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Characterization of capsaicinoid production in recombinant Saccharomyces cerevisiae
No full textKapsaicinoider är ämnen som finns i chilifrukterna och har på senaste tiden fått intresse som läkemedel på grund av sina analgetiska, anti-inflammatoriska och anti-cancer egenskaper. Ett nytt tillvägagångssätt att producera kapsaicinoider kan vara syntesen i rekombinant Saccharomyces cerevisiae med hjälp av metabolisk engineering och rekombinant DNA-tekniker. Gener från Capsicum chinensis, som kodar för enzymerna capsaicinoid-syntas (CS) och acyl-CoA-syntas (ACS), integrerades i S. cerevisiae i tidigare projekt. Den kända laboratoriesträngen CEN.PK modifierades med plasmidtransformation och för vildtyp-stammen ERF 5273 användes den nya CRISPR/Cas9-tekniken. Syftet med detta projekt är att ytterligare karakterisera och jämföra dessa tidigare konstruerade stammar angående deras förmåga att producera nonivamid eller andra jästspecifika kapsaicinoider. Vidare undersöks huruvida kapsaicinoider utsöndras i odlings-medium eller om de ackumuleras intracellulärt. Stammarna odlades i en bioreaktor i lite laboratorieskala. Som odlingsmedium används ett definierat medium med eller utan tillsatser. Odlings-medium kompletterades med vanillyl-amin och nonanoic acid som precursor. För att identifiera de kapsaicinoid-producerande stammarna extraherades supernatanten och cellpelleten och analyserades kromatografisk med HPLC. Resultaten från denna studie visade att jäststammarna, som innehöll båda generna (ACS + CS), sannolikt producerade nonivamid om de odlades i kompletterat medium. Vidare observerades bildning av nonivamid som ackumulerades i själva cellen. Möjligtvis producerades också jästspecifika kapsaicinoider, men topphöjden är nästan inte mätbar. Därför måste dessa resultat bekräftas ytterligare. Framtida arbeten behövs för att säkerställa och förbättra produktionen av kapsaicinoider.Capsaicinoids are compounds found in chili plants and have recently gained interest as pharmaceuticals due to their analgesic, anti-inflammatory and anti-cancer properties. A novel approach producing capsaicinoids could be synthesis in recombinant Saccharomyces cerevisiae with help of metabolic engineering. Genes from Capsicum chinensis, encoding the enzymes capsaicinoid synthase (CS) and acyl-CoA synthase (ACS), were previously inserted into S. cerevisiae. The known laboratory stain CEN.PK was modified with plasmid transformation and the novel CRISPR/Cas9 technology was used for the wild type strain ERF 5273. The aim of this project is to further characterize and compare these previously constructed strains concerning their ability to produce nonivamide or yeast specific capsaicinoids. Furthermore, it is examined whether capsaicinoids are excreted into the broth or accumulated intracellularly. Four different strains were cultivated in bench-scale bioreactors using medium supplemented with or without different precursors (vanillylamine and nonanoic acid). Culture broth supernatants and cell pellets were extracted and analyzed by HPLC in order to identify the capsaicinoid-producing strains. The results from this study revealed that the yeast strains harbouring both genes (ACS+CS) produced most likely nonivamide if they were cultivated in media supplemented with both precursors. Nonivamide formation was equally observed in broth supernatant and cell pellet. Additionally it was shown that yeast specific capsaicinoid production occured, althoug the peak height was close to the limit of detection and these results have to be confirmed further. Future work needs to be done in order to ensure and improve capsaicinoid production
Chemically defined mineral medium for Lactobacillus reuteri
No full textKEMISKT DEFINIERAT MINERALMEDIUM FÖR LACTOBACILLUS REUTERI
ELINA BROMELL
Bromell, E. Kemiskt definierat mineralmedium för Lactobacillus reuteri. Examensarbete i biomedicinsk laboratorievetenskap 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för biomedicinsk laboratorievetenskap, 2020.
Släktet Lactobacillus har flera arter som anses ha probiotisk effekt, däribland Lactobacillus reuteri. L. reuteri har positiva effekter på munhälsa, vid förstoppning, spädbarnskolik och atopiskt eksem. Bakterien behöver vara robust nog att tolerera frystorkning, transport och de syror och enzymer den kan utsättas för i magtarmkanalen och behöver därtill vara livskraftig nog att kunna verka i det tilltänkta området. Viabilitet och tolerans kan påverkas positivt, bl.a. genom att förstärka bakteriens membran. Membranen kan förstärkas genom att förbättra odlingsförhållanden och odlingsmedium. För att kunna förbättra odlingsmediumet på detaljnivå behöver det vara kemiskt definierat. Eftersom det finns ett semi-definierat medium, SD4, för L. reuteri blir det första steget att byta ut de odefinierade komponenterna i SD4 till definierade alternativ. Syftet med studien var att tillverka ett kemiskt definierat mineralmedium för L. reuteri. Metoden var att ersätta Casamino acids i SD4 med alla 20 aminosyror, glutathion och en kvävekälla. One-Factor-At a-Time (OFAT) tillämpades genom att utesluta en aminosyra i taget och på så sätt se vilka som är essentiella för tillväxt. Experimenten resulterade i ett odlingsmedium med di-ammoniumcitrat som kvävekälla och 14 aminosyror, essentiella för tillväxt. Studien konkluderade att ett nytt semidefinierat odlingsmedium för L. reuteri har tagits fram. Mediumet är fortfarande semidefinierat då Tween 80™ inte ersatts med definierade fettsyror. Eftersom kraven på näring kan skilja mellan olika stammar är detta medium än så länge bara definierat för L. reuteri DSM 17938.
Nyckelord: aminosyror, definierat mineralmedium, Lactobacillus reuteri DSM 17938, mediumoptimering, OFAT.CHEMICALLY DEFINED MINERAL MEDIUM FOR LACTOBACILLUS REUTERI
ELINA BROMELL
Bromell, E. Chemically defined mineral medium for Lactobacillus reuteri. Degree project in Biomedical laboratory science 15 Credits. Malmö University: Faculty of Health and Society, Department of Biomedical laboratory science, 2020.
Several species of the genus Lactobacillus have probiotic effect, amongst them Lactobacillus reuteri. L. reuteri has positive effects on periodontal health, constipation, infant colic and atopic eczema. The bacterium needs to be robust enough to tolerate freeze-dry, transport and the acids and enzymes of the gastrointestinal fluids, thereto it needs to be viable enough to colonise the intended area. Viability and tolerance can be increased by strengthening the membrane. A stronger membrane can be achieved by optimizing the environmental conditions and the medium. To optimize the medium on a detailed level, the medium needs to be chemically defined. Since a semi-defined medium for L. reuteri, SD4 already exists, the next step will be to replace the undefined ingredients in SD4 to defined alternatives. The aim of the study was to make a chemically defined mineral medium for L. reuteri. The method chosen was to replace Casamino acids in SD4 with all 20 amino acids, glutathione and a nitrogen source. One-Factor-At a-Time (OFAT) was applied by removing one amino acid at a time and to evaluate which amino acids were essential or not essential for growth. The experiments resulted in a medium with di-ammonium citrate as nitrogen source and 14 amino acids, essential for growth. The study concludes that a new semi-defined medium for L. reuteri has been developed. The medium is still semi-defined since Tween 80™ has not been replaced with defined fatty acids. Growth requirements can differ between strains, therefore this medium is still only regarded as semi-defined for L. reuteri DSM 17938.
Keywords: amino acids, defined mineral medium, Lactobacillus reuteri DSM 17938, medium optimization, OFAT
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Exploring amino acid metabolism in Saccharomyces cerevisiae for improved eco-efficient production of chiral amine
No full textKirala aminer används idag både som aktiva substanser och som bindningsmedel i flertalet läkemedel, dock är dagens produktion med kinetic resolution ineffektiv vilket gör att mer effektiva och miljövänliga produktionssätt eftersträvas. Biotransformation har visat sig både vara miljövänligt och en effektiv metod för att producera kirala aminer. Aminosyror kan användas som aminodonatorer för att producera den kirala aminen 1-methyl-3-phenylpropylamine (MPPA) från prokirala ketonen bensylaceton (BA) med hjälp av aminetransaminas. I denna studie användes metaboliskt konstruerad Saccharomyces cerevisiae med enzymet CV-ωTA för att identifiera vilka aminosyror som var bäst lämpade för MPPA produktion. MPPA produktion kunde detekteras för alla testade aminosyror. Aminosyrans koncentration hade ingen tydlig påverkan på produktionen av MPPA. Alanin vara den aminosyra som gav högst produktionsutbyte följt av lysin. Ingen tydlig relation mellan produktion av MPPA och aminosyrornas koncentrationer kunde ses. Produktionen av MPPA var snabbare än förväntat och var klar redan dag tre för flera av aminosyrorna. Det fanns en antydan att BA kunde vara toxiskt för cellerna i högre koncentrationer och därmed påverka produktionen av MPPA.Chiral amines are used in several types of pharmaceuticals as both active substrates and building blocks, and there is an endeavor to find new and more eco-efficient ways to produce them than today’s production with kinetic resolution. Biotransformation in yeast has shown great potential for production and is also seen as an eco-friendly way to produce chiral amines. Amino acids can be used as an amino donor for the production of chiral amines, e.g. 1-methyl-3-phenylpropylamine (MPPA) from prochiral ketones, e.g. benzylacetone (BA) with aminotransaminase. In this study the production was done with metabolically engineered Saccharomyces cerevisiae, with the gene for the enzyme CV-ωTA transformed. Ten different amino acids were screened in up to three different concentrations for each amino acid. Production of MPPA was observed for all amino acids, with alanine as the most efficient followed by lysine. No clear relationship was seen between amino acid concentration and MPPA production. The production of MPPA for several amino acids were quicker than expected and was completed at day three. Our data indicated a cytotoxic effect of BA at higher concentrations, that negatively affected the production of MPPA
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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