Untersuchung des Einflusses von Rab-GTPasen und Endozytose-Inhibitoren auf die Zellbiologie und Zellschicksalsspezifikation von hämatopoetischen Stamm- und Vorläuferzellen

Ein wichtiger Aspekt der Stammzellbiologie ist es zu verstehen, welche Mechanismen an der Zellschicksalsentscheidung Selbsterhalt oder Differenzierung von Stammzellen beteiligt sind. Vorarbeiten unserer Arbeitsgruppe konnten zeigen, dass hämatopoetische Stamm- und Vorläuferzellen (HSVZ) in der Lage sind sich asymmetrisch zu teilen (Beckmann et al., 2007). Dabei segregierten drei Proteine die mit Endosomen assoziiert waren asymmetrisch in die Tochterzellen, was zu der Vermutung führte, dass eine Verbindung zwischen der endosomalen Maschinerie und Zellschicksalsentscheidungen von HSVZ besteht. Rab-GTPasen sind Hauptregulatoren endosomaler Transportwege und endozytotische Prozesse lassen sich durch verschiedene Inhibitoren blockieren. Durch ektope Expression von wildtypischen, konstitutiv-aktiven und dominant-negativen Varianten von Rab5, Rab7, Rab11 und Rab21 wurde der Einfluss endosomaler Transportwege auf die Zellbiologie und Zellschicksalsspezifikation von HSVZ untersucht. Zusätzlich zu der genetischen Manipulation der endosomalen Wege, wurde die Wirkung von Endozytoseinhibitoren auf die Zellbiologie und das Zellschicksal analysiert. Ähnlich wie in anderen zellulären Systemen führte die Expression der Rab-GTPase Varianten (WT, DN und CA) zu teils drastischen Veränderungen der subzellulären Organisation von endosomalen Kompartimenten. Vor allem zeigte die Expression von Rab5CA eine abnormale Vergrößerung der Endosomen und Reorganisation von späten endosomalen Markerproteinen. Darüber hinaus, zeigte die Expression von Rab11DN eine Verminderung der Ausbildung morphologisch polarisierter CD34+ Zellen. Eine Beeinträchtigung der Motilität durch Rab11DN oder eine andere Rab-GTPase-Variante war nicht erkennbar. Die Behandlung mit Endozytose-Inhibitoren resultierte in einer gestörten Endozytose von Rezeptorliganden und verhinderte teilweise die Ausbildung und den Erhalt der Zellpolarität von CD34+ Zellen. Funktionelle Analysen von genetisch und pharmazeutisch manipulierten CD34+ Zellen in CFC-Ansätzen zeigten, dass endosmale Prozesse, die durch Rab5WT, Rab5DN, Rab7DN und Rab21DN sowie die Inhibitoren Dynasore, MβCD und Spautin-1 verändert werden, das erythrozytäre-myeloische Linienpotenzial der CD34+ Zellen beeinflussen. Rab5DN und Rab21DN sowie Dynasore, MβCD und Spautin-1 verminderten die Bildung von Makrophagen (CFU-G bzw. CFU-GM), Granulozyten (CFU-G bzw. CFU-GM) und Erythrozyten enthaltenen Kolonien (BFU-E und CFU-Mix). Die Effekte von Rab5DN und Rab21DN auf die spezifischen Kolonietypen fanden sich in beiden analysierten Zellpopulationen (CD133+CD34+ und CD133lowCD34+) der CD34+Zellen und verminderten zudem das gesamte Koloniebildungspotenzial der Zellen. Rab5WT und Rab7DN förderten die Bildung von erythrozytären Kolonien (BFU-E und CFU-Mix) innerhalb der CD133lowCD34+ Zellpopulation. Durch die Verwendung von erythrozytären und megakaryozytären Differenzierungsverfahren konnten die fördernden Effekte von Rab5WT und Rab7DN auf die erythrozytäre und megakaryozytäre Differenzierung von CD133lowCD34+ Zellen bestätigt werden.An important aspect of stem cell biology is to understand the mechanisms controlling the decision of self-renewal versus differentiation. Previously, we confirmed the long lasting conjecture that human hematopoietic stem and progenitor cells (HSPCs) have the capability to divide asymmetrically. In our studies we demonstrated that in a proportion of dividing HSPCs endosomal proteins segregate differently into arising daughter cells, pointing towards a relationship between organization of the endosomal compartment and cell fate specification processes in HSPCs. Rab-GTPases are key regulators of endosomal trafficking and endocytic pathways can be interfered with different inhibitors. By means of ectopic expression, we analyzed the impacts of wild type (WT), constitutive active (CA) and dominant-negative (DN) Rab5, Rab7, Rab11 and Rab21 variants on the development of human HSPCs (umbilical cord blood (UCB) derived CD34+ cells). In addition impacts of different endocytic inhibitors were investigate on cell fate specification of HSPCs. According to other cellular systems expression of Rab-GTPase variants (WT, DN and CA) was found to interfere partly drastically with the organization of the endosomal compartment. Especially, Rab5CA expression resulted in the formation of giant endosomes, which contain early as well as late endosomal marker proteins. In addition, expression of Rab11DN results in a reduction of the formation of polarized migration phenotypes. However, we did not recognize any impact on cell motility by this or any other analyzed Rab-GTPases. Treatment of CD34+ cells with endocytic inhibitors result in impaired endocytosis of receptor ligands and affected establishment and maintenance of cell polarization. In contrast, CFC-assays revealed impacts of Rab5WT, Rab5DN, Rab7DN and Rab21DN as well as of the inhibitors Dynasore, MβCD and Spautin-1 on the erythro-myeloid lineage potential of CD34+ cells. Here, Rab5DN and Rab21DN as well as Dynasore, MβCD and Spautin-1 impaired colony formation potential of Macrophage (CFU-G/CFU-GM), granulocytic (CFU-G/CFU-GM) and erythroid colonies (BFU-E and CFU-Mix). Effects of Rab5DN and Rab21DN on the different colony types were detected in both analyzed cell populations (CD133+CD34+ und CD133lowCD34+) of CD34+ cells and decreased the total colony formation potential of the cells. Rab5WT and Rab7DN enhance erythroid colony formation (BFU-E und CFU-Mix). Using special erythroid and megakaryocytic read out systems, we confirmed that Rab5WT and Rab7DN promote erythroid and megakaryocytic lineage differentiation

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic a-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic a-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 a-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of a-dystroglycanopathy

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling

Tissue lectins are emerging (patho) physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N-and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated beta-galactosides. This protein is turned into a new homodimer, i. e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) beta-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N

Influence of protein (human galectin-3) design on aspects of lectin activity

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3′-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity

Chemokines and galectins form heterodimers to modulate inflammation

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted

ESPAD Report 2019: Results From European School Survey Project on Alcohol and Other Drugs

The main purpose of the European School Survey Project on Alcohol and Other Drugs (ESPAD) is to collect comparable data on substance use and other forms of risk behaviour among 15- to 16-year-old students in order to monitor trends within, as well as between, countries. Between 1995 and 2019, seven waves of data collection were conducted across 49 European countries. This report presents selected key results. The full set of data on which the current report is based, including all of the standard tables, is available online (http://www.espad.org). All tables can be downloaded in Excel format and used for further analysi

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs

Survey of Third-Party Parenting Options Associated With Fertility Preservation Available to Patients With Cancer Around the Globe

Purpose: In the accompanying article, “Analysis of Fertility Preservation Options Available to Patients With Cancer Around the Globe,” we showed that specific fertility preservation services may not be offered at various sites around the world because of cultural and legal barriers. We assessed global and regional experiences as well as the legal status of third-party reproduction and adoption to serve as a comprehensive international data set and resource for groups that wish to begin oncofertility interventions. Methods: We provide data on the legalities of third-party assisted reproductive technologies and other family-building options in the 28 oncofertility-practicing countries surveyed. Results: We found regional and country differences that will be important in the development of tailored resources for physicians and for patient brochures that are sensitive to these local restrictions and cultural norms. Conclusion: Because many patients first consult Web-based materials, the formal assessment of the availability of these options provides members of the global oncofertility community with data to which they might otherwise not have ready access to better serve their patients

Survey of third-party parenting options associated with fertility preservation available to patients with cancer around the globe

bstract PURPOSE In the accompanying article, “Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe,” we showed that specific fertility preservation services may not be offered at various sites around the world because of cultural and legal barriers. We assessed global and regional experiences as well as the legal status of third-party reproduction and adoption to serve as a comprehensive international data set and resource for groups that wish to begin oncofertility interventions. METHODS We provide data on the legalities of third-party assisted reproductive technologies and other familybuilding options in the 28 oncofertility-practicing countries surveyed. RESULTS We found regional and country differences that will be important in the development of tailored resources for physicians and for patient brochures that are sensitive to these local restrictions and cultural norms. CONCLUSION Because many patients first consult Web-based materials, the formal assessment of the availability of these options provides members of the global oncofertility community with data to which they might otherwise not have ready access to better serve their patients