Differences in metabolism among Saccharomyces species and their hybrids during wine fermentation.

This study aimed to investigate how parental genomes contribute to yeast hybrid metabolism using a metabolomic approach. Previous studies have explored central carbon and nitrogen metabolism in Saccharomyces species during wine fermentation, but this study analyses the metabolomes of Saccharomyces hybrids for the first time. We evaluated the oenological performance and intra- and extracellular metabolomes, and we compared the strains according to nutrient consumption and production of the main fermentative by-products. Surprisingly, no common pattern was observed for hybrid genome influence; each strain behaved differently during wine fermentation. However, this study suggests that the genome of the S. cerevisiae species may play a more relevant role in fermentative metabolism. Variations in biomass/nitrogen ratios were also noted, potentially linked to S. kudriavzevii and S. uvarum genome contributions. These results open up possibilities for further research using different "omics" approaches to comprehend better metabolic regulation in hybrid strains with genomes from different species.

Divergence in the Saccharomyces Species' Heat Shock Response Is Indicative of Their Thermal Tolerance.

The Saccharomyces species have diverged in their thermal growth profile. Both Saccharomyces cerevisiae and Saccharomyces paradoxus grow at temperatures well above the maximum growth temperature of Saccharomyces kudriavzevii and Saccharomyces uvarum but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature, we measured changes in gene expression in response to a 12 °C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families, we sequenced, assembled, and annotated a complete S. uvarum genome. In response to heat, the cryophilic species showed a stronger stress response than the thermophilic species, and the hybrids showed a mixture of parental responses that depended on the time point. After an initial strong response indicative of high thermal stress, hybrids with a thermophilic parent resolved their heat shock response to become similar to their thermophilic parent. Within the hybrids, only a small number of temperature-responsive genes showed consistent differences between alleles from the thermophilic and cryophilic species. Our results show that divergence in the heat shock response is mainly a consequence of a strain's thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat-induced changes are relevant to thermal divergence in the Saccharomyces species.

Novel findings about the mode of action of the antifungal protein PeAfpA against Saccharomyces cerevisiae.

Antifungal proteins (AFPs) from filamentous fungi offer the potential to control fungal infections that threaten human health and food safety. AFPs exhibit broad antifungal spectra against harmful fungi, but limited knowledge of their killing mechanism hinders their potential applicability. PeAfpA from Penicillium expansum shows strong antifungal potency against plant and human fungal pathogens and stands above other AFPs for being active against the yeast Saccharomyces cerevisiae. We took advantage of this and used a model laboratory strain of S. cerevisiae to gain insight into the mode of action of PeAfpA by combining (i) transcriptional profiling, (ii) PeAfpA sensitivity analyses of deletion mutants available in the S. cerevisiae genomic deletion collection and (iii) cell biology studies using confocal microscopy. Results highlighted and confirmed the role of the yeast cell wall (CW) in the interaction with PeAfpA, which can be internalized through both energy-dependent and independent mechanisms. The combined results also suggest an active role of the CW integrity (CWI) pathway and the cAMP-PKA signalling in the PeAfpA killing mechanism. Besides, our studies revealed the involvement of phosphatidylinositol metabolism and the participation of ROX3, which codes for the subunit 19 of the RNA polymerase II mediator complex, in the yeast defence strategy. In conclusion, our study provides clues about both the killing mechanism of PeAfpA and the fungus defence strategies against the protein, suggesting also targets for the development of new antifungals. KEY POINTS: • PeAfpA is a cell-penetrating protein with inhibitory activity against S. cerevisiae. • The CW integrity (CWI) pathway is a key player in the PeAfpA killing mechanism. • Phosphatidylinositol metabolism and ROX3 are involved in the yeast defence strategy.

Divergence in the Saccharomyces species' heat shock response is indicative of their thermal tolerance.

The Saccharomyces species have diverged in their thermal growth profile. Both S. cerevisiae and S. paradoxus grow at temperatures well above the maximum growth temperature of S. kudriavzevii and S. uvarum, but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature we measured changes in gene expression in response to a 12°C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families we sequenced, assembled and annotated a complete S. uvarum genome. All the strains exhibited a stronger response to heat than cold treatment. In response to heat, the cryophilic species showed a stronger response than the thermophilic species. The hybrids showed a mixture of parental stress responses depending on the time point. After the initial response, hybrids with a thermophilic parent were more similar to S. cerevisiae and S. paradoxus, and the S. cerevisiae × S. paradoxus hybrid showed the weakest heat shock response. Within the hybrids a small subset of temperature responsive genes showed species specific responses but most were also hybrid specific. Our results show that divergence in the heat shock response is indicative of a strain's thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat induced changes are relevant to thermal divergence in the Saccharomyces species.

Saccharomyces cerevisiae wine strains show a wide range of competitive abilities and differential nutrient uptake behavior in co-culture with S. kudriavzevii.

The wine industry has implemented complex starters with multiple yeast species as an efficient method to improve certain wine properties. Strains' competitive fitness becomes essential for its use in such cases. In the present work, we studied this trait in 60 S. cerevisiae strains from different origins, co-inoculated with a S. kudriavzevii strain, and confirmed it to be associated with the strains' origin. To gather deeper knowledge about the characteristics of strains with highly competitive ability versus the rest, microfermentations using representative strains from each group were performed and the carbon and nitrogen sources uptake was analysed. Our results demonstrate that despite wine strains being the subclade with the highest competitive ability, they present a wide range of behaviors as well as nutrient uptake dynamics, which points to a heterogeneous nature of domestication processes. An interesting strategy was observed in the highly competitive strains (GRE and QA23), the nitrogen sources uptake in the competition was accelerated and the sugar fermentation was slowing despite the fermentation finish at the same time. Therefore, this competition study, using particular combinations of strains, expands the knowledge in the field of the usage of mixed starters in wine manufactured products.

The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases.

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate's aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

Genome-wide effect of non-optimal temperatures under anaerobic conditions on gene expression in Saccharomyces cerevisiae.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions. Transcriptomic analysis of the three strains revealed a huge complexity of cellular mechanisms and responses. Overall, cold exerted a stronger transcriptional response in the three strains comparing with heat conditions, with a higher number of down-regulating genes than of up-regulating genes regardless the strain analyzed. The comparison of the transcriptome at both sub- and supra-optimal temperatures showed the presence of common genes up- or down-regulated in both conditions, but also the presence of common genes up- or down-regulated in the three studied strains. More specifically, we have identified and validated three up-regulated genes at sub-optimal temperature in the three strains, OPI3, EFM6 and YOL014W. Finally, the comparison of the transcriptomic data with a previous proteomic study with the same strains revealed a good correlation between gene activity and protein abundance, mainly at low temperature. Our work provides a global insight into the specific mechanisms involved in temperature adaptation regarding both transcriptome and proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid.

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII-XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms.

Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients.

SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0-5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1mut patients is essential for a better prognostic evaluation.

Co-occurrence of cohesin complex and Ras signaling mutations during progression from myelodysplastic syndromes to secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to secondary acute myeloid leukemia (sAML). However, the mutational dynamics and clonal evolution underlying disease progression are poorly understood at present. To elucidate the mutational dynamics of pathways and genes occurring during the evolution to sAML, next generation sequencing was performed on 84 serially paired samples of MDS patients who developed sAML (discovery cohort) and 14 paired samples from MDS patients who did not progress to sAML during follow-up (control cohort). Results were validated in an independent series of 388 MDS patients (validation cohort). We used an integrative analysis to identify how mutations, alone or in combination, contribute to leukemic transformation. The study showed that MDS progression to sAML is characterized by greater genomic instability and the presence of several types of mutational dynamics, highlighting increasing (STAG2) and newly-acquired (NRAS and FLT3) mutations. Moreover, we observed cooperation between genes involved in the cohesin and Ras pathways in 15-20% of MDS patients who evolved to sAML, as well as a high proportion of newly acquired or increasing mutations in the chromatin-modifier genes in MDS patients receiving a disease-modifying therapy before their progression to sAML.

Temperature Shapes Ecological Dynamics in Mixed Culture Fermentations Driven by Two Species of the Saccharomyces Genus.

Mixed culture wine fermentations combining species within the Saccharomyces genus have the potential to produce new market tailored wines. They may also contribute to alleviating the effects of climate change in winemaking. Species, such as S. kudriavzevii, show good fermentative properties at low temperatures and produce wines with lower alcohol content, higher glycerol amounts and good aroma. However, the design of mixed culture fermentations combining S. cerevisiae and S. kudriavzevii species requires investigating their ecological interactions under cold temperature regimes. Here, we derived the first ecological model to predict individual and mixed yeast dynamics in cold fermentations. The optimal model combines the Gilpin-Ayala modification to the Lotka-Volterra competitive model with saturable competition and secondary models that account for the role of temperature. The nullcline analysis of the proposed model revealed how temperature shapes ecological dynamics in mixed co-inoculated cold fermentations. For this particular medium and species, successful mixed cultures can be achieved only at specific temperature ranges or by sequential inoculation. The proposed ecological model can be calibrated for different species and provide valuable insights into the functioning of alternative mixed wine fermentations.

The effect of two antifungal commercial formulations on the metabolism of a commercial Saccharomyces cerevisiae strain and their repercussion on fermentation evolution and phenylalanine catabolism.

The effect of two commercial formulations (incorporating mepanipyrim and tetraconazole as active substances) on the metabolism of Saccharomyces cerevisiae Lalvin T73™, growing on a synthetic grape must, and their influence on the alcoholic fermentation course and the biosynthesis of volatiles derived from phenylalanine catabolism was studied. No relevant effects were observed for mepanipyrim except for glycerol production. On the contrary, in the presence of tetraconazole many genes and some proteins related to cell cycle progression and mitosis were repressed. This fact could explain the lower biomass concentration and the lower sugar consumption registered for tetraconazole at the end of the study. However, the biomass-to-ethanol yield was higher in connection with the overexpression of the ADH1 gene. The presence of tetraconazole residues seems to accelerate the Ehrlich pathway. These results agree with the overexpression of several genes (BAT1, PDC1, PDC5, ADH1, SFA1, ATF2, PFK1, PFK2 and ARO3) and a higher abundance of two proteins (Gap1p and Atf2p) involved in this metabolic pathway.

Dominance of wine Saccharomyces cerevisiae strains over S. kudriavzevii in industrial fermentation competitions is related to an acceleration of nutrient uptake and utilization.

Grape must is a sugar-rich habitat for a complex microbiota which is replaced by Saccharomyces cerevisiae strains during the first fermentation stages. Interest on yeast competitive interactions has recently been propelled due to the use of alternative yeasts in the wine industry to respond to new market demands. The main issue resides in the persistence of these yeasts due to the specific competitive activity of S. cerevisiae. To gather deeper knowledge of the molecular mechanisms involved, we performed a comparative transcriptomic analysis during fermentation carried out by a wine S. cerevisiae strain and a strain representative of the cryophilic S. kudriavzevii, which exhibits high genetic and physiological similarities to S. cerevisiae, but also differences of biotechnological interest. In this study, we report that transcriptomic response to the presence of a competitor is stronger in S. cerevisiae than in S. kudriavzevii. Our results demonstrate that a wine S. cerevisiae industrial strain accelerates nutrient uptake and utilization to outcompete the co-inoculated yeast, and that this process requires cell-to-cell contact to occur. Finally, we propose that this competitive phenotype evolved recently, during the adaptation of S. cerevisiae to man-manipulated fermentative environments, since a non-wine S. cerevisiae strain, isolated from a North American oak, showed a remarkable low response to competition.

New Trends in the Uses of Yeasts in Oenology.

The most important factor in winemaking is the quality of the final product and the new trends in oenology are dictated by wine consumers and producers. Traditionally the red wine is the most consumed and more popular; however, in the last times, the wine companies try to attract other groups of populations, especially young people and women that prefer sweet, whites or rosé wines, very fruity and with low alcohol content. Besides the new trends in consumer preferences, there are also increased concerns on the effects of alcohol consumption on health and the effects of global climate change on grape ripening and wine composition producing wines with high alcohol content. Although S. cerevisiae is the most frequent species in wines, and the subject of most studies, S. uvarum and hybrids between Saccharomyces species such as S. cerevisiae×S. kudriavzevii and S. cerevisiae×S. uvarum are also involved in wine fermentations and can be preponderant in certain wine regions. New yeast starters of non-cerevisiae strains (S. uvarum) or hybrids (S. cerevisiae×S. uvarum and S. cerevisiae×S. kudriavzevii) can contribute to solve some problems of the wineries. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts, while fulfilling the requirements of the commercial yeasts, such as a good fermentative performance and aromatic profiles that are of great interest for the wine industry. In this review, we will analyze different applications of nonconventional yeasts to solve the current winemaking demands.

Basophil-lineage commitment in acute promyelocytic leukemia predicts for severe bleeding after starting therapy.

Severe hemorrhagic events occur in a significant fraction of acute promyelocytic leukemia patients, either at presentation and/or early after starting therapy, leading to treatment failure and early deaths. However, identification of independent predictors for high-risk of severe bleeding at diagnosis, remains a challenge. Here, we investigated the immunophenotype of bone marrow leukemic cells from 109 newly diagnosed acute promyelocytic leukemia patients, particularly focusing on the identification of basophil-related features, and their potential association with severe bleeding episodes and patient overall survival.From all phenotypes investigated on leukemic cells, expression of the CD203c and/or CD22 basophil-associated markers showed the strongest association with the occurrence and severity of bleeding (p ≤ 0.007); moreover, aberrant expression of CD7, coexpression of CD34+/CD7+ and lack of CD71 was also more frequently found among patients with (mild and severe) bleeding at baseline and/or after starting treatment (p ≤ 0.009). Multivariate analysis showed that CD203c expression (hazard ratio: 26.4; p = 0.003) and older age (hazard ratio: 5.4; p = 0.03) were the best independent predictors for cumulative incidence of severe bleeding after starting therapy. In addition, CD203c expression on leukemic cells (hazard ratio: 4.4; p = 0.01), low fibrinogen levels (hazard ratio: 8.8; p = 0.001), older age (hazard ratio: 9.0; p = 0.002), and high leukocyte count (hazard ratio: 5.6; p = 0.02) were the most informative independent predictors for overall survival.In summary, our results show that the presence of basophil-associated phenotypic characteristics on leukemic cells from acute promyelocytic leukemia patients at diagnosis is a powerful independent predictor for severe bleeding and overall survival, which might contribute in the future to (early) risk-adapted therapy decisions.

Saccharomyces cerevisiae and S. kudriavzevii Synthetic Wine Fermentation Performance Dissected by Predictive Modeling.

Wineries face unprecedented challenges due to new market demands and climate change effects on wine quality. New yeast starters including non-conventional Saccharomyces species, such as S. kudriavzevii, may contribute to deal with some of these challenges. The design of new fermentations using non-conventional yeasts requires an improved understanding of the physiology and metabolism of these cells. Dynamic modeling brings the potential of exploring the most relevant mechanisms and designing optimal processes more systematically. In this work we explore mechanisms by means of a model selection, reduction and cross-validation pipeline which enables to dissect the most relevant fermentation features for the species under consideration, Saccharomyces cerevisiae T73 and Saccharomyces kudriavzevii CR85. The pipeline involved the comparison of a collection of models which incorporate several alternative mechanisms with emphasis on the inhibitory effects due to temperature and ethanol. We focused on defining a minimal model with the minimum number of parameters, to maximize the identifiability and the quality of cross-validation. The selected model was then used to highlight differences in behavior between species. The analysis of model parameters would indicate that the specific growth rate and the transport of hexoses at initial times are higher for S. cervisiae T73 while S. kudriavzevii CR85 diverts more flux for glycerol production and cellular maintenance. As a result, the fermentations with S. kudriavzevii CR85 are typically slower; produce less ethanol but higher glycerol. Finally, we also explored optimal initial inoculation and process temperature to find the best compromise between final product characteristics and fermentation duration. Results reveal that the production of glycerol is distinctive in S. kudriavzevii CR85, it was not possible to achieve the same production of glycerol with S. cervisiae T73 in any of the conditions tested. This result brings the idea that the optimal design of mixed cultures may have an enormous potential for the improvement of final wine quality.

