Living Yeast Cells Research Articles

Dominant and genome-wide formation of DNA:RNA hybrid G-quadruplexes in living yeast cells

Guanine-rich DNA forms G-quadruplexes (G4s) that play a critical role in essential cellular processes. Previous studies have mostly focused on intramolecular G4s composed of four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has not been strictly confirmed in the genome of living eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and two other independent G4-specific detections reveal that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of potential G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s readily form and even dominate at G4 sites that are theoretically capable of forming the intramolecular DNA G4s (dG4s) by themselves. Compared to dG4s, hG4s exhibit broader kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing it to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also establishes a sensitive and currently the only method for detecting the structural form of G4s in living cells.

Sterol-lipids enable large-scale, liquid-liquid phase separation in bilayer membranes of only 2 components.

A wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined "sterol-lipid" molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

Dielectrophoretic cell sorting with high velocity enabled by two-layer sidewall microelectrodes extending along the entire channel

Dielectrophoresis has been widely applied in microfluidic cell separation, and it enables label-free and size-independent cell sorting based on their dielectric fingerprints which is prerequisite for downstream biomedical analysis. The linear flow velocity and cross-sectional area are the two key factors proportional to the flow rate and thus directly related to the sample processing throughput. Aiming at higher flow rate, the state-of-art techniques mostly focus on the cross-sectional area, either by using wide chamber with angled electrode digits or 3D volumetric electrodes with expanded channel depth. On the other hand, the velocity is always carefully limited because cells have very squeezed time to interact with the DEP force that drives them to migrate, and the flow drag induced by high velocity potentially poses adverse effect against the DEP-driven cell migration. In this paper, we present DEP-activated cell sorting at high-velocity being 1–2 orders of magnitude higher than the existing DEP separators, which is enabled by easily-fabricated two-layer 3D sidewall electrodes extending along the entire flow channel. Such electrodes project DEP force fully decoupled from the flow drag, and acting on cells continuously, allowing sufficient time for cell migration. The two-layer design leads to cell migration across a shorter distance between the upper and lower layers, with a unique bridge adaptor evolved at the downstream for the collection of the cells vertically separated. The DEP device achieves separation of live and dead yeast cells at high velocity up to 78.3 mm/s, more than a full order of magnitude higher compared to any reported DEP platforms. This velocity corresponds to a competitive flow rate of 3.1 mL/h given the fairly small cross-sectional area, where the averaged collection rate is over 95 %. For the separation between the live and dead Hela cells, our device enables a high velocity up to 50.5 mm/s with an averaged collection rate of ∼85 %. A high throughput of 1.79 × 1 0^(5) cells/min for the live and dead Hela cell separation has been achieved with 89.7 % collection for live cells and a velocity of 30.3 mm/s.

Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

Power loss model of electromagnetic waves propagation in cell solutions by GHz electrical impedance spectroscopy

The propagation of GHz electromagnetic (EM) waves across cells in cell solutions has been analytically modeled and numerically calculated in order to elucidate the power loss in the boundary between dispersed medium and cell by establishing a theoretical model. Living and dead yeast cells are chosen as objects because of the simple cell structure and ease of observation under optical microscope. Through the model, the average power density of the incident wave S avi , reflected wave S avr , transmitted wave S avt , and ratio of the power loss ψ are calculated and compared to analyze the power loss of EM waves inside living and dead yeast cells by considering the impacts of frequency of EM wave, cell viability, concentration, and component structures of the cell. Results demonstrate decreased S avi, S avr , and S avt with rising frequency, especially noticeable below 100 MHz due to enhanced absorption from cell components. EM waves in living yeast cell solutions exhibit faster attenuation and stronger reflection compared to dead yeast cells, attributed to intact organelles and membranes intensifying absorption and scattering. The increasing cells concentration further attenuates EM waves. This work elucidates propagation and power loss of EM waves in cell solutions and provides an effective computational approach to optimize EM wave based biomedical applications.

Retracted: High-Resolution Microscopy to Learn the Nuclear Organization of the Living Yeast Cells.

[This retracts the article DOI: 10.1155/2021/9951114.].

APPLICATION OF THE FEED ADDITIVE PRODUCTIV IN THE FEEDING OF HIGH-YIELDING COWS

As a result of the conducted research, the positive effect of the feed additive "Productiv" based on live yeast cells of Saccharomyces cerevisiae in the amount of 10 grams per head per day on the productivity of cows was established. The use of the feed additive "Productiv" in the diet of cows contributes to obtaining additional 1.8 kg of milk with 3.6% fat content per cow per day, and additional profit for the period of experience in the amount of 131.76 rubles per cow.

PSVI-29 Using Feeding Behavior Assessment to Evaluate the Effects of Beef Cattle Dietary Type Or the Presence of Saccharomyces Cerevisiae

Abstract The effects of live yeast and diet type (grower or finisher) offered to beef steers on feeding behavior were evaluated. Eight ruminally cannulated yearling beef steers (BW = 550 ± 20 kg) were used in a duplicated 4 × 4 Latin Square design and offered four dietary treatments to ad libitum intake, following a 2 × 2 factorial treatment arrangement: A) presence or not of live-yeast Saccharomyces cerevisiae [2 × 1010 CFU/g (CNCM I-1077)]; and B) diet type. Diets (DM basis) contained 3% yellow-grease and mineral/vitamin supplement-adjusted. The grower [40% low-quality alfalfa-hay (LQ-hay), 35% steam-flaked corn (SFC), 20% wet corn gluten feed (WCGF)] and finisher (65% SFC, 20% WCGF, 8% LQ-hay) diets were offered once daily at 0700 h. Live-yeast cells or placebo (soybean meal) were delivered with gel capsules (0.25 g, as is) twice daily directly into the rumen via ruminal cannula. Four, 35 d periods were used, in which feeding behavior assessment was conducted by trained personnel on d 31 (continuous 24 h, every 5 min) while no other major management was performed during the evaluation. The time spent eating, ruminating (standing/laying), resting (stating/laying), active, and drinking were recorded, while chewing was estimated by adding rumination and eating time. The number of meals and time spent on each meal were also assessed. Data were analyzed using the GLIMMIX procedures of SAS, with animal considered as the experimental unit. The statistical model used the fixed effects of diet type, live-yeast presence, and the diet × live-yeast interaction, while square, period, and animal(square) were used as random effects. No diet × live-yeast interactions (P ≥ 0.17) or main effects of live-yeast (P ≥ 0.23) were observed for the variables measured. Feeding behavior was affected by diet type, in which steers offered a grower diet had greater time spent ruminating (341 vs. 231 min/d; P < 0.01), eating (154 vs. 105 min/d; P < 0.01), chewing (494 vs. 336 min/d; P < 0.01), and meal length (13 vs. 10 min/meal; P < 0.01), while less time resting (838 vs. 973 min/d; P < 0.01) compared with those offered the finisher diet. The time spent drinking (15 min/d; P = 0.29) and the number of daily meals (13 meals/d; P = 0.66) were not affected by treatments, while steers offered the finisher diet tended (P = 0.12) to be more active than those offered the grower diet (116 vs. 95 min/d). As expected, grower diets were more prone than finisher diets to stimulate feeding behavior variables of beef steers. Moreover, when directly introduced inside the rumen via ruminal cannula, the live yeast used seemed to not affect the feeding behavior of beef steers consuming a steam-flaked corn-based grower or finisher diet.

Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining.

Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in Saccharomyces cerevisiae and Ogataea polymorpha yeasts. The protocol for the DNA content assay was also optimized to be suitable for both Ogataea and Saccharomyces yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.

Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

Graphene-encapsulated yeast cells in harsh conditions

Yeast, as a versatile microorganism, holds significant importance in various industries and research fields due to its remarkable characteristics. In the pursuit of biotechnological applications, cell-surface engineering including encapsulation has been proposed as a new strategy to interface with individual living yeast cells. While previous researches of yeast encapsulation with materials have shown promise, it often involves complex processes and lacks confirmation of condition-dependent yeast viability under harsh conditions. To address these issues, we present a rational and facile design for graphene-encapsulated yeast cells. Through a straightforward blending technique, yeast cells are encapsulated with graphene layers, demonstrating the unique properties of yeast cells in structural and functional aspects with graphene. We show graphene layer-dependent functions of yeast cells under various conditions, including pH and temperature-dependent conditions. The layer of graphene can induce the delayed lag time without the transfer of graphene-layered membrane. Our findings highlight the high potential of graphene-encapsulated yeast cells for various industrial applications, offering new avenues for exploration in biotechnology.

Aureobasidium pullulans S2 controls tomato gray mold and produces volatile organic compounds and biofilms

Gray mold emergence is detrimental to the production, transportation, and storage of tomatoes. The application of antagonistic microorganisms to control fruit diseases is a potential alternative to fungicides. This study investigated the effect of Aureobasidium pullulans S2 on controlling gray mold in tomatoes and explored the possible mechanisms. The results demonstrated that A. pullulans S2 effectively controlled the incidence of gray mold in tomatoes. The response of Botrytis cinerea to different components of the A. pullulans S2 culture showed that live yeast cells had B. cinerea inhibition ability. Environmental adaptability, host resistance induction, biofilm formation and production of volatile organic compounds (VOCs) with antimicrobial effects could also be important mechanisms of action of A. pullulans S2. The VOCs were identified, among which 2-phenylethanol, 2-heptanol and acetic acid, octyl ester could play an important role in B. cinerea inhibition. Ultimately, A. pullulans S2 may be a possible alternative to fungicides in managing tomato fruit post-harvest diseases.

