This chapter describes the development of a math trail for high school students. In 2016, we developed this trail through Leiden (The Netherlands) during a student project for the Science... Show moreThis chapter describes the development of a math trail for high school students. In 2016, we developed this trail through Leiden (The Netherlands) during a student project for the Science Communication and Society specialization, a track for master students at the Faculty of Science at Leiden University. Our aim was to provide a guided trail through the city that links everyday sights to mathematical concepts within the curriculum of high school students between 13 and 15 years old. The entire project was carried out in 3 weeks. We did background research, consisting of literature reviews, target audience surveys with school children, and focus groups with teachers. Based on the conclusions from this background research, we developed questions that suited both the goal to make the math trail a fun experience that makes math less abstract and the goal to include questions from across the curriculum. In this chapter, we would like to share our insights from the background research and our experiences in developing a math trail. We moreover aim to provide those who are interested in designing a math trail in their city with a practical step-by-step plan and checklist. Show less
Bouma, J.; Soethoudt, M.; Gils, N. van; Xia, L.; Stelt, M, van der; Heitman, L.H. 2022
Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G... Show moreCannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here. Show less
The ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of... Show moreThe ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of an activity assay that is compatible with the sample and selective for the enzyme(s) of interest. Deconvolution of the contributions of different enzymes to an observed catalytic ability further necessitates an often-challenging protein separation. The advent of broadly reactive activity-based probes (ABPs) for retaining glycoside hydrolases (GHs) has enabled an alternative, often complementary, assay for active GHs. Using activity-based protein profiling (ABPP) techniques, many retaining glycoside hydrolases can be separated, detected, and identified with high sensitivity and selectivity. This chapter outlines ABPP methods for the detection and identification of retaining glycoside hydrolases from microbial sources, including protein sample preparation from bacterial lysates and fungal secretomes, enzyme labeling and detection via fluorescence, and enzyme identification using affinity-based enrichment coupled to peptide sequencing following isobaric labeling. Show less
Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of... Show moreCorrelative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples. Show less
Purchase, R.; Cogdell, R.; Breitling, F.; Stadler, V.; Hulst, N. van; Kramer, G.J.; ... ; Groot, J.J.M. de 2019
Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how... Show moreBacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens. Show less
Volbeda, A.G.; Marel, G.A. van der; Codée, J.D.C. 2018
Carbohydrates are the most densely functionalized class of biopolymers in nature. This chapter describes the differences in the reactivity of the various functional groups on a carbohydrate ring... Show moreCarbohydrates are the most densely functionalized class of biopolymers in nature. This chapter describes the differences in the reactivity of the various functional groups on a carbohydrate ring and how to exploit these in the design of effective protecting group strategies. It then describes how protecting group effects can be used to control stereoselective transformations and reactivity‐controlled one‐pot synthesis strategies. The chapter highlights applications and strategies in automated synthesis. Different strategies have been developed to automate oligosaccharide assembly based on either solution‐phase synthesis or solid‐phase techniques, and automated solid‐phase synthesizers are now commercially available. Protecting group chemistry can make or break any (oligo)saccharide synthesis effort. The impact of protecting groups on the stereochemical outcome of a glycosylation reaction is best illustrated by the anchimeric assistance that neighboring groups can provide during a glycosylation reaction. Show less
Koenders, S.T.A.; Gagestein, B.; Stelt, M. van der 2018
Lipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has... Show moreLipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has remained elusive due to their low abundance, high lipophilicity, and inherent instability. Various chemical biology approaches, such as photoaffinity or activity-based protein profiling methods, have been employed to shed light on the biological role of lipids and the lipid-protein interaction profile. In this review, we will summarize the recent developments in the field of chemical probes to study lipid biology, especially in immunology, and indicate potential avenues for future research. Show less
The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that... Show moreThe binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM. Show less
Acoustic Force Spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers... Show moreAcoustic Force Spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations. Show less
DNA looping is important for genome organization in all domains of life. The basis of DNA loop formation is the bridging of two separate DNA double helices. Detecting DNA bridge formation generally... Show moreDNA looping is important for genome organization in all domains of life. The basis of DNA loop formation is the bridging of two separate DNA double helices. Detecting DNA bridge formation generally involves the use of complex single-molecule techniques (atomic force microscopy, magnetic, or optical tweezers). Although DNA bridging can be qualitatively described, quantification of DNA bridging and bridging dynamics using these techniques is challenging. Here, we describe a novel biochemical assay capable of not only detecting DNA bridge formation, but also allowing for quantification of DNA bridging efficiency and the effects of physico-chemical conditions on DNA bridge formation. Show less
Corbeski, I.; Horn, V.; Valk, R.A. van der; Paige, U. le; Dame, R.T.; Ingen, H. van 2018
Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and... Show moreArchitectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique that has seen increasing application in the study of biomolecular interactions thanks to its solution-based nature, its rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events. Here, we describe the use of MST in the study of chromatin interactions, with emphasis on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB; the sequential binding of core histone complexes to histone chaperone APLF; the impact of the nucleosomal context on the recognition of histone modifications; and the binding of a LANA-derived peptide to nucleosome core. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples. Show less
