spectrum of hydrohalite (NaCl⋅2H(2)O), which is formed as the result of the eutectic crystallization of the liquid solution around the cells. We also show that the spatial distribution of hydrohalite in the sample significantly depends on the cooling rate. At lower cooling rate (1°C/min), products of eutectic crystallization form layer on the cell surface which thickness varies for different cells and can reach ∼1μm in thickness. At higher cooling rate (20°C/min), the hydrohalite distribution appears more homogeneous, in the sample, and the eutectic crystallization layer around the cells was estimated to be less than ∼20nm. These experimental results are consistent with scenarios predicted by the two-factor hypothesis for freezing induced cell injury. This work demonstrates a potential of Raman microspectroscopy to study peculiarities of the eutectic crystallization around single cells in vivo with the high spatial resolution. Copyright © 2012 Elsevier Inc. All rights reserved.
Metabolic regulation and maximal reaction optimization in the central metabolism of a yeast cell

NASA Astrophysics Data System (ADS)

Kasbawati, Gunawan, A. Y.; Hertadi, R.; Sidarto, K. A.

2015-03-01

Regulation of fluxes in a metabolic system aims to enhance the production rates of biotechnologically important compounds. Regulation is held via modification the cellular activities of a metabolic system. In this study, we present a metabolic analysis of ethanol fermentation process of a yeast cell in terms of continuous culture scheme. The metabolic regulation is based on the kinetic formulation in combination with metabolic control analysis to indicate the key enzymes which can be modified to enhance ethanol production. The model is used to calculate the intracellular fluxes in the central metabolism of the yeast cell. Optimal control is then applied to the kinetic model to find the optimal regulation for the fermentation system. The sensitivity results show that there are external and internal control parameters which are adjusted in enhancing ethanol production. As an external control parameter, glucose supply should be chosen in appropriate way such that the optimal ethanol production can be achieved. For the internal control parameter, we find three enzymes as regulation targets namely acetaldehyde dehydrogenase, pyruvate decarboxylase, and alcohol dehydrogenase which reside in the acetaldehyde branch. Among the three enzymes, however, only acetaldehyde dehydrogenase has a significant effect to obtain optimal ethanol production efficiently.
The impact of yeast fermentation on dough matrix properties.

PubMed

Rezaei, Mohammad N; Jayaram, Vinay B; Verstrepen, Kevin J; Courtin, Christophe M

2016-08-01

Most studies on dough properties are performed on yeastless dough to exclude the complicating, time-dependent effect of yeast. Baker's yeast, however, impacts dough matrix properties during fermentation, probably through the production of primary (CO2 and ethanol) and secondary (glycerol, acetic acid and succinic acid) metabolites. The aim of this study is to obtain a better understanding of the changes in yeasted dough behavior introduced by fermentation, by investigating the impact of yeast fermentation on Farinograph dough consistency, dough spread, Kieffer rig dough extensibility and gluten agglomeration behavior in a fermented dough-batter gluten starch separation system. Results show that fermentation leads to a dough with less flow and lower extensibility that breaks more easily under stress and strain. The dough showed less elastic and more plastic deformation behavior. Gluten agglomerates were smaller for yeasted dough than for the unyeasted control. These changes probably have to be attributed to metabolites generated during fermentation. Indeed, organic acids and also ethanol in concentrations produced by yeast were previously shown to have similar effects in yeastless dough. These findings imply the high importance of yeast fermentation metabolites on dough matrix properties in industrial bread production. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.
Nonmutagenic carcinogens induce intrachromosomal recombination in dividing yeast cells.

PubMed

Schiestl, R H

1993-12-01

A large number of animal and human carcinogens without apparent genotoxic activity exist (nonmutagenic carcinogens) that are difficult or impossible to detect with the currently used short-term tests. Because of the association of carcinogenesis with genome rearrangement, a system selecting for intrachromosomal recombination (DEL recombination) that results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae. Because DEL recombination is under different genetic control than interchromosomal recombination and meiotic recombination, it is probably due to a different mechanism. It has been found that DEL recombination is readily inducible by 10 mutagenic carcinogens and 17 nonmutagenic carcinogens that are not detectable (false negatives) with the Ames assay. In addition, three out of four mutagens that do not cause cancer (false positives in the Ames assay) do not induce DEL recombination. DEL recombination is inducible by UV only in dividing cells but not in cells synchronized in the G1 or G2 phase of the cell cycle. Interchromosomal recombination, on the other hand, is inducible in G1 but not in G2. The nonmutagenic carcinogens induce DEL recombination only in actively growing cells, which may give some indication as to their mechanism. Further characterization of the mechanism involved in induction of DEL recombination may contribute to the understanding of the biological activity of nonmutagenic carcinogens.
EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

PubMed Central

Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

2014-01-01

Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362
Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

NASA Technical Reports Server (NTRS)

Marina, Diana B.; Santa Maria, Sergio; Bhattacharya, Sharmila

2016-01-01

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate
Fuel cell separator with compressible sealing flanges

DOEpatents

Mientek, A.P.

1984-03-30

A separator for separating adjacent fuel cells in a stack of such cells includes a flat, rectangular, gas-impermeable plate disposed between adjacent cells and having two opposite side margins thereof folded back over one side of the plate to form two first seal flanges and having the other side margins thereof folded back over the opposite side of the plate to form two second seal flanges, each of the seal flanges cooperating with the plate to define a channel in which is disposed a resiliently compressible stack of thin metal sheets. The two first seal flanges cooperate with the electrolyte matrix of one of the cells to form a gas-impermeable seal between an electrode of the one cell and one of two reactant gas manifolds. The second seal flanges cooperate with the electrolyte matrix of the other cell for forming a gas-impermeable seal between an electrode of the other cell and the other of the two reactant gas manifolds. The seal flanges cooperate with the associated compressible stacks of sheets for maintaining a spacing between the plate and the electrolyte matrices while accommodating variation of that spacing.
Fuel cell separator with compressible sealing flanges

DOEpatents

Mientek, Anthony P.

1985-04-30

A separator for separating adjacent fuel cells in a stack of such cells includes a flat, rectangular, gas-impermeable plate disposed between adjacent cells and having two opposite side margins thereof folded back over one side of the plate to form two first seal flanges and having the other side margins thereof folded back over the opposite side of the plate to form two second seal flanges, each of the seal flanges cooperating with the plate to define a channel in which is disposed a resiliently compressible stack of thin metal sheets. The two first seal flanges cooperate with the electrolyte matrix of one of the cells to form a gas-impermeable seal between an electrode of the one cell and one of two reactant gas manifolds. The second seal flanges cooperate with the electrolyte matrix of the other cell for forming a gas-impermeable seal between an electrode of the other cell and the other of the two reactant gas manifolds. The seal flanges cooperate with the associated compressible stacks of sheets for maintaining a spacing between the plate and the electrolyte matrices while accommodating variation of that spacing.
Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe

PubMed Central

Duhig, Trevor; Nam, Miyoung; Palmer, Georgia; Han, Sangjo; Jeffery, Linda; Baek, Seung-Tae; Lee, Hyemi; Shim, Young Sam; Lee, Minho; Kim, Lila; Heo, Kyung-Sun; Noh, Eun Joo; Lee, Ah-Reum; Jang, Young-Joo; Chung, Kyung-Sook; Choi, Shin-Jung; Park, Jo-Young; Park, Youngwoo; Kim, Hwan Mook; Park, Song-Kyu; Park, Hae-Joon; Kang, Eun-Jung; Kim, Hyong Bai; Kang, Hyun-Sam; Park, Hee-Moon; Kim, Kyunghoon; Song, Kiwon; Song, Kyung Bin; Nurse, Paul; Hoe, Kwang-Lae

2014-01-01

SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth. PMID:20473289
Utilization of brewery wastewater for culturing yeast cells for use in river water remediation.

PubMed

Chang, Su-Yun; Sun, Jing-Mei; Song, Shu-Qiang; Sun, Bao-Sheng

2012-01-01

Successful in situ bio-augmentation of contaminated river water involves reducing the cost of the bio-agent. In this study, brewery wastewater was used to culture yeast cells for degrading the COD(Cr) from a contaminated river. The results showed that 15 g/L of yeast cells could be achieved after being cultured in the autoclaved brewery wastewater with 5 mL/L of saccharified starch and 9 g/L of corn steep liquor. The COD(Cr) removal efficiency was increased from 22% to 33% when the cells were cultured using the mentioned method. Based on the market price of materials used in this method, the cost of the medium for remediating 1 m3 of river water was 0.0076 US dollars. If the additional cost of field implementation is included, the total cost is less than 0.016 US dollars for treating 1 m3 of river water. The final cost was dependent on the size of remediation: the larger the scale, the lower the cost. By this method, the nutrient in the brewery wastewater was reused, the cost of brewery wastewater treatment was saved and the cost of the remediation using bio-augmentation was reduced. Hence, it is suggested that using brewery wastewater to culture a bio-agent for bio-augmentation is a cost-effective method.
Micro-Biocidal Activity of Yeast Cells by Needle Plasma Irradiation at Atmospheric Pressure

NASA Astrophysics Data System (ADS)

Kurumi, Satoshi; Takahashi, Hideyuki; Taima, Tomohito; Suzuki, Kaoru; Hirose, Hideharu; Masutani, Shigeyuki

In this study, we report on the biocidal activity technique by needle helium plasma irradiation at atmospheric pressure using borosilicate capillary nozzle to apply for the oral surgery. The diameter of needle plasma was less than 50 µm, and temperature of plasma irradiated area was less than body temperature. Needle plasma showed emission due to OH and O radical. Raman spectra and methylene blue stain showed yeast cells were inactivated by needle plasma irradiation.
Immobilised Sarawak Malaysia yeast cells for production of bioethanol.

PubMed

Zain, Masniroszaime Mohd; Kofli, Noorhisham Tan; Rozaimah, Siti; Abdullah, Sheikh

2011-05-01

Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.

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