The BioVisualSpeech Serious Game with Voice Exercises for People with Parkinson's Disease with Hypokinetic Dysarthria.

People with Parkinson's disease (PD) can have dysarthria, a voice disorder that affects speech intelligibility. To fight this disorder people may resort to speech and language therapy. Unfortunately, weekly speech therapy sessions may not be enough, because to achieve and maintain good voice quality, intensive training is required. Additionally, the COVID-19 pandemic brought attention to the need for alternative speech therapy treatments that complement face-to-face appointments. Here, we propose a serious therapy game to improve voice loudness that can be used for intensive therapy or when face-to-face appointments are not possible. The game integrates three voice exercises used in speech therapy sessions for people with PD and aims to provide motivation for patients to perform the exercises on a daily basis. This application evaluates the vocal intensity, vocal frequency and maximum phonation time, offering real-time visual feedback. It also allows pathologists to customize the exercises difficulty to the needs of each patient.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses.

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7-22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

Metabolic footprint analysis of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations.

INTRODUCTION: There has been a growing interest towards creating defined mixed starter cultures for alcoholic fermentations. Previously, metabolite differences between single and mixed cultures have been explored at the endpoint of fermentations rather than during fermentations. OBJECTIVES: To create metabolic footprints of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations. METHODS: 1H NMR- and GC-MS-based metabolomics was used to identify metabolites that discriminate single and mixed cultures of Lachancea thermotolerans (LT) and Saccharomyces cerevisiae (SC) during alcoholic fermentations. RESULTS: Twenty-two metabolites were found when comparing single LT and mixed cultures, including both non-volatiles (carbohydrate, amino acid and acids) and volatiles (higher alcohols, esters, ketones and aldehydes). Fifteen of these compounds were discriminatory only at the death phase initiation (T1) and fifteen were discriminatory only at the death phase termination (T2) of LT in mixed cultures. Eight metabolites were discriminatory at both T1 and T2. These results indicate that specific metabolic changes may be descriptive of different LT growth behaviors. Fifteen discriminatory metabolites were found when comparing single SC and mixed cultures. These metabolites were all volatiles, and twelve metabolites were discriminatory only at T2, indicating that LT-induced changes in volatiles occur during the death phase of LT in mixed cultures and not during their initial growth stage. CONCLUSIONS: This work provides a detailed insight into yeast metabolites that differ between single and mixed cultures, and these data may be used for understanding and eventually predicting yeast metabolic changes in wine fermentations.

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation.

In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non-Saccharomyces yeast species (Lachancea thermotolerans, Metschnikowia pulcherrima, Hanseniaspora uvarum and Torulaspora delbrueckii) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae), serine (for L. thermotolerans), alanine (for H. uvarum), alanine and asparagine (for M. pulcherrima), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans, H. uvarum and M. pulcherrima, single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.

Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0<∆pH<3). Taken together, these results indicated that this subpopulation was under a viable but non-culturable (VBNC) state, which was further confirmed by recuperation assays. In summary, our study reveals that these AMPs compromise the plasma membrane integrity (and possibly also the vacuole membrane) of H. guilliermondii cells, disturbing the pHi homeostasis and inducing a loss of culturability.

Efficient fermentation of an improved synthetic grape must by enological and laboratory strains of Saccharomyces cerevisiae.

Grape must or freshly pressed grape juice is a complex chemical matrix that impacts the efficiency of yeast fermentation. The composition of natural grape must (NGM) can be variable; thus, to ensure reproducibility, a synthetic grape must (SGM) with defined composition is commonly used. The aim of this work was to create conditions to advance the use of Saccharomyces cerevisiae laboratory strains for wine fermentation studies, considering previous results obtained for enological strains fermenting NGM under simulated winery conditions. We designed a new SGM formulation, ISA-SGM, by introducing specific modifications to a commonly used formulation, putting together previous reports. We added glucose and fructose in equal amounts (125 g/l) and 50 parts per million (ppm) sulfur dioxide (SO2, corresponding to standard enological treatment), and we optimized the concentrations of malic acid (3 g/l), citric acid (0.3 g/l), and tartaric acid (3 g/l). Using ISA-SGM, we obtained similar fermentative profiles for the wine strain ISA1000, the prototrophic strain S288C, and its auxotrophic derivative BY4741. In this case, the concentrations of supplements were optimized to 120 mg/l L-uracil, 80 mg/l L-methionine, 400 mg/l L-leucine, and 100 mg/l L-histidine. All these strains tested in ISA-SGM presented a similar fermentative performance as ISA1000 in NGM. ISA-SGM formulation is a promising new tool to allow the use of the auxotrophic BY strains in the detailed assessment of the alcoholic fermentation process under simulated winery conditions, and it provides a foundation to extract relevant physiological conclusions in future research on enological yeast traits.

Peculiar H⁺ homeostasis of Saccharomyces cerevisiae during the late stages of wine fermentation.

Intracellular pH (pH(in)) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H(+) homeostasis is accomplished by an industrial wine strain of Saccharomyces cerevisiae while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH(in) 5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H(+) via H(+)-ATPase and of keeping H(+) out by means of an impermeable membrane were evaluated as potential mechanisms of H(+) homeostasis. At 30°C, H(+) efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H(+). Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H(+) remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H(+) permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H(+) homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.

Manejo da dor crônica em pacientes terminais / Management of chronic pain in terminal patients

Nesse artigo os autores realizaram uma revisão bibliográfica sobre o manejo da dor crônica em pacientes terminais. Abordando as técnicas anestésicas empregadas, bem como os fármacos utilizados para o alivio total ou parcial da dor crônica.