Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Tissue Metabolic Changes Drive Cytokine Responses to Mycobacterium tuberculosis
Lachmandas, Ekta ; Rios-Miguel, Ana B. ; Koeken, Valerie A.C.M. ; Pasch, Eva van der; Kumar, Vinod ; Matzaraki, Vasiliki ; Li, Yang ; Oosting, Marije ; Joosten, Leo A.B. ; Notebaart, Richard A. ; Noursadeghi, Mahdad ; Netea, Mihai G. ; Crevel, Reinout van; Pollara, Gabriele - \ 2018
The Journal of Infectious Diseases 218 (2018)1. - ISSN 0022-1899 - p. 165 - 170.
cytokines - functional genomics - human challenge model - immune response - immunometabolism - metabolism - microarrays - transcriptomics - tuberculosis

Cellular metabolism can influence host immune responses to Mycobacterium tuberculosis. Using a systems biology approach, differential expression of 292 metabolic genes involved in glycolysis, glutathione, pyrimidine, and inositol phosphate pathways was evident at the site of a human tuberculin skin test challenge in patients with active tuberculosis infection. For 28 metabolic genes, we identified single nucleotide polymorphisms that were trans-acting for in vitro cytokine responses to M. tuberculosis stimulation, including glutathione and pyrimidine metabolism genes that alter production of Th1 and Th17 cytokines. Our findings identify novel therapeutic targets in host metabolism that may shape protective immunity to tuberculosis.

Follicular development of sows at weaning in relation to estimated breeding value for within-litter variation in piglet birth weight
Costermans, N.G.J. ; Teerds, K.J. ; Keijer, J. ; Knol, E.F. ; Koopmanschap, R.E. ; Kemp, B. ; Soede, N.M. - \ 2018
Animal (2018). - ISSN 1751-7311 - 10 p.
lactation - litter uniformity - metabolism - reproduction - sows

In this study we aimed to identify possible causes of within-litter variation in piglet birth weight (birth weight variation) by studying follicular development of sows at weaning in relation to their estimated breeding value (EBV) for birth weight variation. In total, 29 multiparous sows (parity 3 to 5) were selected on their EBV for birth weight variation (SD in grams; High-EBV: 15.8±1.6, N=14 and Low-EBV: −24.7±1.5, N=15). The two groups of sows had similar litter sizes (15.7 v. 16.9). Within 24 h after parturition, piglets were cross-fostered to ensure 13 suckling piglets per sow. Sows weaned 12.8±1.0 and 12.7±1.0 piglets, respectively, at days 26.1±0.2 of lactation. Blood and ovaries were collected within 2 h after weaning. The right ovary was immediately frozen to assess average follicle size and percentage healthy follicles of the 15 largest follicles. The left ovary was used to assess the percentage morphologically healthy cumulus-oocyte complexes (COCs) of the 15 largest follicles. To assess the metabolic state of the sows, body condition and the circulating metabolic markers insulin, IGF1, non-esterified fatty acid, creatinine, leptin, urea and fibroblast growth factor 21 were analysed at weaning. No significant differences were found in any of the measured follicular or metabolic parameters between High-EBV and Low-EBV. A higher weight loss during lactation was related to a lower percentage healthy COCs (β= −0.65, P=0.02). Serum creatinine, a marker for protein breakdown, was negatively related to average follicle size (β= −0.60, P=0.05). Backfat loss during lactation was related to a higher backfat thickness at parturition and to a higher average follicle size (β=0.36, P<0.001) at weaning. In conclusion, we hypothesise that modern hybrid sows with more backfat at the start of lactation are able to mobilise more energy from backfat during lactation and could thereby spare protein reserves to support follicular development.

Food Design to Feed the Human Gut Microbiota
Ercolini, Danilo ; Fogliano, Vincenzo - \ 2018
Journal of Agricultural and Food Chemistry 66 (2018)15. - ISSN 0021-8561 - p. 3754 - 3758.
food ingredients - food processing - Mediterranean diet - melanoidins - metabolism - phytochemicals
The gut microbiome has an enormous impact on the life of the host, and the diet plays a fundamental role in shaping microbiome composition and function. The way food is processed is a key factor determining the amount and type of material reaching the gut bacteria and influencing their growth and the production of microbiota metabolites. In this perspective, the current possibilities to address food design toward a better feeding of gut microbiota are highlighted, together with a summary of the most interesting microbial metabolites that can be made from dietary precursors.
No Adverse Programming by Post-Weaning Dietary Fructose of Body Weight, Adiposity, Glucose Tolerance, or Metabolic Flexibility
Bouwman, Lianne M.S. ; Fernández-Calleja, José M.S. ; Swarts, Hans J.M. ; Stelt, Inge van der; Oosting, Annemarie ; Keijer, Jaap ; Schothorst, Evert M. van - \ 2018
Molecular Nutrition & Food Research 62 (2018)2. - ISSN 1613-4125
carbohydrates - indirect calorimetry - metabolic programming - metabolism - monosaccharides
Scope: Metabolic programming can occur not only in the perinatal period, but also post-weaning. This study aims to assess whether fructose, in comparison to glucose, in the post-weaning diet programs body weight, adiposity, glucose tolerance, metabolic flexibility, and health at adult age. Methods and results: Three-week-old male and female C57BL6/JRccHsd mice are given an intervention diet with 32 energy percent (en%) glucose or fructose for only 3 weeks. Next, all animals are switched to the same 40 en% high fat diet for 9 weeks. Neither body weight nor adiposity differs significantly between the animals fed with glucose or fructose diets at any point during the study in both sexes. Glucose tolerance in adulthood is not affected by the post-weaning diet, nor are activity, energy expenditure, and metabolic flexibility, as measured by indirect calorimetry. At the end of the study, only in females fasting serum insulin levels and HOMA-IR index are lower in post-weaning fructose versus glucose diet (p = 0.02), without differences in pancreatic β-cell mass. Conclusions: Our present findings indicate no adverse programming of body weight, adiposity, glucose tolerance, and metabolic flexibility by dietary (solid) fructose in comparison to glucose in the post-weaning diet in mice.
Age and Sex Effects on Plasma Metabolite Association Networks in Healthy Subjects
Vignoli, Alessia ; Tenori, Leonardo ; Luchinat, Claudio ; Saccenti, Edoardo - \ 2018
Journal of Proteome Research 17 (2018)1. - ISSN 1535-3893 - p. 97 - 107.
differential network analysis - metabolism - metabolomics - network inference - NMR
In the era of precision medicine, the analysis of simple information like sex and age can increase the potential to better diagnose and treat conditions that occur more frequently in one of the two sexes, present sex-specific symptoms and outcomes, or are characteristic of a specific age group. We present here a study of the association networks constructed from an array of 22 plasma metabolites measured on a cohort of 844 healthy blood donors. Through differential network analysis we show that specific association networks can be associated with sex and age: Different connectivity patterns were observed, suggesting sex-related variability in several metabolic pathways (branched-chain amino acids, ketone bodies, and propanoate metabolism). Reduction in metabolite hub connectivity was also found to be associated with age in both sex groups. Network analysis was complemented with standard univariate and multivariate statistical analysis that revealed age- and sex-specific metabolic signatures. Our results demonstrate that the characterization of metabolite-metabolite association networks is a promising and powerful tool to investigate the human phenotype at a molecular level.
Metabolic status, lactation persistency, and udder health of dairy cows after different dry period lengths
Hoeij, Renny van - \ 2017
University. Promotor(en): Bas Kemp; T.J.G.M. Lam, co-promotor(en): Ariette van Knegsel; Jan Dijkstra. - Wageningen : Wageningen University - ISBN 9789463438070 - 285
dairy cattle - animal health - animal behaviour - dry period - metabolism - energy balance - lactation - milk production - udders - cattle feeding - melkvee - diergezondheid - diergedrag - gustperiode - metabolisme - energiebalans - lactatie - melkproductie - uiers - rundveevoeding

Cows traditionally have a 6 to 8 week non-lactating –‘dry period’- before calving and the start of the next lactation in order to maximize milk production in the subsequent lactation. An omitted, compared with a shortened, dry period reduces milk yield and improves energy availability in cows postpartum, but effects on udder health and persistency were unclear. Cows without a dry period fattened and spontaneously dried off due to the improved energy availability. Reducing the energy availability in the feed for cows without a dry period did not affect fattening or lactation persistency in late lactation. Cows with a short or without a dry period did not receive dry cow antibiotics in this study and this did not affect udder health across the dry period or in early lactation, but seemed to impair udder health in late lactation for cows without a dry period.

Photosynthetic efficiency in microalgal lipid production
Remmers, Ilse M. - \ 2017
University. Promotor(en): Rene Wijffels, co-promotor(en): Packo Lamers. - Wageningen : Wageningen University - ISBN 9789463434607 - 200
algae - biofuels - light - triacylglycerols - lipids - metabolism - algae culture - cultural methods - algen - biobrandstoffen - licht - triacylglycerolen - lipiden - metabolisme - algenteelt - cultuurmethoden

Microalgae can contain large amounts of lipids which make them a promising feedstock for sustainable production of food, feed, fuels and chemicals. Various studies, including pilot-scale, have been performed and the knowledge on microalgal processes has advanced quickly. Unfortunately, current production costs for cultivation are still too high for bulk lipid production from microalgae.

One of the major causes for the high costs of bulk lipid production is the reduced solar-to-lipid conversion efficiency. Current research, however, does not provide sufficient insight to identify optimization targets. Therefore, in this thesis we have studied the lipid production in microalgae in depth.

Different TAG-accumulation strategies were investigated from a process engineering and metabolic point of view. The combination of all findings were used in the general discussion to thoroughly evaluate the microalgal lipid accumulation strategies. Current phototrophic microalgal lipid yields are still 10 times lower than the theoretical maximum. There is, however, still an enormous potential for further improvements. Future research should focus on (genetically) improved strains and advanced cultivation strategies, including adaptation to fluctuating outdoor weather conditions.

This thesis was performed within the EU FP7 FUEL4ME project under grand agreement No 308938. Objective of this program is to develop a sustainable and scalable process for biofuels from microalgae and to valorize the by-products.

Metabolic modeling to understand and redesign microbial systems
Heck, Ruben G.A. van - \ 2017
University. Promotor(en): Vitor Martins dos Santos, co-promotor(en): Maria Suarez Diez. - Wageningen : Wageningen University - ISBN 9789463434553 - 239
micro-organismen - modelleren - kooldioxide - biotechnologie - algen - metabolisme - pseudomonas - microorganisms - modeling - carbon dioxide - biotechnology - algae - metabolism

The goals of this thesis are to increase the understanding of microbial metabolism and to functionally (re-)design microbial systems using Genome- Scale Metabolic models (GSMs). GSMs are species-specific knowledge repositories that can be used to predict metabolic activities for wildtype and genetically modified organisms. Chapter 1 describes the assumptions associated with GSMs, the GSM generation process, common GSM analysis methods, and GSM-driven strain design methods. Thereby, chapter 1 provides a background for all other chapters. In this work, there is a focus on the metabolically versatile bacterium Pseudomonas putida (chapters 2,3,4,5,6), but also other model microbes and biotechnologically or societally relevant microbes are considered (chapters 3,4,6,7,8).

GSMs are reflections of the genome annotation of the corresponding organism. For P. putida, the genome annotation that GSMs have been built on is more than ten years old. In chapter 2, this genome annotation was updated both on a structural and functional level using state-of-the-art annotation tools. A crucial part of the functional annotation relied on the most comprehensive P. putida GSM to date. This GSM was used to identify knowledge gaps in P. putida metabolism by determining the inconsistencies between its growth predictions and experimental measurements. Inconsistencies were found for 120 compounds that could be degraded by P. putida in vitro but not in silico. These compounds formed the basis for a targeted manual annotation process. Ultimately, suitable degradation pathways were identified for 86/120 as part of the functional reannotation of the P. putida genome.

For P. putida there are 3 independently generated GSMs, which is not uncommon for model organisms. These GSMs differ in generation procedure and represent different and complementary subsets of the knowledge on the metabolism of the organism. However, the differing generation procedures also makes it extremely cumbersome to compare their contents, let alone to combine them into a single consensus GSM. Chapter 3 addresses this issue through the introduction of a computational tool for COnsensus Metabolic Model GENeration (COMMGEN). COMMGEN automatically identifies inconsistencies between independently generated GSMs and semi-automatically resolves them. Thereby, it greatly facilitates a detailed comparison of independently generated GSMs as well as the construction of consensus GSMs that more comprehensively describe the knowledge on the modeled organism.

GSMs can predict whether or not the corresponding organism and derived mutants can grow in a large variety of different growth conditions. In comparison, experimental data is extremely limited. For example, BIOLOG data describes growth phenotypes for one strain in a few hundred different media, and genome-wide gene essentially data is typically limited to a single growth medium. In chapter 4 GSMs of multiple Pseudomonas species were used to predict growth phenotypes for all possible single-gene-deletion mutants in all possible minimal growth media to determine conditionally and unconditionally essential genes. This simulated data was integrated with genomic data on 432 sequenced Pseudomonas species, which revealed a clear link between the essentiality of a gene function and the persistence of the gene within the Pseudomonas genus.

Chapters 5 and 6 describe the use of GSMs to (re-)design microbial systems. P. putida is, despite its acknowledged versatile metabolism, an obligate aerobe. As the oxygen-requirement limits the potential applications of P. putida, there have been several experimental attempts to enable it to grow anaerobically, which have so far not succeeded. Chapter 5 describes an in silico effort to determine why P. putida cannot grow anaerobically using a combination of GSM analyses and comparative genomics. These analyses resulted in a shortlist of several essential and oxygen-dependent processes in P. putida. The identification of these processes has enabled the design of P. putida strains that can grow anaerobically based on the current understanding of P. putida metabolism as represented in GSMs.

Efficient microbial CO2 fixation is a requirement for the biobased community, but the natural CO2 fixation pathways are rather inefficient, while the synthetic CO2 fixation pathways have been designed without considering the metabolic context of a target organism. Chapter 6 introduces a computational tool, CO2FIX, that designs species-specific CO2 fixation pathways based on GSMs and biochemical reaction databases. The designed pathways are evaluated for their ATP efficiency, thermodynamic feasibility, and kinetic rates. CO2FIX is applied to eight different organisms, which has led to the identification of both species-specific and general CO2 fixation pathways that have promising features while requiring surprisingly few non-native reactions. Three of these pathways are described in detail.

In all previous chapters GSMs of relatively well-understood microbes have been used to gain further insight into their metabolism and to functionally (re-)design them. For complex microbial systems, such as algae (chapter 7) and gut microbial communities (chapter 8), GSMs are similarly useful, but substantially more difficult to create and analyze. Algae are widely considered as potential centerpieces of a biobased economy. Chapter 7 reviews the current challenges in algal genome annotation, modeling and synthetic biology. The gut microbiota is an incredibly complex microbial system that is crucial to our well-being. Chapter 8 reviews the ongoing developments in the modeling of both single gut microbes and gut microbial communities, and discusses how these developments will enable the move from studying correlation to causation, and ultimately the rational steering of gut microbial activity.

Chapter 9 discusses how the previous chapters contribute to the research goals of this thesis. In addition, it provides an extensive discussion on current GSM practices, the issues associated therewith, and how these issues can be tackled. In particular, the discussion focuses on issues related to: (i) The inability to distinguish between biological difference and GSM generation artifacts when using multiple GSMs, (ii) The lack of continuous GSM updates, (iii) The mismatch between what GSM predictions and experimental data represent, (iv) The need for standardization in GSM evaluation, and (v) The lack of experimental validation of GSM-driven strain design for metabolic engineering.

FeedOmics, an approach to evaluate the functional properties of protein containing feed ingredients
Kar, Soumya K. - \ 2017
University. Promotor(en): Mari Smits; Jerry Wells, co-promotor(en): Alfons Jansman; Dirkjan Schokker. - Wageningen : Wageningen University - ISBN 9789463434461 - 254
compound feeds - ingredients - protein sources - proteins - functional properties - metabolism - feed formulation - protein digestion - proteomics - digestive tract - nutrition physiology - animal nutrition - livestock feeding - mengvoer - ingrediënten - eiwitbronnen - eiwitten - functionele eigenschappen - metabolisme - voersamenstelling - eiwitvertering - eiwitexpressieanalyse - spijsverteringskanaal - voedingsfysiologie - diervoeding - veevoeding

This thesis presents FeedOmics approach as a toolkit, to evaluate (novel) protein containing feed ingredients of different origin considering both their nutritional and functional value in terms of their capacity to support or modify nutrient supply, the animal’s physiology, tissue development and functioning. Such knowledge may contribute to introduce novel and/or alternative protein containing feed ingredients in the diet of livestock, thus creating a sustainable food supply for growing human population.

First week nutrition for broiler chickens : effects on growth, metabolic status, organ development, and carcass composition
Lamot, David - \ 2017
University. Promotor(en): Bas Kemp, co-promotor(en): Henry van den Brand; Peter Wijtten. - Wageningen : Wageningen University - ISBN 9789463430777 - 187
broilers - animal nutrition - poultry feeding - feeds - growth - metabolism - carcass composition - nutrition physiology - vleeskuikens - diervoeding - pluimveevoeding - voer - groei - metabolisme - karkassamenstelling - voedingsfysiologie

During the first week of life, broiler chickens undergo various developmental changes that are already initiated during incubation. Ongoing development of organs such as the gastro- intestinal tract and the immune system may affect the nutritional requirements during this age period. Despite the residual yolk that is available at hatch and that may provide nutritional support during the first days after hatch, the growth performance may be affected by the time in between hatch and first feed intake. Furthermore, it remains largely unknown to what extend nutritional composition of a pre-starter diet, as well as feed availability directly after hatch have an effect on physiological development directly after hatch, but also at later age. The aim of this thesis was to determine the impact of feed availability and feed composition provided during the first week of life on short-term physiological development, as well as potential long-term effects on growth performance of broiler chickens. Especially early hatched chickens were suggested to benefit more from direct feed access compared to midterm and late hatched chickens, as they tended to have a higher body weight gain during the first week after hatch. A delay in feed access for 48 h resulted in lowered body weight gain and feed intake when compared to direct feed access, but so did a short (13 to 26 h) delay in feed access after hatch. In the latter case, delayed feed access resulted in a lower weight to length ratio of the jejunum and ileum at 4 d of age compared with chickens with direct feed access. Although delayed feed access after hatch resulted in lower body weight gain during the first week after hatch and thereafter, it can be discussed whether this is truly an impairment of long-term growth or just a delayed onset of growth. With respect to feed composition, the inclusion of fish oil and medium chain fatty acids in a pre-starter diet had minor effects on humoral immune function. Inclusion of medium chain fatty acids did result in higher body weight gain and lowered feed efficiency during the first week of life, but only during the period it was provided. Feeding increased diet densities during the first week of life, obtained by formulating diets with different dietary fat levels, resulted in an increased gain to feed ratio, whereas body weight gain and feed intake decreased. Despite the shift in dietary energy supply from carbohydrates to fat and the perceived lower fat digestibility in young broiler chickens, nitrogen metabolizability and fat digestibility were not affected in the current study by feeding increased diet densities. The relative crop, liver and pancreas weights decreased when feeding increased diet densities, whereas the length of the entire intestinal tract increased. This suggests that broiler chickens repartition visceral organ development in response to feeding more concentrated diets during the first week of life. Interestingly, protein and fat accretion were not affected. Continued feeding of increased diet densities after 7 d of age resulted in increased BW gain, G:F ratio and metabolizable energy intake, but mainly during the periods that these diets were provided. In summary, even short durations of delayed feed access already impact intestinal development of young broiler chickens. However, a delayed feed access up to 48 h after hatch does not result in impaired growth, but only a delayed onset of growth. Even though digestibility of fats and oils may be suboptimal in young broiler chickens, feeding of these diets does not have to result in lowered performance per se. Young broiler chickens appear to adapt themselves towards high density diets with high fat inclusion levels in the first week of life, enabling them to digest and metabolize these diet types despite a suboptimal capacity for fat digestion. High density diets result in higher growth performance, but only for the period these diets are provided and thus carry-over effects at later age appear to be limited.

Physiology and application of sulfur-reducing microorganisms from acidic environments
Florentino, Anna Patrícya - \ 2017
University. Promotor(en): Fons Stams, co-promotor(en): Irene Sanchez Andrea; Jan Weijma. - Wageningen : Wageningen University - ISBN 9789463430975 - 264
bacteria - desulfurella - metabolism - sulfur - reduction - genome analysis - proteomes - bacteriën - metabolisme - zwavel - reductie - genoomanalyse - proteomen

Sulfur cycle is one of the main geochemical cycles on Earth. Oxidation and reduction reactions of sulfur are mostly biotic and performed by microorganisms. In anaerobic conditions – marine and some freshwater systems, dissimilatory sulfur- and sulfate-reducing bacteria and archaea are key players in the decomposition of organic carbon releasing sulfide as the product of their metabolism. Sulfide can then be used as terminal reductant by anoxygenic photosynthetic microorganisms or it can be used as electron donor for aerobic or nitrate-reducing bacteria, etc.

One particular case of the sulfur cycle is the naturally occurring oxidation of metallic sulfide-ores, which produce sulfur-rich waters with low pH and high heavy metals content. Extremophilic sulfur-reducing microorganisms are of scientific and technological interest. They are abundant in natural conditions in extreme environments, so they are environmentally relevant. Although hydrogen sulfide is corrosive and odorous, its production can be beneficial for industrial activities such as the precipitation and recovery of heavy metals. Therefore, sulfur reducers have also potential for extending the range of operating conditions of metal precipitation. This thesis describes the isolation and characterization of acidotolerant sulfur-reducing bacteria, providing a first understanding on their metabolism of sulfur compounds and insights on the beneficial microbial interactions for biotechnological purposes.

In Chapter 2, the ecology and physiology of sulfur-reducing prokaryotes is investigated. The ability of sulfur reduction is wide-spread phylogenetically over the microbial tree of life, found in more than 70 genera. Elemental sulfur reduction can occur via direct cell attachment to the solid substrate or with polysulfide as an intermediate. At least four different enzymes are described to be involved in sulfur reduction pathways, and these enzymes were also detected in several microorganisms that are potential sulfur reducers, but were not reported as such in literature so far. The ecological distribution of sulfur respiration seems to be more widespread at high temperatures with neutral pH values. However, some sulfur reducers can grow at pH as low as 1 and the strategies adopted by microorganisms to face high proton concentrations in the environment were commented in this chapter. The sulfide produced from sulfur reduction can be used to selectively precipitate metals by varying the pH values from 2 to 7, depending on the target metal. Economic calculations were presented to show that sulfur reduction is more advantageous then sulfate reduction due to the cost savings of the electron donor needed. Therefore, acidophilic sulfur reducers are of particular interest for application in selective precipitation and recovery of heavy metals from metalliferous waste streams and the suitable technologies for that purpose are also discussed.

Enrichments for sulfur reducers with various electron donors at low pH and mesophilic conditions were performed from sediments of the acidic Tinto river (Spain). A solid-media with colloidal sulfur was developed to facilitate the isolation of true elemental sulfur reducers at low pH. This strategy resulted in the isolation of a sulfur-reducing bacterium, strain TR1, belonging to the Desulfurella genus. The enrichment and isolation procedure were described in Chapter 3. The growth and activity of the isolate was tested at different pH values, temperature conditions, utilization of electron donors, and growth in the presence of heavy metals in solution. The isolate showed tolerance to metals, and growth in a broad temperature and pH, revealing its feasibility to precipitate and recover heavy metals from acidic wastewater and mining water, without the need to neutralize the water before treatment. In Chapter 4, the morphological, biochemical and physiological characterization of the isolate is provided, for which the name Desulfurella amilsii TR1 sp. nov. was proposed. D. amilsii is affiliated to the Deltaproteobacteria class showing 97% of 16S rRNA gene identity to the four species described in the Desulfurella genus. In the presence of elemental sulfur, D. amilsii utilized acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2 as substrates, completely oxidizing them to H2S and CO2. Besides elemental sulfur, thiosulfate was used as an electron acceptor and the isolate also grew in the absence of external electron donor, by disproportionation of elemental sulfur into sulfide and sulfate.

The draft genome sequence of Desulfurella amilsii TR1 and a comparative genomic analysis with the members of Desulfurellaceae family are reported in Chapter 5. Based on average nucleotide identity and in silico DNA hybridization values, D. multipotens and D. acetivorans were revealed to belong to the same species. Reclassification was therefore suggested. Regarding sulfur metabolism, the analysed genomes encode different sulfur-reducing enzymes per genus. Hippea species encode polysulfide reductase and a sulfide dehydrogenase. The analysed genomes of Desulfurella especies do not possess the polysulfide reductase but possess the sulfide dehydrogenase. D. amilsii is the only member of the family encoding sulfur reductase. Since D. amilsii is able to grow at the lowest pH value, this enzyme was suggested to play a role in sulfur reduction when the microorganism grows in acidic conditions. Genes encoding resistance to acidic conditions were reported for all Desulfurellaceae members, countering physiological tests that showed ability to grow at low pH only for D. amilsii and D. acetivorans. Sulfur respiration by D. amilsii was studied in more detail in Chapter 6, in which the requirement for cell-sulfur interaction at acidic (pH 3.5) and circumneutral (pH 6.5) conditions was evaluated. D. amilsii was shown to benefit from contact with the insoluble substrate, as activity and number of cells decreased when sulfur was sequestered from the medium in dialysis bags of 6-8 kDa pore size. Besides, the abundance of enzymes possibly involved in sulfur respiration, acid resistance and chemolithotrophic growth were investigated by proteomics. Sulfur reductases were not detected in the dataset, but the limitations of the method might leave membrane-bound proteins underrepresented in the study. Different rhodanese-like proteins were detected in high abundance at low and neutral pH, while sulfide dehydrogenase seems to function as a ferredoxin:NADP oxidoreductase. We suggest that the sulfurtransferases might play a key role in sulfur/polysulfide reduction in D. amilsii. Proteomic data also showed that genes involved in acid resistance are constitutively expressed in this microorganism. Some proteins were exclusively detected at low pH, but with very few overlapping with proteins reported to be involved in acid resistance. Moreover, analysis of the proteome revealed the involvement of the hydrogenase HydABC for oxidation of hydrogen during chemolitotrophic growth, as well as the complete pathway for CO2 fixation via the reductive TCA cycle.

More aspects of the sulfur metabolism by D. amilsii were investigated in Chapter 7. Cultures grown on acetate with sulfur or thiosulfate as electron acceptor and cultures grown by disproportionation of elemental sulfur, all at pH 6.5, had their proteomes compared. Rhodanese-like sulfurtransferases were abundant in all the analyzed conditions, with specific differences in the sequences. In sulfur respiration and disproportionation, sulfurtransferases were the only sulfur enzymes detected and so, they are likely to play a central role in the process. The respiration of thiosulfate is likely to happen via a thiosulfate reductase and a dissimilatory sulfite reductase, highly abundant in this specific condition. Analysis on the heterotrophic cultures revealed the ability of D. amilsii to activate acetate to acetyl-CoA via the acetyl-CoA synthetase enzyme and its oxidation via the TCA cycle being this the first report of acetate activation happening via acetyl-CoA synthetase in sulfur-reducing bacteria.

The isolation and characterization of another acidotolerant sulfur respirer, Lucifera butyrica strain ALE, and its growth in co-culture with D. amilsii were described in Chapter 8. L. butyrica was shown to use a wide range of substrate, such as glucose, lactose, ethanol, glycerol glycogen, peptone, etc. When growing on glycerol, a cheap substrate, by fermentation or by respiration of elemental sulfur, L. butyrica produced acetate, ethanol and 1,3-propanediol as major products. Elemental sulfur reduction by this bacterium, however, was not efficient and led to the production of maximum 2.5 mM of sulfide. When L. butyrica grew in a co-culture with D. amilsii, the acetate produced by the first was consumed by the latter and the production of sulfide was boosted in the culture. As D. amilsii is not able to degrade glycerol, the co-culture represents a strategy to broaden the applicability of sulfur reduction at low pH with different sources of electron donors.

Dynamic photosynthesis under a fluctuating environment: a modelling-based analysis
Morales Sierra, Alejandro - \ 2017
University. Promotor(en): Paul Struik; Jaap Molenaar, co-promotor(en): Xinyou Yin; Jeremy Harbinson. - Wageningen : Wageningen University - ISBN 9789463430456 - 282
photosynthesis - modeling - analysis - environmental factors - light - canopy - leaves - crop physiology - metabolism - fotosynthese - modelleren - analyse - milieufactoren - licht - kroondak - bladeren - gewasfysiologie - metabolisme

In their natural environment, leaves are exposed to rapid fluctuations of irradiance. Research on CO2 assimilation under fluctuating irradiance often relies on measurements of gas exchange during transients where irradiance is rapidly increased or decreased, after the leaf has adapted to a particular set of environmental conditions. In the field, such increases and decreases occur mostly because of sunflecks (rapid increases in irradiance on a low irradiance background) created by gaps in the canopy and plant movement by wind, and cloudflecks (rapid decreases in irradiance on a high irradiance background) generated by clouds that transiently block the sun.

In this dissertation, the metabolic regulation of photosynthesis and how this may limit dynamic CO2 assimilation is studied in silico with the development and application of simulation models. In order to support the development of the models, a review of the literature was performed as well as an experiment designed to generate data on dynamic CO2 assimilation for different photosynthetic mutants of Arabidopsis thaliana. In addition to providing these models to the research community, this dissertation also identifies multiple targets that may be used for improving dynamic CO2 assimilation in plants. It further demonstrates that the dynamic responses of CO2 assimilation to changes in irradiance has a significant effect on canopy CO2 assimilation, even for dense canopies exposed to open skies, resembling the conditions of commercial crops.

In Chapter 1, the context of this dissertation is presented. The societal relevance of this research is argued, making reference to the role that photosynthesis could play in addressing global problems such as food and energy security. The necessary background on the physiology of photosynthesis is provided, with special emphasis on the terminology and concepts required to understand the rest of the dissertation, with the aim of making the contents more accessible to a wider audience. Then, prior literature on the specific topics of this dissertation (i.e., photosynthesis in a dynamic environment and its mathematical modelling) is presented, with a chronological approach that analyses the evolution of ideas and methodologies up to the present.

In Chapter 2, the current literature on dynamic CO2 assimilation is reviewed, with an emphasis on the effects of environmental conditions ([CO2], temperature, and air humidity) on the rates of photosynthetic induction and loss of induction. This review reveals major knowledge gaps, especially on the loss of induction. The little data available indicates that rates of photosynthetic induction increase with [CO2], which could be explained by a weak effect on Rubisco activation and a strong effect on stomatal opening. Increases in temperature also increase the rates of photosynthetic induction, up to an optimum, beyond which a strong negative effect can be observed, which could be attributed to deactivation of Rubisco activase.

In Chapter 3, an experiment is presented that makes use of several photosynthetic mutants of A. thaliana. Downregulating non-photochemical quenching and sucrose synthesis did not have any significant effect on dynamic CO2 assimilation, whereas CO2 diffusion and Rubisco activation exerted stronger limitations. Further analysis reveals that whether stomatal opening limits CO2 assimilation after an increase in irradiance depends on the stomatal conductance prior to the change in irradiance. A threshold value of 0.12 mol m−2 s−1 (defined for fluxes of water vapour) could be defined, above which stomata did not affect the rates of photosynthetic induction. The comparison of measurements across irradiance levels also indicated that the apparent rate constant of Rubisco activation is irradiance-dependent, at least for irradiance levels below 150 μmol m−2 s−1.

In Chapter 4, a phenomenological model of leaf-level CO2 assimilation is presented. The model is described in detail and all the parameters are first estimated with published data, and later refined by fitting the model to the data from Chapter 3. Additional data from the experiment in Chapter 3 is used to validate predictions of CO2 assimilation under lightflecks for the different photosynthetic mutants. The model predicts accurately dynamic CO2 assimilation for the different photosynthetic mutants by only modifying those parameters that are affected by the mutation. This demonstrates that the model has a high predictive power and that the equations, although phenomenological in nature, have a solid physiological basis.

The model is further used to analyse, in silico, the limitations imposed by different photosynthetic processes on dynamic CO2 assimilation at the leaf and canopy level, allowing a more in depth analysis than in Chapter 3. The analysis demonstrates that results obtained at the leaf level should not be extrapolated directly to the canopy level, as the spatial and temporal distribution of irradiance within a canopy is more complex than what is achieved in experimental protocols. Both at the leaf and canopy level, CO2 diffusion is strongly limiting, followed by photoinhibition, chloroplast movements and Rubisco activation.

In Chapter 5, a mechanistic model of the dynamic, metabolic regulation of the electron transport chain is presented. The model is described in detail and all the parameters are estimated from published literature, using measurements on A. thaliana when available. Predictions of the model are tested with steady-state and dynamic measurements of gas exchange, chlorophyll fluorescence and absorbance spectroscopy on A. thaliana, with success.

The analysis in silico indicates that a significant amount of alternative electron transport is required to couple ATP and NADPH production and demand, and most of it is associated with nitrogen assimilation and export of redox power through the malate shuttle. The analysis also reveals that the relationship between ATP synthesis and the proton motive force is highly regulated by the concentrations of substrates (ADP, ATP and inorganic phosphate), and this regulation facilitates an increase in non-photochemical quenching under conditions of low metabolic activity in the stroma.

In Chapter 6, the findings of Chapters 2–5 are summarised and employed to answer in detail the four research questions formulated in Chapter 1. Of great interest is the identification of six potential targets that may be used to improve dynamic CO2 assimilation. These targets are: (i) regulation of Rubisco activity through changes in the amount or regulation of Rubisco activase, (ii) acceleration of stomatal opening and closure, (iii) a lower /ATP for ATP synthesis, (iv) faster relaxation of non-photochemical quenching, (v) reduced chloroplast movements, and (vi) reduced photoinhibition by increased rates of repair of Photosystem II.

A maternal Western diet during gestation and lactation modifies offspring’s microbiota activity, blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs
Val-Laillet, David ; Besson, M. ; Guérin, S. ; Coquery, N. ; Randuineau, G. ; Kanzari, A. ; Quesnel, H. ; Bonhomme, N. ; Bolhuis, J.E. ; Kemp, B. ; Blat, S. ; Huërou-Luron, I. Le; Clouard, C.M. - \ 2017
FASEB Journal 31 (2017)5. - ISSN 0892-6638 - p. 2037 - 2049.
early nutrition - development - eating behavior - metabolism - brain
A suboptimal early nutritional environment (i.e., excess of energy, sugar, and fat intake) can increase susceptibility to diseases and neurocognitive disorders. The purpose of this study was to investigate in nonobese Yucatan minipigs (Sus scrofa) the impact of maternal diet [standard (SD) vs. Western (WD) diet] during gestation and 25 d of lactation on milk composition, blood metabolism, and microbiota activity of sows (n = 17) and their piglets (n = 65), and on spatial cognition (n = 51), hippocampal plasticity (n = 17), and food preferences/motivation (n = 51) in the progeny. Milk dry matter and lipid content, as well as plasma total cholesterol and free fatty acid (FFA) concentrations (P < 0.05) were higher in WD than in SD sows. Microbiota activity decreased in both WD sows and 100-d-old piglets (P < 0.05 or P < 0.10, depending on short-chain FAs [SCFAs]). At weaning [postnatal day (PND) 25], WD piglets had increased blood triglyceride and FFA levels (P < 0.01). Both SD and WD piglets consumed more of a known SD than an unknown high-fat/-sucrose (HFS) diet (P < 0.0001), but were quicker to obtain HFS rewards compared with SD rewards (P < 0.01). WD piglets had higher working memory (P = 0.015) and reference memory (P < 0.001) scores, which may reflect better cognitive abilities in the task context and a higher motivation for the food rewards. WD piglets had a smaller hippocampal granular cell layer (P = 0.03) and decreased neurogenesis (P < 0.005), but increased cell proliferation (P < 0.001). A maternal WD during gestation and lactation, even in the absence of obesity, has significant consequences for piglets’ blood lipid levels, microbiota activity, gut–brain axis, and neurocognitive abilities after weaning.—Val-Laillet, D., Besson, M., Guérin, S., Coquery, N., Randuineau, G., Kanzari, A., Quesnel, H., Bonhomme, N., Bolhuis, J. E., Kemp, B., Blat, S., Le Huërou-Luron, I., Clouard, C. A maternal Western diet during gestation and lactation modifies offspring’s microbiota activity, blood lipid levels, cognitive responses, and hippocampal neurogenesis in Yucatan pigs.
Shortening or omitting the dry period in dairy cows : effects on milk yield, energy balance, metabolic status, and fertility
Chen, Juncai - \ 2016
University. Promotor(en): Bas Kemp, co-promotor(en): Ariette van Knegsel. - Wageningen : Wageningen University - ISBN 9789462579088 - 205
dairy cows - dairy cattle - dry period - milk yield - energy balance - metabolism - lactation - melkkoeien - melkvee - gustperiode - melkopbrengst - energiebalans - metabolisme - lactatie

During early lactation, dairy cows typically experience negative energy balance (EB) caused by the high energy requirement for milk yield, which cannot be met by feed intake. Severity of negative EB has been associated with an increased incidence of metabolic disorders and infectious diseases, subfertility, and increased culling rates. Shortening or omitting the dry period (DP) and feeding glucogenic diet could possibly improve EB in dairy cows. The objective of this thesis was to study the effects of shortening or omitting the DP on milk yield, energy balance, metabolism, and fertility over two subsequent lactations in dairy cows fed a lipogenic or glucogenic diet during early lactation. In the current study, 167 cows were assigned to three DP lengths (0, 30, or 60 days) and two early lactation diets (glucogenic or lipogenic diet), and cows were planned to have same DP length and diet over two subsequent lactations. In the first lactation after DP length and dietary treatments, shortening or omitting the DP improved EB due to a decreased milk yield in the early lactation compared with a conventional DP of 60 days. Omitting the DP or feeding a glucogenic diet improved metabolic status in early lactation. Moreover, omitting the DP increased the percentage of cows with normal resumption of ovarian cyclicity. Shortening the DP to 30 d did not influence metabolic status and fertility compared with conventional DP in dairy cows. In the first lactation, the cows with a 0-d DP had less milk but similar energy intake, leading to excessive weight gain and, therefore, high body condition score (BCS) at onset of the second lactation after DP length and dietary treatments. In the second lactation, improvement of EB in cows with a 0-d DP was less pronounced than the first lactation, which could be related to the high BCS at onset of lactation and reduced milk yield losses. Shortening or omitting the DP did not influence uterine health status, ovarian activity, and reproductive performance in the second lactation. In second lactation, feeding a glucogenic diet improved metabolic status and shortened the interval from calving to first ovulation compared with a lipogenic diet without affecting EB independent of DP length. Furthermore, shortening or omitting the DP decreased peak yield but did not influence lactation persistency in both lactations after implementation of DP treatment. In conclusion, omitting the DP improved metabolic status and resumption of ovarian activity, which was related to an improved EB in early lactation. Shortening the DP for two subsequent lactations could be achieved for most cows with limited milk yield losses. Independent of DP length, glucogenic diet improved EB and metabolic status compared with lipogenic diet in early lactation.

Biotransformation and bioactivation reactions – 2015 literature highlights
Baillie, Thomas A. ; Dalvie, Deepak ; Rietjens, Ivonne M.C.M. ; Cyrus Khojasteh, S. - \ 2016
Drug Metabolism Reviews 48 (2016)2. - ISSN 0360-2532 - p. 113 - 138.
2015 review - Bioactivation - biotransformation - literature highlights - metabolism

Since 1972, Drug Metabolism Reviews has been recognized as one of the principal resources for researchers in pharmacological, pharmaceutical and toxicological fields to keep abreast of advances in drug metabolism science in academia and the pharmaceutical industry. With a distinguished list of authors and editors, the journal covers topics ranging from relatively mature fields, such as cytochrome P450 enzymes, to a variety of emerging fields. We hope to continue this tradition with the current compendium of mini-reviews that highlight novel biotransformation processes that were published during the past year. Each review begins with a summary of the article followed by our comments on novel aspects of the research and their biological implications. This collection of highlights is not intended to be exhaustive, but rather to be illustrative of recent research that provides new insights or approaches that advance the field of drug metabolism. AbbreviationsNAPQIN-acetyl-p-benzoquinoneimineALDH aldehyde dehydrogenaseAO aldehyde oxidaseAKR aldo-keto reductaseCES carboxylesteraseCSB cystathionine β-synthaseCSE cystathionine γ-lyaseP450 cytochrome P450DHPO 2,3-dihydropyridin-4-oneESI electrosprayFMO flavin monooxygenaseGSH glutathioneGSSG glutathione disulfideICPMS inductively coupled plasma mass spectrometryi.p. intraperitonealMDR multidrug-resistantNNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanolNNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanoneoaTOF orthogonal acceleration time-of-flightPBK physiologically based kineticPCP pentachlorophenolSDR short-chain dehydrogenase/reductaseSULT sulfotransferaseTB tuberculosis

Are all eggs equal? : embryonic development and nutrient metabolism in chicken eggs of different origins
Nangsuay, A. - \ 2016
University. Promotor(en): Bas Kemp, co-promotor(en): Henry van den Brand; R. Meijerhof. - Wageningen : Wageningen University - ISBN 9789462577749 - 213 p.
eggs - hens - broilers - characteristics - strains - embryonic development - nutrients - metabolism - hatcheries - poultry - nutrition physiology - eieren - hennen - vleeskuikens - karakteristieken - stammen (biologisch) - embryonale ontwikkeling - voedingsstoffen - metabolisme - broedinstallaties - pluimvee - voedingsfysiologie

Hatching eggs, supplied to hatcheries are originating from different origins varying in breed, strain, and breeder age. These hatching eggs can be different in size, composition and eggshell properties, which might influence nutrient and O2 availability and consequently could affect embryonic development and nutrient metabolism. The aim of this thesis was therefore 1) to investigate effects of egg origin on nutrient and O2 availability, 2) to investigate effects of egg origins on nutrient metabolism and embryonic development and 3) to investigate consequences of different egg origins on the incubation process and hatching characteristics. In five studies, effects of different egg origins on nutrient and O2 availability, nutrient metabolism, embryo development and hatching characteristics were investigated. The first and second study focused on breeder age and egg size. The third study on breed; broilers and layers. The fourth study on broiler strain and the fifth study on breeder age, strain and eggshell temperature (EST). The overall findings in this thesis suggest that hatching eggs from different origins are not equal in availability of nutrients and O2. Nutrient availability is altered through variation in yolk size, especially by the effects of breeder age and breed. O2 availability is altered by differences in eggshell properties, which is influenced by especially breed and broiler strain. The availability of both nutrients and O2 plays a role on nutrient metabolism measured as embryonic heat production (HP) and consequently on embryonic development. Between incubation day (E) E7 and E14, both nutrient and O2 availability might affect nutrient metabolism as shown in the results of the broiler and layer comparison. Between E14 and hatching, the availability of O2 becomes the most determinant factor for nutrient metabolism and consequently for embryonic development. An increase in EST from 37.8 to 38.9°C from E7 onward resulted in an acceleration of nutrient metabolism and embryonic development until E16, but thereafter a high EST resulted in reduced yolk free body mass development. Embryos with an accelerated metabolic speed at an early stage of incubation, caused by an increased EST, might reach limited O2 availability at a higher magnitude than the embryos at a normal EST. As a result, nutrient metabolism is restricted and embryonic development is depressed. It can be concluded that not only the HP, but also the availability of O2 is crucial to be taken into account for developing incubator temperature. The principle is to obtain an optimal EST, which could maintain the balance between O2 requirement (driven by nutrient metabolism) and O2 availability for a continuing optimal nutrient metabolism to generate sufficient energy for embryonic development throughout incubation.

Metabolic engineering of biosynthesis and sequestration of artemisinin
Wang, B. - \ 2016
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789462576728 - 210 p.
artemisinin - nicotiana benthamiana - arabidopsis - biosynthesis - malaria - drugs - genetic engineering - metabolism - artemisinine - biosynthese - geneesmiddelen - genetische modificatie - metabolisme

The sesquiterpenoid artemisinin (AN) is the most important medicine for the treatment of malaria in humans. The industrial production of AN still mainly depends on extraction from the plant Artemisia annua. However, the concentration of AN in A. annua is low. Although different engineering strategies have been used in both A. annua and heterologous plant and yeast production platforms, the worldwide capacity and production costs for AN are not in balance with its demand (Chapter 1). Although the genes encoding for the entire AN biosynthesis pathway (AN-PW) of the AN precursor dihydroartemisinic acid (DHAA) have been identified, the application of these genes in pathway engineering seem to be limited by lack of control over product transport and sequestration. At the onset of this thesis project there was no information on transport in the AN-PW. However, it was known that DHAA is converted into AN outside the glandular trichome cells of A. annua. Therefore, in this thesis I tried to gain more knowledge on transport within the AN-PW and the use of different metabolic engineering strategies to improve the production of AN.

At the onset of my PhD project, the AN-PW genes from two different A. annua chemotypes were compared to understand the basis of different relative activities in the two branches of the AN-PW (Chapter 2). For these assays we used transient expression in N. benthamiana. In the AN-PW, artemisinic aldehyde (AAA) is at a branch point as it can be converted to artemisinic acid (AA) by amorphadiene oxidase (AMO), or to dehydroartemisinic aldehyde (DHAAA) by artemisinic aldehyde Δ11 (13) reductase (DBR2). AA is the precursor for arteannuin B (AB) while DHAAA may be converted by a CYP71AV1 or an ALDH1 to dehydroartemisinic acid (DHAA), the precursor for AN. In this chapter we demonstrate that the CYP71AV1 from a high AN production (HAP) chemotype has reduced activity in the AB branch of the pathway compared to the CYP71AV1 from a low AN production (LAP) chemotype. In addition, we show that the relative expression levels of DBR2 and ALDH1 also affect the AN/AB chemotype. The low catalytic efficiency of AMO from the HAP chemotype may be caused by a deletion of seven amino acids at the N-terminus of the protein compared to CYP71AV1 from LAP. Ectopic expression of the AN-PW genes in N. benthamiana showed that the bulk of the PW products are modified by glycosylation and glutathione conjugations. These side reactions therefore compete with the biosynthesis flux towards the AN precursor DHAA. At this point in my thesis the ectopic expression of AN-PW genes in N. benthamiana had not yielded any AN. At a later stage it became clear that this was due to harvest of leaves at 5-7 days post agro-infiltration (dpi), while AN in N. benthamiana leaves expressing AN-PW genes only becomes detectable after 7 dpi.

Glycosylation of the bulk of the AN-PW products in N. benthamiana stresses the need for an efficient transport of (DH)AA to the outside of cells in order to escape from the glycosylation reactions. In Chapter 3, transport and sequestration of AN precursors was investigated by studying the effect of membrane transporters (PDRs) and lipid transfer proteins (LTPs). Hereto, two membrane transporters with activity towards AN-PW products were made available by the group of Prof. Marc Boutry and we isolated three LTP genes from Artemisia annua which showed expression in the glandular trichomes. In this chapter we show that AaLTP3 displays specific activity, together with AaPDR2 towards transport of (DH)AA to the apoplast in N. benthamiana. Moreover, infiltration experiments with (DH)AA in N. benthamiana leaves revealed that these compounds are rapidly taken up by the cells and that inside the cells there is a strong reverse flux in the AN-PW by conversion of (DH)AA towards (DH)AAA and (DH)AAOH. Subsequently we demonstrated that AaLTP3 has a stronger activity in keeping products in the apoplast than the AaPDR2 membrane transporter. Therefore, I suggest that by removal of (DH)AA from the cytosol through transport over the plasma membrane by AaPDR2 and subsequent sequestration in the apoplast by AaLTP3, AaLTP3 creates sink activity which prevents reflux of (DH)AA from the apoplast back into the cells. AaLTP3 therefore contributes to a directional flux through the AN-PW towards the end product (DH)AA. Finally, in this work we could also for the first time detect AN and AB in N. benthamiana leaves by extraction of necrotic leaves at 13 dpi.

Because in A. annua glandular trichome cells both the AN sesquiterpene biosynthesis pathway and the flavonoid biosynthesis pathway are active, we explored whether there is a functional interaction between these two major secondary metabolite biosynthesis pathways. In Chapter 4 we describe how we manipulate the flavonoid biosynthesis pathway in N. benthamiana leaves using the Antirhinum majus transcription factor Rosea1 (ROS) and test coexpression of ROS with AN-PW genes. The co-expression of ROS stimulates AN-PW product accumulation. Subsequent analysis indicates that this is most likely from transcriptional activation of the enzyme Mevalonate Kinase (MVK) in the mevalonate pathway, which provides precursors for the sesquiterpene biosynthesis pathway. In addition, we demonstrate that production of flavonoids competes with AN-PW product accumulation, as co-expression of AN-PW genes with ROS, but simultaneous inhibition of chalcone synthase (CHS) by a CHSRNAi construct, results in higher AN-PW product levels. However, accumulation of the end products AN and AB was not affected significantly. Finally, the combined expression of AN-PW+ROS+AaPDR2+AaLTP3+ CHSRNAi results in highest sequestration of (DH)AA in the apoplast and highest accumulation of the end products AN and AB in N. benthamiana.

During my thesis work, in a related project it was found that expression of another sesquiterpene biosynthesis gene (caryophyllene synthase; CST) in transgenic Arabidopsis resulted in higher caryophyllene emission for a transformant expressing a genomic DNA of CST, compared with a similar transformant expressing a CST cDNA described in literature. This suggested that ectopic expression of intron containing genes is more efficient than ectopic expression of cDNAs. To test whether in the context of metabolic engineering the use of genomic (intron-containing) genes is more efficient than the use of the corresponding cDNA we generated a set of stable transformed Arabidopsis lines with either genomic CST (gCST), cDNA CST (cCST), genomic amorphadiene synthesis (gADS) and cDNA ADS (cADS). In chapter 5 we show that indeed the lines with overexpression of the genomic clones yield higher levels of the anticipated products (caryophyllene or amorphadiene) than the lines with overexpression of the corresponding cDNAs. Transcript analysis showed that for gCST the increase in caryophyllene production was higher than can be explained solely by the increase in CST transcription. In the context of transient expression in N. benthamiana leaves the intron-mediated-enhancement effect was less pronounced.

In the final discussion chapter 6 I review limitations and potential solutions to metabolic engineering of the AN-PW in plants, and I discuss the impact of our findings on AN production capacity using transient expression versus natural production in A. annua. Moreover, I discuss how the finding of this thesis go beyond just insights into the AN-PW as especially the identification of the role of LTPs in sesquestration of (sesqui)terpenes into the apoplast may have an impact on the metabolic engineering efforts of many other (sesqui)terpene pathways. Because some plant hormones are also terpenoid products the newly identified role of LTPs may also have impact on a deeper understanding of hormone signalling in plants. I have already started exploring this path by generating a set of Arabidopsis plants with overexpression of different Arabidopsis LTP genes to test whether any hormone related traits are altered (Chapter 6). Preliminary results do indeed confirm a role of LTPs in endogenous plant hormone balance, something worthwhile to be further explored in future research.

Replacing lactose from calf milk replacers : effects on digestion and post-absorptive metabolism
Gilbert, M.S. - \ 2015
University. Promotor(en): Wouter Hendriks, co-promotor(en): Walter Gerrits; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576032 - 171
vleeskalveren - lactose - kunstmelk - polysacchariden - glucose - fructose - glycerol - zetmeelvertering - metabolisme - fermentatie - kalvervoeding - diervoeding - voedingsfysiologie - veal calves - filled milk - polysaccharides - starch digestion - metabolism - fermentation - calf feeding - animal nutrition - nutrition physiology

Summary PhD thesis Myrthe S. Gilbert

Replacing lactose from calf milk replacers – Effects on digestion and post-absorptive metabolism

Veal calves are fed milk replacer (MR) and solid feed. The largest part of the energy provided to veal calves originates from the MR. Calf MR contains 40 to 50% lactose, originating from whey, a by-product from cheese production. High and strongly fluctuating dairy prices are a major economic incentive to replace lactose from the calf MR by alternative energy sources. The objective of this thesis was to study the effects of replacing lactose from calf MR on nutrient digestion and fermentation and post-absorptive metabolism.

In Chapter 2 and 3, four starch products (SP) were evaluated for replacing lactose. The four SP differed in size and branching, and consequently required different ratios of starch-degrading enzymes for their complete hydrolysis to glucose. Gelatinized starch required α-amylase and (iso)maltase; maltodextrin required (iso)maltase and α-amylase; maltodextrin with α-1,6-branching required isomaltase, maltase and α-amylase and maltose required maltase. In Chapter 2, adaptation to these SP was assessed during 14 weeks, using a within-animal titration study. Forty male Holstein-Friesian calves (n = 8 per treatment) were assigned to either a lactose control MR or one of four titration strategies, each testing the stepwise exchange of lactose for one of the SP. For control calves, fecal dry matter (DM) content and fecal pH did not change over time. The response in fecal DM content and fecal pH in time did not differ between SP treatments and decreased linearly with 0.57% and 0.32 per week, respectively, where one week corresponded to an increase in SP inclusion of 3%. This indicates that the capacity for starch digestion was already exceeded at low inclusion levels, resulting in SP fermentation. All SP required maltase to achieve complete hydrolysis to glucose and it was, therefore, suggested that maltase is the rate-limiting enzyme in starch digestion in milk-fed calves.

Following the titration, a fixed inclusion level of 18% of the SP in the MR was applied. Effects on starch-degrading enzyme activity, nutrient disappearance, SP fermentation and jugular glucose appearance were measured (Chapter 3). Lactase activity in the brush border was high in the proximal small intestine of all calves, resulting in a high apparent ileal disappearance of lactose (≥ 99% of intake). Maltase and isomaltase activities in the brush border were not increased for any of the SP treatments. Luminal α-amylase activity was lower in the proximal small intestine but greater in the distal small intestine of SP-fed calves compared to control calves. This amylase activity in the distal small intestine of SP-fed calves might have been of microbial origin. Apparent SP disappearance did not differ between SP treatments. The difference between apparent ileal (62%) and total tract (99%) SP disappearance indicated substantial SP fermentation in the large intestine (37% of intake). In addition, total tract SP fermentation was quantified using fecal 13C excretion which originated from the naturally 13C-enriched corn SP. Total tract SP fermentation averaged 89% of intake, regardless of SP treatment. MR leaking into the reticulorumen was measured as the recovery of Cr in the reticulorumen at slaughter after feeding MR pulse-dosed with Cr 4h prior to slaughter. MR leaking into the reticulorumen averaged 11% for SP-fed calves. By difference, this leaves 41% of the SP intake fermented in the small intestine. This coincided with increased fecal nitrogen (N) and DM losses for SP-fed calves. However, apparent total tract crude fat disappearance tended to increase when replacing lactose with SP. The substantial SP fermentation indicates that only 10% of the SP intake was enzymatically hydrolyzed and absorbed as glucose. This was in agreement with the marginal increase in 13C enrichment in peripheral plasma glucose after feeding naturally 13C-enriched gelatinized starch and maltose, compared to a clear increase after feeding naturally 13C-enriched lactose to control calves. It was concluded that fermentation, rather than enzymatic digestion, is the main reason for small intestinal starch disappearance in milk-fed calves. The expected decrease in growth performance with such extensive SP fermentation is partially compensated by the greater crude fat digestion and possibly by a reduced urinary glucose excretion when replacing lactose with SP.

Glucose, fructose and glycerol do not require enzymatic hydrolysis and can be absorbed directly from the small intestine. However, these lactose replacers might differentially affect glucose and insulin metabolism and with that energy partitioning. The effects of partly replacing lactose with glucose, fructose or glycerol on energy and N partitioning and glucose homeostasis and insulin sensitivity were, therefore, studied in Chapter 4 and 5. Forty male Holstein-Friesian calves either received a lactose control MR or a MR in which one third of the lactose was replaced with glucose, fructose or glycerol (n = 10 per treatment). Energy and N retention were not affected by MR composition. Fructose absorption from the small intestine was incomplete resulting in fructose fermentation. This resulted in fecal losses of DM, energy and N and the lowest numerical energy and N retention for fructose-fed calves. Postprandial plasma concentrations of glucose exceeded the renal threshold for glucose in glucose-fed calves and control calves, which resulted in urinary glucose excretion. Glycerol was likely excreted with the urine of glycerol-fed calves. Oxidation of glucose, fructose and glycerol was quantified by feeding a single dose of [U-13C]glucose, [U-13C]fructose or [U-13C]glycerol with the MR and subsequently measuring 13CO2 production. Oxidation of lactose replacers did not differ between lactose replacers and averaged 72% of intake. However, the time at which the maximum rate of oxidation was reached was delayed for fructose-fed compared to glucose-fed and glycerol-fed calves, indicating that fructose was converted into other substrates before being oxidized. Conversion of fructose and glycerol into glucose was confirmed by an increase in 13C enrichment of peripheral plasma glucose after feeding [U-13C]fructose and [U-13C]glycerol, respectively. Insulin sensitivity did not differ between MR treatments, but was already low at the start of the experiment at 15 weeks of age and remained low throughout the experiment. It was concluded that glucose and glycerol can replace one third of the lactose from the calf MR, but that inclusion of fructose should be lower to prevent incomplete absorption from the small intestine.

In literature and the studies in this thesis, high inter-individual variation in growth performance was found in veal calves. The experiment described in Chapter 6 was, therefore, designed to assess the predictability of later life growth performance by charactering calves in early life. In addition, it was examined whether the ability of calves to cope with MR in which lactose is partially replaced by alternative energy sources can be predicted. From 2 to 11 weeks of age, male Holstein-Friesian calves were fed a lactose control MR and solid feed according to a practical feeding scheme and were characterized individually using targeted challenges related to feeding motivation, digestion, post-absorptive metabolism, immunology, behavior and stress. Based on the results in Chapter 4, a combination of glucose, fructose and glycerol in a 2:1:2 ratio was used to replace half of the lactose from the MR (GFG). From 11 to 27 weeks of age, calves received a lactose control MR or the GFG MR (n = 65 per treatment). Growth performance from 11 to 27 weeks of age tended to be lower for GFG-fed than for control calves (-25 g/d). Measurements in early life explained 12% of the variation in growth performance in later life. However, this was mainly related to variation in solid feed refusals. When growth performance was adjusted to equal solid feed intake, only 4% of the variation in standardized growth performance in later life, reflecting feed efficiency, could be explained by early life measurements. This indicates that > 95% of the variation in feed efficiency in later life could not be explained by early life characterization. It is hypothesized that variation in health status explains substantial variation in feed efficiency in veal calves. Significant relations between fasting plasma glucose concentrations, fecal dry matter and fecal pH in early life and feed efficiency in later life depended on MR composition. These measurements are, therefore, potential tools for screening calves in early life on their ability to cope with a MR in which half of the lactose is replaced by glucose, fructose and glycerol (in a 2:1:2 ratio).

The studies reported in this thesis demonstrate that glycerol, glucose and a combination of glucose, fructose and glycerol in a 2:1:2 ratio are promising lactose replacers. The effects of replacing lactose by other carbohydrate or energy sources described in this thesis are required to evaluate the potential of lactose replacers for inclusion in calf milk replacers and provide input for feed evaluation for calves and ruminants.

Phosphorus metabolism in dairy cattle : literature study on recent developments and gaps in knowledge
Goselink, R.M.A. ; Klop, G. ; Dijkstra, J. ; Bannink, A. - \ 2015
Wageningen : Wageningen UR Livestock Research (Livestock Research rapport 910) - 36
dairy cattle - phosphorus - nutrient requirements - animal nutrition - metabolism - melkvee - fosfor - voedingsstoffenbehoeften - diervoeding - metabolisme
Goal of this literature study is to define the uncertainties related to P requirement models and ways to make them more precise and more reliable than current systems and to find biomarkers to monitor P balance in living animals. Milk P is an important factor in P balance, and concentration may vary between individual cows. Bone represents a large P reserve and can have a profound impact on cow P balance and plasma P regulation. Plasma P concentration is still the most commonly used diagnostic measurement available to judge the P status of an animal, with all restrictions for good interpretation; new biomarkers defining P status are needed. Further research into mechanisms and quantification of the is needed to improve our understanding of P metabolism.
Evaluation of the interindividual human variation in bioactivation of methyleugenol using physiologically based kinetic modeling and Monte Carlo simulations
Al-Subeihi, A.A. ; Alhusainy, W. ; Kiwamoto, R. ; Spenkelink, A. ; Bladeren, P.J. van; Rietjens, I.M.C.M. ; Punt, A. - \ 2015
Toxicology and Applied Pharmacology 283 (2015)2. - ISSN 0041-008X - p. 117 - 126.
human liver-microsomes - in-vitro data - alkenylbenzene methyleugenol - methyl eugenol - rat - glucuronidation - estragole - detoxification - derivatives - metabolism
The present study aims at predicting the level of formation of the ultimate carcinogenic metabolite of methyleugenol, 1'-sulfooxymethyleugenol, in the human population by taking variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Depending on the metabolic route, variation was simulated based on kinetic constants obtained from incubations with a range of individual human liver fractions or by combining kinetic constants obtained for specific isoenzymes with literature reported human variation in the activity of these enzymes. The results of the study indicate that formation of 1'-sulfooxymethyleugenol is predominantly affected by variation in i) P450 1A2-catalyzed bioactivation of methyleugenol to 1'-hydroxymethyleugenol, ii) P450 2B6-catalyzed epoxidation of methyleugenol, iii) the apparent kinetic constants for oxidation of 1'-hydroxymethyleugenol, and iv) the apparent kinetic constants for sulfation of 1'-hydroxymethyleugenol. Based on the Monte Carlo simulations a so-called chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived by dividing different percentiles by the 50th percentile of the predicted population distribution for 1'-sulfooxymethyleugenol formation. The obtained CSAF value at the 90th percentile was 3.2, indicating that the default uncertainty factor of 3.16 for human variability in kinetics may adequately cover the variation within 90% of the population. Covering 99% of the population requires a larger uncertainty factor of 6.4. In conclusion, the results showed that adequate predictions on interindividual human variation can be made with Monte Carlo-based PBK modeling. For methyleugenol this variation was observed to be in line with the default variation generally assumed in risk assessment.
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