Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

<p>Abstract</p> <p>Background</p> <p>Acetic acid is a byproduct of <it>Saccharomyces cerevisiae </it>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 <it>S. cerevisiae </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Attomolar label-free detection of DNA hybridization with electrolyte-gated Graphene field-effect transistors

In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UID/FIS/04650/2013 and project POCI01-0145-FEDER-031069 (PORTGRAPHE). G. Machado Jr. acknowledges a PhD grant (no. 237630/2012–5) from CNPq– Brazil. J.B. acknowledges European funding from NBFS project under contract NORTE-01-0145-FEDER-00001

On the development of selective chelators for Cadmium: Synthesis, structure and chelating properties of 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide, a novel Thiadiazolyl Saccharinate

Aquatic contamination by heavy metals is a major concern for the serious negative consequences it has for plants, animals, and humans. Among the most toxic metals, Cd(II) stands out since selective and truly efficient methodologies for its removal are not known. We report a novel multidentate chelating agent comprising the heterocycles thiadiazole and benzisothiazole. 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide (AL14) was synthesized from cheap saccharin and characterized by different techniques, including single crystal X-ray crystallography. Our studies revealed the efficiency and selectivity of AL14 for the chelation of dissolved Cd(II) (as compared to Cu(II) and Fe(II)). Different spectral changes were observed upon the addition of Cd(II) and Cu(II) during UV-Vis titrations, suggesting different complexation interactions with both metals.UIDB/04326/2020, UIDB/04564/2020, ALG-01-0145-FEDER-022121, SFRH/BD/130407/2017info:eu-repo/semantics/publishedVersio

Mitochondrial proteomics of the acetic acid – induced programmed cell death response in a highly tolerant Zygosaccharomyces bailii – derived hybrid strain

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.Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledge

Tuning the Biological Activity of Camphorimine Complexes through Metal Selection

This research was funded by FCT—Fundação para a Ciência e a Tecnologia, through projects CQE (UIDB/00100/2020 and UIDP/00100/2020) and C2TN (UID/MULTI/04349/2019), the projects of the Research Unit Institute for Bioengineering and Biosciences—iBB (UIDB/04565/2020 and UIDP/04565/2020), the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, and a PhD grant to J.P.C. (UI/BD/152244/2021).The cytotoxic activity of four sets of camphorimine complexes based on the Cu(I), Cu(II), Ag(I), and Au(I) metal sites were assessed against the cisplatin-sensitive A2780 and OVCAR3 ovarian cancer cells. The results showed that the gold complexes were ca. one order of magnitude more active than the silver complexes, which in turn were ca. one order of magnitude more active than the copper complexes. An important finding was that the cytotoxic activity of the Ag(I) and Au(I) camphorimine complexes was higher than that of cisplatin. Another relevant aspect was that the camphorimine complexes did not interact significantly with DNA, in contrast with cisplatin. The cytotoxic activity of the camphorimine complexes displayed a direct relationship with the cellular uptake by OVCAR3 cells, as ascertained by PIXE (particle-induced X-ray emission). The levels of ROS (reactive oxygen species) formation exhibited an inverse relationship with the reduction potentials for the complexes with the same metal, as assessed by cyclic voltammetry. In order to gain insight into the toxicity of the complexes, their cytotoxicity toward nontumoral cells (HDF and V79 fibroblasts) was evaluated. The in vivo cytotoxicity of complex 5 using the nematode Caenorhabditis elegans was also assessed. The silver camphorimine complexes displayed the highest selectivity coefficients (activity vs. toxicity).publishersversionpublishe

Cobaltabis(dicarbollide) ([o-COSAN]&minus;) as Multifunctional Chemotherapeutics: A Prospective Application in Boron Neutron Capture Therapy (BNCT) for 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&prime;-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&prime;-I2-o-COSAN], whereas no apparent cell-killing effect was observed for untreated cells. Conclusions: These small molecules, particularly [8,8&prime;-I2-o-COSAN]&minus;, are serious candidates for BNCT now that the facilities of accelerator-based neutron sources are more accessible, providing an alternative treatment for resistant glioblastoma

Molecular Imprinting of Complex Matrices at Localized Surface Plasmon Resonance Biosensors for Screening of Global Interactions of Polyphenols and Proteins

Molecular imprinting polymers (MIP) have been applied to capture and stabilize complex protein matrices at plasmonic sensor surfaces. Ultrathin MIP layers at the surface of gold nanodisks enable the label free quantification of global interactions of polyphenols with protein mixtures. Separate polyphenols (catechin, procyanidin B3- catechin dimer, and PGG-pentagalloyl glucose) give specific and different binding levels to the MIP supported saliva plasmonic sensor. The demonstrated biosensor has application to study bioavailability of polyphenols or evaluation of local retention of small drug molecules.info:eu-repo/semantics/publishedVersio

The Mossbauer effect using Fe-57-ferrabisdicarbollide ([o-(57)FESAN](-)): a glance into the potential of a low-dose approach for glioblastoma radiotherapy

Although a variety of cancers are initially susceptible to chemotherapy, they eventually develop multi-drug resistance. To overcome this situation, more effective and selective treatments are necessary using anti-tumour agents that act in two or more ways and offer greater therapeutic benefits over single-mechanism entities. In this study, we report on treating cancer with Na[3,3′-57Fe(1,2-C2B9H11)2], which offers the possibility of dual action (radiation-drug combinations) to improve the clinical benefits and reduce healthy tissue toxicity. An approach to evaluating the potential of [o-57FESAN]− to treat glioblastoma using the Mössbauer effect is presented. As the therapeutic outcomes rely on the amount and distribution of [o-57FESAN]− inside the cells, several studies, using magnetization, Mössbauer spectroscopy and nuclear microscopy techniques, were performed to ascertain the uptake of [o-57FESAN]− in U87 glioblastoma cells. [o-57FESAN]− was found to be within the cells; 29% of its uptake was in the nuclear fraction, which is a particularly desirable target, because the nucleus is the cell's control centre where DNA and the transcription machinery reside. Irradiation studies with 2D and 3D cellular models of U87 cells showed that the growth inhibition effect observed was more pronounced when [o-57FESAN]− was used in combination with the Mössbauer effect in low total dose regimens, suggesting that this procedure either alone or as adjuvant may be useful for glioblastoma treatment.This work was supported by the Spanish Ministerio de Economía y Competitividad (PID2019-106832RB-100) and the Generalitat de Catalunya (2017SGR1720) and by the Fundação para a Ciência e Tecnologia (FCT/MEC) for projects UID/MULTI/04349/2020, PTDC/BTM-TEC/29256/2017, UIDB/04565/2020, UIDP/04565/2020 (iBB/IST), LA/P/0140/2020 (Associate Laboratory Institute for Health and Bioeconomy – i4HB), PTDC/QUI-QIN/32240/2017, LISBOA-01-0145-FEDER-022096 (National Infrastructure Roadmap, LTHMFL-NECL) and GCT grant to A. C. Cerdeira (BL156/2019_IST-ID). A. B. Buades was enrolled in the PhD program of the UAB. C. I. G. Pinto is enrolled in the PhD scholarship 689 DFA/BD/07119/2020. The authors thank Dr Moulay Sougrati, Charles Gerhardt Institute, ICGM UMR 5253, Montpellier, France for a kind gift of 57FeCl2. The LMRI (Metrology Laboratory of Ionizing Radiation) team is acknowledged for their support in the X-ray irradiation setup.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Boron clusters (ferrabisdicarbollides) shaping the future as radiosensitizers for multimodal (chemo/radio/PBFR) therapy of glioblastoma

Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, and is highly resistant to conventional radiotherapy and chemotherapy. Therefore, the development of multidrug resistance and tumor recurrence are frequent. Given the poor survival with the current treatments, new therapeutic strategies are urgently needed. Radiotherapy (RT) is a common cancer treatment modality for GBM. However, there is still a need to improve RT efficiency, while reducing the severe side effects. Radiosensitizers can enhance the killing effect on tumor cells with less side effects on healthy tissues. Herein, we present our pioneering study on the highly stable and amphiphilic metallacarboranes, ferrabis(dicarbollides) ([o-FESAN]- and [8,8'-I2-o-FESAN]-), as potential radiosensitizers for GBM radiotherapy. We propose radiation methodologies that utilize secondary radiation emissions from iodine and iron, using ferrabis(dicarbollides) as iodine/iron donors, aiming to achieve a greater therapeutic effect than that of a conventional radiotherapy. As a proof-of-concept, we show that using 2D and 3D models of U87 cells, the cellular viability and survival were reduced using this treatment approach. We also tested for the first time the proton boron fusion reaction (PBFR) with ferrabis(dicarbollides), taking advantage of their high boron (11B) content. The results from the cellular damage response obtained suggest that proton boron fusion radiation therapy, when combined with boron-rich compounds, is a promising modality to fight against resistant tumors. Although these results are encouraging, more developments are needed to further explore ferrabis(dicarbollides) as radiosensitizers towards a positive impact on the therapeutic strategies for GBM.The authors received support from the Spanish Ministerio de Economía y Competitividad (PID2019-106832RB-100), the Generalitat de Catalunya (2017SGR1720), FCT - Fundação para a Ciência e a Tecnologia, in the scope of the project UID/Multi/04349/2019 and the projects LISBOA-01-0247-FEDER-045904 and UTAP-EXPL/FMT/0020/2021 of Centro de Ciências e Tecnologias Nucleares/IST, PTDC/BTM-TEC/29256/2017, UIDP/04565/2020 of iBB/IST, UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences – UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB. The Metrology Laboratory of Ionizing Radiation team of Centro Tecnológico e Nuclear, Instituto Superior Técnico (CTN/IST) is acknowledged for their support in the irradiation setups. Miquel Nuez-Martínez is enrolled in the PhD program of the UAB. MQM and VMA acknowledge financial support by the Spanish Government MCIN/AEI/10.13039/501100011033 (project 2019-108434GB-I00 to VMA and project IJC2018-035283-I to MQM), and Universitat Jaume I (project UJI-B2018-53 to V. M. A. and project UJI-A2020-21 to MQM). SV thanks Croatian Science Foundation (project IP-2018-01-3168). Catarina I.G. Pinto is enrolled in the PhD scholarship 689 DFA/BD/07119/2020.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Quality of life, psychological morbidity and family stress in elderly residing in the community

Este estudo procurou investigar as relações existentes entre morbilidade psicológica, stress familiar e qualidade de vida (QV) da pessoa idosa. A amostra foi constituída por 126 idosos. Os instrumentos utilizados foram: The Lawton Instrumental Activities of Daily Living (IADL), Quality of Life (WHOQOL-Bref), Geriatric Anxiety Inventory (GSI), Geriatric Depression Scale (GDS); e Index of Family Relations (IFR). Os resultados revelaram a importância da idade, estado civil, escolaridade e número de patologias assim como o género na capacidade funcional, morbilidade, stress familiar e QV. Ao nível dos preditores, a depressão foi a variável que mais contribuiu para a QV. Não foram encontradas variáveis moderadoras no modelo. A discussão e implicações dos resultados são abordadas bem como a intervenção psicológica nesta população.This study sought to understand the relationships among psychological morbidity, family stress and quality of life (QL) of elderly. The sample consisted of 126 elderly. The following instruments were used: the Lawton Instrumental Activities of Daily Living (IADL); Quality of Life (WHOQOL-Bref), Geriatric Anxiety Inventory (GSI), Geriatric Depression Scale (GDS), and the Index of Family Relations (IFR). Results revealed the importance of age, marital status, education and number of pathologies as well as gender on functional capacity, morbidity, family stress and QV. In terms of predictors, depression was the variable that contributed the most to QL. There were no moderating variables in the model. Discussion and implications of results are addressed as well as psychological interventions.(undefined