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

Simon Wisnovsky, Leonhard Möckl, Stacy A. Malaker, Kayvon Pedram, Gaelen T. Hess, Nicholas M. Riley, Melissa A. Gray, Benjamin A. H. Smith, Michael C. Bassik, et al.

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface<br>antigens, are emerging as attractive targets for cancer immunotherapy.<br>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<br>cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9.<br>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<br>cells relieves Siglec-7-mediated inhibition of immune killing activity.<br>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.

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

Leonhard Möckl, W. E. Moerner

Single-molecule super-resolution microscopy has developed from a specialized technique into one of the most versatile and powerful imaging methods of the nanoscale over the past two decades. In this perspective, we provide a brief overview of the historical development of the field, the fundamental concepts, the methodology required to obtain maximum quantitative information, and the current state of the art. Then, we will discuss emerging perspectives and areas where innovation and further improvement are needed. Despite the tremendous progress, the full potential of single-molecule super-resolution microscopy is yet to be realized, which will be enabled by the research ahead of us.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Marjoke F. Debets, Omur Y. Tastan, Simon P. Wisnovsky, Stacy A. Malaker, Nikolaos Angelis, Leonhard Möckl, Junwon Choi, Helen Flynn, Lauren J. S. Wagner, et al.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

From small Beginnings to steep Ascent The Fraunhofer Society

Jürgen Evers, Christiane Herzog, Leonhard Möckl

The Fraunhofer-Gesellschaft (FhG) was founded by the Secretary of the Baverian Ministry of Economics, Hugo Geiger. He intended to create a research organisation that should unite science and economics to foster applied research. The new institution was named after Joseph von Fraunhofer, a hint to its intended orientation as Fraunhofer was both an ingenious scientist and a successful businessman. His inventions were developed into products which were sold all over the word. The FhG as new organisation would become the third pillar of German Research next to the Max-Planck-Society (MPG) and the German Research Foundation (DFG), but initially, the MPG and the DFG caused some problems for the newly founded FhG. However, when the German Ministry for Research guaranteed funding, the FhG quickly rose to international recognition.

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

Nada Raddaoui, Stefano Croce, Florian Geiger, Alexander Borodavka, Leonhard Möckl, Samuele Stazzoni, Bastien Viverge, Christoph Braeuchle, Thomas Frischmuth, et al.

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.

The Emerging Role of the Mammalian Glycocalyx in Functional Membrane Organization and Immune System Regulation

Leonhard Möckl

All cells in the human body are covered by a dense layer of sugars and the proteins and lipids to which they are attached, collectively termed the "glycocalyx." For decades, the organization of the glycocalyx and its interplay with the cellular state have remained enigmatic. This changed in recent years. Latest research has shown that the glycocalyx is an organelle of vital significance, actively involved in and functionally relevant for various cellular processes, that can be directly targeted in therapeutic contexts. This review gives a brief introduction into glycocalyx biology and describes the specific challenges glycocalyx research faces. Then, the traditional view of the role of the glycocalyx is discussed before several recent breakthroughs in glycocalyx research are surveyed. These results exemplify a currently unfolding bigger picture about the role of the glycocalyx as a fundamental cellular agent.

Deep learning in single-molecule microscopy: fundamentals, caveats, and recent developments [Invited]

Leonhard Möckl, Anish R. Roy, W. E. Moerner

Deep learning-based data analysis methods have gained considerable attention in all fields of science over the last decade. In recent years, this trend has reached the single-molecule community. In this review, we will survey significant contributions of the application of deep learning in single-molecule imaging experiments. Additionally, we will describe the historical events that led to the development of modern deep learning methods, summarize the fundamental concepts of deep learning, and highlight the importance of proper data composition for accurate, unbiased results. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet

Leonhard Möckl, Anish R. Roy, Petar N. Petrov, W. E. Moerner

Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3-dimensional localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point-spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, for both simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of superresolution reconstructions of biological structures.

Accurate phase retrieval of complex 3D point spread functions with deep residual neural networks

Leonhard Möckl, Petar N. Petrov, W. E. Moerner

Phase retrieval, i.e., the reconstruction of phase information from intensity information, is a central problem in many optical systems. Imaging the emission from a point source such as a single molecule is one example. Here, we demonstrate that a deep residual neural net is able to quickly and accurately extract the hidden phase for general point spread functions (PSFs) formed by Zernike-type phase modulations. Five slices of the 3D PSF at different focal positions within a two micrometer range around the focus are sufficient to retrieve the first six orders of Zernike coefficients.

