Improved biocatalytic cascade conversion of CO2 to methanol by enzymes Co-immobilized in tailored siliceous mesostructured cellular foams

CO2 can be enzymatically reduced to methanol in a cascade reaction involving three enzymes: formate-, formaldehyde- and alcohol dehydrogenase (FateDH, FaldDH, ADH). We report an improvement in the yield of this reaction by co-immobilizing the three dehydrogenases in siliceous mesostructured cellular foams (MCF). This material consists of large mesopores suitable for the co-immobilization of these comparatively large enzymes. To improve the interaction between the enzymes and support, the host silica material was functionalized with mercaptopropyl groups (MCF-MP). The enzymes were fluorescently labelled to independently monitor their uptake and spatial distribution into the particle. The three dehydrogenases were co-immobilized using two sequential methods. In the first one, the enzymes were immobilized according to the reaction order (FateDH -> FaldDH -> ADH) and secondly in order of increasing enzyme size (FateDH -> ADH -> FaldDH). Two protein loadings were also tested: 50 and 150 mg(enzymes) g(support)(-1). We could observe a 4.5-fold higher methanol yield in comparison to enzymes free in solution when the enzymes were immobilized in order of size and with a loading of 50 mg(enzymes) g(support)(-1). The results of this work show that by using MCF-MP, a simple method of immobilization can be applied to significantly increase the activity of the enzymes for the cascade reaction

Platelet lamellipodium formation is not required for thrombus formation and stability

