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Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual el

Cichorium intybus var. sativum genome sequencing, Cichorium intybus var. sativum genome sequencing

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus causing a respiratory disease that emerged in 2019, leading to a public health emergency situation which continues to date. The treatment options are still very limited and vaccines available are less effective against new variants. SARS-CoV-2 enzymes, namely main protease (Mpro) and papain-like protease (PLpro), play a pivotal role in the viral life cycle, making them a putative drug target. Here, we described for the first time the potential inhibitory activity of chicory extract against both proteases. Besides, we have identified that the four most abundant sesquiterpene lactones in chicory inhibited these proteases, showing an effective binding in the active sites of Mpro and PLpro. This paper provides new insight for further drug development or food-based strategies for the prevention of SARS-CoV-2 by targeting viral proteases.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Een chirurgische resectie van een slokdarmcarcinoom is nog steeds de belangrijkste curatieve behandelmodaliteit. Deze gaat echter gepaard met een aanzienlijk risico op complicaties. Het doel van dit proefschrift was om risicofactoren te identificeren die gerelateerd zijn aan morbiditeit en mortaliteit gedurende de behandeling van een patiënt met een slokdarmcarcinoom. Om een correcte inschatting te maken van het individuele perioperatieve risico, zijn verschillende risico-inschattende modellen ontwikkeld. Uit dit proefschrift blijkt dat O-POSSUM een relatief correcte voorspelling geeft, maar nog steeds ongeschikt is voor individuele risico-inschatting in de dagelijkse praktijk. Een mogelijk risicovolle groep wordt gevormd door de oudere patiënten (> 70 jaar). Uit dit proefschrift blijkt echter dat er geen significant verschil bestaat in korte –en lange termijn overleving tussen oudere en jongere patiënten. Wel kwamen enkele complicaties vaker voor bij oudere patiënten. Neoadjuvante chemoradiotherapie (CRT) wordt tegenwoordig standaard toegepast in de behandeling van patiënten met een slokdarmcarcinoom. Ondanks laag gerapporteerde toxiciteit, toont dit proefschrift aan dat neoadjuvante CRT een verhoogd risico geeft op preoperatieve trombo-embolische processen. Tevens werden er meer postoperatieve longontstekingen en hartritmestoornissen geconstateerd na neoadjuvante CRT. Laboratorium waarden zijn een belangrijk hulpmiddel in de vroege identificatie van patiënten met een gecompliceerd postoperatief beloop. Dit proefschrift toont aan dat zowel bekende waarden (C-reactief protein en lactaat dehydrogenase), als minder bekende waarden (intestinal fatty acid binding protein en angiopoietin) een belangrijke rol spelen in de vroege identificatie van postoperatieve complicaties. Daarnaast zijn er aanwijzingen dat cytokinen concentraties gerelateerd zijn aan de mate van pathologische respons na neoadjuvante CRT. Mogelijk spelen ze ook een rol bij geïndiv, Surgical resection of the esophagus remains the main curative treatment modality in patients with esophageal cancer, but is accompanied by a significant risk of complications. The aim of this thesis was to identify risk-factors for perioperative morbidity and mortality in patients with esophageal cancer. In order to assess perioperative risk, several risk-prediction models have been developed. In this thesis we concluded that postoperative mortality after esophagectomy was best predicted by O-POSSUM, which was still unsuitable in daily practice. In general it is assumed that elderly patients (> 70 years) are at an increased risk for perioperative complications. However, we could not demonstrate significant differences in short -or long-term survival between older and younger patients. Nevertheless, complications were frequently observed in elderly patients. Neoadjuvant chemoradiotherapy (CRT) is nowadays standard treatment of care in patients with esophageal cancer. Despite low reported toxicity, this thesis shows that neoadjuvant CRT is associated with an increased risk of preoperative thromboembolic processes. In addition, we observed more postoperative pneumonia and cardiac arrhythmias in patients treated with neoadjuvant CRT. Laboratory values are an important tool in early identification of patients with a complicated postoperative course. This thesis shows that well known values (C- reactive protein and lactate dehydrogenase), but also less known values (intestinal fatty acid binding protein and angiopoietin) are both important in early identification of postoperative complications. Furthermore, there are indications that cytokine concentrations are related to the degree of pathologic response after neoadjuvant CRT, which could be used to individualize treatment strategy.

Deficiency of a-galactosidase A (a-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal a-N-acetyl-galactosaminidase (a-NAGAL) with increased a-galactosidase activity (a-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional a-GAL, a-NAGAL, and a-NAGALEL enzymes for research purposes. All enzymes could be visualized with activity-based probes covalently binding in their catalytic pocket. Characterization of purified proteins indicated that a-NAGALEL is improved in activity toward artificial 4MU-a-galactopyranoside. Recombinant a-NAGALEL and a-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than a-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of a-NAGALEL, and to a lesser extent that of a-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified a-NAGALEL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in Nicotiana benthamiana and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.

Een chirurgische resectie van een slokdarmcarcinoom is nog steeds de belangrijkste curatieve behandelmodaliteit. Deze gaat echter gepaard met een aanzienlijk risico op complicaties. Het doel van dit proefschrift was om risicofactoren te identificeren die gerelateerd zijn aan morbiditeit en mortaliteit gedurende de behandeling van een patiënt met een slokdarmcarcinoom. Om een correcte inschatting te maken van het individuele perioperatieve risico, zijn verschillende risico-inschattende modellen ontwikkeld. Uit dit proefschrift blijkt dat O-POSSUM een relatief correcte voorspelling geeft, maar nog steeds ongeschikt is voor individuele risico-inschatting in de dagelijkse praktijk. Een mogelijk risicovolle groep wordt gevormd door de oudere patiënten (> 70 jaar). Uit dit proefschrift blijkt echter dat er geen significant verschil bestaat in korte –en lange termijn overleving tussen oudere en jongere patiënten. Wel kwamen enkele complicaties vaker voor bij oudere patiënten. Neoadjuvante chemoradiotherapie (CRT) wordt tegenwoordig standaard toegepast in de behandeling van patiënten met een slokdarmcarcinoom. Ondanks laag gerapporteerde toxiciteit, toont dit proefschrift aan dat neoadjuvante CRT een verhoogd risico geeft op preoperatieve trombo-embolische processen. Tevens werden er meer postoperatieve longontstekingen en hartritmestoornissen geconstateerd na neoadjuvante CRT. Laboratorium waarden zijn een belangrijk hulpmiddel in de vroege identificatie van patiënten met een gecompliceerd postoperatief beloop. Dit proefschrift toont aan dat zowel bekende waarden (C-reactief protein en lactaat dehydrogenase), als minder bekende waarden (intestinal fatty acid binding protein en angiopoietin) een belangrijke rol spelen in de vroege identificatie van postoperatieve complicaties. Daarnaast zijn er aanwijzingen dat cytokinen concentraties gerelateerd zijn aan de mate van pathologische respons na neoadjuvante CRT. Mogelijk spelen ze ook een rol bij geïndiv, Surgical resection of the esophagus remains the main curative treatment modality in patients with esophageal cancer, but is accompanied by a significant risk of complications. The aim of this thesis was to identify risk-factors for perioperative morbidity and mortality in patients with esophageal cancer. In order to assess perioperative risk, several risk-prediction models have been developed. In this thesis we concluded that postoperative mortality after esophagectomy was best predicted by O-POSSUM, which was still unsuitable in daily practice. In general it is assumed that elderly patients (> 70 years) are at an increased risk for perioperative complications. However, we could not demonstrate significant differences in short -or long-term survival between older and younger patients. Nevertheless, complications were frequently observed in elderly patients. Neoadjuvant chemoradiotherapy (CRT) is nowadays standard treatment of care in patients with esophageal cancer. Despite low reported toxicity, this thesis shows that neoadjuvant CRT is associated with an increased risk of preoperative thromboembolic processes. In addition, we observed more postoperative pneumonia and cardiac arrhythmias in patients treated with neoadjuvant CRT. Laboratory values are an important tool in early identification of patients with a complicated postoperative course. This thesis shows that well known values (C- reactive protein and lactate dehydrogenase), but also less known values (intestinal fatty acid binding protein and angiopoietin) are both important in early identification of postoperative complications. Furthermore, there are indications that cytokine concentrations are related to the degree of pathologic response after neoadjuvant CRT, which could be used to individualize treatment strategy.