A Photoswitchable Trivalent Cluster Mannoside to Probe the Effects of Ligand Orientation in Bacterial Adhesion

Guillaume Despras, Leonhard Möckl, Anne Heitmann, Insa Stamer, Christoph Braeuchle, Thisbe K. Lindhorst

We have recently demonstrated, by employing azobenzene glycosides, that bacterial adhesion to surfaces can be switched through reversible reorientation of the carbohydrate ligands. To investigate this phenomenon further, we have turned here to more complex-that is, multivalent-azobenzene glycoclusters. We report on the synthesis of a photosensitive trivalent cluster mannoside conjugated to an azobenzene hinge at the focal point. Molecular dynamics studies suggested that this cluster mannoside, despite the conformational flexibility of the azobenzene-glycocluster linkage, offers the potential for reversibly changing the glycocluster's orientation on a surface. Next, the photoswitchable glycocluster was attached to human cells, and adhesion assays with type 1 fimbriated Escherichia coli bacteria were performed. They showed marked differences in bacterial adhesion, dependent on the light-induced reorientation of the glycocluster moiety. These results further underline the importance of orientational effects in carbohydrate recognition and likewise the value of photoswitchable glycoconjugates for their study.

Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx

Leonhard Möckl, Kayvon Pedram, Anish R. Roy, Venkatesh Krishnan, Anna-Karin Gustavsson, Oliver Dorigo, Carolyn R. Bertozzi, W. E. Moerner

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

Physical Principles of Membrane Shape Regulation by the Glycocalyx

Carolyn R. Shurer, Joe Chin-Hun Kuo, LaDeidra Monet Roberts, Jay G. Gandhi, Marshall J. Colville, Thais A. Enoki, Hao Pan, Jin Su, Jade M. Noble, et al.

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.

Bisacylphosphane oxides as photo-latent cytotoxic agents and potential photo-latent anticancer drugs

Andreas Beil, Friederike A. Steudel, Christoph Braeuchle, Hansjorg Grutzmacher, Leonhard Möckl

Bisacylphosphane oxides (BAPOs) are established as photoinitiators for industrial applications. Light irradiation leads to their photolysis, producing radicals. Radical species induce oxidative stress in cells and may cause cell death. Hence, BAPOs may be suitable as photolatent cytotoxic agents, but such applications have not been investigated yet. Herein, we describe for the first time a potential use of BAPOs as drugs for photolatent therapy. We show that treatment of the breast cancer cell lines MCF-7 and MDA-MB-231 and of breast epithelial cells MCF-10A with BAPOs and UV irradiation induces apoptosis. Cells just subjected to BAPOs or UV irradiation alone are not affected. The induction of apoptosis depend on the BAPO and the irradiation dose. We proved that radicals are the active species since cells are rescued by an antioxidant. Finally, an optimized BAPO-derivative was designed which enters the cells more efficiently and thus leads to stronger effects at lower doses.

Zur logischen Position der Hypothese

Leonhard Möckl

The concept of the hypothesis is omnipresent across all discipline of science, be it natural sciences or humanities. Despite the prominent role in everyday scientific work, its true meaning and logical position are remarkably elusive. In this essay, I argue that within the concept of the hypothesis, a twofold collision of categories occurs. First, the hypothesis needs to be connected to current, already achieved knowledge. At the same time, it may not be directly deduced from this knowledge: Therefore, its formulation has a necessary non-rational aspect. Second, in this dichotomic state, the hypothesis is in the moment of its formulation immediately revoked because the hypothesis is not meant to be permanent. Either, its connection to present knowledge survives if the hypothesis is later shown to be true. Or, its non-rational aspect survives if the hypothesis is disproven. Both these cases are thought as potential outcomes when the hypothesis is formulated. Hence, in this formulation, rationality and non-rationality collide twofold. The collision of these two categories cannot be resolved within the position of the categories rationality and non-rationality. If either of the two colliding categories are emphasised, paradoxes arise, for example in the question of responsibility for scientific research (ethics of conviction vs. ethics of responsibility). The hypothesis must, therefore, be traced back to its radically individual origin: The researcher who formulated it – nature itself does not know any hypothesis.

Von Kautschuk zu Metallen: ein Werkslabor mit Weltgeltung

Leonhard Möckl, Jürgen Evers, Christiane Herzog

Anfang des 20. Jahrhunderts leitete zunächst der Chemiker Salomon Axelrod das chemische Laboratorium im Kabelwerk Oberspree. Sein Hauptforschungsgebiet war Kautschuk. Nach seinem Tod übernahm der Ingenieur Wichard von Moellendorff die Leitung des Labors, und Forschungsschwerpunkt wurden Metalle. Der Name eines seiner Laboranten steht noch heute in Chemiebüchern: Jan Czochralski.

Quantitative super-resolution microscopy reveals the architecture of the mammalian glycocalyx and its changes during cancer progression

Leonhard Möckl, Kayvon Pedram, Anish Roy, Anna-Karin Gustavsson, Carolyn Bertozzi, William Moerner

Invasiveness of Cells Leads to Changes in Their Interaction Behavior with the Glycocalyx

Ellen Broda, Adriano A. Torrano, Laura Loebbert, Leonhard Möckl, Christoph Braeuchle, Hanna Engelke

Transendothelial migration is a crucial step during metastasis. Before circulating tumor cells enter the endothelium, they face the glycocalyx. While invasive migration of cancer cells is well studied, few investigations exist regarding their interaction with the glycocalyx. Here, the interaction of three breast cell lines with an endothelial glycocalyx is studied. Benign MCF-10A, noninvasive malign MCF-7, and invasive MDA-MB-231 cells penetrate the glycocalyx, just adhere to it or approach without even attaching to it. Remarkable fluctuations in these interaction modes are detected by time-resolved interaction profiles. Adhesion, migration, and invasion characteristics as well as combinations of interaction modes, cell shapes, and cell extensions are studied. The motility and penetration depth into the glycocalyx are analyzed. The invasive cells are the most flexible, penetrating the glycocalyx mostly with a round shape and feet-like membrane extensions. Noninvasive cancer cells penetrate the glycocalyx the deepest over time and benign cells integrate more likely into the endothelial cell layer underneath the glycocalyx.

Quantitative Super-Resolution Imaging Reveals Mammalian Glycocalyx Dynamics

Leonhard Möckl, Kayvon Pedram, Anish Roy, Carolyn Bertozzi, William Esco Moerner

Die neue Macht des Forschers

Leonhard Möckl

Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ

Nada Raddaoui, Samuele Stazzoni, Leonhard Möckl, Bastien Viverge, Florian Geiger, Hanna Engelke, Christoph Braeuchle, Thomas Carell

The in vivo incorporation of alkyne-modified bases into the genome of cells is today the basis for the efficient detection of cell proliferation. Cells are grown in the presence of ethinyl-dU (EdU), fixed and permeabilised. The incorporated alkynes are then efficiently detected by using azide-containing fluorophores and the Cu-I-catalysed alkyne-azide click reaction. For a world in which constant improvement in the sensitivity of a given method is driving diagnostic advancement, we developed azide- and alkyne-modified dendrimers that allow the establishment of sandwich-type detection assays that show significantly improved signal intensities and signal-to-noise ratios far beyond that which is currently possible.

Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates

Katharina Kolbe, Leonhard Möckl, Victoria Sohst, Julius Brandenburg, Regina Engel, Sven Malm, Christoph Braeuchle, Otto Holst, Thisbe K. Lindhorst, et al.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

Back Cover: Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates (ChemBioChem 13/2017)

Katharina Kolbe, Leonhard Möckl, Victoria Sohst, Julius Brandenburg, Regina Engel, Sven Malm, Christoph Bräuchle, Otto Holst, Thisbe K. Lindhorst, et al.

The back cover picture shows a new metabolic labeling method for Mycobacterium tuberculosis that uses azido pentoses. Mtb clinical isolates can be effectively illuminated in the presence of these synthetic carbohydrate derivatives, and this allows insight into the unknown worlds of the Mtb cell wall and its arabinan metabolism.

New insights into the intracellular distribution pattern of cationic amphiphilic drugs

Magdalena Vater, Leonhard Möckl, Vanessa Gormanns, Carsten Schultz Fademrecht, Anna M. Mallmann, Karolina Ziegart-Sadowska, Monika Zaba, Marie L. Frevert, Christoph Braeuchle, et al.