During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development

Hochauflösende Mikroskopie von Plasmamembran Rezeptoren

Plasma membrane receptors are the most crucial and most commonly studied components of cells, since they not only ensure communication between the extracellular space and cells, but are also responsible for the regulation of cell cycle and cell division. The composition of the surface receptors, the so-called "Receptome", differs and is characteristic for certain cell types. Due to their significance, receptors have been important target structures for diagnostic and therapy in cancer medicine and often show aberrant expression patterns in various cancers compared to healthy cells. However, these aberrations can also be exploited and targeted by different medical approaches, as in the case of personalized immunotherapy. In addition, advances in modern fluorescence microscopy by so-called single molecule techniques allow for unprecedented sensitive visualization and quantification of molecules with an attainable spatial resolution of 10-20 nm, allowing for the detection of both stoichiometric and expression density differences. In this work, the single molecule sensitive method dSTORM was applied to quantify the receptor composition of various cell lines as well as in primary samples obtained from patients with hematologic malignancies. The focus of this work lies on artefact free quantification, stoichiometric analyses of oligomerization states and co localization analyses of membrane receptors. Basic requirements for the quantification of receptors are dyes with good photoswitching properties and labels that specifically mark the target structure without generating background through non-specific binding. To ensure this, antibodies with a predefined DOL (degree of labeling) were used, which are also standard in flow cytometry. First background reduction protocols were established on cell lines prior analyses in primary patient samples. Quantitative analyses showed clear expression differences between the cell lines and the patient cells, but also between individual patients. An important component of this work is the ability to detect the oligomerization states of receptors, which enables a more accurate quantification of membrane receptor densities compared to standard flow cytometry. It also provides information about the activation of a certain receptor, for example of FLT3, a tyrosine kinase, dimerizing upon activation. For this purpose, different well-known monomers and dimers were compared to distinguish the typical localization statistics of single bound antibodies from two or more antibodies that are in proximity. Further experiments as well as co localization analyses proved that antibodies can bind to closely adjacent epitopes despite their size. These analytical methods were subsequently applied for quantification and visualization of receptors in two clinically relevant examples. Firstly, various therapeutically relevant receptors such as CD38, BCMA and SLAMF7 for multiple myeloma, a malignant disease of plasma cells, were analyzed and quantified on patient cells. Furthermore, the influence of TP53 and KRAS mutations on receptor expression levels was investigated using the multiple myeloma cell lines OPM2 and AMO1, showing clear differences in certain receptor quantities. Secondly, FLT3 which is a therapeutic target receptor for acute myeloid leukemia, was quantified and stoichiometrically analyzed on both cell lines and patient cells. In addition, cells that have developed resistance against midostaurin were compared with cells that still respond to this type I tyrosine-kinase-inhibitor for their FLT3 receptor expression and oligomerization state.Plasmamembranrezeptoren sind die wohl wichtigsten und meist untersuchten Komponenten einer Zelle, da sie nicht nur die Kommunikation zwischen dem extrazellulären Bereich und den Zellen gewährleisten, sondern auch für die Regulierung des Zellzyklus und der Zellteilung zuständig sind. Dabei unterscheidet sich die Zusammensetzung der Oberflächenrezeptoren, das sogenannte „Rezeptom“, und ist charakteristisch für bestimme Zelltypen. Aufgrund ihrer Bedeutsamkeit sind Rezeptoren wichtige Zielstrukturen für Diagnose und Therapie in der Krebsmedizin, welche häufig bei verschiedensten Krebserkrankungen im Vergleich zu gesunden Zellen aberrante Expressionsmuster aufweisen. Diese Abweichungen können sich allerdings auch zu Nutze gemacht werden und zum Ziel verschiedener medizinischer Behandlungsmethoden, wie es bei der personalisierten Immuntherapie der Fall ist, werden. Zusätzlich hat der Fortschritt in der modernen Fluoreszenzmikroskopie durch sogenannte Einzelmolekültechniken, es auch erlaubt, eine noch nie dagewesene empfindliche Visualisierung und Quantifizierung von Molekülen mit einer räumlichen Auflösung von 10-20 nm zu erreichen, wodurch sowohl stöchiometrische Unterschiede, als auch Unterschiede in der Expressionsdichte detektiert werden können. In dieser Arbeit wurde die einzelmolekülsensitive Methode dSTORM genutzt, um die Rezeptorkomposition von verschiedenen Zelllinien aber auch von primären Patientenzellen mit zugrundeliegenden hämatologischen Erkrankungen zu quantifizieren. Schwerpunkte dieser Arbeit sind dabei die artefaktfreie Quantifizierung, stöchiometrische Analysen von Oligomerisierungszuständen, sowie die Kolokalisationsanalyse von Membranrezeptoren. Grundvoraussetzung für die Quantifizierung von Rezeptoren sind dabei gut schaltbare Farbstoffe, sowie Label, welche die Zielstruktur spezifisch markieren ohne dabei Hintergrund durch unspezifische Bindung zu generieren. Um dies zu gewährleisten, kamen Antikörper mit einem vordefinierten DOL (degree of labeling; engl. für: Markierungsgrad) zum Einsatz, welche auch in der Durchflusszytometrie standardmäßig eingesetzt werden. Protokolle zur Hintergrundreduktion wurden dabei an Zelllinien etabliert, bevor Primärzellen von Krebspatienten analysiert wurden. Durch quantitative Analysen konnten dabei deutliche Expressionsunterschiede zwischen den Zelllinien und den Patientenzellen, aber auch zwischen den verschiedenen Patienten gezeigt werden. Ein wichtiger Bestandteil dieser Arbeit ist die Fähigkeit, den Oligomerisierungszustand von Rezeptoren zu erkennen, was eine genauere Quantifizierung der Membran-rezeptordichten im Vergleich zur Durchflusszytometrie ermöglicht. Allerdings können diese Oligomerisierungszustände auch Informationen über die Aktivierung eines Rezeptors beinhalten, wie zum Beispiel von FLT3, einer Tyrosinkinase, welche zur Aktivierung dimerisieren muss. Hierfür wurden verschiedene bekannte Monomere und Dimere verglichen, um die typische Lokalisationsstatistik von vereinzelten gebundenen Antikörpern mit der von zwei oder mehr Antikörpern, welche nah beieinanderliegen, zu vergleichen. Durch weitere Etablierungsexperimente sowie Kolokalisationsanalysen konnte außerdem bewiesen werden, dass Antikörper trotz ihrer Größe auch an nah benachbarte Epitope binden können. Diese Analyseverfahren wurden im weiteren Verlauf zur Quantifizierung und Visualisierung von Rezeptoren an zwei klinisch relevanten Beispielen angewendet. Zum einen wurden verschiedene therapeutisch relevante Rezeptoren wie z.B. CD38, BCMA und SLAMF7 für das Multiple Myelom, einer malignen Erkrankung von Plasmazellen, auf Patientenzellen analysiert und quantifiziert. Zusätzlich wurde der Einfluss von TP53 und KRAS Mutationen auf die Rezeptorexpressionen anhand der Multiplen Myelom Zelllinien OPM2 und AMO1 untersucht, bei denen eindeutige Unterschiede in der Rezeptorexpression detektiert wurden. Zum anderen wurde FLT3, welches ein therapeutischer Zielrezeptor für die akute myeloische Leukämie ist, sowohl auf Zelllinien als auch auf Patientenzellen quantifiziert und stöchiometrisch analysiert. Hierbei wurden auch Zellen welche eine Midostaurinresistenz entwickelt haben mit Zellen, welche auf diesen Typ I Tyrosinkinase Inhibitor ansprechen, auf ihre FLT3 Rezeptorexpression und ihren Oligomerisierungszustand verglichen

Glucose and inositol transporters, SLC5A1 and SLC5A3, in glioblastoma cell migration

(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment

Targetable Conformationally Restricted Cyanines Enable Photon-Count Limited Applications

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to parent species in a range of contexts including their use on a DNA-nano-antenna, in single-molecule Förster resonance energy transfer (FRET) applications, far-red fluorescence lifetime imaging microscopy (FLIM), and single-molecule localization microscopy. These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance

Targetable conformationally restricted cyanines enable photon-count-limited applications

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance