Lipidomic Profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii Reveals Critical Changes in Lipid Composition in Response to Acetic Acid Stress

When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L−1, while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L−1 acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large rearrangements in its lipid profile

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Matematiken i yrkesutbildningen : möjligheter och begränsningar

The aim of this thesis is to contribute to knowledge regarding affordances and constraints of mathematics in vocational education. Different theories, i.e. theory-guided bricolage, has been used for the analyses. In the different articles grounded theory, hermeneutics and Kennewell's model about affordances and constraints were applied. Data has been collected from documents and reports.The first article analyses what methods and theories that has been used by researchers to uncover mathematics in vocational education. The development is going in the direction to use many methods at the same time and the technical development has made this possible. Results from earlier research shows that mathematics is invisible, hidden in black boxes and something existing as silent knowledge. In order to uncover mathematics the researcher has to know the subject.The second article gives an historical overview of the role of mathematics in vocational education to this day from the days of the guild when the master was responsible for the education to the upper secondary school of today when the pupils are being taught in Mathematics, course A, by the mathematics teacher. The third article is an analysis of a development project in a vocational program with the purpose to develop cooperation between teachers of mathematics and teachers in the vocational subjects. The results indicate that the historical overview can help to explain that even today it is hard to make cooperation happen between categories of teachers with different conditions and educational background. The analysis of the development project that took place after the reform in 1994 pointed at possibilities, but that it takes time and support is required from the school leaders. The pupils had deficient preknowledge in mathematics but became more motivated and achieved better when they were taught by teachers who had cooperated before and in class with the other teachers.The results indicate that it is relevant to carry out in-service training with different categories of teachers to be prepared for the possibility to get time to meet and discuss content and teaching in mathematics and in the vocational subjects. It is important to document results from in-service training and to link researchers for support and to disseminate results to other schools and groups nationally and internationally through publications and conferences.Syftet med denna avhandling är att bidra med kunskap om matematikens möjligheter och begränsningar i yrkesutbildningar. Vid analysen har flera olika teorier använts s.k. theory-guided bricolage. I de olika artiklarna har grounded theory, hermeneutik respektive teorin om affordances och constraints använts. Data har hämtats från dokument och rapporter.Den första artikeln analyserar vilka metoder och teorier som använts av forskare för att få syn på matematiken i yrkesutbildningar. Utvecklingen har gått mot att använda flera metoder samtidigt och den tekniska utvecklingen har gjort detta möjligt. Resultat från tidigare forskning visar att matematiken är osynlig, gömd i svarta lådor eller något som bara finns som tyst kunskap. För att kunna "se" matematiken måste forskaren vara ämneskunnig.Den andra artikeln ger en historisk genomgång av matematikens roll i yrkesutbildningar fram till i dag, från skråväsendets dagar då mäster hade hand om hela utbildningen inklusive matematiken till dagens gymnasieskola där alla elever undervisas i Matematik, kurs A av matematiklärare.Den tredje artikeln är en analys av ett utvecklingsprojekt på ett yrkesprogram som handlade om att utveckla samarbete mellan lärare i karaktärsämnet och matematikämnet. Resultatet pekade på att den historiska genomgången från skråväsende fram till våra dagar kan förklara att det fortfarande är svårt att få till stånd samarbete eftersom lärarkategorierna har olika förutsättningar och utbildningsbakgrund. Analysen av utvecklingsprojektet som genomfördes efter gymnasiereformen 1994 pekade på möjligheter, men att det tar tid och att det kräver stöd av skolans ledning. Eleverna hade bristande förkunskaper i matematik men blev mer motiverade och presterade något bättre när de undervisades av lärare som hade samverkat före och i genomförande av undervisning.Resultaten indikerar att det är relevant att genomföra fortbildning för lärare av olika kategorier för att dessa skall beredas möjlighet att få tid att mötas och diskutera innehåll och undervisning i matematik och karaktärsämnena. Det är viktigt att dokumentera resultat från forbildning och att knyta forskare som stöd vid fortbildning samt att kunna sprida resultaten utanför den egna skolan både nationellt och internationellt genom publikationer och konferenser.Godkänd; 2010; 20101019 (ysko); LICENTIATSEMINARIUM Ämnesområde: Matematik och lärande Examinator: Professor Lars-Erik Persson, Luleå tekniska universitet Extern opponent: Professor Astrid Pettersson PRIM-gruppen, Stockholms Universitet Tid: Torsdag den 18 november, klockan 10.00 Plats: D2214-15, Luleå tekniska universitet</p

Assessment in mathemathics teacher education

Authentic tasks can promote higher-order learning. In the student task discussed in this paper the student teachers developed, tried, graded, discussed and revised mathematics test questions, in connection to an amusement park visit. The task was designed in an effort to bring the teacher educators in the mathematics and educational departments and those in the schools closer to each other through participation in a joint project, which was part of the "VFU" - the time that student teachers spend in school as part of the subject studies. The task involved different aspects of authenticity, both in the mathematics questions created in and in the creation of questions.Qualities in students' problems are discussed, and also student reflections on pupil and teacher reactions in relation to the authentic contexts. Traditional textbook problems were found to have a strong influence, as evidenced by many of the student-created problems and some of the supervisor reactions. Students' descriptions of teacher comments indicated that these focused more on classroom management than on mathematics content, consistent with traditional expectations on the VFU

Fundamental studies reveal membrane engineering as strain engineering target for enhanced robustness towards lignocellulose hydrolysate inhibitors.

