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Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

Simple Summary: A 9-years-old cat presenting signs of chronic respiratory disease was evaluated though rhinoscopy, and samples for histopathology and microbiological cultures were collected. Histopathology revealed chronic infiltration of mature lymphocytes and plasma cells, leading to a diagnosis of chronic lymphoplasmacytic rhinitis. No fungal growth was observed, but bacterial cultures revealed growth of an atypical bacterium mistakenly identified with conventional bacterial identification methods. This isolate was later identified as P. aestus by sequencing followed by homologous sequences analysis, corresponding to an environmental strain of Pseudomonas used in agricultural settings as a biocide. Antimicrobial susceptibility testing was performed, and this P. aestus isolate presented a multidrug resistant profile. Pseudomonas are ubiquitous bacteria frequently described as animal and human opportunistic pathogens, and P. aeruginosa is the principal species related with animal disease. This work aims to report the first case of animal disease related with P. aestus, to the author's best knowledge. Furthermore, it highlights the need to establishing protocols aiming at the identification and characterization of non-traditional, multidrug-resistant Pseudomonas in the clinical setting. The Pseudomonas genus includes ubiquitous bacteria frequently described as animal and human opportunistic pathogens. A 9-year-old cat was referred for rhinoscopy at the Veterinary Hospital of the Faculty of Veterinary Medicine, University of Lisbon, Portugal, for an investigation of the chronic respiratory signs. Upon rhinoscopy, nasal and nasopharyngeal discharge were observed, and the nasal turbinates showed signs of inflammation. The nasal biopsies were evaluated by histopathology and mycological and bacterial cultures. The histopathology revealed chronic lymphoplasmacytic inflammation. The mycological culture was negative, but the bacterial culture revealed the growth of a bacterial isolate in the pure culture, identified as P. aestus by the sequencing of a 1750 bp PCR amplicon obtained with BCR1 and BCR2 primers, followed by homologous sequences analysis using the NCBI database. The isolate's susceptibility profile towards 14 antimicrobials was evaluated through the disk diffusion method, being observed that it presented a multidrug resistance profile. The studies available on this environmental Pseudomonas strain focused on its potential use for biocide production and application in agricultural settings, and, to the authors' best knowledge, there are no reports describing its association with infectious diseases in humans or animals, highlighting the importance of establishing protocols aiming at the identification and characterization of non-traditional, multidrug-resistant Pseudomonas in the clinical setting. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma is an extremely aggressive malignant tumor with a very poor prognosis. Due to the increased proliferation rate of glioblastoma, there is the development of hypoxic regions, characterized by an increased concentration of copper (Cu). Considering this, 64Cu has attracted attention as a possible theranostic radionuclide for glioblastoma. In particular, [64Cu]CuCl2 accumulates in glioblastoma, being considered a suitable agent for positron emission tomography. Here, we explore further the theranostic potential of [64Cu]CuCl2, by studying its therapeutic effects in advanced three-dimensional glioblastoma cellular models. First, we established spheroids from three glioblastoma (T98G, U373, and U87) and a non-tumoral astrocytic cell line. Then, we evaluated the therapeutic responses of spheroids to [64Cu]CuCl2 exposure by analyzing spheroids' growth, viability, and cells' proliferative capacity. Afterward, we studied possible mechanisms responsible for the therapeutic outcomes, including the uptake of 64Cu, the expression levels of a copper transporter (CTR1), the presence of a cancer stem cell population, and the production of reactive oxygen species (ROS). Results: Results revealed that [64Cu]CuCl2 is able to significantly reduce spheroids' growth and viability, while also affecting cells' proliferation capacity. The uptake of 64Cu, the presence of cancer stem-like cells and the production of ROS were in accordance with the therapeutic response. However, expression levels of CTR1 were not in agreement with uptake levels, revealing that other mechanisms could be involved in the uptake of 64Cu. Conclusions: Overall, our results further support [64Cu]CuCl2 potential as a theranostic agent for glioblastoma, unveiling potential mechanisms that could be involved in the therapeutic response. [ABSTRACT FROM AUTHOR]

Abstract Background Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic engineering to obtain more robust yeast strains against acetic acid toxicity. Among these genes there are number of transcription factors that are documented regulators of a large percentage of the genes found to exert protection against acetic acid thus being considered interesting targets for subsequent genetic engineering. The increase of potassium concentration in the growth medium was found to improve the expression of maximal tolerance to acetic acid, consistent with the idea that the adequate manipulation of nutrient concentration of industrial growth medium can be an interesting strategy to surpass the deleterious effects of this weak acid in yeast cells.

In this work, it is described the sequencing and annotation of the genome of the yeast strain ISA1307, isolated from a sparkling wine continuous production plant. This strain, formerly considered of the Zygosaccharomyces bailii species, has been used to study Z. bailii physiology, in particular, its extreme tolerance to acetic acid stress at low pH. The analysis of the genome sequence described in this work indicates that strain ISA1307 is an interspecies hybrid between Z. bailii and a closely related species. The genome sequence of ISA1307 is distributed through 154 scaffolds and has a size of around 21.2 Mb, corresponding to 96% of the genome size estimated by flow cytometry. Annotation of ISA1307 genome includes 4385 duplicated genes (∼90% of the total number of predicted genes) and 1155 predicted single-copy genes. The functional categories including a higher number of genes are ‘Metabolism and generation of energy’, ‘Protein folding, modification and targeting’ and ‘Biogenesis of cellular components’. The knowledge of the genome sequence of the ISA1307 strain is expected to contribute to accelerate systems-level understanding of stress resistance mechanisms in Z. bailii and to inspire and guide novel biotechnological applications of this yeast species/strain in fermentation processes, given its high resilience to acidic stress. The availability of the ISA1307 genome sequence also paves the way to a better understanding of the genetic mechanisms underlying the generation and selection of more robust hybrid yeast strains in the stressful environment of wine fermentations. [ABSTRACT FROM PUBLISHER]

Simple Summary: Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors. Although a variety of GBMs is initially susceptible to chemotherapy, the development of multi-drug resistance and recurrence is frequent. Therefore, there is an urgent need for more efficient treatment modalities for GBM. Boron neutron capture therapy (BNCT) is a cancer therapy based on the potential of 10B atoms to produce α particles that cross tissues in which the 10B accumulates without damaging the surrounding healthy tissues after irradiation with low energy thermal neutrons. The aim of our study was to assess that the sodium salt of cobaltabis(dicarbollide) and its di-iodinated derivative could be good candidates for dual anti-cancer treatment (chemotherapy + BNCT). Our results strongly suggest that these small molecules, in particular [8,8′-I2-o-COSAN]−, are serious candidates to be taken into account for BNCT now that the accelerator-based neutron source facilities are more accessible, providing an alternative treatment for resistant glioblastoma. Purpose: The aim of our study was to assess if the sodium salt of cobaltabis(dicarbollide) and its di-iodinated derivative (Na[o-COSAN] and Na[8,8′-I2-o-COSAN]) could be promising agents for dual anti-cancer treatment (chemotherapy + BNCT) for GBM. Methods: The biological activities of the small molecules were evaluated in vitro with glioblastoma cells lines U87 and T98G in 2D and 3D cell models and in vivo in the small model animal Caenorhabditis elegans (C. elegans) at the L4-stage and using the eggs. Results: Our studies indicated that only spheroids from the U87 cell line have impaired growth after treatment with both compounds, suggesting an increased resistance from T98G spheroids, contrary to what was observed in the monolayer culture, which highlights the need to employ 3D models for future GBM studies. In vitro tests in U87 and T98G cells conclude that the amount of 10B inside the cells is enough for BNCT irradiation. BNCT becomes more effective on T98G after their incubation with Na[8,8′-I2-o-COSAN], whereas no apparent cell-killing effect was observed for untreated cells. Conclusions: These small molecules, particularly [8,8′-I2-o-COSAN]−, are serious candidates for BNCT now that the facilities of accelerator-based neutron sources are more accessible, providing an alternative treatment for resistant glioblastoma. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma is an extremely aggressive malignant tumor with a very poor prognosis. Due to the increased proliferation rate of glioblastoma, there is the development of hypoxic regions, characterized by an increased concentration of copper (Cu). Considering this, 64 Cu has attracted attention as a possible theranostic radionuclide for glioblastoma. In particular, [ 64 Cu]CuCl 2 accumulates in glioblastoma, being considered a suitable agent for positron emission tomography. Here, we explore further the theranostic potential of [ 64 Cu]CuCl 2 , by studying its therapeutic effects in advanced three-dimensional glioblastoma cellular models. First, we established spheroids from three glioblastoma (T98G, U373, and U87) and a non-tumoral astrocytic cell line. Then, we evaluated the therapeutic responses of spheroids to [ 64 Cu]CuCl 2 exposure by analyzing spheroids' growth, viability, and cells' proliferative capacity. Afterward, we studied possible mechanisms responsible for the therapeutic outcomes, including the uptake of 64 Cu, the expression levels of a copper transporter (CTR1), the presence of a cancer stem cell population, and the production of reactive oxygen species (ROS)., Results: Results revealed that [ 64 Cu]CuCl 2 is able to significantly reduce spheroids' growth and viability, while also affecting cells' proliferation capacity. The uptake of 64 Cu, the presence of cancer stem-like cells and the production of ROS were in accordance with the therapeutic response. However, expression levels of CTR1 were not in agreement with uptake levels, revealing that other mechanisms could be involved in the uptake of 64 Cu., Conclusions: Overall, our results further support [ 64 Cu]CuCl 2 potential as a theranostic agent for glioblastoma, unveiling potential mechanisms that could be involved in the therapeutic response., (© 2024. The Author(s).)

Very high concentrations of acetic acid at low pH induce programmed cell death (PCD) in both the experimental model Saccharomyces cerevisiae and in Zygosaccharomyces bailii , the latter being considered the most problematic acidic food spoilage yeast due to its remarkable intrinsic resistance to this food preservative. However, while the mechanisms underlying S. cerevisiae PCD induced by acetic acid have been previously examined, the corresponding molecular players remain largely unknown in Z. bailii . Also, the reason why acetic acid concentrations known to be necrotic for S. cerevisiae induce PCD with an apoptotic phenotype in Z. bailii remains to be elucidated. In this study, a 2-DE-based expression mitochondrial proteomic analysis was explored to obtain new insights into the mechanisms involved in PCD in the Z. bailii derived hybrid strain ISA1307. This allowed the quantitative assessment of expression of protein species derived from each of the parental strains, with special emphasis on the processes taking place in the mitochondria known to play a key role in acetic acid - induced PCD. A marked decrease in the content of proteins involved in mitochondrial metabolism, in particular, in respiratory metabolism (Cor1, Rip1, Lpd1, Lat1 and Pdb1), with a concomitant increase in the abundance of proteins involved in fermentation (Pdc1, Ald4, Dld3) was registered. Other differentially expressed identified proteins also suggest the involvement of the oxidative stress response, protein translation, amino acid and nucleotide metabolism, among other processes, in the PCD response. Overall, the results strengthen the emerging concept of the importance of metabolic regulation of yeast PCD., Competing Interests: Conflict of interest: The authors declare that there are no conflicts of interest.