Bee venom genotoxicity on Saccharomyces cerevisiae cells - The role of mitochondria and YAP1 transcription factor.

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated. Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro.

Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.

Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Recurrent copy number variations in the human fungal pathogen Candida parapsilosis.

Candida parapsilosis is an opportunistic fungal pathogen with increasing incidence in hospital settings worldwide; however, we lack a comprehensive understanding of the mechanisms promoting its virulence and drug resistance. Bergin et al. systematically quantify the frequency and effect of copy number variation (CNV) across 170 diverse clinical and environmental isolates of C. parapsilosis (Bergin SA, Zhao F, Ryan AP, Müller CA, Nieduszynski CA, Zhai B, Rolling T, Hohl TM, Morio F, Scully J, Wolfe KH, Butler G, 2022, mBio, https://doi.org/10.1128/mbio.01777-22). Using a combination of both short- and long-read whole genome sequencing techniques, they determine the structure and copy number of two CNVs that arose recurrently throughout the evolution of these isolates. Each CNV predominantly amplifies one coding sequence (ARR3 or RTA3); however, the amplitude and recombination breakpoints are variable across the isolates. Amplification of RTA3 correlates with drug resistance and deletion causes drug susceptibility. This study highlights the need for further research into the mechanisms and dynamics of CNV formation and the impact of these CNVs on virulence and drug resistance across diverse fungal pathogens.

Phenotypic and comparative transcriptomics analysis of RDS1 overexpression reveal tolerance of Saccharomyces cerevisiae to furfural.

The yeast Saccharomyces cerevisiae able to tolerate lignocellulose-derived inhibitors like furfural. Yeast strain performance tolerance has been measured by the length of the lag phase for cell growth in response to the furfural inhibitor challenge. The aims of this work were to obtain RDS1 yeast tolerant strain against furfural through overexpression using a method of in vivo homologous recombination. Here, we report that the overexpressing RDS1 recovered more rapidly and displayed a lag phase at about 12 h than its parental strain. Overexpressing RDS1 strain encodes a novel aldehyde reductase with catalytic function for reduction of furfural with NAD(P)H as the co-factor. It displayed the highest specific activity (24.8 U/mg) for furfural reduction using NADH as a cofactor. Fluorescence microscopy revealed improved accumulation of reactive oxygen species resistance to the damaging effects of inhibitor in contrast to the parental. Comparative transcriptomics revealed key genes potentially associated with stress responses to the furfural inhibitor, including specific and multiple functions involving defensive reduction-oxidation reaction process and cell wall response. A significant change in expression level of log2 (fold change >1) was displayed for RDS1 gene in the recombinant strain, which demonstrated that the introduction of RDS1 overexpression promoted the expression level. Such signature expressions differentiated tolerance phenotypes of RDS1 from the innate stress response of its parental strain. Overexpression of the RDS1 gene involving diversified functional categories is accountable for stress tolerance in yeast S. cerevisiae to survive and adapt the furfural during the lag phase.

Lipid biosynthesis perturbation impairs endoplasmic reticulum-associated degradation.

The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

Structural and mechanistic insights into fungal ß-1,3-glucan synthase FKS1.

The membrane-integrated synthase FKS is involved in the biosynthesis of ß-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal ß-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity.

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

Elesclomol elevates cellular and mitochondrial iron levels by delivering copper to the iron import machinery.

Copper (Cu) and iron (Fe) are redox-active metals that serve as cofactors for many essential cellular enzymes. Disruption in the intracellular homeostasis of these metals results in debilitating and frequently fatal human disorders, such as Menkes disease and Friedreich's ataxia. Recently, we reported that an investigational anticancer drug, elesclomol (ES), can deliver Cu to critical mitochondrial cuproenzymes and has the potential to be repurposed for the treatment of Cu deficiency disorders. Here, we sought to determine the specificity of ES and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments. Using a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry-based metal measurements, we showed that ES and ES-Cu treatment results in an increase in cellular and mitochondrial Fe content, along with the expected increase in Cu. Using yeast mutants of Cu and Fe transporters, we demonstrate that ES-based elevation in cellular Fe levels is independent of the major cellular Cu importer but is dependent on the Fe importer Ftr1 and its partner Fet3, a multicopper oxidase. As Fet3 is metalated in the Golgi lumen, we sought to uncover the mechanism by which Fet3 receives Cu from ES. Using yeast knockouts of genes involved in Cu delivery to Fet3, we determined that ES can bypass Atx1, a metallochaperone involved in Cu delivery to the Golgi membrane Cu pump, Ccc2, but not Ccc2 itself. Taken together, our study provides a mechanism by which ES distributes Cu in cells and impacts cellular and mitochondrial Fe homeostasis.

In vitro antiaging activity of polyphenol rich Polyalthia longifolia (Annonaceae) leaf extract in Saccharomyces cerevisiae BY611 yeast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polyalthia longifolia var. angustifolia Thw. (Annonaceae) is commonly used in traditional medicine as a tonic for rejuvenation and exhibiting good antioxidant activities. AIM OF THE STUDY: To evaluate P. longifolia methanolic leaf extract (PLME) antiaging activity at 1 mg/mL in Saccharomyces cerevisiae BY611 yeast. MATERIALS AND METHODS: The antiaging effect of PLME was studied via replicative lifespan assay, antioxidative stress assays, reactive oxygen species (ROS) determination, reduced glutathione (GSH) determination, superoxide dismutase (SOD) and Sirtuin 1 (SIRT1) genes regulation studies and SOD and SIRT1 proteins activities. RESULTS: The PLME treatment increased the growth and prolonged the lifespan of the yeast significantly (p < 0.05) compared to the untreated yeast group. Besides, the PLME also protected the yeast from oxidative stress induced by 4-mM-H2O2 via decreasing (p < 0.05) the ROS from 143.207 to 127.223. The antioxidative action of PLME was proved by spot assay. Phloxine B staining was further confirmed the PLME antioxidative action of PLME, where more whitish-pink live yeast cells were observed. In addition, the PLME also enhanced GSH content significantly (p < 0.05) in yeast treated with PLME from 16.81 to 25.31 µmol. Furthermore, PLME increased the SOD and SIRT1 genes expression significantly (p < 0.05) with ΔCt values of 1.11 and 1.15, respectively. The significantly (p < 0.05) elevated SOD and SIRT1 protein activities were recorded as 51.54 U/mg Prot and 1716 ng/mL, respectively. CONCLUSIONS: PLME exhibited good antiaging activities in S. cerevisiae, by modulating oxidative stress, enhancing GSH content, and increasing SOD and SIRT1 genes expression.

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells.

Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast.

Involvement of Gtr1p in the oxidative stress response in yeast Saccharomyces cerevisiae.

Yeast Gtr1p is a GTPase that forms a heterodimer with Gtr2p, another GTPase; it is involved in regulating TORC1 activity in nutrient signaling, including amino acid availability and growth control. Gtr1p is a positive regulator of TORC1, a kinase that regulates various cellular functions (e.g., protein synthesis and autophagy) under specific nutrient and environmental conditions, including oxidative stress. In this study, we examined the roles of Gtr1p in oxidative stress responses. We found that yeast cells expressing guanosine diphosphatase (GDP)-bound Gtr1p (Gtr1-S20Lp) were resistant to hydrogen peroxide (H2O2), whereas guanosine triphosphate (GTP)-bound Gtr1p (Gtr1-Q65Lp) was sensitive to H2O2 compared with the wild type. Consistent with these findings, yeast cells lacking Iml1p, a component of the GTPase-activating protein complex for Gtr1p, exhibited the H2O2-sensitive phenotype. In gtr1S20L cells, autophagy was highly induced under oxidative stress. gtr1Q65L cells showed decreased expression of the SNQ2 gene, which encodes a multidrug transporter involved in resistance to oxidative stress, and the overexpression of SNQ2 rescued the oxidative stress sensitivity of gtr1Q65L cells. These results suggest that Gtr1p is involved in oxidative stress responses through mechanisms that include autophagy and SNQ2 expression.

Site-specific Phosphorylation of Histone H3K36 Methyltransferase Set2p and Demethylase Jhd1p is Required for Stress Responses in Saccharomyces cerevisiae.

Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of almost all phosphosites remain unknown. Here, we comprehensively analyse the phosphoregulation of the yeast histone methylation network by functionally investigating 40 phosphosites on six enzymes. A total of 82 genomically-edited S. cerevisiae strains were generated through mutagenesis of sites to aspartate as a phosphomimetic or alanine as a phosphonull. These phosphosite mutants were screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. For methyltransferase Set2p, we found that phosphorylation at threonine 127 significantly decreased H3K36 methylation in vivo, and that an N-terminal phosphorylation cluster at serine residues 6, 8, and 10 is required for the diamide stress response. Proteomic analysis of Set2p phosphosite mutants revealed a specific downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion and the sensitivity of mutants to diamide. For demethylase Jhd1p, we found that its sole phosphorylation site at serine 44 is required for the cold stress response. This study represents the first systematic investigation into the phosphoregulation of the epigenetic network in any eukaryote, and shows that phosphosites on histone methylation enzymes are required for a normal cellular response to stress in S.cerevisiae.

Radiolytic synthesis and characterization of selenium nanoparticles: comparative biosafety evaluation with selenite and ionizing radiation.

The goal of this work is use a green chemistry route to synthesize selenium nanoparticles (SeNPs) that do not trigger oxidative stress, typical of metallic, oxide metallic and carbonaceous nanostructures, and supply the same beneficial effects as selenium nanostructures. SeNPs were synthesized using a radiolytic method involving irradiating a solution containing sodium selenite (Se4+) as the precursor in 1% Yeast extract, 2% Peptone, 2% Glucose (YPG) liquid medium with gamma-rays (60Cobalt). The method did not employ any hazardous reducing agents. Saccharomyces cerevisiae cells were incubated with 1 mM SeNPs for 24 h and/or then challenged with 400 Gy of ionizing radiation were assessed for viability and biomarkers of oxidative stress: lipid peroxidation, protein carbonylation, free radical generation, and total sulfhydryl content. Spherical SeNPs with variable diameters (from 100 to 200 nm) were formed after reactions of sodium selenite with hydrated electrons (eaq-) and hydrogen radicals (H·). Subsequent structural characterizations indicated an amorphous structure composed of elemental selenium (Se0). Compared to 1 mM selenite, SeNPs were considered safe and less toxic to Saccharomyces cerevisiae cells as did not elicit significant modifications in cell viability or oxidative stress parameters except for increased protein carbonylation. Furthermore, SeNPs treatment afforded some protection against ionizing radiation exposure. SeNPs produced using green chemistry attenuated the reactive oxygen species generation after in vitro ionizing radiation exposure opens up tremendous possibilities for radiosensitizer development.

Transcriptomic analysis of formic acid stress response in Saccharomyces cerevisiae.

Formic acid is a representative small molecule acid in lignocellulosic hydrolysate that can inhibit the growth of Saccharomyces cerevisiae cells during alcohol fermentation. However, the mechanism of formic acid cytotoxicity remains largely unknown. In this study, RNA-Seq technology was used to study the response of S. cerevisiae to formic acid stress at the transcriptional level. Scanning electron microscopy and Fourier transform infrared spectroscopy were conducted to observe the surface morphology of yeast cells. A total of 1504 genes were identified as being differentially expressed, with 797 upregulated and 707 downregulated genes. Transcriptomic analysis showed that most genes related to glycolysis, glycogen synthesis, protein degradation, the cell cycle, the MAPK signaling pathway, and redox regulation were significantly induced under formic acid stress and were involved in protein translation and synthesis amino acid synthesis genes were significantly suppressed. Formic acid stress can induce oxidative stress, inhibit protein biosynthesis, cause cells to undergo autophagy, and activate the intracellular metabolic pathways of energy production. The increase of glycogen and the decrease of energy consumption metabolism may be important in the adaptation of S. cerevisiae to formic acid. In addition, formic acid can also induce sexual reproduction and spore formation. This study through transcriptome analysis has preliminarily reveal the molecular response mechanism of S. cerevisiae to formic acid stress and has provided a basis for further research on methods used to improve the tolerance to cell inhibitors in lignocellulose hydrolysate.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Oxidative stress alleviating potential of galactan exopolysaccharide from Weissella confusa KR780676 in yeast model system.

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.

Metabolic remodeling maintains a reducing environment for rapid activation of the yeast DNA replication checkpoint.

Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.

The agonistic bioanalytical equivalent concentration: A novel tool for assessing the endocrine activity of environmental mixtures.

Cell-based bioassays are very sensitive and allow integrative effect screening of the whole environmental sample, which is usually composed of a mixture of agonists and antagonists. Measured toxicity is usually expressed as a bioanalytical equivalent concentration. So far, it is not possible to distinguish which part of this value is caused by the agonists and which by the antagonists. In this article, we present a simple method to analyze the dose-response curve of a mixture and to determine an agonistic bioanalytical equivalent concentration: a concentration of a reference chemical that would elicit the same effect as do only agonists in an unknown mixture. The method has been validated using several artificially prepared mixtures of agonists and competitive antagonists measured in a recombinant yeast assay. No difference was observed between the calculated equivalent concentrations and the used concentrations of the agonist in the mixture.

Effects of a sulfated glycosaminoglycan from Sepia esculenta ink on transcriptional and metabolic profiles of Saccharomyces cerevisiae.

Four fractions of water-extracted Sepia esculenta ink polysaccharides (SIP) were separated by dicthylaminoethy (DEAE) cellulose chromatography. The eluted fraction with the highest yield was characterized as a sulfate-rich glycosaminoglycan named SIP-IV. According to the analysis of laser scattering and refractive index signals, SIP-IV was determined to be 14.4 kDa and spherical molecular conformation in salt solution. SIP-IV is composed of fucose, galactosamine, glucosamine, mannose and glucuronic acid with a molar ratio of 5.1:7.3:3.8:1:4.4, which is obviously different from reported SIPs. SIP-IV promoted yeast proliferation and intercellular antioxidant level. Based on multi-omics strategy, data of transcriptome analysis suggested that growth promotion of SIP-IV on Saccharomyces cerevisiae might be attributed to regulation of Rho protein signal transduction, nuclear autophagy and nitrogen utilization. Combined with the metabolome results, SIP-IV also re-profiled metabolism of amino acids and phospholipids in yeast cells.