Characterization and engineering of epimerases for the production of rare sugars

Tagatose is a rare sugar that can be applied for multiple reasons in different industries, for instance as a low-caloric sweetener in dietary food, as well as additive in detergents, cosmetics, and pharmaceutical formulations, but also as drug molecule itself in diabetes treatment. As for other rare sugars, tagatose is not abundantly present in nature and therefore it has to be made from more available sugars in order to use it. It is currently being produced starting from galactose. However, to be able to compete with the current predominant sweeteners (like sucrose and high fructose corn syrup), it should be produced in much higher quantities than is possible when starting from galactose. In order to overcome this issue, tagatose production should start from a more widely available (and cheaper) substrate. Fructose and glucose are two such very abundant substrates; however, no (bio)catalysts are available to convert fructose or glucose into tagatose. Nevertheless, some enzymes were found to perform similar reactions and therefore are promising to one day become biocatalysts for tagatose production. In this work, the cloning and analysis of two totally different C4-epimerases is described in respect to their capability of tagatose production. The first enzyme is L-ribulose-5-phosphate 4-epimerase from Geobacillus thermodenitrificans, an aldolase-related epimerase that would require an adaptation of the substrate binding site around the phosphate moiety of the substrate. The second C4-epimerase is naturally active on nucleotide activated sugars, namely the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis. The major challenge in the engineering of this UDP-hexose 4-epimerase is trying to get rid of the necessity of the UDP-group of the substrate and making it active on free monosaccharides. At first, the Geobacillus L-ribulose-5-phosphate 4-epimerase gene was cloned in an appropriate expression vector and expressed in E. coli. The recombinant enzyme was first characterized with respect to affinity for L-ribulose-5-phosphate, metal ion activation and stability at 37 °C. To that end, its natural substrate had to be produced first, which was accomplished by phosphorylation of L-ribulose using ATP as phosphate donor and recombinantly expressed L-ribulokinase as biocatalyst. After characterization, mutagenesis was achieved both randomly and semi-rationally by error-prone PCR and site saturation mutagenesis, respectively. To be able to detect enzyme variants harboring (improved) tagatose 4-epimerase activity among the thousands mutant enzymes, two ‘identification’ systems were developed. Two selection strains were developed that can be used for the Darwinian selection of improved enzyme variants, while also a colorimetric screening assay has been created. Although several millions and thousands of mutants were analyzed using the selection strains and screening assay, respectively, no variants were confirmed to possess (improved) tagatose 4-epimerase activity. Secondly, the UDP-hexose 4-epimerase from Marinithermus hydrothermalis was also cloned and heterologously expressed in E. coli. A thorough characterization of this second epimerase was performed, revealing that it belongs to the type 2 UDP-hexose 4-epimerases. As expected for a type 2 epimerase, its substrate specificity could easily be altered by mutagenesis of a single residue, namely the so-called gatekeeper. This also confirms the previously reported hypothesis about substrate specificity in type 1 and type 2 epimerases. Mutational analysis of the UDP-hexose 4-epimerase uncovered two new features that can be found in these epimerases. The Marinithermus enzyme was found to possess a TxnYx3K catalytic triad, rather than the usual serine containing triad (SxnYx3K). The presence of the threonine’s methyl function was found to be of more importance for the enzyme’s affinity for N-acetylated UDP-sugars than for non-acetylated substrates. As such, the TxnYx3K triad might be a new substrate specificity determinant for type 2 UDP-hexose 4-epimerases. The second new feature was the presence of two consecutive glycine residues next to the catalytic threonine, which were found to be important for activity of the enzyme with non-acetylated and even bigger importance for activity on N-acetylated substrates. In an attempt to identify new determinants for specificity towards UDP-GlcNAc, two loop mutants were created but they were found to be inactive, most likely due to dispositioning of the catalytic tyrosine, which results in the disruption of the subtle catalytic chemistry. In addition, the Marinithermus UDP-hexose 4-epimerase was also tested for its ability to convert the free monosaccharides fructose/tagatose, glucose/galactose and the phosphorylated α-Glc-1-P. Furthermore, also the E. coli UDP-hexose 4-epimerase was cloned and also here no epimerase activity could be detected on free monosaccharides, in contrast to what has previously been reported

Friends, acquaintances, pupils and patrons: Japanese intellectual life in the late eighteenth century: a prosopographical approach

This study of the social circumstances of Japanese intellectuals in the last quarter of the eighteenth century is based on biographical data concerning 173 individuals. It deals with the image of intellectual life of that period in current scholarship, and with the self-image and ethos of scholars, authors, poets and artists. That self-image and ethos, however, often clash with the realities of their everyday lives. This prosopographical investigation offers a new look at intellectual life on a basic level. The current image of intellectual life in the Tokugawa period is one of dissatisfaction and withdrawal, whereas the image that results from this study is one of dynamism and interaction

Improving disaster response evaluations : Supporting advances in disaster risk management through the enhancement of response evaluation usefulness

Future disasters or crises are difficult to predict and therefore hard to prepare for. However, while a specific event might not have happened, it can be simulated in an exercise. The evaluation of performance during such an exercise can provide important information regarding the current state of preparedness, and used to improve the response to future events. For this to happen, evaluation products must be perceived as useful by the end user. Unfortunately, it appears that this is not the case. Both evaluations and their products are rarely used to their full extent or, in extreme cases, are regarded as paper-pushing exercises.The first part of this research characterises current evaluation practice, both in the scientific literature and in Dutch practice, based on a scoping study, document and content analyses, and expert judgements. The findings highlight that despite a recent increase in research attention, few studies focus on disaster management exercise evaluation. It is unclear whether current evaluations achieve their purpose, or how they contribute to disaster preparedness. Both theory and practice tend to view, and present evaluations in isolation. This limited focus creates a fragmented field that lacks coherence and depth. Furthermore, most evaluation documentation fails to justify or discuss the rational underlying the selected methods, and their link to the overall purpose or context of the exercise. The process of collecting and analysing contextual, evidence-based data, and using it to reach conclusions and make recommendations lacks methodological transparency and rigour. Consequently, professionals lack reliable guidance when designing evaluations.Therefore, the second part of this research aimed to gain an insights into what make evaluations useful, and suggest improvements. In particular, it highlights the values associated with the methodology used to record and present evaluation outcomes to end users. The notion of an ‘evaluation description’ is introduced to support the identification of four components that are assumed to influence the usefulness of an evaluation: its purpose, object description, analysis and conclusion. Survey experiments identified that how these elements – notably, the analysis and/ or conclusions – are documented significantly influences the usefulness of the product. Furthermore, different components are more useful depending on the purpose of the report (for learning or accountability). Crisis management professionals expect the analysis to go beyond the object of the evaluation, and focus on the broader context. They expect a rigorous evaluation to provide them with evidence-based judgements that deliver actionable conclusions and support future learning.Overall, this research shows that the design and execution of evaluations should provide systematic, rigorous, evidence-based and actionable outcomes. It suggests some ways to manage both the process and the products of an evaluation to improve its usefulness. Finally, it underlines that it is not the evaluation itself that leads to improvement, but its use. Evaluation should, therefore, be seen as a means to an end

Vulnerability Analysis of Transformer-based Optical Character Recognition to Adversarial Attacks

Recent advancements in Optical Character Recognition (OCR) have been driven by transformer-based models. OCR systems are critical in numerous high-stakes domains, yet their vulnerability to adversarial attack remains largely uncharted territory, raising concerns about security and compliance with emerging AI regulations. In this work we present a novel framework to assess the resilience of Transformer-based OCR (TrOCR) models. We develop and assess algorithms for both targeted and untargeted attacks. For the untargeted case, we measure the Character Error Rate (CER), while for the targeted case we use the success ratio. We find that TrOCR is highly vulnerable to untargeted attacks and somewhat less vulnerable to targeted attacks. On a benchmark handwriting data set, untargeted attacks can cause a CER of more than 1 without being noticeable to the eye. With a similar perturbation size, targeted attacks can lead to success rates of around $25\%$ -- here we attacked single tokens, requiring TrOCR to output the tenth most likely token from a large vocabulary