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT).

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, formore consistent expression compared to plasmid-borne constructs. mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Non-invasive monitoring of microbial triterpenoid production using nonlinear microscopy techniques.

Introduction: Bioproduction of plant-derived triterpenoids in recombinant microbes is receiving great attention to make these biologically active compounds industrially accessible as nutraceuticals, pharmaceutics, and cosmetic ingredients. So far, there is no direct method for detecting triterpenoids under physiological conditions on a cellular level, information yet highly relevant to rationalizing microbial engineering. Methods: Here, we show in a proof-of-concept study, that triterpenoids can be detected and monitored in living yeast cells by combining coherent anti-Stokes Raman scattering (CARS) and second-harmonic-generation (SHG) microscopy techniques. We applied CARS and SHG microscopy measurements, and for comparison classical Nile Red staining, on immobilized and growing triterpenoid-producing, and non-producing reference Saccharomyces cerevisiae strains. Results and Discussion: We found that the SHG signal in triterpenoid-producing strains is significantly higher than in a non-producing reference strain, correlating with lipophile content as determined by Nile red staining. In growing cultures, both CARS and SHG signals showed changes over time, enabling new insights into the dynamics of triterpenoid production and storage inside cells.

Two phenanthro[9,10-d]imidazole-based fluorescence probes for distinguishable detection of Cys and Fe3+ and their applications in food and water as well as living cells monitoring

Accurately designing and developing the dual responsive fluorescence probe with simultaneous recognition of intracellular biothiols and metal ions is of great significance. Here, we reported two phenanthrene[9,10-d]imidazole derivatives (PIP-CHO and NO2-PIP-CHO) modified with aldehyde fragment as chemosensor for synchronous detection of Cys and Fe3+. PIP-CHO and NO2-PIP-CHO performed the hydrophilic nature despite the fact that they possessed the distinct aggregation behavior with the increasing of water contents. In aqueous medium (water/DMF), PIP-CHO predominantly exhibited high sensitivity and selectivity for “on–off” fluorescent recognition of Fe3+ with a limit of detection (LOD) of 213.74 nmol·L–1, and Cys with a LOD of 116.66 nmol·L–1, respectively. Correspondingly, the detection limits of NO2-PIP-CHO toward Fe3+ and Cys were calculated in DMF as 347.68 and 215.64 nmol·L–1, respectively. The fluorescent recognition mechanism of cycloaddition and coordination for PIP-CHO with Cys and Fe3+ was rationally explained by SEM, 1H NMR and ESI-MS, respectively. Noteworthy, the naked-eye strips encompassing PIP-CHO enabled distinguishable visualization of Cys and Fe3+ with the color change under visible or UV light. In addition, PIP-CHO can be successfully applied to detect Cys and Fe3+ in food and water samples by fluorescence method, as well as the living Yeast cells via fluorescence dual channel.

Biocompatible Silica-Polyethylene Glycol-Based Composites for Immobilization of Microbial Cells by Sol-Gel Synthesis.

Biocatalysts based on the methylotrophic yeast Ogataea polymorpha VKM Y-2559 immobilized in polymer-based nanocomposites for the treatment of methanol-containing wastewater were developed. The organosilica composites with different matrix-to-filler ratios derived from TEOS/MTES in the presence of PEG (SPEG-composite) and from silicon-polyethylene glycol (STPEG-composite) differ in the structure of the silicate phase and its distribution in the composite matrix. Methods of fluorescent and scanning microscopy first confirmed the formation of an organosilica shell around living yeast cells during sol-gel bio-STPEG-composite synthesis. Biosensors based on the yeast cells immobilized in STPEG- and SPEG-composites are characterized by effective operation: the coefficient of sensitivity is 0.85 ± 0.07 mgO2 × min-1 × mmol-1 and 0.87 ± 0.05 mgO2 × min-1 × mmol-1, and the long-term stability is 10 and 15 days, respectively. The encapsulated microbial cells are protected from UV radiation and the toxic action of heavy metal ions. Biofilters based on the developed biocatalysts are characterized by high effectiveness in the utilization of methanol-rich wastewater-their oxidative power reached 900 gO2/(m3 × cycle), and their purification degree was up to 60%.

Live Yeast Cells in Nutrition of Monogastric Animals

Live Yeast Cells in Nutrition of Monogastric Animals

Abstract 2356: Malate dehydrogenase-citrate synthase multienzyme complex formation is associated with the pH changes in the mitochondrial matrix in living yeast cells

Abstract 2356: Malate dehydrogenase-citrate synthase multienzyme complex formation is associated with the pH changes in the mitochondrial matrix in living yeast cells

Self-decorating cells via surface-initiated enzymatic controlled radical polymerization.

Through the innovative use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of Saccharomyces cerevisiae cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant change in yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering.