Cells Of Yeast Saccharomyces Cerevisiae Research Articles

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast

BackgroundHuman hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication–coupled epigenetic inheritance process, and chromatin stability.ResultsBy comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability.ConclusionOur findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.

Study of the adjuvant properties of beta-glucans from <i>Saccharomyces Cerevisiae</i> yeast

To increase the effectiveness and immunogenicity of modern vaccines, especially subunit ones, it is required to use adjuvants. Polysaccharides, due to their safety and biocompatibility, are desirable candidates for the creation of vaccine adjuvants. The aim of our study was to develop a method for obtaining beta-Glucans from the yeast Saccharomyces cerevisiae cell wall, and evaluate their adjuvant properties. The high purity and non-toxicity of the resulting preparation was achieved by using enzyme complexes of cellulase and protease in combination with ultrasound (22 kHz) at the purification stage. The developed scheme allows for the yield of beta-Glucans up to 2 g from 100 g of the biomass of wet cells. The adjuvant properties of beta-Glucans were studied in 50 male BALB/c mice, weighing 16–18 g. Immunization was performed twice, with a 14-day interval, intramuscularly, 200 μl per animal. The recombinant receptor-binding domain (RBD) of the surface S protein of the SARS-CoV-2 virus (Wuhan-Hu-1 and B.1.617.2 (Delta)) was used as an antigen, at a dose of 50 μg per animal. A positive control group was administered with the antigen combined with aluminum hydroxide. As a negative control, mice injected with the saline solution were used. The titers of specific antibodies in the blood sera were determined by ELISA assays. RBD (Wuhan-Hu-1 and Delta), and S protein (Wuhan-Hu-1, Delta and Omicron) were used as antigens. The titers of virus-neutralizing antibodies were measured in neutralization tests using SARS-CoV-2 virus strains Wuhan-Hu-1, Delta (B.1.617.2) and Omicron (B.1.1.529). The results of the study have shown that beta-Glucans have the ability to enhance the production of specific and virus-neutralizing antibodies in mice immunized with RBD. The titers of specific and virus neutralizing antibodies are comparable to their levels in the group immunized with RBD and Al(OH)₃. It has been found in the experiments in white outbred ICR mice that the preparation belongs to practically non-toxic substances. Therefore, it can be concluded that the use of beta-Glucans could become a preferable alternative to the conventional adjuvants based on aluminum salts, being biocompatible, biodegradable and non-toxic substances of low labor-intensive production.

The plant rhabdovirus viroporin P9 facilitates insect-mediated virus transmission in barley.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of glycine 14 to threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-GFP (BYGFP) virus, a P9-deleted mutant (BYGFPΔP9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFPΔP9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission.

Reduction of Ferric Chloride in Yeast Growth Media, by Sugars and Aluminum

Iron compounds can be used in antimicrobial applications by exploiting the toxicity of divalent iron to living organisms due to the Fenton reaction. In this study, the growth inhibitory effects of ferrous sulfate FeSO4·7H2O and ferric chloride FeCl3·6H2O were observed on Metschnikowia clade and Saccharomyces cerevisiae yeast cells. The relatively high amount of reduced Fe3+ to Fe2+ in the growth medium determined by Mössbauer spectroscopy may contribute to the antimicrobial activity of ferric chloride. In order to test the reducing ability of sugars in the growth media of yeasts, the reaction of ferric chloride FeCl3·6H2O with sugars was investigated. In mixtures of FeCl3·6H2O and fructose, approximately two thirds of Fe3+ can be reduced to Fe2+. When the mixture of FeCl3·6H2O and fructose is placed on the surface of aluminum foil, an iron film is formed on the surface of the aluminum due to the reduction by both fructose and aluminum. The relative amount of Fe3+ which was reduced to Fe0 reached 68%.

Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Abscisic acid root-to-shoot translocation by transporter AtABCG25 mediates stomatal movements in Arabidopsis.

The phytohormone abscisic acid (ABA) plays a central role in regulating stomatal movements under drought conditions. The root-derived peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 25 (CLE25) moves from the root to shoot for activating ABA biosynthesis under drought conditions. However, the root-to-shoot translocation of root-derived ABA and its regulation of stomatal movements in the shoot remain to be clarified. Here, we reveal that the ABA transporter ATP-binding cassette subfamily G member 25 (AtABCG25) mediates root-to-shoot translocation of ABA and ABA-glucosyl ester (ABA-GE) in Arabidopsis (Arabidopsis thaliana). Isotope-labeled ABA tracer experiments and hormone quantification in xylem sap showed that the root-to-shoot translocation of ABA and ABA-GE was substantially impaired in the atabcg25 mutant under nondrought and drought conditions. However, the contents of ABA and ABA-GE in the leaves were lower in the atabcg25 mutant than in the wild type (WT) under nondrought but similar under drought conditions. Consistently, the stomatal closure was suppressed in the atabcg25 mutant under nondrought but not under drought conditions. The transporter activity assays showed that AtABCG25 directly exported ABA and ABA-GE in planta and in yeast (Saccharomyces cerevisiae) cells. Thus, we proposed a working model in which root-derived ABA transported by AtABCG25 via xylem mediates stomatal movements in the shoot under nondrought conditions but might exhibit little effect on stomatal movements under drought conditions. These findings extend the functions of AtABCG25 and provide insights into the long-distance translocation of ABA and its role in stomatal movements.

Encapsulation and characterization of catechin and epicatechin microcapsules using yeast cell biocarriers

In this study, catechin and epicatechin compounds were encapsulated using Saccharomyces cerevisiae yeast cell and fabricated yeast cell microcarriers were subjected to bioactive analysis, conformational and morphological characterization. Encapsulation efficiency values of the yeast cell microcarriers were 24.74 and 31.87% for the epicatechin and catechin loaded yeast cell microcapsules, respectively. Total flavonoid content of the epicatechin and catechin loaded yeast cell microcapsules were determined to be 79.67 and 61.86 mg CE/g while the antiradical activity of the samples was in the range of 4.16-39.24%. FT-IR and XRD spectrum revealed that the catechin and epicatechin were encapsulated by the yeasts cell successfully.

Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae.

The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.

Chalcone derivatives disrupt cell membrane integrity of Saccharomyces cerevisiae cells and alter their biochemical composition.

Fungal infections can be serious or life threatening in severe cases, and the need to discover and find novel antifungal agents persists. Chalcones are plant-derived aromatic compounds that have been appealing synthons for pharmaceutical industry as they have good anticancer, antibacterial, antifungal and anti-inflammatory properties. Although there are few structure-activity relationship studies on chalcones, studies that link the structural features of these compounds to their mode of action are scant. Thus, in this study, we aim to clarify the relationship between chalcone derivatives and their cellular target within the yeast cell Saccharomyces cerevisiae. We observed that some chalcone compounds lead to disruption of cell membrane and cause ion leakage out of the cell. Moreover, chalcones alter the biochemical composition of yeast cells detectable by FTIR spectroscopy and bind to the DNA as shown by our titration experiments based on UV-Vis absorbance spectroscopy. Thus, their interaction with the DNA may be the major impact of these compounds on yeast cells.

Comparison of radiation protection effects between epigallocatechin gallate and ascorbic acid

Ionizing radiation can originate from naturally occurring radiation sources on the earth or it can be from man-made sources. When interacting with cells and living organisms, ionizing radiation produces free radicals, impacting biological molecules such as proteins, lipids, and DNA in the cell nucleus and membrane, leading to cell death or causing cell mutations. Epigallocathecin gallate (EGCG) and ascorbic acid (AA) are well-known natural antioxidants that have been studied and applied as potential radical scavengers. In this study, the radiation protection effects in the presence of EGCG and AA via the scavenging process of free radicals (mainly hydroxyl radicals) were examined. Saccharomyces cerevisiae yeast cells were grown in YDP liquid medium containing yeast extract, peptone, and dextrose/glucose that supplemented with EGCG and AA at different concentrations. Then, the cell cultures were irradiated with both low (gamma) and high (helium ion beam) linear energy transfer (LET) radiations to evaluate the radiation effect on the survival of the yeast cell. Both of EGCG and AA play effectively important roles as radiation-protective agent for yeast cells and the effectiveness in radiation protection of EGCG and AA at the same concentration was almost the same between these two additives.

Single Infrared Spectrum Enables Simultaneous Identification of (Bio)Chemical Nature and Particle Size of Microorganisms and Synthetic Microplastic Beads.

Populations of nearly identical chemical and biological microparticles include the synthetic microbeads used in cosmetic, biomedical, agri-food, and pharmaceutical industries as well as the class of living microorganisms such as yeast, pollen, and biological cells. Herein, we identify simultaneously the size and chemical nature of spherical microparticle populations with diameters larger than 1 μm. Our analysis relies on the extraction of both physical and chemical signatures from the same optical spectrum recorded using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy. These signatures are the spectral resonances caused by the microparticles, which depend on their size and the absorption peaks revealing their chemical nature. We validate the method first on separated and mixed groups of spherical microplastic particles of two different diameters, where the method is used to calculate the diameter of the microspherical particles. Then, we apply the method to correctly identify and measure the diameter of Saccharomyces cerevisiae yeast cells. Theoretical simulations to help in understanding the effect of size distribution and dispersion support our results.

Cellular Stress Impact on Yeast Activity in Biotechnological Processes-A Short Overview.

The importance of Saccharomyces cerevisiae yeast cells is known worldwide, as they are the most used microorganisms in biotechnology for bioethanol and biofuel production. Also, they are analyzed and studied for their similar internal biochemical processes to human cells, for a better understanding of cell aging and response to cell stressors. The special ability of S. cerevisiae cells to develop in both aerobic and anaerobic conditions makes this microorganism a viable model to study the transformations and the way in which cellular metabolism is directed to face the stress conditions due to environmental changes. Thus, this review will emphasize the effects of oxidative, ethanol, and osmotic stress and also the physiological and genetic response of stress mitigation in yeast cells.

Dual-Pulsed Laser Ablation of Oyster Shell Producing Novel Thin Layers Deposed to Saccharomyces cerevisiae.

Dual-pulsed (DPL) laser deposition using oyster shells as targets was studied in order to find out if this method can replace the use of high-power pulsed lasers. Aspects related to changes in the morphological structure of the thin layer but also to the chemical composition of the obtained thin layer were analyzed and compared with the target as well as with the thin layers obtained with a higher power pulsed laser in a single-pulsed (SPL) regime. Orthorhombic structures were noticed with Scanning Electron Microscopy for the thin film obtained in DPL mode compared to the irregular particles obtained in SPL mode. The deacetylation process during ablation was evidenced by Fourier Transform Infrared spectroscopy, resulting in chitosan-based thin films. The effect of the obtained thin films of chitosan on the cells of baker's yeast (Saccharomyces cerevisiae) was studied. Restoration of the yeast paste into initial yeast was noticed mainly when the hemp fabric was used as support for the coating with yeas which was after that coated with chitosan thin film produced by DPL method.

Continuous Flow Separation of Live and Dead Cells Using Gravity Sedimentation.

The separation of target cell species is an important step for various biomedical applications ranging from single cell studies to drug testing and cell-based therapies. The purity of cell solutions is critical for therapeutic application. For example, dead cells and debris can negatively affect the efficacy of cell-based therapies. This study presents a cost-effective method for the continuous separation of live and dead cells using a 3D resin-printed microfluidic device. Saccharomyces cerevisiae yeast cells are used for cell separation experiments. Both numerical and experimental studies are presented to show the effectiveness of the presented device for the isolation of dead cells from cell solutions. The experimental results show that the 3D-printed microfluidic device successfully separates live and dead cells based on density differences. Separation efficiencies of over 95% are achieved at optimum flow rates, resulting in purer cell populations in the outlets. This study highlights the simplicity, cost-effectiveness, and potential applications of the 3D-printed microfluidic device for cell separation. The implementation of 3D printing technology in microfluidics holds promise for advancing the field and enabling the production of customized devices for biomedical applications.

Formation of β-structures and fibrillar associates by NovoRapid insulin molecules in the presence of Saccharomyces cerevisiae yeast cell walls

Formation of β-structures and fibrillar associates by NovoRapid insulin molecules in the presence of Saccharomyces cerevisiae yeast cell walls

A Matlab-Based Approach for Estimating the Area Taken Up by Cells Attached to Micropatterned Optically Opaque Surfaces

Abstract Cell attachment is of paramount importance in implant design, bioreactor design, tissue engineering and the design of non-fouling surfaces. Surface roughness is a significant factor that affects cell attachment. To explore the impact of roughness characteristics, micromachining approaches can be used to fabricate surfaces with controlled microscale topography. When optical microscopy is employed to study cell attachment to optically opaque micropatterned surfaces, one needs to separate the area of an image coated with cells from the background. Manual cell counting can be used to assess the amount of attached cells. However, this process is very time consuming, when the studied surface is larger than several square millimeters. This paper describes an approach for the automatic estimation of the area of cells attached to the surfaces of micro-patterned optically opaque platforms. Saccharomyces cerevisiae yeast cells were used to test the developed approach. The approach uses image registration and segmentation tools available in MathWorks MATLAB R2020b Image Processing Toolbox. The factors that affect the accuracy of the developed approach (magnification, contrast and focus) as well as the ways of improving the results are discussed.

