Programmed cell death processes in Saccharomyces cerevisiae are altered by GUP1 deletion

During the past years, yeast has been successfully established as a model to study mechanisms of programmed cell death regulation. Saccharomyces cerevisiae commits to cell death showing typical hallmarks of metazoan apoptosis, in response to different stimuli. Gup1p, an O-acyltransferase, is required for several cellular processes that are related to apoptosis development, such as rafts integrity and stability, lipid metabolism including GPI anchor correct remodeling, proper mitochondrial and vacuole function, bud site selection and actin dynamics. We used two known apoptosis inducing conditions, chronological aging and acetic acid, to assess several apoptotic markers in gup1∆ mutant strain. We found that this mutant presents a significantly reduced chronological life span, comparing to Wt and it is also highly sensitive to acetic acid treatment. Although both chronological aging and acetic acid lead to identical effects, the differences between the strains in the levels/types of apoptotic markers are notorious. In addition, ROS levels of gup1∆ mutant strain were extremely high. According to our results, cells lacking GUP1 seem to be incapable of undergoing apoptosis. Instead this mutant appears to be experiencing a necrotic cell death process. Gup1p has been described to have an important function on lipid rafts assembly/integrity as well as on cell lipid profile. On the other hand, in the literature, rafts have been increasingly implicated on apoptotic signaling. The present results reinforce such idea.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201

First molecular and biochemical characterization of the extracellular matrix of Saccharomyces cerevisiae

Programa Doutoral em Biologia Molecular e Ambiental (área de especialização em Biotecnologia Molecular)A levedura Saccharomyces cerevisiae, tal como todos os microrganismos, é usualmente considerada como um organismo unicelular. Contudo, os microrganismos formam mais frequentemente comunidades multicelulares macroscópicas que apresentam diferenciação celular, e são coordenadas por um complexo sistema de comunicação, e suportadas por uma matriz extracelular (MEC). A presença deste tipo de suporte das comunidades multicelulares de S. cerevisiae foi descrita no início deste século. Apesar disso, a informação relacionada com a sua composição e organização tridimensional é escassa. Assim, o principal objetivo deste trabalho foi realizar a primeira abordagem sistemática aos principais componentes da MEC de levedura. Para o efeito, foram desenvolvidas metodologias para (1) obter de forma reprodutível uma considerável e homogénea biomassa de leveduras produtora de MEC, e (2) extrair e fracionar a MEC produzida de forma a obter frações analiticamente puras de proteínas e polissacáridos, compatíveis com a aplicação de metodologias analíticas de alto-débito como o GC-MS e o DIGE. A análise detalhada da fração proteica permitiu a identificação de mais de 600 proteínas. A maioria destas tem função e localização intracelulares, e é aqui identificada extracelularmente pela primeira vez, o que pode indicar um moonlighting surpreendentemente elevado. A presença de todas as enzimas associadas à glicólise e à fermentação, assim como ao ciclo do glioxilato, levanta suspeitas sobre a possibilidade de haver metabolismo extracelular. Além disso, um grande número de proteínas associadas à síntese, remodelação e degradação de outras proteínas foi identificado, incluindo elementos da família HSP70 e várias proteases. De realçar a presença das exopeptidases Lap4, Dug1 e Ecm14, e das metaloproteinases Prd1, Ape2 e Zps1, que partilham um domínio funcional zincin com as metaloproteinases da MEC de Eucariotas superiores. A presença adicional de proteínas intervenientes em várias vias de sinalização, como as Bmh1 e Bmh2, e da homing endonuclease Vde, que partilha o domínio Hedgehog/inteína com os morfogenos de Eucariotas superiores, sugere que a MEC de levedura poderá, tal como nesses organismos, mediar sinalização intercelular. As análises cromatográfica e eletroforética da fração glicosídica revelaram claramente a presença de dois polissacáridos. A análise por espectrometria de massa identificou glucose, manose e galactose na composição destes polissacáridos. Foram ainda observados indícios da presença de ácido urónico. A indução de metacromasia sugeriu que os polissacáridos detetados apresentam substituição química. A possibilidade desta corresponder a sulfatação foi testada através de um teste de atividade anticoagulante. Das diversas amostras de MEC de diferentes estirpes de levedura usadas, o duplo mutante gup1Δgup2Δ apresentou, ao contrário da estirpe Wt, razoável atividade anticoagulante indicadora da presença de grupos sulfato. Os efeitos da deleção do gene GUP1 na composição da MEC de levedura proporcionaram uma perspectiva mais detalhada da composição molecular e mecanismos a ela associados. Observaram-se alterações nas frações protéica e glicosídica. A deleção resultou na ausência de várias proteínas, associadas principalmente com o metabolismo de fontes de carbono, defesa e resgate da célula, bem como síntese, modificação e degradação de proteínas, e organização celular. Adicionalmente, a deleção deste gene também teve um grande impacto na composição glicosídica da matriz, levando ao desaparecimento do polissacárido de maior peso molecular detetado na estirpe Wt. Globalmente, os efeitos da deleção do GUP1 na MEC mostram que a estrutura desta é muito dinâmica e que se encontra sob controlo apertado das células que compõem o agregado multicelular. As funções sugeridas para as proteínas ortólogas das Gup1 e Gup2 de levedura, respetivamente Hhatl e Hhat, nas vias de sinalização de Eucariotas superiores esteve na origem da construção de uma bateria de estirpes de levedura recombinantes transformadas com os ortólogos da via Hedgehog de ratinho, mosca e homem, para futura avaliação. Da mesma forma, foram clonados em S. cerevisiae os recetores de mamífero para o ácido hialurónico (AH), CD44 e HMMR. Estes transformantes foram submetidos ao crescimento na presença de AH de diferentes tamanhos moleculares. As estirpes exprimindo ambos os recetores foram igualmente sensíveis à presença de AH de elevado peso molecular, mas foram diferentemente sensíveis à presença de AH de tamanho molecular intermédio. As células expressando o recetor CD44 mostraram-se, tal como em Eucariotas superiores, sensíveis à presença de AH 50 kDa, apresentando uma forte redução da taxa específica de crescimento. Isto indica a expressão funcional dos recetores de AH em levedura e a provável conservação da maquinaria celular de resposta a este componente da MEC dos Eucariotas superiores. Este trabalho é o primeiro a apresentar um estudo detalhado sobre as frações protéica e glicosídica secretadas para a matriz extracelular de S. cerevisiae durante o seu crescimento em comunidades multicelulares, oferecendo a primeira abordagem proteómica e glicómica da sua composição e organização. Globalmente, este trabalho permite prever que a MEC de levedura exerça funções equivalentes às conhecidas da MEC de Eucariotas superiores.The yeast Saccharomyces cerevisiae, as all microbes, is generally regarded as a unicellular organism. However, microorganisms live more frequently in macroscopic multicellular aggregates, presenting cellular differentiation, coordinated by complex communication, and supported by an extracellular matrix (ECM). The presence of this type of structure supporting multicellular life-style of S. cerevisiae was first described early this century. However, the information available on the yeast ECM components and three-dimensional spatial organization is scarce. Hence, this work aimed to provide a first methodical insight into the molecular composition of the yeast ECM major components. A methodology was developed capable of reproducibly obtaining ECMproducing homogenous yeast mats, and extracting and fractionating the yeast ECM into analytical-grade fractions. This was developed in order to be fully compatible with the application of high-throughput analytical techniques, like GC-MS and DIGE. The in-depth analysis of the proteins in the yeast ECM identified more than 600 proteins, most of which being ascribed to intracellular functions and localization, and therefore found extracellularly for the first time. This might indicate unexpectedly extensive moonlighting. The entire sets of enzymes from glycolysis and fermentation, as well as gluconeogenesis through glyoxylate cycle were highly represented, raising considerable reason for doubt as whether extracellular metabolism might exist. Moreover, a large number of proteins associated with protein fate and remodelling were found. These included several proteins from the HSP70 family, and proteases, importantly, the exopeptidases Lap4, Dug1 and Ecm14, and the metalloproteinases Prd1, Ape2 and Zps1, sharing a functional zincin domain with higher Eukaryotes ECM metalloproteinases. The further presence of the broad signalling cross-talkers Bmh1 and Bmh2, as well as the homing endonuclease Vde that shares a Hedgehog/intein domain with the Hh morphogens from higher Eukaryotes, suggest that analogously to the tissues in these organisms, yeast ECM is mediating signalling events. The chromatographic and electrophoretic analysis of the sugar fraction revealed the clear presence of two distinct polysaccharides. Mass spectrometry identified glucose, mannose and galactose in their composition. Evidence was also obtained of the presence of uronic acids. Both polysaccharides showed chemical substitution, as indicated by metachromasia, and the existence of sulphate groups was assessed through an anticoagulant activity test. From several ECM samples from different yeasts strains surveyed, the double mutant gup1Δgup2Δ displayed a relatively high anticoagulant activity, which was not observed in Wt, likely related to the presence of sulphate groups. The effects of the deletion of GUP1 gene in the composition of yeast ECM were also assessed, providing a more in-depth perspective of the ECM components and molecular mechanisms associated. Alterations in both protein and sugar fractions were observed. The deletion of GUP1 led to the absence of several ECM proteins, mainly associated with the carbon metabolism, cell rescue and defence, protein fate and cellular organization. Additionally, the disruption of this gene impacted in the composition of the ECM sugar fraction, through the disappearance of the higher molecular weight polysaccharide that had been detected in the Wt sample. The effects of GUP1 deletion on the ECM show that its structure is very dynamic, and that it is under the tight control of the cells composing the aggregate. S. cerevisiae Gup1 and Gup2 orthologues have suggested regulatory roles in the Hedgehog signalling pathway from higher Eukaryotes, in which organisms these proteins are known as Hhatl and Hhat, respectively. This led to the engineering the yeast mutants defective on either or both GUP1 and GUP2 by expressing these genes orthologues from fly, human and mouse, yielding a collection of transformants for future assessment. Similarly, the mammalian receptors of hyaluronic acid (HA), CD44 and HMMR, were cloned into the yeast S. cerevisiae. The engineered strains were subjected to growth in the presence of different molecular sizes of HA, and were identically and differentially sensitive to, respectively, high and intermediate molecular weight HA. The strain expressing CD44 presented a high growth sensitivity to the presence of 50 kDa HA as in high Eukaryotes. The HA receptors are therefore functional in the yeast cell, and the cellular machinery to respond to HA stimuli appears to be fairly conserved. The present work is the first to present a comprehensive detailed study on the protein and polysaccharide fractions secreted during growth in S. cerevisiae multicellular aggregates. Overall, this work gives a first insight of the multicellular communities of S. cerevisiae proteomics and glycomics, ascertaining yeast ECM with putative roles derived from its components that resemble ECM from higher Eukaryotes

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia

Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast\ua0Candida intermedia\ua0is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for\ua0C. intermedia, based on electroporation and gene deletion cassettes containing the\ua0Candida albicans NAT1\ua0dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the\ua0ADE2\ua0gene originally resulted in\ua0<1% targeting efficiencies, suggesting that\ua0C. intermedia\ua0mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for\ua0C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in\ua0C. intermedia, which opens the possibility to uncover and enhance its cell factory potential

Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p

<p>Abstract</p> <p>Background</p> <p><it>GUP1 </it>gene was primarily identified in <it>Saccharomyces cerevisiae </it>being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes.</p> <p>Results</p> <p>We show that <it>Candida albicans </it>Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to <it>S. cerevisiae</it>, Ca<it>gup1Δ </it>null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane.</p> <p>Conclusions</p> <p>This work is the first study in the opportunistic yeast <it>Candida albicans</it>, showing a role for the <it>GUP1 </it>gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about <it>GUP1 </it>gene (from <it>S. cerevisiae </it>and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.</p

Construção e validação de cenário de simulação clínica sobre o cuidado com o coto umbilical

Objetivo: Desenvolver e validar cenário de simulação clínica para ensino de puérperas e familiares sobre cuidados com o coto umbilical do recém-nascido.Método: Estudo metodológico em duas fases: 1 - construção de checklist do procedimento de higienização do coto umbilical, elaboração e validação semântica do caso clínico com 11 experts em simulação e área materno-infantil; 2 - desenvolvimento e validação do cenário junto a 11 experts em simulação e em neonatologia/pediatria. O Índice de Validade de Conteúdo de 0,8 foi adotado como relevante alcance de concordância.Resultados: Após construção do caso, realizou-se validação, aprovada por 100% dos experts. Todos itens do cenário simulado obtiveram escores de concordância superiores a 0,91.Conclusão: O cenário validado pode ser utilizado em diferentes contextos: ensino da Enfermagem Neonatal, capacitação de equipes e, aprendizagem de puérperas e familiares, alvos do estudo. Vistos benefícios da simulação, acredita-se na sua contribuição para melhoria assistencial e cuidado seguro. Palavras-chave: Enfermagem neonatal. Educação em saúde. Simulação. Alta do paciente. Recém-nascido. Cordão umbilical

Molecular characterization of Saccharomyces cerevisiae extracellular matrix and yeast response to different sizes of hyaluronan

Yeast, Saccharomyces cerevisiae, presents itself as the best studied eukaryotic model, with the possibility to modify its genetic information with highly advanced molecular techniques. This microorganism displays a high degree of similarity and conservation of processes with higher eukaryotes. Several fundamental mechanisms such as cell cycle regulation, DNA replication, recombination and repair were first uncovered in this microorganism. From areas as cancer to neurological diseases, as Parkinson, yeast has provided vital information. Different kinds of diseases in humans are connected to the Extracellular Matrix (ECM), like fibrosis and scleroderma. Therefore, the study of ECM components function as well as its regulation has been receiving lots of attention. Several of these, such as the structural molecule Hyaluronic Acid (HA) and diffusible growth factors, have the capacity to signal and redirect the behavior of the surrounding cells. HA has the ability to give different signal inputs depending solely on its size and concentration. Yeast is a unicellular eukaryotic with the remarkable capacity to live as individual, small aggregates and colonies. Within a colony, yeast cells live or die according to their relative position, being able to differentiate into hyphae, as well as pseudohyphae, and stalks. All these accomplished through the communication between the cells within the colony, which are embedded on a yet uncharacterized ECM. Besides ECM tridimensional structure aspect, none is known regarding its composition and organization. So an efficient method for the extraction, analysis and identification of S. cerevisiae ECM components, proteins and sugars, in colonies is currently in the last phase of implementation. The knowledge of the main constituents of the ECM will be a milestone in the establishment of this microbe as a model organism for ECM-related processes. Yeast doesn’t present the necessary enzymes and receptors to produce a response from HA. As yeast lacks the capacity to produce, degrade or “understand” the HA molecules, it is an excellent model for the manipulation of all aspects regarding the eukaryotic transduction of information from HA. The heterologous expression of HA receptors in yeast (CD44 and HMMR),the effect of this glycosaminoglycan on the main pathways, HOG, PKC or TOR, as well as changes in duplication time, chronological aging or life span, are also currently underway. The main asset of our work resides on the use of a simpler and better understood model, the yeast, for the study of HA effects on eukaryotic cells, and how the information is transduced. The characterization of yeast own ECM, and the understanding of the HA effect on this microorganism could lead yeast to a privileged position as a model organism for the study of ECM-related pathologies.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201

Austenite reversion kinetics and stability during tempering of an additively manufactured maraging 300 steel

Reverted austenite is a metastable phase that can be used in maraging steels to increase ductility via transformation-induced plasticity or TRIP effect. In the present study, 18Ni maraging steel samples were built by selective laser melting, homogenized at 820¿°C and then subjected to different isothermal tempering cycles aiming for martensite-to-austenite reversion. Thermodynamic simulations were used to estimate the inter-critical austenite¿+¿ferrite field and to interpret the results obtained after tempering. In-situ synchrotron X-ray diffraction was performed during the heating, soaking and cooling of the samples to characterize the martensite-to-austenite reversion kinetics and the reverted austenite stability upon cooling to room temperature. The reverted austenite size and distribution were measured by Electron Backscattered Diffraction. Results showed that the selected soaking temperatures of 610¿°C and 650¿°C promoted significant and gradual martensite-to-austenite reversion with high thermal stability. Tempering at 690¿°C caused massive and complete austenitization, resulting in low austenite stability upon cooling due to compositional homogenization.Peer ReviewedPostprint (author's final draft

Yeasts, the man’s best friend

1. Introduction In most cultures, bread making depends on a fermentation step. The flour leavening ability was, at first, most probably dependent on spontaneous fermentation. It became a controlled process by the maintenance of fresh innocula from one preparation to the next and this kind of environmental constraints eventually generated a particular type of yeast and bacteria biodiversity, adapted to ferment a certain brand of flour mixture, yielding specific organoleptic characteristics to the dough. Nowadays, although the baking industry generally uses commercially available strains of Saccharomyces cerevisiae for bread making, some types of bread are still prepared using dough carried over from previous makings as a starter. This trend decreased worldwide bread diversity and the cultural values associated, simultaneously increasing the dependence of local producers on world-scale yeast producers. Sustainability demands assessing yeast biodiversity, as well as devising simple and cheap methods for maintaining dough and multiply yeast.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201

The Role of Yeast and Lactic Acid Bacteria in the Production of Fermented Beverages in South America

Fermentation is one of the oldest forms of food preservation in the world. In South America, most fermented beverages are nondairy products featuring several other food raw materials such as cereals, fruits, and vegetables. Generally, natural fermentations are carried out by yeast and lactic acid bacteria forming a complex microbiota that acts in cooperation. Yeast have a prominent role in the production of beverages, due to the ability to accumulate high levels of ethanol and to produce highly desirable aroma compounds, but lactic acid bacteria are particularly important in fermentation because they produce desirable acids, flavor compounds, and peptides that inhibit the growth of undesirable organisms. Among the South America beverages based on cereals and vegetables, the fermented beverages chicha, caxiri, cauim and champús, and cachaça, a fermented and distilled beverage, could be cited. Genetic and physiological analyses of Saccharomyces cerevisiae strains isolated from cachaça have been shown to present interesting traits for beer production, such as flocculation and production of aroma compounds, fundamental to high-quality beer. The study of these traditional beverages allows the identification of new microorganism strains displaying enhanced resistance or new flavor and aroma profiles that could lead to applications in several industries and ultimately new products