Yeast Research Articles

Instability of natural deep eutectic solvents (NADESs) induced by Amadori rearrangement and its effects on cryopreservation of yeast cells

Natural deep eutectic solvents (NADESs) showing higher cryoprotective effects are attracting concerns, because during the storage, system browning always occurs in aldose/amino acid-based NADESs, which generated brown substances remarkably weaken the cryoprotective effects. In this study, proline/glucose-based (PG) and proline/sorbitol-based (PS) NADESs were prepared, of which storage stability, browning profile, brown substance, and cryoprotective effects were investigated. Results showed that PG at molar ratios of 1:1, 2:1, and 3:1, as well as PS at 1:1, and 2:1 can form NADESs, among which only the PG-based ones could get browning after storage. The predominant brown substance was identified as 1-deoxy-1-L-proline-d-fructose (C11H19O7N, 278 m/z), which was subsequently verified to show cytotoxicity and decrease Saccharomyces cerevisiae cells viability after cryopreservation, suggesting that the brown substance could take a negative effect on cryopreservation. This study may help to attract more concerns to the storage and cryopreservation stabilities of the NADESs in food-related applications.

Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors

BackgroundLipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery.ResultsHydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids.ConclusionThis work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration.Graphical abstract

Evaluation of dietary yeast derived mannan oligosaccharide for pacific whiteleg shrimp Penaeus vannamei: Effects on growth performance, immune response, hepatopancreas morphology and resilience to infection against Vibrio parahaemolyticus

With trends towards low fishmeal and higher plant ingredient-based diets for shrimp, there is consequently greater demand on feed additives to bolster the protein and nutrient profile quality for achieving more efficiency. Also, there is a need to enhance innate immunity and resilience to infection under intensive shrimp production scenarios. This investigation tested incremental levels 0, 0.2; 0.4; 0.6 and 0.8 % of a commercial yeast cell wall extract mainly comprising β-glucan and mannan oligosaccharides (MOS) (YM, YeaMOS, Angel Yeast, Yichang, Hubei, China) for post-larval shrimp Penaeus vannamei in isonitrogenous and iso-lipidic diets with proteinaceous plant ingredients amounting to 50 % contribution of the diet formulation. After 60 d, growth and feed utilization performance were not significantly affected in shrimp in terms of weight gain or FCR, but due to much higher survival, total biomass in groups of shrimps fed with dietary YM was higher compared to the control treatment. A sequential specific pathogen challenge test with Vibrio parahaemolyticus at final dose of 104 CFU mL−1 resulted in a marked improvement in survival against the control group with much enhancement at the inclusion level of 0.4 % YM (p<0.05). Before challenged, the total haemocyte count (THC) and the relative gene expression of Prophenoloxidase (ProPO) were higher in the group of shrimps fed with YM. Likewise, after infection, the amount of THC and ProPO remained better in the group of shrimps fed with YM. However, the 0.8 % YM treatment did not significantly differ from the control for phagocytic activity and index. The histomorphology condition of the hepatopancreas of shrimp before the infection for all treatments appears normal without significant numbers of hepatopancreatic tubules (T) and epithelial cell vacuoles (V). After pathogen challenge, all shrimp exhibited severe necrotic cell damage and massive sloughing of hepatopancreatic tubule epithelial cells into the lumen. Overall, giving YM as feed additives can improve the shrimp biomass and health, with the use of 0.4 % inclusion levels in practical diets for shrimp P. vannamei become the optimum dose to develop more efficient aquafeed production.

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations.

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be converted into mutations that promote carcinogenesis or the development of genetically inherited diseases. As a result, researchers and clinicians require tools that can detect DNA damage and mutations with exceptional sensitivity. In this study, we describe a massively parallel sequencing tool termed Mutation And DNA Damage Detection-seq (MADDD-seq) that is capable of detecting O6-methyl guanine lesions and mutations simultaneously, with a single assay. To illustrate the dual capabilities of MADDD-seq, we treated WT and DNA repair deficient yeast cells with the DNA-damaging agent MNNG and tracked DNA lesions and mutations over a 24-htime period. This approach allowed us to identify thousands of DNA adducts and mutations in a single sequencing run and gain deep insight into the kinetics of DNA repair and mutagenesis.

A Droplet-Based Microfluidic Platform for High-Throughput Culturing of Yeast Cells in Various Conditions.

Yeast plays a significant role in a variety of fields. In particular, it is extensively used as a model organism in genetics and cellular biology studies, and is employed in the production of vaccines, pharmaceuticals, and biofuels. Traditional "bulk"-based studies on yeast growth often overlook cellular variability, emphasizing the need for single-cell analysis. Micro-droplets, tiny liquid droplets with high surface-area-to-volume ratios, offer a promising platform for investigating single or a small number of cells, allowing precise control and monitoring of individual cell behaviors. Microfluidic devices, which facilitate the generation of micro-droplets, are advantageous due to their reduced volume requirements and ability to mimic in vivo micro-environments. This study introduces a custom-designed microfluidic device to encapsulate yeasts in micro-droplets under various conditions in a parallel manner. The results reveal that optimal glucose concentrations promoted yeast growth while cycloheximide and Cu2+ ions inhibited it. This platform enhances yeast cultivation strategies and holds potential for high-throughput single-cell investigations in more complex organisms.

Advances in Flavonoid and Derivative Biosynthesis: Systematic Strategies for the Construction of Yeast Cell Factories.

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Membrane potential dynamics unveil the promise of bioelectrical antimicrobial susceptibility Testing (BeAST) for anti-fungal screening.

Rapid assessment of the efficacy of antimicrobials is important for optimizing treatments, avoiding misuse and facilitating the screening of new antimicrobials. The need for rapid antimicrobial susceptibility testing (AST) is growing with the rise of antimicrobial resistance. Here, we present bioelectrical AST (BeAST). Combining time-lapse microscopy and mathematical modeling, we show that electrically induced membrane potential dynamics of yeast cells correspond to the strength of growth inhibition. Furthermore, we demonstrate the utility of BeAST for assessing antimicrobial activities of novel compounds using biogenic silver nanoparticles.

Functional suppression of a yeast maf1 deletion mutant by overdose of the N-terminal fragment of the largest RNA polymerase III subunit, C160

Maf1 is a general and global negative regulator of RNA polymerase III (Pol III) transcription. Under repressive conditions, Maf1 binds directly to the Pol III complex and sequesters Pol III elements that are involved in transcription initiation. To further understand Pol III regulation, we searched for genetic bypass suppressors of a maf1 deletion mutant (maf1Δ) of Saccharomyces cerevisiae. Strains that carried maf1Δ were temperature-sensitive on media that contained nonfermentable carbon sources and showed the antisuppressor phenotype. Suppressors allowed colonies to grow at the restrictive temperature on glycerol media and partially complemented the antisuppressor phenotype of maf1Δ. DNA plasmids that were identified as overdose suppressors encoded N-terminal fragments of the largest Pol III subunit, C160 of various lengths. The shortest fragment, 372 amino acids long, the overdose of which partially complemented the antisuppressor phenotype and temperature-sensitive respiratory growth of maf1Δ, was named C160-NTF. In this study, we showed that the expression of HA-tagged C160-NTF resulted in accumulation of approximately 40 kDa protein that was distributed throughout the yeast cell, in the cytoplasm and nucleus. The overdose of C160-NTF led to decrease of tRNA transcription in maf1Δ mutant cells, demonstrating functional suppression. Levels of newly synthesized individual tRNAs and Pol III occupancies on tRNA genes were decreased by C160-NTF in the maf1Δ mutant. Additionally, we analyzed the effect of C160-NTF overproduction and the presence of Maf1 on the associations among Pol III subunits. Previous structural analyzes of Pol III have indicated that the N-terminal region of C160 interacts with the C82-34-31 heterotrimeric Pol III subcomplex. We suggest that the negative effect of C160-NTF overdose on tRNA transcription is related to defective Pol III assembly, because overproduction of C160-NTF altered C160 interactions with C34 and C82 in the maf1Δ mutant.

β-TrCP-Mediated Proteolysis of Mis18β Prevents Mislocalization of CENP-A and Chromosomal Instability.

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.

Genetic Strategies and Mechanisms for Improving Ribonucleic Acid Levels of Saccharomyces pastorianus.

Developing microorganisms with a high ribonucleic acid (RNA) content is crucial for the RNA industry. Numerous studies have been conducted to enhance RNA production in yeast cells through genetic engineering, yet precise mechanisms remain elusive. Previously, upregulation of TAL1 or PGM2 and deleting PRS5 or DBP8 individually could increase the RNA content in Saccharomyces pastorianus. In this study, within these genetically modified strains, the intracellular nucleotide levels notably increased following cell fragmentation. Deletion of PRS5 and DBP8 within the strain prompted the upregulation of genes sharing similar functions, consequently augmenting the flow of the gene pathway. Furthermore, the upregulation of genes encoding cell-cycle-dependent protein kinases (CDK) was observed in the G03-△PRS5 strain. The influence of TAL1 and PGM2 on RNA content was attributed to the pentose phosphate pathway (PPP). The RNA content of polygenic recombinant strains, G03-△PRS5+△DBP8 and G03-△PRS5+△DBP8+PGM2, displayed the most significant improvement, increasing by 71.8 and 80.1% when compared to the parental strain. Additionally, the maximum specific growth rate of cells increased in these strains. This study contributes valuable insights into the genetic mechanisms underlying high nucleic acid synthesis in S. pastorianus.

Brewing with Sea Vegetable: The Effect of Spirulina (Arthrospira platensis) Supplementation on Brewing Fermentation Kinetics, Yeast Behavior, and the Physiochemical Properties of the Product

Spirulina is a highly nutritious microalgae commonly used as a food additive. During fermentation, different adjuncts are incorporated to act as a nutrient source for yeast and fortify or modify the sensory attributes of the final product. In this study, the effect of Spirulina on the characteristics of controlled yeast fermentation and the production of volatile organic compounds (VOCs) was analyzed. Spirulina was added to malted barley during mashing and fermented under standard conditions. An unaltered mash (negative control) and yeast extract (positive control) were also fermented. The addition of Spirulina resulted in an increased fermentation rate (~14% faster) and bigger yeast cells (~34% larger) in comparison to the negative control. There were differences in color (determined as SRM) between treatments; however, there were only minor differences in VOCs, with no statistical differences observed between chemical compound groups. No differences were observed in the pH, total number of yeast cells, or final attenuation between treatments. The primary mechanism for the observed differences is believed to be an increase in amino acids available to yeast that were contributed by the Spirulina. This shows both that Spirulina has a high potential as a fermentation adjunct and that the amino acid profile of an adjunct can significantly impact fermentation.

One-pot synthesis of cellobiose from sucrose using sucrose phosphorylase and cellobiose phosphorylase co-displaying Pichia pastoris as a reusable whole-cell biocatalyst

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production.

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

Geraniol Potentiates the Effect of Fluconazole against Planktonic and Sessile Cells of Azole-Resistant Candida tropicalis: In Vitro and In Vivo Analyses.

Candida tropicalis is regarded as an opportunistic pathogen, causing diseases ranging from superficial infections to life-threatening disseminated infections. The ability of this yeast to form biofilms and develop resistance to antifungals represents a significant therapeutic challenge. Herein, the effect of geraniol (GER), alone and combined with fluconazole (FLZ), was evaluated in the planktonic and sessile cells of azole-resistant C. tropicalis. GER showed a time-dependent fungicidal effect on the planktonic cells, impairing the cell membrane integrity. Additionally, GER inhibited the rhodamine 6G efflux, and the molecular docking analyzes supported the binding affinity of GER to the C. tropicalis Cdr1 protein. GER exhibited a synergism with FLZ against the planktonic and sessile cells, inhibiting the adhesion of the yeast cells and the viability of the 48-h biofilms formed on abiotic surfaces. C. tropicalis biofilms treated with GER, alone or combined with FLZ, displayed morphological and ultrastructural alterations, including a decrease in the stacking layers and the presence of wilted cells. Moreover, neither GER alone nor combined with FLZ caused toxicity, and both treatments prolonged the survival of the Galleria mellonella larvae infected with azole-resistant C. tropicalis. These findings indicate that the combination of GER and FLZ may be a promising strategy to control azole-resistant C. tropicalis infections.

Spermidine is essential for fasting-mediated autophagy and longevity

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine–hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.

Yeast cell wall supplementation affects the Salmonella enteretidis load in the ceca and ovaries of layer pullets

Salmonella enteretidis (SE) has a great propensity to translocate from the cecum into internal organs such as the spleen and liver. However, a major concern is the ability of SE to colonize the ovaries. This study aimed to evaluate the efficacy of cell walls from Saccharomyces cerevisiae to control the Salmonella load in the ceca and ovaries of commercial layer pullets. Ten-week-old layer pullets were divided into 2 groups: one group was fed a control diet with commercial feed without additives, and another group was fed the same diet supplemented with 0.5 kg/metric ton of yeast cell walls (YCWs). At 16 wk of age, the birds in both groups were challenged with 3.0 × 109 CFU/mL SE by oral gavage. The birds were euthanized on d 7 and 14 postchallenge to collect the ceca and ovaries for Salmonella load determination. The results demonstrated that there were no statistical differences in ovary SE infection rates. The trend in the prevalence of SE positivity in the ovaries was similar at 14 d, with 2.1% (YCW pullets) to 4.2% positive for the ovaries of the nontreated pullets. There was also no significant difference in the SE log10 MPN/gram between the YCW and the control groups. In the ceca, the high level of SE (3.0 × 109 cfu/pullet), which results in ovarian transmission, causes high intestinal tract inflammation. There was a significant difference in the prevalence of SE in the ceca at 7 d postchallenge but not at 14 d postchallenge. In conclusion, the reduction in Salmonella load observed in the ceca on d 7 in this study shows the potential of YCW supplementation for reducing Salmonella colonization in poultry.

Effect of Partial Elimination of Mitochondrial DNA on Genome-Wide Identified AOX Gene Family in Chlamydomonas reinhardtii

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho0 or rho− cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family.

Synergistic antifungal effect of thiophene derivative as an inhibitor of fluconazole-resistant Candida spp. biofilms.

Candida species resistant to fluconazole have raised concern in the scientific medical community due to high mortality in patients with invasive disease. In developing countries, such as Brazil, fluconazole is the most commonly used antifungal, and alternative treatments are expensive or not readily available. Furthermore, the occurrence of biofilms is common, coupled with their inherent resistance to antifungal therapies and the host's immune system, these microbial communities have contributed to making infections caused by these yeasts an enormous clinical challenge. Therefore, there is an urgent need to develop alternative medicines, which surpass the effectiveness of already used therapies, but which are also effective against biofilms. Therefore, the present study aimed to describe for the first time the antifungal and antibiofilm action of the derivative 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-isopropyl carboxylate (2AT) against clinical strains of Candida spp. resistant to fluconazole (FLZ). When determining the minimum inhibitory concentrations (MIC), it was found that the compound has antifungal action at concentrations of 100 to 200µg/mL, resulting in 100% inhibition of yeast cells. Its synergistic effect with the drug FLZ was also observed. The antibiofilm action of the compound in subinhibitory concentrations was detected, alone and in association with FLZ. Moreover, using scanning electron microscopy, it was observed that the compound 2AT in isolation was capable of causing significant ultrastructural changes in Candida. Additionally, it was also demonstrated that the compound 2AT acts by inducing characteristics compatible with apoptosis in these yeasts, such as chromatin condensation, when visualized by transmission electron microscopy, indicating the possible mechanism of action of this molecule. Furthermore, the compound did not exhibit toxicity in J774 macrophage cells up to a concentration of 4000µg/mL. In this study, we identify the 2AT derivative as a future alternative for invasive candidiasis therapy, in addition, we highlighted the promise of a strategy combined with fluconazole in combating Candida infections, especially in cases of resistant isolates.

Fdo1, Fkh1, Fkh2, and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

Length control emerges from cytoskeletal network geometry

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures.