Proteomanalyse neuronaler Stammzellen

In bestimmten Regionen des Gehirns von Säugetieren und somit auch des Menschen entstehen zeitlebens neue Zellen, die sich zu Nervenzellen und Gliazellen entwickeln. Die neu entstandenen Zellen scheinen sich entsprechend ihrer physiologischen Funktion in das Gewebe zu integrieren und elektrisch aktiv zu sein. Die Ursprungszellen werden als neuronale Stammzellen bezeichnet; sie können ihre Stammzelleigenschaften über die gesamte Lebensspanne des Organismus beibehalten. Zu diesen Eigenschaften gehören Teilungsfähigkeit, Selbsterneuerung und die Fähigkeit, sich zu Gehirnzellen zu differenzieren. Die Signalwege, die zur Aufrechterhaltung der Stammzelleigenschaften aktiviert werden, sind bislang unbekannt. Wir haben die Proteomanalyse basierend auf der zweidimensionalen Gelelektrophorese in Kombination mit der Massenspektrometrie dazu eingesetzt, zum einen das Proteom neuronaler Stammzellen zu erfassen (Proteomprofilierung) und zum anderen die Veränderungen des Proteoms unter Differenzierungsvorgängen zu untersuchen (funktionelle Proteomanalyse). Dazu haben wir neuronale Stammzellen aus drei Regionen des Gehirns ausgewachsener Ratten isoliert, in denen spontane Neurogenese beschrieben wurde, nämlich dem Hippokampus, der Subventrikulärzone -einer Wachstumszone entlang der Seitenventrikel- und dem Bulbus olfaktorius. Die Stammzellen haben wir über sechs Wochen in Kultur vermehrt und Proteinlysate angefertigt. Um das Proteom der in vitro differenzierten Zellen mit demjenigen undifferenzierter Zellen vergleichen zu können, haben wir ein Protokoll zur in vitro Differenzierung der neuronalen Stammzellen entwickelt. Im Rahmen der Proteinprofilierung konnten wir in undifferenzierten neuronalen Stammzellen etwa 2500 Proteinspots anfärben. Nicht jeder dieser Spots entspricht einem einzelnen Protein; durch das Vorhandensein von Proteinisoformen und posttranslationalen Modifikationen ist die Zahl der Proteine geringer. Im Rahmen der funktionellen Proteomanalyse haben wir die Veränderungen der Proteinzusammensetzung der Stammzellen während der Differenzierungsvorgänge der Stammzellen gemessen. Die Stammzellen durchlaufen hierbei eine Reihe tiefgreifender morphologischer und funktioneller Veränderungen, die sich auch in der Veränderung der Proteinzusammensetzung widerspiegeln. Wir fanden nach Differenzierung mehr als 350 Proteinspots mit veränderter Proteinkonzentration, die wir zahlreichen regulierten Signal- und Stoffwechselwegen zuordnen konnten. Die grössten Veränderungen waren im Zytoskelet und dem zellulären Stoffwechsel nachzuweisen, aber auch in transkriptionsregulierenden Proteinen und Protein-Faltungshilfen, den sogenannten molekularen Chaperonen. Aufgrund der Proteomanalysen erhielten wir Hinweise, dass der Wnt-Signalweg ein Signalweg ist, der die Eigenschaften neuronaler Stammzellen aufrechterhält. Der Wnt-Signalweg wurde bisher in anderen Zellen als einer der verantwortlichen Signalwege bei der Regulation von Zellwachstum und -größe, der Zellorientierung im Raum und der Transkriptionskontrolle beschrieben. Durch ergänzende biochemische Verfahren wie z. B. Western Blot, Immunzytochemie und Polymerase-Kettenreaktion und Gabe des Inhibitors Genistein konnten wir nachweisen, dass die Aktivierung dieses Signalwegs wesentlich für die Differenzierung neuronaler Stammzellen in Nervenzellen und Gliazellen ist und gleichzeitig die Selbsterneuerung und Teilungsfähigkeit neuronaler Stammzellen unterbrechen kann. Zusammenfassend konnten wir mit Hilfe der zweidimensionalen Gel-Elektrophorese in Kombination mit der Massenspektrometrie durch Proteomprofilierung ein Proteininventar von etwa 2500 Proteinen adulter neuronaler Stammzellen erstellen, die aus den neurogenen Zonen des Rattenhirns isoliert wurden, nämlich aus Hippokampus, Subventrikulärzone und Bulbus olfaktorius. Die so entstandene Proteomdatenbank haben wir über das Internet zugänglich gemacht und damit den Grundstein für weitere Vergleiche des Proteoms neuronaler Stammzellen mit dem anderer (Stamm-) Zelltypen eröffnet. Gleichzeitig konnten wir statistische und grafische Werkzeuge bereitstellen, die zu einer weiteren Standardisierung der Proteomanalyse beitragen