Expression of heterologous transporters in Saccharomyces kudriavzevii: A strategy for improving yeast salt tolerance and fermentation performance.

S. kudriavzevii has potential for fermentations and other biotechnological applications, but is sensitive to many types of stress. We tried to increase its tolerance and performance via the expression of various transporters from different yeast species. Whereas the overexpression of Z. rouxii fructose uptake systems (ZrFfz1 and ZrFsy1) or a glycerol importer (ZrStl1) did not improve the ability of S. kudriavzevii to consume fructose and survive osmotic stress, the expression of alkali-metal-cation exporters (ScEna1, ScNha1, YlNha2) improved S. kudriavzevii salt tolerance, and that of ScNha1 also the fermentation performance. The level of improvement depended on the type and activity of the transporter suggesting that the natural sensitivity of S. kudriavzevii cells to salts is based on a non-optimal functioning of its own transporters.

The Use of Mixed Populations of Saccharomyces cerevisiae and S. kudriavzevii to Reduce Ethanol Content in Wine: Limited Aeration, Inoculum Proportions, and Sequential Inoculation.

Saccharomyces cerevisiae is the most widespread microorganism responsible for wine alcoholic fermentation. Nevertheless, the wine industry is currently facing new challenges, some of them associate with climate change, which have a negative effect on ethanol content and wine quality. Numerous and varied strategies have been carried out to overcome these concerns. From a biotechnological point of view, the use of alternative non-Saccharomyces yeasts, yielding lower ethanol concentrations and sometimes giving rise to new and interesting aroma, is one of the trendiest approaches. However, S. cerevisiae usually outcompetes other Saccharomyces species due to its better adaptation to the fermentative environment. For this reason, we studied for the first time the use of a Saccharomyces kudriavzevii strain, CR85, for co-inoculations at increasing proportions and sequential inoculations, as well as the effect of aeration, to improve its fermentation performance in order to obtain wines with an ethanol yield reduction. An enhanced competitive performance of S. kudriavzevii CR85 was observed when it represented 90% of the cells present in the inoculum. Furthermore, airflow supply of 20 VVH to the fermentation synergistically improved CR85 endurance and, interestingly, a significant ethanol concentration reduction was achieved.

Multidimensional assessment of patient condition and mutational analysis in peripheral blood, as tools to improve outcome prediction in myelodysplastic syndromes: A prospective study of the Spanish MDS group.

The International Prognostic Scoring System and its revised form (IPSS-R) are the most widely used indices for prognostic assessment of patients with myelodysplastic syndromes (MDS), but can only partially account for the observed variation in patient outcomes. This study aimed to evaluate the relative contribution of patient condition and mutational status in peripheral blood when added to the IPSS-R, for estimating overall survival and the risk of leukemic transformation in patients with MDS. A prospective cohort (2006-2015) of 200 consecutive patients with MDS were included in the study series and categorized according to the IPSS-R. Patients were further stratified according to patient condition (assessed using the multidimensional Lee index for older adults) and genetic mutations (peripheral blood samples screened using next-generation sequencing). The change in likelihood-ratio was tested in Cox models after adding individual covariates. The addition of the Lee index to the IPSS-R significantly improved prediction of overall survival [hazard ratio (HR) 3.02, 95% confidence interval (CI) 1.96-4.66, P < 0.001), and mutational analysis significantly improved prediction of leukemic evolution (HR 2.64, 1.56-4.46, P < 0.001). Non-leukemic death was strongly linked to patient condition (HR 2.71, 1.72-4.25, P < 0.001), but not to IPSS-R score (P = 0.35) or mutational status (P = 0.75). Adjustment for exposure to disease-modifying therapy, evaluated as a time-dependent covariate, had no effect on the proposed model's predictive ability. In conclusion, patient condition, assessed by the multidimensional Lee index and patient mutational status can improve the prediction of clinical outcomes of patients with MDS already stratified by IPSS-R.