Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer''s Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer's yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.     

Scent of a killer: How could killer yeast boost its dispersal?

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.     

Detailed Analysis of the Microbial Population in Malaysian Spontaneous Cocoa Pulp Fermentations Reveals a Core and Variable Microbiota

The fermentation of cocoa pulp is one of the few remaining large-scale spontaneous microbial processes in today''s food industry. The microbiota involved in cocoa pulp fermentations is complex and variable, which leads to inconsistent production efficiency and cocoa quality. Despite intensive research in the field, a detailed and comprehensive analysis of the microbiota is still lacking, especially for the expanding Asian production region. Here, we report a large-scale, comprehensive analysis of four spontaneous Malaysian cocoa pulp fermentations across two time points in the harvest season and two fermentation methods. Our results show that the cocoa microbiota consists of a “core” and a “variable” part. The bacterial populations show a remarkable consistency, with only two dominant species, Lactobacillus fermentum and Acetobacter pasteurianus. The fungal diversity is much larger, with four dominant species occurring in all fermentations (“core” yeasts), and a large number of yeasts that only occur in lower numbers and specific fermentations (“variable” yeasts). Despite this diversity, a clear pattern emerges, with early dominance of apiculate yeasts and late dominance of Saccharomyces cerevisiae. Our results provide new insights into the microbial diversity in Malaysian cocoa pulp fermentations and pave the way for the selection of starter cultures to increase efficiency and consistency.     

A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.     

Genomic Insights into the Saccharomyces sensu stricto Complex

The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical “domestication” of these yeasts for baking, brewing, and winemaking.     

Commentary: Personal Renewal

After John Gardner''s presentation on “Self-Renewal” to THE WESTERN JOURNAL OF MEDICINE Editors'' Meeting, ^* Joseph Murphy, MD, Special Editor for Wyoming, asked the former Secretary of Health, Education, and Welfare, “Where are you in your life''s cycle?” Dr Gardner, who is 80 years old, answered, “When Chief Justice Oliver Wendell Holmes, Jr, was in his 90s, he was asked a similar question and said, `I''m like a race horse cantering along after the race is over, cooling down.'' Well, I''m nowhere near cantering! I''m still in the race, pushing the world.” race, pushing the world.”John Gardner, who received his undergraduate degree from Stanford and PhD from the University of California, Berkeley, taught at the college level for several years before he joined the Carnegie Foundation. As president of Carnegie Corporation and Carnegie Foundation for the Advancement of Teaching, he began to “push the world” toward education and in 1964 received the country''s highest civilian honor, the Presidential Medal of Freedom. He has also pushed it toward political reform by founding Common Cause, toward grass-roots political action by founding the Urban Coalition, toward leadership training by founding the White House Fellows program, and toward volunteerism by founding the Independent Sector (a coalition of for-profit and not-for-profit organizations and foundations). His books, including Excellence, Self-Renewal, No Easy Victories, and On Leadership, have pushed readers to new understanding of themselves and of organizations to higher levels of creativity and energy to get important work done. His current research focuses on discovering and defining the characteristics of healthy, vital communities. His call to “keep on keeping on,” indeed, to push the world, leads to constructive change. Active people become effective people, infused with the energy and optimism that good hard work inspires. I think you will find this paper as invigorating to read as it was to hear.     

Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer

The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed.This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between “vital”, “sublethal” and “dead” subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.     

A statistical anomaly indicates symbiotic origins of eukaryotic membranes

Compositional analyses of nucleic acids and proteins have shed light on possible origins of living cells. In this work, rigorous compositional analyses of ∼5000 plasma membrane lipid constituents of 273 species in the three life domains (archaea, eubacteria, and eukaryotes) revealed a remarkable statistical paradox, indicating symbiotic origins of eukaryotic cells involving eubacteria. For lipids common to plasma membranes of the three domains, the number of carbon atoms in eubacteria was found to be similar to that in eukaryotes. However, mutually exclusive subsets of same data show exactly the opposite—the number of carbon atoms in lipids of eukaryotes was higher than in eubacteria. This statistical paradox, called Simpson''s paradox, was absent for lipids in archaea and for lipids not common to plasma membranes of the three domains. This indicates the presence of interaction(s) and/or association(s) in lipids forming plasma membranes of eubacteria and eukaryotes but not for those in archaea. Further inspection of membrane lipid structures affecting physicochemical properties of plasma membranes provides the first evidence (to our knowledge) on the symbiotic origins of eukaryotic cells based on the “third front” (i.e., lipids) in addition to the growing compositional data from nucleic acids and proteins.     

Excitability of the Primary Motor Cortex Increases More Strongly with Slow- than with Normal-Speed Presentation of Actions

Introduction

The aim of the present study was to investigate how the speed of observed action affects the excitability of the primary motor cortex (M1), as assessed by the size of motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS).

Methods

Eighteen healthy subjects watched a video clip of a person catching a ball, played at three different speeds (normal-, half-, and quarter-speed). MEPs were induced by TMS when the model''s hand had opened to the widest extent just before catching the ball (“open”) and when the model had just caught the ball (“catch”). These two events were locked to specific frames of the video clip (“phases”), rather than occurring at specific absolute times, so that they could easily be compared across different speeds. MEPs were recorded from the thenar (TH) and abductor digiti minimi (ADM) muscles of the right hand.

Results

The MEP amplitudes were higher when the subjects watched the video clip at low speed than when they watched the clip at normal speed. A repeated-measures ANOVA, with the factor VIDEO-SPEED, showed significant main effects. Bonferroni''s post hoc test showed that the following MEP amplitude differences were significant: TH, normal vs. quarter; ADM, normal vs. half; and ADM, normal vs. quarter. Paired t-tests showed that the significant MEP amplitude differences between TMS phases under each speed condition were TH, “catch” higher than “open” at quarter speed; ADM, “catch” higher than “open” at half speed.

Conclusions

These results indicate that the excitability of M1 was higher when the observed action was played at low speed. Our findings suggest that the action observation system became more active when the subjects observed the video clip at low speed, because the subjects could then recognize the elements of action and intention in others.     

Relationship Between the Sterol Content of Yeast Cells and Their Fermentation Activity in Grape Must

In grape must of high sugar concentration, yeast growth, the viability rate of “resting” yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols added in the medium acted as (i) growth factors, (ii) fermentation inhibitors, and (iii) survival factors for the yeast.     

A Study on the Fundamental Mechanism and the Evolutionary Driving Forces behind Aerobic Fermentation in Yeast

Baker’s yeast Saccharomyces cerevisiae rapidly converts sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. The later phenomenon is called Crabtree effect and has been described in two forms, long-term and short-term effect. We have previously studied under fully controlled aerobic conditions forty yeast species for their central carbon metabolism and the presence of long-term Crabtree effect. We have also studied ten steady-state yeast cultures, pulsed them with glucose, and followed the central carbon metabolism and the appearance of ethanol at dynamic conditions. In this paper we analyzed those wet laboratory data to elucidate possible mechanisms that determine the fate of glucose in different yeast species that cover approximately 250 million years of evolutionary history. We determine overflow metabolism to be the fundamental mechanism behind both long- and short-term Crabtree effect, which originated approximately 125–150 million years ago in the Saccharomyces lineage. The “invention” of overflow metabolism was the first step in the evolution of aerobic fermentation in yeast. It provides a general strategy to increase energy production rates, which we show is positively correlated to growth. The “invention” of overflow has also simultaneously enabled rapid glucose consumption in yeast, which is a trait that could have been selected for, to “starve” competitors in nature. We also show that glucose repression of respiration is confined mainly among S. cerevisiae and closely related species that diverged after the whole genome duplication event, less than 100 million years ago. Thus, glucose repression of respiration was apparently “invented” as a second step to further increase overflow and ethanol production, to inhibit growth of other microbes. The driving force behind the initial evolutionary steps was most likely competition with other microbes to faster consume and convert sugar into biomass, in niches that were semi-anaerobic.     

Research of a holiday kind: A clinical trial gone awry: the Chocolate Happiness Undergoing More Pleasantness (CHUMP) study

The randomized controlled trial is the “gold standard” for evaluating the benefits and harms of interventions. The Chocolate Happiness Undergoing More Pleasantness (CHUMP) study was designed to compare the effects of dark chocolate, milk chocolate and normal chocolate consumption on happiness. Although the intention-to-treat analysis showed that participants who received either dark or milk chocolate were happier than those who received no additional chocolate, the actual-consumption analysis showed that there were no differences between any of the groups. The reason for this result is that many participants switched groups mid-study because of their personal chocolate preferences. Although the CHUMP study was pleasurable, it demonstrated the difficulties associated with performing a truly blinded clinical trial.The randomized controlled trial is the “gold standard” for testing the beneficial and harmful effects of interventions. There has been a growing concern in the literature about the potential for bias during randomization of participants in clinical trials.^1–5 I designed the Chocolate Happiness Undergoing More Pleasantness (CHUMP) study to compare the effects of dark chocolate, milk chocolate and “normal” chocolate consumption on happiness. The CHUMP study was a double-blinded clinical trial, and it demonstrated the difficulties associated with performing a truly blinded clinical trial.     

The Metabolism of the Volatile Amines: VII. The Clinical Significance of Two Components of the Blood Ammonia Level

Blood ammonia levels consist of two components: ammonia present in blood at the time of shedding, termed “free” ammonia, and ammonia produced by the deamidating action of the alkali reagents. Blood of healthy people contained little or no “free” ammonia while blood of patients with chronic liver disease occasionally showed levels up to 1.2 μg./ml. Patients with hepatic encephalopathy had significantly elevated levels which usually fell to zero following therapy. Levels of “free” ammonia above 0.6 μg./ml. were diagnostic of hepatic encephalopathy in patients suffering from unexplained neurological disorders.The rate of formation of ammonia by the alkali reagents was increased in patients with hepatic necrosis and was depressed in those with chronic hepatitis. The ammonia appeared to arise from the deamidation of glutamine and asparagine, present in blood in both the free and peptide forms.     

How Often Are Antibiotic-Resistant Bacteria Said to “Evolve” in the News?

Media plays an important role in informing the general public about scientific ideas. We examine whether the word “evolve,” sometimes considered controversial by the general public, is frequently used in the popular press. Specifically, we ask how often articles discussing antibiotic resistance use the word “evolve” (or its lexemes) as opposed to alternative terms such as “emerge” or “develop.” We chose the topic of antibiotic resistance because it is a medically important issue; bacterial evolution is a central player in human morbidity and mortality. We focused on the most widely-distributed newspapers written in English in the United States, United Kingdom, Canada, India, and Australia. We examined all articles that focused primarily on the evolution of antibiotic resistance, were published in 2014 or earlier, and were accessible in online archives, for a total of 1639 articles. The total years examined per newspaper ranged from 5 to 37 years with a median of 27 years, and the overall range was 1978–2014. We quantified how many articles included the term “evolve” and analyzed how this varied with newspaper, country, and time. We found that an overall rate of 18% of articles used the term “evolve” but with significant variation among countries. Newspapers in the United Kingdom had the highest rate (24%), more than double of those in India (9%), the country with the lowest rate. These frequencies were lower than those found in scientific papers from both evolutionary journals and biomedical journals. There were no statistically significant changes in frequency and no trends when “evolve” usage was compared against variables such as newspaper circulation, liberal/conservative bias, time, and state evolution acceptance in U.S. newspapers. This study highlights the globally low usage of the word “evolve” in the popular press. We suggest this low usage may affect public understanding and acceptance of evolutionary concepts.     

Practitioner attitudes in the United States and United Kingdom toward decisions at the end of life: are medical ethicists out of touch?

Objective To assess whether UK and US health care professionals share the views of medical ethicists about medical futility, withdrawing or withholding treatment, ordinary or extraordinary interventions, and the doctrine of double effect. Design, subjects, and setting Answers to a 138-item attitudinal questionnaire completed by 469 UK nurses studying the Open University course on “Death and Dying” were compared with those of a similar questionnaire administered to 759 US nurses and 687 US physicians taking the Hastings Center course on “Decisions Near the End of Life.” Results Practitioners accept the relevance of concepts widely disparaged by bioethicists: double effect, medical futility, and the distinctions between heroic and ordinary interventions and withholding and withdrawing treatment. Within the UK nurses'' group, the responses of a “rationalist” axis of respondents who describe themselves as having “no religion” are closer to the bioethics consensus on withholding and withdrawing treatment. Conclusions Professionals'' beliefs differ substantially from the recommendations of their professional bodies and from majority opinion in bioethics. Bioethicists should be cautious about assuming that their opinions will be readily accepted by practitioners.     

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

Continuous directed evolution using the yeast OrthoRep system is a powerful way to improve enzymes for use in plant engineering as illustrated by “plantizing” a bacterial thiamin synthesis enzyme.     

Adaptive Response to DNA-Damaging Agents in Natural Saccharomyces cerevisiae Populations from “Evolution Canyon”, Mt. Carmel,Israel

Background

Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the “Evolution Canyon” microsite (Nahal Oren, Mt. Carmel, Israel). The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%–800% more on the “African” slope) caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid “African” environment while the north-facing slope is represented by temperate, “European” forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS) in order to evaluate the interslope effect on the strains'' ability to withstand DNA-damaging agents.

Methodology/Principal Findings

We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the “African” slope were more resilient to both UVA and MMS than the strains from the “European” slope. In contrast, we found that there was almost no difference between strains (with similar ploidy) from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the “African” strains are more adapted to higher solar radiation than the “European” strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation.

Conclusions/Significance

Our results and the results of parallel studies with several other organisms, suggest that natural selection appears to select, at a microscale, for adaptive complexes that can tolerate the higher UV radiation on the “African” slope.     

Different Views about Work-Hour Limitations in Medicine: A Qualitative Content Analysis of Surgeons', Lawyers', and Pilots' Positive and Negative Arguments

Background

Whereas work-hour regulations have been taken for granted since 1940 in other occupational settings, such as commercial aviation, they have been implemented only recently in medical professions, where they lead to a lively debate. The aim of the present study was to evaluate arguments in favour of and against work-hour limitations in medicine given by Swiss surgeons, lawyers, and pilots.

Methods

An electronic questionnaire survey with four free-response items addressing the question of what arguments speak in favour of or against work-hour limitations in general and in medicine was sent to a random sample of board-certified surgeons, lawyers in labour law, and pilots from SWISS International Airlines Ltd.

Results

In all, 279/497 (56%) of the respondents answered the survey: 67/117 surgeons, 92/226 lawyers, and 120/154 pilots. Support for work-hour limitations in general and in medicine was present and higher among lawyers and pilots than it was in surgeons (p<0.001). The latter agreed more with work-hour limitations in general than in medicine (p<0.001). The most often cited arguments in favour of work-hour limitations were “quality and patient safety,” “health and fitness,” and “leisure and work-family balance,” whereas the lack of “flexibility” was the most important argument against. Surgeons expected more often that their “education” and the “quality of their work” would be threatened (p<0.001).

Conclusions

Work-hour limitations should be supported in medicine also, but a way must be found to reduce problems resulting from discontinuity in patient care and to minimise the work in medicine, which has no education value.