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.

The Endothelial Glycocalyx Controls Interactions of Quantum Dots with the Endothelium and Their Translocation across the Blood-Tissue Border

Bernd Uhl, Stephanie Hirn, Roland Immler, Karina Mildner, Leonhard Möckl, Markus Sperandio, Christoph Braeuchle, Christoph A. Reichel, Dagmar Zeuschner, et al.

Advances in the engineering of nanoparticles (NPs), which represent particles of less than 100 nm in one external dimension, led to an increasing utilization of nanomaterials for biomedical purposes. A prerequisite for their use in diagnostic and therapeutic applications, however, is the targeted delivery to the site of injury. Interactions between blood-borne NPs and the vascular endothelium represent a critical step for nanoparticle delivery into diseased tissue. Here, we show that the endothelial glycocalyx, which constitutes a glycoprotein polysaccharide meshwork coating the luminal surface of vessels, effectively controls interactions of carboxyl-functionalized quantum dots with the micro vascular endothelium. Glycosaminoglycans of the endothelial glycocalyx were found to physically cover endothelial adhesion and signaling molecules, thereby preventing endothelial attachment, uptake, and translocation of these nanoparticles through different layers of the vessel wall. Conversely, degradation of the endothelial glycocalyx promoted interactions of these nanoparticles with microvascular endothelial cells under the pathologic condition of ischemia-reperfusion, thus identifying the injured endothelial glycocalyx as an essential element of the blood-tissue border facilitating the targeted delivery of nanomaterials to diseased tissue.

The glycocalyx regulates the uptake of nanoparticles by human endothelial cells in vitro

Leonhard Möckl, Stephanie Hirn, Adriano A. Torrano, Bernd Uhl, Christoph Braeuchle, Fritz Krombach

Aim: To assess the role of the endothelial glycocalyx (eGCX) for the uptake of nanoparticles by endothelial cells. Methods: The expression of the eGCX on cultured human umbilical vein endothelial cells was determined by immunostaining of heparan sulfate. Enzymatic degradation of the eGCX was achieved by incubating the cells with eGCX-shedding enzymes. The uptake of 50-nm polystyrene nanospheres was quantified by confocal microscopy. Results: Human umbilical vein endothelial cells expressed a robust eGCX when cultured for 10 days. The uptake of both carboxylated and aminated polystyrene nanospheres was significantly increased in cells in which the glycocalyx was enzymatically degraded, while it remained at a low level in cells with an intact glycocalyx. Conclusion: The eGCX constitutes a barrier against the internalization of blood-borne nanoparticles by endothelial cells.

More Than 50 Years after Its Discovery in SiO2 Octahedral Coordination Has Also Been Established in SiS2 at High Pressure

Juergen Evers, Leonhard Möckl, Gilbert Oehlinger, Ralf Koeppe, Hansgeorg Schnoeckel, Oleg Barkalov, Sergey Medvedev, Pavel Naumov

SiO2 exhibits a high-pressure high-temperature polymorphism, leading to an increase in silicon coordination number and density. However, for the related compound SiS2 such pressure-induced behavior has not been observed with tetrahedral coordination yet. All four crystal structures of SiS2 known so far contain silicon with tetrahedral coordination. In the orthorhombic, ambient-pressure phase these tetrahedra share edges and achieve only low space filling and density. Up to 4 GPa and 1473 K, three phases can be quenched as metastable phases from high-pressure high-temperature to ambient conditions. Space occupancy and density are increased first by edge and corner sharing and then by comer sharing alone. The structural situation of SiS2 up to the current study resembles that of SiO2 in 1960: Then, in its polymorphs only Si-O-4 tetrahedra were known. But in 1961, a polymorph with rutile structure was discovered: octahedral Si-O-6 coordination was established. Now, 50 years later, we report here on the transition from 4 fold to 6-fold coordination in SiS2, the sulfur analogue of silica.

100 Jahre Einkristallzucht aus der Schmelze: Vom Spreeknie ins Silicon Valley

Jürgen Evers, Christiane Herzog, Leonhard Möckl, Christoph von Plotho, Peter Stallhofer, Rudolf Staudigl

Im August 1916 reichte der Deutsch‐Pole Jan Czochralski eine Publikation über die Wachstumsgeschwindigkeit von Metallkristallen bei der Zeitschrift für physikalische Chemie ein. Daher wird 1916 als das Jahr der Entdeckung des Czochralski‐Kristallzuchtverfahrens angesehen. Im Metall‐Laboratorium der AEG, wo Czochralski zunächst arbeitete, wurden seine Forschungsarbeiten nicht gebührend anerkannt, sodass er zur Metallbank und Metallurgischen Gesellschaft (später: Metallgesellschaft) nach Frankfurt wechselte, wo er bald zum Laborleiter und Oberingenieur aufstieg. In Frankfurt machte er sich mit Forschungen zu Metallen und technischen Legierungen rasch einen Namen. Die Entdeckung des Czochralski‐Kristallzuchtverfahrens gehört zu den wichtigsten technologischen Erfindungen des 20. Jahrhunderts, die aber erst in dessen zweiten Hälfte mit dem Aufstieg der Halbleiterindustrie ökonomische Bedeutung erlangte. Heute werden 95 % der Weltproduktion an Siliciumeinkristallen nach dem Czochralski‐Verfahren hergestellt. Der Umsatz der Halbleiter‐Industrie betrug 2015 etwa 335 Milliarden US‐Dollar, wobei die Kristallzucht nach dem Czochralski‐Verfahren jeweils der erste Schritt bei der Herstellung ist. Der größte Teil der heute gefertigten Siliciumeinkristalle hat einen Durchmesser von 300 mm. Die industrielle Produktion von Einkristallen mit 450 mm Durchmesser scheitert bisher nicht an technologischen, sondern an ökonomischen Problemen.

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

Leonhard Möckl, Claudia Fessele, Guillaume Despras, Christoph Braeuchle, Thisbe K. Lindhorst

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors. (C) 2016 Elsevier B.V. All rights reserved.

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow General subjects

Leonhard Möckl, Claudia Fessele, Guillaume Despras, Christoph Bräuchle, Thisbe K. Lindhorst

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.

Inside Cover: Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study (ChemPhysChem 6/2016)

Leonhard Möckl, Thisbe K. Lindhorst, Christoph Bräuchle

The trajectories of membrane proteins, which are artificially interconnected by biotin and streptavidin, are reminiscent of the work of action painters. This provided the inspiration to think of the different membrane biotin concentrations as colors that can be used to paint various networks onto cells by using the streptavidin brush. More information can be found in the Full Paper by C. Bräuchle et al. on page 829 in Issue 6, 2016 (DOI: 10.1002/cphc.201500809)

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study

Leonhard Möckl, Thisbe K. Lindhorst, Christoph Braeuchle

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.

Switching first contact: photocontrol of E. coli adhesion to human cells

Leonhard Möckl, A. Mueller, C. Braeuchle, T. K. Lindhorst

We have shown previously that carbohydrate-specific bacterial adhesion to a non-physiological surface can be photocontrolled by reversible E/Z isomerisation using azobenzene-functionalised sugars. Here, this approach is applied to the surface of human cells. We show not only that bacterial adhesion to the azobenzene glycoside-modified cell surface is higher in the E than in the Z state, but add data about the specific modulation of the effect.

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins

Leonhard Möckl, Andrea K. Horst, Katharina Kolbe, Thisbe K. Lindhorst, Christoph Braeuchle

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.

Inside Cover: Microdomain Formation Controls Spatiotemporal Dynamics of Cell‐Surface Glycoproteins (ChemBioChem 14/2015)

Leonhard Möckl, Andrea K. Horst, Katharina Kolbe, Thisbe Lindhorst, Christoph Bräuchle

The inside cover picture shows a metaphoric representation of the key idea of our paper. We tagged membrane glycoproteins by metabolic labeling, interconnected them in a physiological or artificial way, and employed high-resolution fluorescence microscopy to investigate the effects of this network formation on the spatiotemporal dynamics of the interconnected membrane glycoproteins. More details can be found in the Full Paper by T. K. Lindhorst, C. Bräuchle et al. on page 2023 in Issue 14, 2015. (DOI: 10.1002/cbic.201500361).

Wichard von Moellendorff: Mit logischer Schärfe und systematischer Unbeugsamkeit

Jürgen Evers, Leonhard Möckl

Untersuchungen über die Vorgänge, die beim Verformen von Metallen und Legierungen ablaufen, waren im ersten Drittel des 20. Jahrhunderts zentrales Forschungsthema. Wichard von Moellendorff baute dazu im Kabelwerk Oberspree (KWO) der “Allgemeinen Elektricitätsgesellschat” (AEG) ein neues Industrie‐Labor auf. So konnte er sich mit systematischen metallographischen und mechanischen Untersuchungen an der wissenschaftlichen Auseinandersetzung über die Verformungsvorgänge beteiligen. 1913 leitete er zusammen mit Czochralski erste Vorstellungen über die Auswirkungen der Kristallinität auf die Verformungvorgänge ab. Seine Deutungen waren zunächst im Widerspruch zu gängigen wissenschaftlichen Auffassungen, setzten sich dann aber als richtig durch. Nach einer Auszeit während des Ersten Weltkriegs setzte er diese Forschung als Direktor des Staatlichen Materialprüfungsamtes und gleichzeitig des Kaiser‐Wilhelm‐Instituts für Metallforschung in Berlin‐Dahlem fort und untersuchte die Form der Fließkegel, die beim mechanischen Zerreißen von Metallstäben entstehen. Diese Vorgänge ließen sich 1929 durch “Drehung, Verzerrung und Gleitung kristallographischer Gleitebenen” deuten. Polany befasste sich ab 1923 mit röntgenographischen und mechanischen Untersuchungen der Verformungsvorgänge. 1932 gelang ihm der wissenschaftliche Durchbruch über einen Versetzungsmechanismus. Demnach werden einzelne Atome auf Zwischenpositionen verschoben und bewegen sich dann schrittweise durch den Kristall. 1933 verließ Polanyi wegen seiner jüdischen Abstammung Nazi‐Deutschland; Moellendorff gehört zu den wenigen der deutschen wissenschaftlichen und technischen Elite, die sich mutig dem Nazi‐Regime entgegengestellten.

Two High-Pressure Phases of SiS2 as Missing Links between the Extremes of Only Edge-Sharing and Only Corner-Sharing Tetrahedra

Juergen Evers, Peter Mayer, Leonhard Möckl, Gilbert Oehlinger, Ralf Koeppe, Hansgeorg Schnoeckel

The ambient pressure phase of silicon disulfide (NP-SiS2), published in 1935, is orthorhombic and contains chains of distorted, edge-sharing SiS4 tetrahedra. The first high pressure phase, HP3-SiS2, published in 1965 and quenchable to ambient conditions, is tetragonal and contains distorted corner-sharing SiS4 tetrahedra. Here, we report on the crystal structures of two monoclinic phases, HP1-SiS2 and HP2-SiS2, which can be considered as missing links between the orthorhombic and the tetragonal phase. Both monoclinic phases contain edge- as well as corner-sharing SiS4 tetrahedra. With increasing pressure, the volume contraction (-Delta V/V) and the density, compared to the orthorhombic NP-phase, increase from only edge-sharing tetrahedra to only corner-sharing tetrahedra. The lattice and the positional parameters of NP-SiS2, HP1-SiS2, HP2-SiS2, and HP3-SiS2 were derived in good agreement with the experimental data from group-subgroup relationships with the CaF2 structure as aristotype. In addition, the Raman spectra of SiS2 show that the most intense bands of the new phases HP1-SiS2 and HP2-SiS2 (408 and 404 cm(-1), respectively) lie between those of NP-SiS2 (434 cm(-1)) and HP3-SiS2 (324 cm(-1)). Density functional theory (DFT) calculations confirm these observations.

Dendritische Nanostrukturen zur rezeptorvermittelten Aufnahme von siRNA in neurale Zellen

Korbinian Brunner, Johannes Harder, Tobias Halbach, Julian Willibald, Fabio Spada, Felix Gnerlich, Konstantin Sparrer, Andreas Beil, Leonhard Möckl, et al.

Wir berichten über die Entwicklung von dendritischen siRNA‐Nanostrukturen, die das Einbringen von siRNA in schwierig zu transfizierende Zellen wie Neuronen mithilfe eines speziellen Rezeptorliganden ermöglichen. Die Nanopartikel rufen trotz ihrer dendritischen Struktur eine starke siRNA‐Antwort hervor. Durch siRNA‐Dendrimere gegen das Nukleoprotein (N‐Protein) und das Phosphoprotein (P‐Protein), zwei Schlüsselproteine des Tollwutvirus (RABV), konnte der Virustiter in Neuronen unter die Nachweisgrenze gesenkt werden. Die mithilfe von Klick‐Chemie aufgebauten siRNA‐Dendrimere weisen den Weg für eine neuartige Heilungsmöglichkeit dieser tödlichen Krankheit.

Cell-Penetrating and Neurotargeting Dendritic siRNA Nanostructures

Korbinian Brunner, Johannes Harder, Tobias Halbach, Julian Willibald, Fabio Spada, Felix Gnerlich, Konstantin Sparrer, Andreas Beil, Leonhard Möckl, et al.

We report the development of dendritic siRNA nanostructures that are able to penetrate even difficult to transfect cells such as neurons with the help of a special receptor ligand. The nanoparticles elicit strong siRNA responses, despite the dendritic structure. An siRNA dendrimer directed against the crucial rabies virus (RABV) nucleoprotein (Nprotein) and phosphoprotein (Pprotein) allowed the suppression of the virus titer in neurons below the detection limit. The cell-penetrating siRNA dendrimers, which were assembled using click chemistry, open up new avenues toward finding novel molecules able to cure this deadly disease.

Super-resolved Fluorescence Microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William E. Moerner

Leonhard Möckl, Don C. Lamb, Christoph Braeuchle

A big honor for small objects: The Nobel Prize in Chemistry 2014 was jointly awarded to Eric Betzig, Stefan Hell, and William E. Moerner “for the development of super‐resolved fluorescence microscopy”. This Highlight describes how the field of super‐resolution microscopy developed from the first detection of a single molecule in 1989 to the sophisticated techniques of today.

Der Wittelsbacher und der Hope‐Diamant: Optische Untersuchungen klären ein Rätsel

Jürgen Evers, Leonhard Möckl, Heinrich Nöth

Diamanten entstehen aus Kohlenstoff bei hohen Drücken und hohen Temperaturen im Erdinneren. Dotierung mit geringen Mengen Bor erzeugt ihre blaue Farbe. Zwei der berühmtesten blauen historischen Diamanten, der Wittelsbacher und der Hope‐Diamant, stammen aus der indischen Kollur‐Mine. Letzteren brachte der französische Diamantenhändler Tavernier im 17. Jahrhundert nach Europa. Heute ist er im Smithsonian Institute in Washington ausgestellt. Der Wittelsbacher Diamant befand sich lange Zeit im Besitz des Hauses Wittelsbach, bis er 1951 heimlich in Antwerpen verkauft wurde. 2008 ersteigerte ihn der Juwelier Graff und ließ ihn umschleifen. 2011 wurde er an einen unbekannten Besitzer weiterverkauft. Aufgrund der gemeinsamen Herkunft und Farbe der Diamanten wurde lange Zeit vermutet, die beiden Edelsteine könnten Bruchstücke eines gemeinsamen größeren Kristalles sein. Durch optische Untersuchungen konnte nun eindeutig geklärt werden, dass beide Diamanten zwar ähnliche optische Eigenschaften besitzen, in bestimmten Merkmalen jedoch so stark voneinander abweichen, dass sie nicht einem gemeinsamen größeren Kristall entstammen können.

Optical Investigations to clear up a Mystery The Wittelsbach and the Hope Diamond

Juergen Evers, Leonhard Möckl, Heinrich Noeth

Diamonds are formed from carbon at high pressures and high temperatures in the inner part of the earth. Doping with very small amounts of boron leads to diamonds with blue colour. Two of the most famous historical blue diamonds, the Wittelsbach and Hope Diamond, were found in the Indian Kollur mine. The latter was brought to Europe by the French gem merchant Tavernier. Today it is displayed in the Smithsonian Institute. The Wittelsbach Diamond was for a long time in the possession of the House Wittelsbach until it was secretly sold in Antwerp in 1951. In 2008, it was purchased by auction by the jeweller Graff who recut the gem. In 2011, it was sold to an unknown buyer. As the Wittelsbach and the Hope diamond share origin and colour, it was assumed for a long time that both are pieces from a larger crystal. By optical investigation it was now shown that they have indeed some similar optical properties, but differ strikingly in other ones. Hence, they cannot originate from the same crystal.

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

Frauke M. Mickler, Leonhard Möckl, Nadia Ruthardt, Manfred Ogris, Ernst Wagner, Christoph Braeuchle

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.