Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion... Show moreConventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research. Show less
Martianez-Vendrell, X.; Kikkert, M.; Gerold, G. 2021
Proteases precisely and irreversibly catalyze the hydrolysis of peptide bonds, regulating the fate, localization, and activity of many proteins. Consequently, proteolytic activity plays an... Show moreProteases precisely and irreversibly catalyze the hydrolysis of peptide bonds, regulating the fate, localization, and activity of many proteins. Consequently, proteolytic activity plays an important role in fundamental cellular processes such as differentiation and migration, immunological and inflammatory reactions, apoptosis and survival. During virus infection, host proteases are involved in several processes, from cell entry to initiation, progression and resolution of inflammation. On the other hand, many viruses encode their own highly specific proteases, responsible for the proteolytic processing of viral proteins, but, at the same time, to cleave host proteins to corrupt antiviral host responses and adjust protein activity to favor viral replication. Traditionally, protease substrate identification has been addressed by means of hypothesis-driven approaches, but recent advances in proteomics have made a toolkit available to uncover the extensive repertoire of host proteins cleaved during infection, either by viral or host proteases. Here, we review the currently available proteomics-based methods that can and have contributed to the systematic and unbiased identification of new protease substrates in the context of virus-host interactions. The role of specific proteases during the course of virus infections will also be highlighted. 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
In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review... Show moreIn recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease. Show less
In vitro cellular assays analyzing antigen-specific T cells are characterized by their high complexity and require controlled conditions to lower experimental variations. Without standard cellular... Show moreIn vitro cellular assays analyzing antigen-specific T cells are characterized by their high complexity and require controlled conditions to lower experimental variations. Without standard cellular reagents, it is difficult to compare results over time and across institutions. To overcome this problem, a simple and robust technology was developed to generate TCR-engineered reference samples (TERS) containing defined numbers of antigen-specific T cells. Utilization of TERS enables performance control of three main T-cell assays: MHC-peptide multimer staining, IFN-gamma ELISpot and cytokine flow cytometry. TERS continuously deliver stable results and can be stored for longer periods of time. Here, an optimized manufacturing protocol, based on the electroporation of stable T-cell receptor in vitro-transcribed mRNA, is provided for versatile in-house production of TERS. Included are a guideline to optimize the electroporation settings on locally available electroporation devices and a step-by-step protocol for the production process. Show less
C. difficile is a major nosocomial pathogen, but is also increasingly recognised as an important diarrhoeal pathogen in the community, not always associated with antibiotics. The European Society... Show moreC. difficile is a major nosocomial pathogen, but is also increasingly recognised as an important diarrhoeal pathogen in the community, not always associated with antibiotics. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Clostridium difficile (ESGCD) is a group of clinicians and scientists from many European countries and further afield, who share a common interest in C. difficile. The aims of the Study Group are centred around raising the profile of CDI in humans and animals, fostering collaboration amongst centres in different European countries and providing a forum for discussing and disseminating information. One of the principal aims of the Study Group is to raise awareness of C. difficile infections in European hospitals. ESGCD has a particular interest in the development and dissemination of European guidance on prevention, diagnosis and treatment of CDI. This chapter will discuss the organisation of ESGCD within the ESCMID Study Group structure, the origins of the Study Group, the aims and objectives of the group, and will highlight some of the past and present activities of ESGCD in relation to these. Show less
Cardiac progenitor cells (CPCs) have emerged as potential therapy to improve cardiac repair and prevent damage in cardiac diseases. CPCs are a promising cell source for cardiac therapy as they can... Show moreCardiac progenitor cells (CPCs) have emerged as potential therapy to improve cardiac repair and prevent damage in cardiac diseases. CPCs are a promising cell source for cardiac therapy as they can generate all cardiovascular lineages in vitro and in vivo. Originating from the heart itself, CPCs may be destined to activate endogenous repair mechanisms. These CPCs release paracrine molecules that are able to stimulate cardiac repair mechanisms, including stimulation of vessel formation and inhibition of cardiomyocyte apoptosis. In addition to proteins and growth factors, CPCs release extracellular membrane vesicles, such as exosomes, which have gained increasing interest in recent years. Exosomal-derived miRNAs have been indicated to play an important role in these processes. Hereby, CPC exosomes can be considered as potential off-the-shelf therapeutics, as they are able to stimulate the regenerative capacity of the heart by increasing vessel density and lowering apoptosis of cardiomyocytes. Show less
Glycans on proteins and lipids are known to alter with malignant transformation. The study of these may contribute to the discovery of biomarkers and treatment targets as well as understanding of... Show moreGlycans on proteins and lipids are known to alter with malignant transformation. The study of these may contribute to the discovery of biomarkers and treatment targets as well as understanding of cancer biology. We here describe the change of glycosylation specifically defining colorectal cancer with view on N-glycans, O-glycans, and glycosphingolipid glycans in colorectal cancer cells and tissues as well as patient sera. Glycan alterations observed in colon cancer include increased beta 1,6-branching and correlating higher abundance of (poly-)N-acetyllactosamine extensions of N-glycans as well as an increase in (truncated) high-mannose type glycans, while bisected structures decrease. Colorectal cancer-associated O-glycan changes are predominated by reduced expression of core 3 and 4 glycans, whereas higher levels of core 1 glycans, (sialyl) T-antigen, (sialyl) Tn-antigen, and a generally higher density of O-glycans are observed. Specific changes for glycosphingolipid glycans are lower abundances of disialylated structures as well as globo-type glycosphingolipid glycans with exception of Gb3. In general, alterations affecting all discussed glycan types are increased sialylation, fucosylation as well as (sialyl) Lewis-type antigens and type-2 chain glycans. As a consequence, interactions with glycan-binding proteins can be affected and the biological function and cellular consequences of the altered glycosylation with regard to tumorigenesis, metastasis, modulation of immunity, and resistance to antitumor therapy will be discussed. Finally, analytical approaches aiding in the field of glycomics will be reviewed with focus on binding assays and mass spectrometry. Show less
Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three... Show moreLight microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. Show less