Investigation of antifungal activity mechanisms of alpha-pinene, eugenol, and limonene

Plant essential oils are preferred in cosmetics, medicine, food, and beverage industries for various purposes. α-Pinene is found mainly in eucalyptus oils, eugenol is the active ingredient in clove oil, and limonene is the major component in the oil of citrus fruit peels. In this study, we aimed to determine the antifungal activity of α-pinene, eugenol, and limonene against Saccharomyces cerevisiae yeast cells. Besides, we focused on revealing the target side of the compounds on the yeast cells. Firstly, the antifungal activity of compounds was tested via minimum inhibitory concentration (MIC) measurement. After that, we performed a sorbitol effect assay to understand whether it acts on the cell wall or not. With sorbitol, the MIC values were not changed. It means that they are not effective on the yeast cell wall. Then, we measured the extracellular conductivity increase upon treatment with the compounds to understand the effect on the cell membrane. Eugenol and limonene were not changed the extracellular conductivity, and there was no ion leakage from the cell membrane. On the other hand, α-pinene damaged the yeast cell membrane causing a sudden increase in conductivity due to ion leakage. An ergosterol effect assay with α-pinene was performed to detect cell membrane disruption via ergosterol or not. With ergosterol, the MIC value was not changed. α-Pinene must have another target than the ergosterol in the yeast cell membrane. Finally, revealing the mode of action of compounds against yeast cells will provide new insights into their usage in various fields.

The synthesis of Broccoli RNA fluorescent aptamer in <i>Saccharomyces cerevisiae</i> yeast cells

BACKGROUND: RNA aptamers are short, single-stranded oligonucleotides, with remarkable binding ability to target molecules characterized by high specificity and affinity. Such targets are vastly diverse and range from specific ions to entire cells. RNA aptamers are widely used in biology and medicine for basic research, as well as for practical purposes as in therapy and diagnostics. At present, chemical or in vitro methods of synthesis are mainly used to obtain RNA aptamers. However, such methods are expensive and time-consuming with low productivity. Therefore, in vivo methods are becoming more attractive to researchers working on optimizing high-scale production of RNA aptamers.
 AIM: The aim of this work is to develop a reporter system for optimizing the synthesis of small RNA molecules in Saccharomyces cerevisiae yeast cells.
 MATERIALS AND METHODS: We used the Broccoli fluorescent RNA aptamer to develop a reporter system allowing us to optimize the conditions for in vivo short RNA synthesis in yeast cells. This aptamer is about 112 bp in size and binds to the fluorogenic dye DFHBI-1T. Only upon binding, the aptamer-dye complex exhibits fluorescence properties. After excitation using light with a wavelength of 482 nm, the aptamer-dye complex emission is observed with a peak at 505 nm.
 RESULTS: We have designed a reporter system providing the synthesis of the fluorescent Broccoli RNA aptamer in S. cerevisiae yeast cells. Transcription of RNA molecules containing the aptamer is carried out by the regulated promoter of the GAL1 gene. The synthesized transcripts contain the HH and HDV ribozymes to ensure precise cleavage of the RNA aptamer sequences.
 CONCLUSIONS: This reporter system is based on the Broccoli RNA aptamer, and it can be used to optimize the in vivo synthesis of RNA aptamers in S. cerevisiae yeast cells. This work serves an urgent task in connection with the active use of such aptamers in scientific research, biotechnology and medicine.

Optical Oxygen Sensing and Clark Electrode: Face-to-Face in a Biosensor Case Study.

In the last decade, there has been continuous competition between two methods for detecting the concentration of dissolved oxygen: amerometric (Clark electrode) and optical (quenching of the phosphorescence of the porphyrin metal complex). Each of them has obvious advantages and disadvantages. This competition is especially acute in the development of biosensors, however, an unbiased comparison is extremely difficult to achieve, since only a single detection method is used in each particular study. In this work, a microfluidic system with synchronous detection of the oxygen concentration by two methods was created for the purpose of direct comparison. The receptor element is represented by Saccharomyces cerevisiae yeast cells adsorbed on a composite material, previously developed by our scientific group. To our knowledge, this is the first work of this kind in which the comparison of the oxygen detection methods is carried out directly.

Intracellular trehalose accumulation via the Agt1 transporter promotes freeze-thaw tolerance in Saccharomyces cerevisiae.

This study is to investigate the use of a constitutively expressed trehalose transport protein to directly control intracellular trehalose levels and protect baker's yeast (Saccharomyces cerevisiae) cells against freeze-thaw stress in vivo. We used a constitutively overexpressed Agt1 transporter to investigate the role of trehalose in the freeze-thaw tolerance of yeast cells by regulating intracellular trehalose concentrations independently of intracellular biosynthesis. Using this method, we found that increasing intracellular trehalose in yeast cells improved cell survival rate after 8 days of freezing at -80 and -20°C. We also observed that freeze-thaw tolerance promoted by intracellular trehalose only occurs in highly concentrated cell pellets rather than cells in liquid suspension. Trehalose is sufficient to provide freeze-thaw tolerance using our Agt1 overexpression system. Freeze-thaw tolerance can be further enhanced by deletion of genes encoding intracellular trehalose degradation enzymes. These findings are relevant to improving the freeze-thaw tolerance of baker's yeast in the frozen baked goods industry through engineering strains that can accumulate intracellular trehalose via a constitutively expressed trehalose transporter and inclusion of trehalose into the growth medium.