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Saccharomyces pastorianus, which is responsible for the production of bottom-fermented lager beer, is a hybrid species that arose from the mating of the top-fermenting ale yeast Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus around the start of the 17th century. Based on detailed analysis of Central European brewing records, we propose that the critical event for the hybridization was the introduction of top-fermenting S. cerevisiae into an environment where S. eubayanus was present, rather than the other way around. Bottom fermentation in parts of Bavaria preceded the proposed hybridization date by a couple of hundred years and we suggest that this was carried out by mixtures of yeasts, which may have included S. eubayanus. A plausible case can be made that the S. cerevisiae parent came either from the Schwarzach wheat brewery or the city of Einbeck, and the formation of S. pastorianus happened in the Munich Hofbräuhaus between 1602 and 1615 when both wheat beer and lager were brewed contemporaneously. We also describe how the distribution of strains from the Munich Spaten brewery, and the development by Hansen and Linder of methods for producing pure starter cultures, facilitated the global spread of the Bavarian S. pastorianus lineages.

The study of nitrogen assimilation in yeast is of interest from genetic, evolutionary, and biotechnological perspectives. Over the course of evolution, yeasts have developed sophisticated control mechanisms to regulate nitrogen metabolism, with domesticated lineages sometimes displaying particular specialisation. The focus of this study was on assimilation of asparagine, which is a significant nutritional source for some alcoholic fermentations. We were particularly interested in ASP3, which encodes a periplasmic asparaginase and that was proposed to have been acquired relatively recently in S. cerevisiae by horizontal gene transfer. We examined 1680 S. cerevisiae genome assemblies to evaluate the distribution and evolutionary trajectory of ASP3. Our findings suggest an alternative hypothesis that ASP3 is an ancient Saccharomyces gene that has generally been lost over the course of evolution but has been retained in certain fermentative environments. As asparagine is the major nitrogen source in apple juice, we explored whether the presence of ASP3 would confer a growth advantage. Interestingly, we found that although ASP3 enhances growth when asparagine is the sole nitrogen source, the same effect is not seen in apple juice. These data indicate that growth in pure culture may not reflect the original selective environment for ASP3+ strains and highlight the role that complex regulation may play in optimising nitrogen assimilation in yeasts.

Following a whole genome duplication (WGD) event approximately 100 million years ago, the yeast lineage from which the model Saccharomyces cerevisiae derives maintained two copies of genes where it was necessary to synthesise proteoforms with different sub-cellular localisation. In contrast, yeasts that did not undergo the WGD event have a single gene that must encode both proteoforms. We adopted an integrated in silico and experimental approach to study how this is achieved with BAT1, a gene that encodes mitochondrial and cytosolic forms of a branched chain aminotransferase (BCAT) in pre-WGD yeast such as Kluyveromyces marxianus. We determined that condition-specific regulation of alternative transcription sites gives rise to mRNA isoforms that differ at the 5’end and that, when decoded, generate a mitochondrial or cytosolic proteoform. Furthermore, targeted mutants lacking specific transcription factors were generated to establish how this differentiation was regulated. As in S. cerevisiae, Gcn4 and Leu3 activated expression of the mRNA encoding the mitochondrial proteoform under conditions when branched chain amino acid synthesis was required. Unlike S. cerevisiae, however, K. marxianus lacked tight regulation of the mRNA encoding the cytosolic proteoform supporting the hypothesis that maintaining paralogous genes in post-WGD yeasts facilitated development of more sophisticated expression control mechanisms.

The coding potential of the eukaryotic genome can be greatly expanded by the regulated use of mechanisms that generate more than one protein product from a gene. We combined techniques for mapping 5’ and 3’ ends of RNA transcripts with ribosome profiling to study the organisation of protein coding gene expression in the yeast Kluyveromyces marxianus. We uncovered over 1000 cases of novel proteoforms due to use of alternative transcription or translation start sites, identified 800 translated upstream open reading frames, observed surprising translation of antisense RNAs, and discovered a novel case of programmed ribosomal frameshifting. In some cases, features are conserved across yeast species, whereas others are species-specific. This offers new possibilities to explore the evolution of genomes and gene regulation in budding yeasts. Our analysis also enabled us to improve the genome annotation of K. marxianus by adding or correcting annotations of over 300 protein coding genes. The processed data has been made available on the GWIPS-viz and Trips-Viz browsers, thus providing an accurate data-driven annotation of transcripts and their protein coding regions along with quantitative information on their transcription and translation.

It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as

Kluyveromyces marxianus is an interesting and important yeast because of particular traits such as thermotolerance and rapid growth, and for applications in food and industrial biotechnology. For both understanding its biology and developing bioprocesses, it is important to understand how K. marxianus responds and adapts to changing environments. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. We report here the development of a ribosome profiling method for K. marxianus, which allows codon resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs. To aid in the analysis and sharing of ribosome profiling data, we added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. We also provide a set of step-by-step protocols for the experimental and bioinformatic methods that we developed.

Kluyveromyces marxianus is an interesting and important yeast because of particular traits like thermotolerance and rapid growth, and applications in food and industrial biotechnology. Knowing how K. marxianus responds and adapts to changing environments is important to achieve a full understanding of the its biology and to develop bioprocesses. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. Whereas transcriptome analysis by RNA-Seq quantifies mRNA abundance, ribosome profiling allows codon-resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs and is emerging as a valuable tool to study translation control of gene expression. We report here the development of a ribosome profiling method for K. marxianus and we make the procedure available as a step by step protocol. To aid in the analysis and sharing of ribosome profiling data, we also added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. As many studies only focus on the use of RNA-Seq to study K. marxianus in different environments, the availability of ribosome profiling is a powerful addition to the K. marxianus toolbox.Graphical abstractDevelopment of a Ribosome Profiling protocol to study gene expression in the thermotolerant yeast Kluyveromyces marxianus.

Bonded contact models have been increasingly used in the discrete element method (DEM) to study cemented and sintered particulate materials in recent years. Several popular DEM bond models have been proposed in the literature; thus it is beneficial to assess the similarities and differences between the different bond models before they are used in simulations. This paper identifies and discusses two fundamental types of bond models: the Spring Bond Model where two bonded particles are joined by a set of uniform elastic springs on the bond’s cross-section, and the Beam Bond Model in which a beam is used to connect the centres of two particles. A series of cantilever beam bending simulation cases were carried out to verify the findings and assess the strength and weakness of the bond models. Despite the numerous bond models described in the literature, they can all be considered as a variation of these two fundamental model types. The comparative evaluation in this paper also shows that all the bond models investigated can be unified to a general form given at a predefined contact point location.

In this study, a new approach to measure metabolic activity of yeast via the Crabtree effect is described. BARDS is an analytical technique developed to aid powder and tablet characterisation by monitoring changes in the compressibility of a solvent during solute dissolution. It is a rapid and simple method which utilises a magnetic stir bar to mix added solute and induce the acoustic resonance of a vessel containing a fixed volume of solvent. In this study it is shown that initiation of fermentation in a yeast suspension, in aqueous buffer, is accompanied by reproducible changes in the frequency of induced acoustic resonance. These changes signify increased compressibility of the suspension due to CO2 release by the yeast. A simple standardised BARDS protocol reveals yeast carbon source preferences and can generate quantitative kinetic data on carbon source metabolism which are characteristic of each yeast strain. The Crawford-Woods equation can be used to quantify total gaseous CO2 produced by a given number of viable yeast when supplied with a fixed amount of carbon source. This allows for a value to be calculated for the amount of gaseous CO2 produced by each yeast cell. The approach has the potential to transform the way in which yeast metabolism is tracked and potentially provide an orthogonal or surrogate method to determining viability, vitality and attenuation measurements in the future.

The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies., New yeast screening platform to identify pentose transporters.

This paper presents a generic framework of Model Driven Design (MDD) with its application for a twin screw granulation process using a mechanistic-based population balance model (PBM). The process kernels including nucleation, breakage, layering and consolidation are defined in the PBM. A recently developed breakage kernel is used with key physics incorporated in the model formulation. Prior to granulation experiments, sensitivity analysis of PBM parameters is performed to investigate the variation of model outputs given the input parameter variance. The significance of liquid to solid ratio (L/S ratio), nucleation and breakage parameters is identified by sensitivity analysis. The sensitivity analysis dramatically reduces the number of fitting parameters in PBM and only nine granulation experiments are required for model calibration and validation. A model validation flowchart is proposed to elucidate the evolution of kinetic rate parameters associated with L/S ratio and screw element geometry. The presented MDD framework for sensitivity analysis, parameter estimation, model verification and validation can be generalized and applied for any particulate process.

Free-access link to the article: "Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter" Pacheco, Andreia; Donzella, Lorena; Hernández-López, M. José; Almeida, Maria Judite; Prieto, José Antonio; Rández Gil, Francisca; Morrisey, John P.; Sousa, Maria Joao. FEMS Yeast Research 20(1): foaa004 (2020). Available online at: https://doi.org/10.1093/femsyr/foaa004, Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km = 6.5 ± 2.0 mM) and a high-affinity (Km = 0.10 ± 0.01 mM) component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5 ± 1.5 mM). This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry., AP was supported by PhD fellowship [BD/13 282/2003] from Fundação para a Ciência e para a Tecnologia (FCT), Portugal. LD is supported by the YEASTDOC Marie Curie ITN project which received funding from the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement no 764 927. This work was supported by the strategic programme UID/BIA/0 4050/2013 (POCI-01-0145-FEDER-0 07569) and project PTDC/BIA-MIC/32 059/2017 funded by national funds through the FCT,I.P. and by the ERDF through the COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and Sistema de Apoio à Investigação Científica e Tecnológica (SAICT).

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology., Fermentation parameters of industrial processes are often not the ideal growth conditions for industrial microbes. Here, the authors reveal that young genes are more responsive to environmental stress than ancient genes using a new gene age assignment method and provide targeted genes for metabolic engineering.

Introduction Self-administered in-home digital therapeutics could expand access to cognitive rehabilitation for individuals with multiple sclerosis (MS), over half of whom experience cognitive impairment (CI). However, feasibility in an MS population must be clarified. This study was conducted to assess the feasibility of deploying a videogame-like digital treatment for CI in MS, including initial efficacy and barriers to adherence. Methods In this pilot study, 21 participants with MS completed an in-clinic baseline neurological evaluation. Cognitive tests included paper-and-pencil Brief International Cognitive Assessment for Multiple Sclerosis [BICAMS—which included the Symbol Digit Modalities Test (SDMT)] and other unsupervised tablet-based tests (including Match: an unsupervised test of executive functions and processing speed, developed at UCSF; and the Cogstate MS Battery). Participants then completed an in-home, tablet-based, videogame-like investigational digital treatment (Project: EVO™) for 25 min daily, 5 days weekly, for 4 weeks. This was followed by a repeat in-clinic evaluation. Results Of the 21 participants (mean [standard deviation, SD] age 53.8 [11.6] years, median Expanded Disability Status Scale (EDSS) 2.5 [SD 2.0, IQR [2–3.5]]) enrolled to use the digital therapeutic at home (mean [SD] SDMT z score: − 0.21 [1.16]), 18 completed the study, during which they completed an average of 19.7 days (median [SD]: 20.5 [8.4]). Overall, 78% of these 18 participants completed 75% of prescribed days (i.e., at least 15), and 50% completed all 20 days or more. Over the 4-week period, scores of processing speed improved significantly (based on one-sided t test), including SDMT (p = 0.003) and Match (p = 0.006). The Cogstate DET test (psychomotor function) also increased (p = 0.006). Mean increase in SDMT was 3.6 points. Male sex, not being employed, and higher baseline anxiety all were significantly associated with greater improvement in SDMT over the 4-week period. Interestingly, lower baseline cognitive scores were associated with greater number of sessions completed (e.g., SDMT: p = 0.003, R2 = 0.44). Adjusting for employment, a proxy for time available, did not significantly improve the model fit. Discussion Deploying an in-home digital tool to improve processing speed in MS is feasible, and shows preliminary efficacy. A larger, randomized controlled clinical trial is ongoing. Electronic supplementary material The online version of this article (10.1007/s40120-018-0121-0) contains supplementary material, which is available to authorized users.

Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.

Envisioning societal futures for systemic low carbon transition is key to supporting the emergence of new configurations of the energy system. Such visions or ‘imaginaries’ facilitate wider societal support for system change. Given the urgency of the decarbonisation agenda, societal understandings of transitions processes and community ideas and visions about pathways for change assume a key role. The paper addresses an important gap in knowledge of place-based visions of energy system transition by soliciting views of community residents through a survey instrument, on what their perspectives on energy transition are. A case-study of Liverpool UK is presented, conducted as part of the EU Horizon 2020 ENTRUST project. Through a cluster analysis approach of study respondent perspectives, this study identities 4 distinct energy visions: (1) Community Affordability; (2) The Centre Ground; (3) Security First; and (4) Green Affordability. Identified visions present different perspectives on how energy is framed, with each vision characterised by distinct preferences on the extent to which stakeholder groups may influence the energy system as well as different views on the role of specific energy generation methods. Further, visions differ on the role of different actors and institutions in the energy transition. Identified energy visions provide insight into the ways in which grassroots communities consider how the energy system should transition in the coming years. The study contributes to understanding of spatially-situated and socially differentiated views of energy system transition, providing insight into contrasting viewpoints on current and future energy system priorities of residents of Liverpool, UK.

Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that their ancestral role was in a combined lactose and cellobiose assimilation system, which used Lac12 to import both of these sugars into the cell, and then hydrolyzed lactose using Lac4, and cellobiose using a cellobiase, Cel2. A second cellobiose transporter Cel1 was also present ancestrally. In the K. lactis lineage, however, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. Among its four LAC12 genes, one became neofunctionalized as an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus where it formed a LAC4-LAC12-CEL2 gene cluster, while another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired by K. lactis var. lactis by introgression of a 15 kb subtelomeric region containing LAC12 and LAC4 from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

Summary Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis [1]. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose [2]. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that the ancestral Lac12 was bifunctional, able to import both lactose and cellobiose into the cell. These disaccharides were then hydrolyzed by Lac4 in the case of lactose or Cel2 in the case of cellobiose. A second cellobiose transporter, Cel1, was also present ancestrally. In the K. lactis lineage, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. One of these LAC12 genes became neofunctionalized to encode an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus, where it formed a LAC4-LAC12-CEL2 gene cluster, although another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired very recently by K. lactis var. lactis by introgression of LAC12 and LAC4 on a 15-kb subtelomeric region from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers., Graphical Abstract, Highlights • Early farmers domesticated the milk yeast Kluyveromyces lactis • A dairy lineage of Kluyveromyces marxianus donated the LAC genes to K. lactis • A new Kluyveromyces-specific gene cluster for utilizing cellobiose was identified • K. marxianus carries an integrated cellobiose and lactose utilization gene cluster, It is well known that humans domesticated brewer’s yeast, and now, Varela et al. report that another yeast is also the product of human activity. They show that an insect-associated, lactose-negative progenitor of the milk yeast Kluyveromyces lactis acquired the genes that enable lactose fermentation from a dairy-adapted population of K. marxianus.

To address the impact of microbiome on disease development, it is essential to go beyond a descriptive study and evaluate the physiological importance of microbiome changes. Our study integrates computational analysis with in vitro and in vivo exploration of inflammatory properties of spore-forming microbial communities, revealing novel functional correlations. We specifically show that while small differences exist between the microbiomes of MS patients and healthy subjects, these differences are exacerbated in the chloroform-resistant fraction. We further demonstrate that, when purified from MS patients, this fraction is correlated with impaired immunomodulatory responses in vitro., Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by adaptive and innate immune system dysregulation. Recent work has revealed moderate alteration of gut microbial communities in subjects with MS and in experimental, induced models. However, a mechanistic understanding linking the observed changes in the microbiota and the presence of the disease is still missing. Chloroform-resistant, spore-forming bacteria, which primarily belong to the classes Bacilli and Clostridia in the phylum Firmicutes, have been shown to exhibit immunomodulatory properties in vitro and in vivo, but they have not yet been characterized in the context of human disease. This study addresses the community composition and immune function of this bacterial fraction in MS. We identify MS-associated spore-forming taxa (primarily in the class Clostridia) and show that their presence correlates with impaired differentiation of IL-10-secreting, regulatory T lymphocytes in vitro. Colonization of antibiotic-treated mice with spore-forming bacteria allowed us to identify some bacterial taxa favoring IL-10+ lymphocyte differentiation and others inducing differentiation of proinflammatory, IFN-γ+ T lymphocytes. However, when fed into antibiotic-treated mice, both MS and control-derived spore-forming bacteria were able to induce similar IL-10-expressing Treg immunoregulatory responses, thus ameliorating symptoms of experimental allergic encephalomyelitis (EAE). Our analysis also identified Akkermansia muciniphila as a key organism that may interact either directly or indirectly with spore-forming bacteria to exacerbate the inflammatory effects of MS-associated gut microbiota. Thus, changes in the spore-forming fraction may influence T lymphocyte-mediated inflammation in MS. This experimental approach of isolating a subset of microbiota based on its functional characteristics may be useful to investigate other microbial fractions at greater depth. IMPORTANCE To address the impact of microbiome on disease development, it is essential to go beyond a descriptive study and evaluate the physiological importance of microbiome changes. Our study integrates computational analysis with in vitro and in vivo exploration of inflammatory properties of spore-forming microbial communities, revealing novel functional correlations. We specifically show that while small differences exist between the microbiomes of MS patients and healthy subjects, these differences are exacerbated in the chloroform-resistant fraction. We further demonstrate that, when purified from MS patients, this fraction is correlated with impaired immunomodulatory responses in vitro.

Background: Remote assessment of neurological disability in people with multiple sclerosis (MS) could improve access to clinical care and efficiency of clinical research. Objective: To develop and validate a telemedicine-based MS disability examination that does not require an in-home examiner. Methods: Adults with MS were recruited after a standardized in-person Expanded Disability Status Scale (EDSS) evaluation, and within 1 week underwent a blinded televideo-enabled EDSS examination with a different clinician. EDSS and tele-EDSS scores were compared. Results: Overall, 41 adults participated (mean (standard deviation (SD)) age: 47.0 years (11.6); median EDSS: 2 (range: 0–7)); 37 required no in-home assistance for the tele-EDSS evaluation (e.g. help positioning camera). Mean difference between EDSS and tele-EDSS was 0.34 (95% confidence interval (CI): 0.07–0.61). For 88% of evaluations, tele-EDSS and EDSS scores were within 1 point (similar to reported in-person inter-rater differences). Unweighted kappa for agreement within 0.5 point was 0.72. Correlation for individual functional systems (FS) ranged from modest (vision: 0.37) to high (bowel/bladder: 0.79). Overall correlation between EDSS and tele-EDSS was 0.89 ( p Conclusion: In this proof of principle study, disability evaluation in mild to moderate MS is feasible using telemedicine without an aide at the patient’s location.

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae . Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.

The transition of food-production and consumption systems to a sustainable, low carbon future presents a dauntingly complex issue, involving technical, political, social and theoretical aspects. Such a transition necessitates an exploration of new ways of production and consumption, new technologies and innovations and new regulatory and institutional infrastructures to co-ordinate the change. Appropriate sustainability assessment has the following advantages: the conceptualisation of complex system functioning; the identification of the need for multi-dimensional, strategic approaches; the development of appropriate policy responses; and the targeting and framing of action (assessment) for sustainability. The paper presents a number of key findings and directions for further research in the context of agri-food systems sustainability.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

Short- and medium-chain fatty acids (SMCFA) are important platform chemicals currently produced from nonsustainable resources. The engineering of microbial cells to produce SMCFA, however, lacks high-throughput methods to screen for best performing cells. Here, we present the development of a whole-cell biosensor for easy and rapid detection of SMCFA. The biosensor is based on a multicopy yeast plasmid containing the SMCFA-responsive PDR12 promoter coupled to GFP as the reporter gene. The sensor detected hexanoic, heptanoic and octanoic acid over a linear range up to 2, 1.5, and 0.75 mM, respectively, but did not show a linear response to decanoic and dodecanoic acid. We validated the functionality of the biosensor with culture supernatants of a previously engineered Saccharomyces cerevisiae octanoic acid producer strain and derivatives thereof. The biosensor signal correlated strongly with the octanoic acid concentrations as determined by gas chromatography. Thus, this biosensor enables the high throughput screening of SMCFA producers and has the potential to drastically speed up the engineering of diverse SMCFA producing cell factories.

While there is growing consensus that human behaviours need to change to a more sustainable paradigm, community driven approaches, such as social enterprise, have yet to be explored as serious instruments of sustainability transition. Social enterprises sit within the third sector of the economy, typically where market or governmental failures exist in the provision of social welfare, and have increasingly become a key driver of social progress. The autonomous nature of the social-economic model applied by such organisations can represent a viable means to reduce state social welfare dependence, and is a proven model for social change. The capability of social enterprises to create both social and economic value is considered a ‘win-win’. Yet there are clear potentials for social enterprise models to be more extensively applied to address contemporary ecological challenges of neo-liberal market economies, moving towards ‘win-win-win’ outcomes across social, economic and ecological domains. This paper investigates the value of social enterprises as drivers of low-carbon transition at the community level, with an emphasis on the energy sector. Evidence from seven organisations in the UK is presented and a socio- technical transitions conceptual framework is applied to analyse these social enterprise operations as a form of social innovation. © 2018 The Author(s)

Doctoral training has changed in recent years with most PhDs now performed in structured programmes operated by university graduate schools. These schools generally superimpose a training framework onto the traditional research project to improve the education experience of the students and to prepare them for their careers. Many graduates progress to the commercial sector, where there is demand for highly skilled employees. The European Union (EU) promotes the development of transnational, training-focused, PhD programmes called Innovative Training Networks (ITNs) through Marie Skłodowska-Curie Actions. ITNs share many features of thematic PhD programmes, but they only recruit a single cohort of students, and they align with EU policy goals. These training networks are prestigious and very well regarded within European academia. The authors of this article were participants in a yeast biotechnology ITN, YEASTCELL, which finished in 2017. Some interesting insights into the more and less successful aspects of the project arose during discussions at the final project workshop. The views of the participants are distilled here in a discussion of how an ITN could be structured to maximise the benefits for the three main stakeholders: students, supervisors and industry partners.

The yeast Kluyveromyces marxianus possesses advantageous traits like rapid growth, GRAS (generally regarded as safe) status and thermotolerance that make it very suitable for diverse biotechnological applications. Although physiological studies demonstrate wide phenotypic variation within the species, there is only limited information available on the genetic diversity of K. marxianus. The aim of this work was to develop a multilocus sequence typing (MLST) method for K. marxianus to improve strain classification and selection. Analysis of housekeeping genes in a number of sequenced strains led to the selection of five genes, IPP1, TFC1, GPH1, GSY2 and SGA1, with sufficient polymorphic sites to allow MLST analysis. These loci were sequenced in an additional 76 strains and used to develop the MLST. This revealed wide diversity in the species and separation of the culture collection and wild strains into multiple distinct clades. Two subsets of strains that shared sources of origin were subjected to MLST and split decomposition analysis. The latter revealed evidence of recombination, indicating that this yeast undergoes mating in the wild. A public access web-based portal was established to allow expansion of the database and application of MLST to additional K. marxianus strains. This will aid understanding of the genetic diversity of the yeast and facilitate biotechnological exploitation.

Despite often stated sustainability goals, much of traditional planning practice remains concerned with facilitating the market and maintaining the status quo rather than challenging and transforming it. In this chapter, the planning system is the focus of a sociotechnical systems perspective analysis. This chapter examines strategic spatial planning at regional and city scales through the lens of sociotechnical transitions concepts to provide insight into the role and capacity of spatial plans and planning processes to challenge the status quo and achieve sustainable urban transitions. We present two cases of strategic planning during the first decade of the 2000s at a national and metropolitan scale in Ireland and Melbourne (Australia), respectively – two cases where strategic spatial plans aimed to achieve sustainable land-use outcomes but where planning failed to act as a brake on booming housing markets and related urban sprawl. This chapter also reflects on the lessons from spatial planning processes to inform sociotechnical systems research pointing to the need to incorporate conceptualisations of space, place and context-specific governance in problem framing particularly in considering the challenges of long-term sustainable land-use transitions. We query whether the prevailing planning system common in most developed contexts can be treated as a stable regime, and if so, what benefit this perspective may provide to planning practitioners.

With the rapid development of high-speed railway over the world in recent years, more and more researchers are focused on the strength, stiffness and stability of the embankment. It’s more and more popular to use the discrete element method (DEM) to study cemented granular materials (e.g. rocks and concrete). This paper verified the Timoshenko beam bond model (TBBM) presented by the University of Edinburgh, then compared the bending and dynamic response of a simply supported beam under the effects of moving load with the analytical prediction. It’s ideally suited to use DEM to model the railroad ballast layer considering the naturally discrete inhomogeneous structure. A simplified railway embankment model composed of equal-sized ballast particles was built and the loading response of the simplified embankment was then investigated. The results show the feasibility of modeling train-induced dynamic load through adding moving load to railway track made up with the bonded particles. This paper presents one way to take the discrete features of the ballast layer and the characteristics of railway track into consideration in the same DEM framework at the same time. Apart from that, the mechanical characteristics of the ballast layer and the interactions between the ballast and railway track can be easily studied.