Bio-processes for the production of fuels and chemicals will contribute to the so-called bioeconomy, where biomass will represent an important source of hydrocarbons. A new concept of bio-based industry is now under development, where lignocellulosic material is the primary source of sugars to be converted in a biorefinery concept not to ethanol or fuels only, but to a portfolio of chemicals. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose bioconversion.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. The goal is to identify successful strain engineering strategies to confer yeast higher robustness. Zygosaccharomyces bailii is a yeast specie that tolerates low pH and high concentrations of weak organic acids. Thus, in order to elucidate a possible link between lipid composition and acetic acid tolerance, a comparative lipidomic profiling of the major lipid species found in the plasma membrane of S. cerevisiae and Z. bailii was performed. The study revealed remarkable changes in glycerolphospholipids and sphingolipids pools in Z. bailii compared to S. cerevisiae, suggesting lipid saturation, high sphingolipid levels as possible determinants of acetic acid tolerance

Comparative lipidomic profiling of Saccharomyces cerevisiae to reveal lipid composition changes in the plasma membrane upon exposure to lignocellulose inhibitors

During pretreatment of lignocellulose raw material, compounds that severely inhibit microbial activity including Saccharomyces cerevisiae strains are released [1]. These compounds, which include furaldehydes and weak organic acids, inhibit yeast metabolism and affect yeast viability and, as a consequence, reduces the overallproductivity of an ethanol production process [2].Elucidation of the molecular mechanisms behind inhibition can suggest new strategies to prevent the inhibitory effect. In the present study, the possible effect on the plasma membrane in S. cerevisiae is studied as a response to inhibitors present in lignocellulose raw material.A comparative lipidomic profiling will be carried out on S. cerevisiae cultured in the absence and presence of lignocellulose inhibitors. LC-CAD and GC-MS will be used toextensively characterize the composition of the plasma membrane. Changes in membrane composition will be correlated with the presence of specific inhibitors.References1. Palmqvist E, Hahn-H\ue4gerdal B: Fermentation of lignocellulosic hydrolysates. II: Inhibitorsand mechanisms of inhibition. Bioresource Technology 2000, 74(1):25-33.2. Klinke HB, Thomsen AB, Ahring BK: Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. AppliedMicrobiology and Biotechnology 2004, 66(1):10-2

Investigation of weak organic acid tolerance mechanisms by lipidomic profiling of Saccharomyces cerevisiae and Zygosaccaromyces bailii

During pretreatment of lignocellulose raw material, compounds such as furaldehydes, phenolics and weak organic acids, severely inhibiting Saccharomyces cerevisiae, are released. Decrease of intracellular pH after diffusion through the plasma membrane is thought to be one of the effects mediating the cellular toxicity of weak organic acids.The aim of the present study is to investigate the relationship between plasma membrane composition and acid tolerance, in order to develop a strategy for engineering a S. cerevisiae strain more tolerant to acetic acid. Zygosaccharomyces bailii, a well-known food spoilage yeast, is highly tolerant to acetic acid and will be used as a model for weak organic acid tolerance.A complete lipidomic profiling of S. cerevisiae and Z. bailii in the presence and absence of acetic acid will be carried out using LC-MS/MS. Similarities and differences in the two profiles will be correlated with acid tolerance

Fundamental studies reveal membrane engineering as strain engineering target for enhanced robustness towards lignocellulose hydrolysate inhibitors.

Bio-processes for the production of fuels and chemicals will contribute to the so-called bioeconomy, where biomass will represent an important source of hydrocarbons. A new concept of bio-based industry is now under development, where lignocellulosic material is the primary source of sugars to be converted in a biorefinery concept not to ethanol or fuels only, but to a portfolio of chemicals. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose bioconversion.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. The goal is to identify successful strain engineering strategies to confer yeast higher robustness. Zygosaccharomyces bailii is a yeast specie that tolerates low pH and high concentrations of weak organic acids. Thus, in order to elucidate a possible link between lipid composition and acetic acid tolerance, a comparative lipidomic profiling of the major lipid species found in the plasma membrane of S. cerevisiae and Z. bailii was performed. The study revealed remarkable changes in glycerolphospholipids and sphingolipids pools in Z. bailii compared to S. cerevisiae, suggesting lipid saturation, high sphingolipid levels as possible determinants of acetic acid tolerance