Small is beautiful? - The Baltic States and Germany in the Greek Debt Crisis

Abstract Over the course of the European Sovereign Debt Crisis members of the euro area have put up significant resources to stabilize the financial situation of a few fellow member states. In Germany, this support is subject to a controversial discussion. One aspect in that is the extent of support provided. Using the financial assistance provided to Greece as an example, this paper sheds some light on the financial burden for Germany in comparison to other member states of the euro area, especially Estonia, Latvia and Lithuania. This implies not only an interesting comparison of strains between large and small economies but also between original and later euro area members. Keywords: euro area, debt crisis, exposure, Greece, Baltic states, GermanyZusammenfassung Im Verlauf der Europäischen Staatsschuldenkrise haben die Mitgliedsländer der Eurozone signifikante Beiträge zur Stabilisierung der öffentlichen Haushalte in einigen anderen Mitgliedsländern geleistet. Diese Unterstützung ist nicht zuletzt wegen ihres Umfangs in Deutschland sehr umstritten. Am Beispiel der Finanzhilfen für Griechenland ordnet das vorliegende Papier den Umfang der Unterstützungsleistungen ein durch einen Vergleich der Belastungen zwischen Deutschland und den anderen Euro-Mitgliedsländern, insbesondere Estland, Lettland und Litauen. Es handelt sich damit um einen interessanten Vergleich nicht nur zwischen großen und kleinen Volkswirtschaften sondern auch zwischen einem Gründungsmitglied der Währungsunion und Mitgliedern, die erst später der Währungsunion beigetreten sind. Stichworte: Eurozone, Schuldenkrise, Haftung, Griechenland, Baltische Staaten, Deutschlan

Quantum authentication with key recycling

We show that a family of quantum authentication protocols introduced in [Barnum et al., FOCS 2002] can be used to construct a secure quantum channel and additionally recycle all of the secret key if the message is successfully authenticated, and recycle part of the key if tampering is detected. We give a full security proof that constructs the secure channel given only insecure noisy channels and a shared secret key. We also prove that the number of recycled key bits is optimal for this family of protocols, i.e., there exists an adversarial strategy to obtain all non-recycled bits. Previous works recycled less key and only gave partial security proofs, since they did not consider all possible distinguishers (environments) that may be used to distinguish the real setting from the ideal secure quantum channel and secret key resource.Comment: 38+17 pages, 13 figures. v2: constructed ideal secure channel and secret key resource have been slightly redefined; also added a proof in the appendix for quantum authentication without key recycling that has better parameters and only requires weak purity testing code

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb

<p>Abstract</p> <p>Background</p> <p>Neural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time.</p> <p>Results</p> <p>We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures.</p> <p>Conclusion</p> <p>In this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.</p

EU-Recht und nationale Umsetzungen der Untauglichkeitsgründe beim Schwein: Vergleich europäischer Befundsysteme

Die Fleischuntersuchung und damit zusammenhängend die Schlachtbefundsysteme dienen der Erfassung und Optimierung der Lebensmittelsicherheit und des Verbraucherschutzes, der Tiergesundheit sowie dem Tierwohl. Die Fleischuntersuchung ist immer wieder Neuerungen unterlegen (Blagojevic et al. 2021. Buncic et al. 2019, Riess &amp; Hölzer 2020). Die Regelungen sind europaweit einheitlich festgelegt (EU-Kommission 2019), sodass sowohl bei der Untersuchung als auch bei den Befundsystemen ein Standard garantiert sein sollte. Im Rahmen einer online-Befragung innerhalb des COST Action Networks RIBMINS (Risk-based meat inspection and integrated meat safety assurance; CA18105) zum Vergleich der existierenden Schlachtbefundsysteme innerhalb Europas zeigte sich allerdings, dass zum Teil große Unterschiede zwischen den nationalen Befundsystemen bestehen (Alban et al. 2021, Vieira-Pinto et al. in Bearbeitung). Ziel der vorliegenden Untersuchung war es, die unterschiedlichen nationalen Befundsysteme zu beschreiben und einerseits untereinander sowie andererseits mit den in der VO (EU) 2019/627 (EU-Kommission 2019) vorgegebenen Untauglichkeitsgründen zu vergleichen. Zusätzlich sollte ein Vorschlag einer Harmonisierung der Befunde für Schweine erarbeitet werden

Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Fourier Magnetic Imaging with Nanoscale Resolution and Compressed Sensing Speed-up using Electronic Spins in Diamond

Optically-detected magnetic resonance using Nitrogen Vacancy (NV) color centres in diamond is a leading modality for nanoscale magnetic field imaging, as it provides single electron spin sensitivity, three-dimensional resolution better than 1 nm, and applicability to a wide range of physical and biological samples under ambient conditions. To date, however, NV-diamond magnetic imaging has been performed using real space techniques, which are either limited by optical diffraction to 250 nm resolution or require slow, point-by-point scanning for nanoscale resolution, e.g., using an atomic force microscope, magnetic tip, or super-resolution optical imaging. Here we introduce an alternative technique of Fourier magnetic imaging using NV-diamond. In analogy with conventional magnetic resonance imaging (MRI), we employ pulsed magnetic field gradients to phase-encode spatial information on NV electronic spins in wavenumber or k-space followed by a fast Fourier transform to yield real-space images with nanoscale resolution, wide field-of-view (FOV), and compressed sensing speed-up.Comment: 31 pages, 10 figure

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context

On the Query Complexity of Constructing PRFs from Non-adaptive PRFs

This paper studies constructions of pseudorandom functions (PRFs) from non-adaptive PRFs (naPRFs), i.e., PRFs which are secure only against distinguishers issuing all of their queries at once. Berman and Haitner (Journal of Cryptology, \u2715) gave a one-call construction which, however, is not hardness preserving -- to obtain a secure PRF (against polynomial-time distinguishers), they need to rely on a naPRF secure against superpolynomial-time distinguishers; in contrast, all known hardness-preserving constructions require $\omega(1)$ calls. This leaves open the question of whether a stronger superpolynomial-time assumption is necessary for one-call (or constant-call) approaches. Here, we show that a large class of one-call constructions (which in particular includes the one of Berman and Haitner) cannot be proved to be a secure PRF under a black-box reduction to the (polynomial-time) naPRF security of the underlying function. Our result complements existing impossibility results (Myers, EUROCRYPT \u2704; Pietrzak, CRYPTO \u2705) ruling out natural specific approaches, such as parallel and sequential composition. Furthermore, we show that our techniques extend to rule out a natural class of constructions making parallel but arbitrary number of calls which in particular includes parallel composition and the two-call, cuckoo-hashing based construction of Berman et al.\ (Journal of Cryptology, \u2719)

Different competing risks models applied to data from the Australian Orthopaedic Association National Joint Replacement Registry

Purpose: Here we describe some available statistical models and illustrate their use for analysis of arthroplasty registry data in the presence of the competing risk of death, when the influence of covariates on the revision rate may be different to the influence on the probability (that is, risk) of the occurrence of revision. Patients and methods: Records of 12,525 patients aged 75–84 years who had received hemiarthroplasty for fractured neck of femur were obtained from the Australian Orthopaedic Association National Joint Replacement Registry. The covariates whose effects we investigated were: age, sex, type of prosthesis, and type of fixation (cementless or cemented). Extensions of competing risk regression models were implemented, allowing the effects of some covariates to vary with time. Results: The revision rate was significantly higher for patients with unipolar than bipolar prostheses (HR = 1.38, 95% CI: 1.01–1.89) or with monoblock than bipolar prostheses (HR = 1.45, 95% CI: 1.08–1.94). It was significantly higher for the younger age group (75–79 years) than for the older one (80–84 years) (HR = 1.28, 95% CI: 1.05–1.56) and higher for males than for females (HR = 1.37, 95% CI: 1.09–1.71). The probability of revision, after correction for the competing risk of death, was only significantly higher for unipolar prostheses than for bipolar prostheses, and higher for the younger age group. The effect of fixation type varied with time; initially, there was a higher probability of revision for cementless prostheses than for cemented prostheses, which disappeared after approximately 1.5 years. Interpretation: When accounting for the competing risk of death, the covariates type of prosthesis and sex influenced the rate of revision differently to the probability of revision. We advocate the use of appropriate analysis tools in the presence of competing risks and when covariates have time-dependent effects.Marianne H Gillam, Amy Salter, Philip Ryan, and Stephen E Grave