Gluten proteins from wheat have the unique property to interact with each other and form a network in dough preparation. In this gluten network gas bubbles can be retained that are produced by yeast, which is added during dough preparation. The result is a voluminous, viscoelastic dough for bread making. Besides these network forming properties, gluten proteins contain remarkably high amounts of the amino acids proline and glutamine. Therefore, these proteins can not be degraded completely in the human gastrointestinal tract and so called epitopes are generated in the small intestine after ingestion of gluten containing foods such as bread and cookies. Intolerance to gluten proteins can cause inflammation of the small intestine, which leads to villous atrophy (flatten mucosa) and malabsorption. This intolerance to gluten proteins is also called celiac disease. The with celiac disease occurring inflammation can lead to many symptoms such as malnutrition, weight loss, stomach pain, diarrhea, dermatitis and in the worst case lymphoma. In children, celiac disease can cause growth retardation. Gluten proteins from rye and barley, because of their similarity to gluten proteins from wheat and high proline and glutamine content, can also cause celiac disease. Celiac disease patients therefore have to avoid life-long all food products containing wheat, rye, and barley (gluten-free diet). Not only gluten protein intake but also genetic susceptibility is necessary to develop celiac disease. Therefore, relatives of celiac disease patients have a highly increased risk to develop celiac disease. About 0.52% of the Western population suffers from celiac disease of which 7097% is undiagnosed, and the prevalence of celiac disease is still increasing. The reason for this may be the consumption of many bread, cookie and pasta products, but also because wheat constituents, such as gluten and starch, are increasingly used as food additive in soups, sauces, sausage, candy, ice-cream, a

In this thesis we set out to increase our knowledge of N-glycosylation in plants with a dual aim: (1) to develop tools for general and cell-specific glycoproteomics by which differences in the glycoproteome of plants in different specified cell types or under different conditions can be studied and (2) to increase our general understanding of N-glycosylation by analysis of the biosynthesis pathway and dissection of N-glycan function in plants. (1) Identification of the glycoproteome from specific cell types during different physiological or developmental conditions provides valuable biological information. For the detection of glycoproteins with plant complex type glycans anti-HRP polyclonal serum is available. Using these antibodies differences were identified in the complex glycoproteome of leaf epidermal and mesophyll cells and it was demonstrated that these differences vary in other plant tissues. Cell specific tagging with complex glycans was accomplished by cell specific complementation of the cgl mutant, demonstrating the power of this technique to detect subtle differences within a tissue that are lost in whole tissue protein extracts of wild-type plants. With anti-HRP we were not able to purify and validate the complex glycoproteome as predicted by bioinformatics. For glycoproteomics, the availability of probes that interact with specific plant N-glycans would be very helpful for the isolation and subsequent analysis of specific sub-pools of glycoproteins. In addition, such probes can aid the analysis of glycan modification on target proteins. For this reason, we used two alternative approaches to isolate monoclonal antibodies that can recognise plant complex N-glycans, neither of which yielded a probe with the desired properties. Suggestions are given on how strategies to select specific plant N-glycan antibodies may be improved. (2) N-glycan synthesis and processing is performed by sequential activity of enzymes in the ER and Golgi. Two of these enzymes w

The central aim of this project was the total elimination of allergenic sequences from the wheat genome while retaining the industrial quality of the gluten proteins.

The experiments carried out in this study fill up a gap in our knowledge of the actions of MNU and ENU, as no systematic, morphological investigations of the short term effects of these compounds were carried out as yet. The numerous data available are derived almost exclusively from biochemical and radiochemical studies on one hand, and from tumourinduction experiments on the other. Zie: Summary

The experiments carried out in this study fill up a gap in our knowledge of the actions of MNU and ENU, as no systematic, morphological investigations of the short term effects of these compounds were carried out as yet. The numerous data available are derived almost exclusively from biochemical and radiochemical studies on one hand, and from tumourinduction experiments on the other. Zie: Summary