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Aims In this study, we aimed to investigate whether body composition analysis (BCA) derived from bioelectrical impedance vector analysis (BIVA) could be used to monitor the hydration status of patients with acute heart failure (AHF) during intensified diuretic therapy. Methods and results This observational, single‐centre study involved a novel, validated eight‐electrode segmental body composition analyser to perform BCA derived from BIVA with an alternating current of 100 μA at frequencies of 5, 7.5, 50, and 75 kHz. The BCA‐derived and BIVA‐derived parameters were estimated and compared with daily body weight measurements in hospitalized patients with AHF. A total of 867 BCA and BIVA assessments were conducted in 142 patients (56.3% men; age 76.8 ± 10.7 years). Daily changes in total body water (TBW) and extracellular water (ECW) were significantly associated with changes in body weight in 62.2% and 89.1% of all measurements, respectively (range, ±1 kg). Repeated measures correlation coefficients between weight loss and TBW loss resulted with rho 0.43, P 0.01, CI [0.67, 0.75] for ECW loss. Between the first and last assessments, the mean weight loss was −2.5 kg, compared with the −2.6 L mean TBW loss and −1.7 L mean ECW loss. BIVA revealed an increase in mean Resistance R and mean Reactance Xc across all frequencies, with the subsequent reduction in body fluid (including corresponding body weight) between the first and last assessments. Conclusions Body composition analysis derived from BIVA with a focus on ECW is a promising approach to detect changes in hydration status in patients undergoing intensified diuretic therapy. Defining personalized BIVA reference values using bioelectrical impedance devices is a promising approach to monitor hydration status.

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Rights and a tolerance of diversity are central to a democratic polity, and for over 60 years scholars have viewed education as a powerful wellspring of liberal attitudes on these issues. But recent concerns with selection bias raise questions about whether exposure to education indeed shapes attitudes. This study offers new perspective on the influence of education on rights and tolerance attitudes in the United States. We use a larger and wider-ranging array of items (63) than has been considered in recent scholarship on this topic. Analyzing General Social Surveys panel data, we apply the Morgan/Winship model to address selection bias concerns. We find novel evidence that education shapes rights and tolerance attitudes. It is exposure to college education, not high school, that appears to be most consequential, suggesting the importance of higher-educational institutions to the diffusion of liberal attitudes. We discuss study limitations and directions for further investigation.

Background The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid. Results For yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L. Conclusions Our results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates. Electronic supplementary material The online version of this article (10.1186/s12934-018-0925-8) contains supplementary material, which is available to authorized users.

In biotechnological applications, cyanobacteria are employed for conversion of CO2 into bioproducts with sunlight as sole energy source. We describe the isolation of motile filamentous cyanobacteria from rockpools of the North Sea or the Mediterranean Sea and their characterization by physiological assays and genome sequencing. The five isolated lines are genetically highly similar, we regard them as strains of the same species. Phylogenetic studies placed the strains in the genus Phormidium; the species is termed Phormidium lacuna. Under liquid media growth conditions or in photobioreactors, Phormidium growth rates were comparable with the single celled model cyanobacterium Synechocystis PCC6803. However, Phormidium strains tolerate different media that can contain up to 3.7× the salt concentration of seawater and grows at temperatures up to 50 °C. Growth in medium free of NH3 or NO3− suggests that Phormidium can fix atmospheric dinitrogen by nitrogenase even in the presence of light. Genome data confirmed the presence of nitrogenase and revealed its evolutionary position close to anoxygenic δ-proteobacteria. Genes for photosynthesis, photoreceptors, nitrogen metabolism, hydrogenases, tryptophan synthesis, glucose uptake, and fermentative pathways are discussed in the context of biotechnological applications.

In the early stages of dementia, care often takes place at home. In the project Dementia Things we form competence teams consisting of designers, professional caregivers, persons with dementia and their care giving relatives. We ask for the everyday problems of persons with dementia and their relatives in private environments and use methods of field research and participatory design to understand the specific contexts of dementia and improve the quality of life. From the ongoing field work we were able to identify four factors influencing the participatory design process: building up trustful relations in the field, the role of the professional caregiver, the surplus of overcoming verbal barriers with design methods, and the overall benefit of changing perspectives and knowledge transfer within the competence team.

Background Saccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker’s yeast. Results We first designed a two-step heterologous pathway, starting with the endogenous amino acid l-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional l-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional l-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol. Conclusions Our results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0667-z) contains supplementary material, which is available to authorized users.

The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5′-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.

Background Saccharomyces cerevisiae can be engineered to perform a multitude of different chemical reactions that are not programmed in its original genetic code. It has a large potential to function as whole-cell biocatalyst for one-pot multistep synthesis of various organic molecules, and it may thus serve as a powerful alternative or complement to traditional organic synthetic routes for new chemical entities (NCEs). However, although the selectivity in many cases is high, the catalytic activity is often low which results in low space-time-yields. In the case for NADH-dependent heterologous reductive reactions, a possible constraint is the availability of cytosolic NADH, which may be limited due to competition with native oxidative enzymes that act to maintain redox homeostasis. In this study, the effect of increasing the availability of cytosolic NADH on the catalytic activity of engineered yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. Results A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b) reduction of the produced acetophenone to (S)-1-phenylethanol. For the gpd1Δgpd2Δ strain, cell metabolism was effectively used for the supply of both amine acceptors and the co-factor pyridoxal-5′-phosphate (PLP) for the ω-transaminase, as well as for regenerating NADH for the reduction. In contrast, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over-expressing SADH. Conclusions Overall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i.e. without addition of external co-factors or prosthetic groups. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0430-x) contains supplementary material, which is available to authorized users.

Background The conversion of vanillin to vanillylamine is a key step in the biosynthetic route towards capsaicinoids in pungent cultivars of Capsicum sp. The reaction has previously been annotated to be catalysed by PAMT (putative aminotransferase; [GenBank: AAC78480.1, Swiss-Prot: O82521]), however, the enzyme has previously not been biochemically characterised in vitro. Results The biochemical activity of the transaminase was confirmed by direct measurement of the reaction with purified recombinant enzyme. The enzyme accepted pyruvate, and oxaloacetate but not 2-oxoglutarate as co-substrate, which is in accordance with other characterised transaminases from the plant kingdom. The enzyme was also able to convert (S)-1-phenylethylamine into acetophenone with high stereo-selectivity. Additionally, it was shown to be active at a broad pH range. Conclusions We suggest PAMT to be renamed to VAMT (vanillin aminotransferase, abbreviation used in this study) as formation of vanillin from vanillylamine could be demonstrated. Furthermore, due to high stereoselectivity and activity at physiological pH, VAMT is a suitable candidate for biocatalytic transamination in a recombinant whole-cell system.

The association of nationalist consciousness and feminist ideology in Slovakia in the late nineteenth century was a protracted and uneven process. This conclusion rests upon the results of this study which examines the feminist and nationalist views of Slovak women intelligentsia who were at the forefront of Slovak nationalist efforts. It explores responses of leading Slovak women to the following issues of nationalist concern: traditional Slovak patriarchy, women's education, and Western feminism. It demonstrates that in Slovakia, gender was not the primary factor determining women's loyalties; there were other connecting allegiances and loyalties to the nation and the community. Slovak women developed their own unique concept of gender equality that aided Slovak nationalist efforts. In doing so they employed the language of motherhood, domestic duties, and religious commitment.Around the turn of the century, a small group of Slovak women intelligentsia attempted to reconcile their own agenda with contemporary nationalist, social, and political currents. Spurred by nationalist efforts of the Slovak male intelligentsia, middle-class women tried to determine what type of new nationalist woman should replace the traditional woman. This question was answered by five women, in four very distinct ways: (1) Ľudmila Ríznerová-Podjavorinská portrayed the goals of Western feminism as a danger to Slovaks; (2) Elena Maróthy-Šolthésová and Terézia Medvecká Vansová encouraged the growth of Christian feminism; (3) Marína Ormisová-Maliaková favored the introduction of pragmatic feminism in Slovak nationalist efforts; and (4) Hana Lilge-Gregorová argued for the establishment of Western feminism as the basis of social and national development. Although the personal lives of these five women represent the social and national distress of the Slovak people, they also show women's fight for the acceptance of new ideas which would improve the fate of their sisters and their nation. Yet this small collection of feminist intellectuals could not and did not effect Slovak public opinion in any substantial way. Their influence, except perhaps that of Hana Lilge-Gregorová, did not stretch beyond the Slovak urban middle-class milieu.

Bei einem Besuch des belgischen Thronfolgerpaares in Korea fragte die junge und hubsche Prinzessin Mathilde meine Frau, was sie eigentlich als Gattin eines Botschafters tue. In moglichst einem Satz eine griffige Antwort zu geben, ist nicht einfach: Einmal weil eine solche — ebenso wie die Antwort auf die Frage nach der Tatigkeit eines Botschafters selbst — die Darstellung von Vielfaltigkeit erfordert, jedenfalls wenn man es nicht bei Floskeln belassen will. Zum anderen verbietet es die Hoflichkeit, bei einem Empfang seinem Gesprachspartner gegenuber zu enzyklopadischen Ausfuhrungen anzusetzen. Andere Gaste mochten auch noch ein Wort mit dem Ehrengast wechseln! Also doch nur mit einer Floskel antworten?

Background One-pot multi-step biocatalysis is advantageous over step-by-step synthesis as it reduces the number of process operation units, leading to significant process intensification. Whole-cell biocatalysis with metabolically active cells is especially valuable since all enzymes can be co-expressed in the cell whose metabolism can be exploited for supply of co-substrates and co-factors. Results In this study, a heterologous enzymatic system consisting of ω-transaminase and ketone reductase was introduced in Saccharomyces cerevisiae, and evaluated for one-pot stereo-selective conversion of amines to alcohols. The system was applied for simultaneous kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine and reduction of the ketone intermediate to (R)-1-phenylethanol. Glucose was used as sole co-substrate for both the supply of amine acceptor and the regeneration of NADPH in the reduction step. Conclusions The whole-cell biocatalyst was shown to sustain transaminase-reductase-catalyzed enantioselective conversion of amines to alcohols with glucose as co-substrate. The transamination catalyzed by recombinant vanillin aminotransferase from Capsicum chinense proved to be the rate-limiting step as a three-fold increase in transaminase gene copy number led to a two-fold increased conversion. The (R)-selective NADPH-dependent alcohol dehydrogenase from Lactobacillus kefir proved to be efficient in catalyzing the reduction of the acetophenone generated in the transamination reaction.

Background Whole-cell biocatalysis based on metabolically active baker’s yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. Results Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5′-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using l-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. Conclusions Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0615-3) contains supplementary material, which is available to authorized users.