Supersensitive Multifluorophore RNA‐FISH for Early Virus Detection and Flow‐FISH by Using Click Chemistry

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid‐19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single‐fluorophore‐containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off‐target binding effects that create background noise. Here, we used click chemistry and alkyne‐modified DNA oligonucleotides to prepare multiple‐fluorophore‐containing probes. We found that these multiple‐dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection

New insights into the intracellular distribution pattern of cationic amphiphilic drugs

Cationic amphiphilic drugs (CADs) comprise a wide variety of different substance classes such as antidepressants, antipsychotics, and antiarrhythmics. It is well recognized that CADs accumulate in certain intracellular compartments leading to specific morphological changes of cells. So far, no adequate technique exists allowing for ultrastructural analysis of CAD in intact cells. Azidobupramine, a recently described multifunctional antidepressant analogue, allows for the first time to perform high-resolution studies of CADs on distribution pattern and morphological changes in intact cells. We showed here that the intracellular distribution pattern of azidobupramine strongly depends on drug concentration and exposure time. The mitochondrial compartment (mDsRed) and the late endolysosomal compartment (CD63-GFP) were the preferred localization sites at low to intermediate concentrations (i.e. 1 mu M, 5 mu M). In contrast, the autophagosomal compartment (LC3-GFP) can only be reached at high concentrations (10 mu M) and long exposure times (72 hrs). At the morphological level, LC3-clustering became only prominent at high concentrations (10 mu M), while changes in CD63 pattern already occurred at intermediate concentrations (5 mu M). To our knowledge, this is the first study that establishes a link between intracellular CAD distribution pattern and morphological changes. Therewith, our results allow for gaining deeper understanding of intracellular effects of CADs

Tuning Nanoparticle Uptake: Live-Cell Imaging Reveals Two Distinct Endocytosis Mechanisms Mediated by Natural and Artificial EGFR Targeting Ligand

Therapeutic nanoparticles can be directed to cancer cells by incorporating selective targeting ligands. Here, we investigate the epidermal growth factor receptor (EGFR)mediated endocytosis of gene carriers (polyplexes) either targeted with natural EGF or GE11, a short synthetic EGFR-binding peptide. Highly sensitive live-cell fluorescence microcopy with single particle resolution unraveled the existence of two different uptake mechanisms; EGF triggers accelerated nanoparticle endocytosis due to its dual active role in receptor binding and signaling activation, For GE11, an alternative EGFR signaling independent, actin-driven pathway is presented

Detailed study of electromigration induced damage in Al and AlCuSi interconnects

Because of the continuing miniaturization, electromigration (EM) phenomena are still a key issue in reliability of VLSI metallizations. The present study of EM induced voiding and hillocking was performed on unpassivated conductor lines with various widths and current densities. Stressed and unstressed interconnects were carefully exymined with SEM and TEM techniques, espcially with regard to void densities, void sizes and characterisitic lengths between void and hillock. The fatal void shape was related to current density and line width indicating that the failure mechanism changes with decreasing line width and decreasing current density

Detailed study of electromigration induced damage in Al and AlCuSi interconnects

Because of the continuing miniaturization, electromigration (EM) phenomena are still a key issue in reliability of VLSI metallizations. The present study of EM induced voiding and hillocking was performed on unpassivated conductor lines with various widths and current densities. Stressed and unstressed interconnects were carefully exymined with SEM and TEM techniques, espcially with regard to void densities, void sizes and characterisitic lengths between void and hillock. The fatal void shape was related to current density and line width indicating that the failure mechanism changes with decreasing line width and decreasing current density

Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells

Physical Principles of Membrane Shape Regulation by the Glycocalyx

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx.Mucin biopolymers and long-chein polysaccharides within the glycocalyx can generates entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix