Bioorthogonal labeling tools to study pathogenic intracellular bacteria

In this thesis, bioorthogonal chemistry is combined with correlative light-electron microscopy to selectively label and study pathogenic intracellular bacteria within the host immune cell. This technique combines the ultrastructural information of transmission electron microscopy with the functional information of fluorescence light microscopy in order to investigate the host-pathogen interactions that contribute to the diseases caused by pathogenic intracellular bacteria such as Salmonella Typhimurium and Mycobacterium tuberculosis. The technique is further expanded with super-resolution microscopy by combining stochastic optical reconstruction microscopy with transmission electron microscopy. Additionally, the bioorthogonal labeling method for the study of intracellular bacteria is validated through a bead-based stability assay, demonstrating the compatibility of alkyne and azide groups to label bacterial proteins within the degradative lysosomal environment. The technique developed in this thesis may contribute to a better understanding of the mechanisms behind bacterial diseases, as well as the development of novel antibiotics and other therapies to fight these important infectious diseases.Bio-organic Synthesi

Dissipation of angular momentum in light heavy ion collision

The inclusive energy distributions of fragments (4$\leq$Z$\leq$7) emitted in the reactions $^{16}$O (116 MeV) + $^{27}$Al, $^{28}$Si, $^{20}$Ne (145 MeV) + $^{27}$Al, $^{59}$Co have been measured in the angular range $\theta_{lab}$= 10$^\circ$ - 65$^\circ$. Fusion-fission and deep inelastic components of the fragment emission have been extracted from the experimental data. The angular mometum dissipations in fully damped deep inelastic collisions have been estimated assming exit channel configuration similar to those for fusion-fission process. It has been found that, the angular momentum dissipations are more than those predicted by the empirical sticking limit in all cases. The deviation is found to increase with increasing charge transfer (lighter fragments). Qualitatively, this may be due to stronger friction in the exit channel. Moreover, for the heavier system $^{20}$Ne + $^{59}$Co, the overall magnitude of deviation is less as compared to those for the lighter systems, {\it i.e.}, $^{16}$O + $^{27}$Al, $^{28}$Si, $^{20}$Ne + $^{27}$Al. This may be due to lesser overlap in time scales of fusion and deep inelastic time scales for heavier systems.Comment: 15 pages, 9 figures, accepted for publication in Phys. Rev.

Real‐Time NMR recording of fermentation and lipid metabolism processes in live microalgae cells

Non-invasive and real-time recording of processes in living cells has been limited to detection of small cellular components such as soluble proteins and metabolites. Here we report a multiphase NMR approach using Magic-Angle Spinning NMR to synchronously follow microbial processes of fermentation, lipid metabolism and structural dynamic changes in live microalgae cells. Chlamydomonas reinhardtii green algae were highly concentrated, introducing dark fermentation and anoxia conditions. Single-pulse NMR experiments were applied to obtain temperature-dependent kinetic profiles of the formed fermentation products. Through dynamics-based spectral editing NMR, simultaneous conversion of galactolipids into TAG and free fatty acids was observed and rapid loss of rigid lipid structures. This suggests that lipolysis under dark and anoxia conditions finally results in the breakdown of cell and organelle membranes, which could be beneficial for recovery of intracellular microbial useful products.NWO680.91.15.19Solid state NMR/Biophysical Organic ChemistryMedical Biochemistr

Metabolic labeling probes for interrogation of the host-pathogen interaction

Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.Microbial Biotechnolog

Magnetic-activated cell sorting using coiled-coil peptides: an alternative strategy for isolating cells with high efficiency and specificity

NWOSupramolecular & Biomaterials Chemistr

Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Bio-organic Synthesi

Application of a highly selective Cathepsin S two-step activity-based probe in multicolor bio-orthogonal correlative light-electron microscopy

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.Microscopic imaging and technolog

Application of a highly selective cathepsin S two-step activity-based probe in multicolor bio-orthogonal correlative light-electron microscopy

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.Bio-organic Synthesi

Effects of Hybrid Cycle and Handcycle Exercise on Cardiovascular Disease Risk Factors in People with Spinal Cord Injury:A Randomized Controlled Trial

Objective: To examine the effects of a 16-week exercise programme, using either a hybrid cycle or a handcycle, on cardiovascular disease risk factors in people with spinal cord injury.Participants: Nineteen individuals with spinal cord injury &gt;= 8 years.Design: Multicentre randomized controlled trial. Both the hybrid cycle group (n = 9) and the handcycle group (n = 10) trained twice a week for 16 weeks on the specific cycle. Outcome measures obtained pre and post the programme were: metabolic syndrome components (waist circumference, systolic and diastolic blood pressure, high-density lipoprotein cholesterol, triglycerides and insulin resistance), inflammatory status (C-reactive protein (CRP), interleukin (1)-6 and -10), and visceral adiposity (trunk and android fat).Results: For all outcome measures, there were no significant differences over time between the 2 training groups. Overall significant reductions were found for waist circumference (p = 0.001), diastolic blood pressure (p = 0.03), insulin resistance (p = 0.006), CRP (p = 0.05), IL-6 (p = 0.04), IL-6/IL-10 ratio (p = 0.03), and trunk (p = 0.04) and android (p = 0.02) fat percentage. No significant main effects for time were observed for systolic blood pressure, triglycerides, high-density lipoprotein cholesterol, IL-10, and trunk and android fat mass.Conclusion: The 16-week exercise programme, using either a hybrid cycle or a handcycle, found similar beneficial effects on metabolic syndrome components, inflammatory status and visceral adiposity, indicating that there were no additional benefits of functional electrical stimulation-induced leg exercise over handcycle exercise alone.</p

Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking

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 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.Microscopic imaging and technolog