Receptor oligomerization and beyond: a case study in bone morphogenetic proteins

BACKGROUND: Transforming growth factor (TGF)β superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system

Sedimentary ancient DNA reveals past ecosystem and biodiversity changes on the Tibetan Plateau: Overview and prospects

Alpine ecosystems on the Tibetan Plateau are being threatened by ongoing climate warming and intensified human activities. Ecological time-series obtained from sedimentary ancient DNA (sedaDNA) are essential for understanding past ecosystem and biodiversity dynamics on the Tibetan Plateau and their responses to climate change at a high taxonomic resolution. Hitherto only few but promising studies have been published on this topic. The potential and limitations of using sedaDNA on the Tibetan Plateau are not fully understood. Here, we (i) provide updated knowledge of and a brief introduction to the suitable archives, region-specific taphonomy, state-of-the-art methodologies, and research questions of sedaDNA on the Tibetan Plateau; (ii) review published and ongoing sedaDNA studies from the Tibetan Plateau; and (iii) give some recommendations for future sedaDNA study designs. Based on the current knowledge of taphonomy, we infer that deep glacial lakes with freshwater and high clay sediment input, such as those from the southern and southeastern Tibetan Plateau, may have a high potential for sedaDNA studies. Metabarcoding (for microorganisms and plants), metagenomics (for ecosystems), and hybridization capture (for prehistoric humans) are three primary sedaDNA approaches which have been successfully applied on the Tibetan Plateau, but their power is still limited by several technical issues, such as PCR bias and incompleteness of taxonomic reference databases. Setting up high-quality and open-access regional taxonomic reference databases for the Tibetan Plateau should be given priority in the future. To conclude, the archival, taphonomic, and methodological conditions of the Tibetan Plateau are favorable for performing sedaDNA studies. More research should be encouraged to address questions about long-term ecological dynamics at ecosystem scale and to bring the paleoecology of the Tibetan Plateau into a new era

Dynamics of the primary recognition steps of BMP receptors

Bone Morphogenetic Proteins (BMPs) bilden zusammen mit den Activinen, Growth and Differentiation Factors (GDFs) und Transforming Growth Factor β (TGF-β) die Transforming Growth Factor β-Superfamilie von sekretierten Signalproteinen. Sie spielen eine wichtige Rolle in der Entwicklung, Erhaltung und Regeneration von Geweben und Organen. Die Signalvermittlung dieser Proteine erfolgt durch die Bindung von zwei verschiedenen Typen von Serin-/Threonin-Kinaserezeptoren, die als Typ-I- und Typ-II-Rezeptoren bezeichnet werden. Im ersten Schritt erfolgt die Bindung an den hochaffinen Rezeptor (im Fall von BMP-2 der Typ-I-Rezeptor), im nächsten Schritt wird der niederaffine Rezeptor in den Komplex rekrutiert. Bis heute sind lediglich sieben Typ-I- und fünf Typ-II-Rezeptoren bekannt, was auf eine Promiskuität in der Liganden-Rezeptor-Interaktion schließen lässt. Die Architektur beider Rezeptorsubtypen ist dabei relativ ähnlich. Beide bestehen aus einer ligandenbindenden extrazellulären Domäne, einer Transmembrandomäne sowie einer intrazellulären Kinasedomäne. Eine nacheinander ablaufende Transphosphorylierung der intrazellulären Domänen führt zu einer Phosphorylierung von SMAD-Proteinen, die dann als nachgeschaltete Vermittler fungieren und die Transkription regulierter Gene auslösen. Im Hauptteil dieser Arbeit wurden die initialen Schritte der Rezeptorkomplexformierung sowie die Mobilität der Rezeptoren mit Hilfe von fluoreszenzmikroskopischen Methoden untersucht. Dabei konnte festgestellt werden, dass für die Bildung eines Signalkomplexes eine bestimmte Schwellenkonzentration des Liganden nötig ist und dass der Mechanismus nach einem Alles-oder-Nichts-Prinzip wie ein Schalter funktioniert. Außerdem konnten Unterschiede in der Nutzung der gleichen Rezeptoren durch verschiedene Liganden festgestellt werden. Die anderen Teile der Arbeit befassen sich mit der Funktionalität der verschiedenen Rezeptordomänen in der Signalübermittlung, der Analyse von hoch- und niederaffinen Ligandenbindestellen auf ganzen Zellen sowie dem Einfluss des SMAD- und des MAPK-Signalwegs auf die Induktion der Alkalischen Phosphatase. Dabei konnte gezeigt werden, dass die Art der SMAD-Phosphorylierung allein vom Typ der Kinasedomäne abhängig ist, dass auf einer Zelle verschiedene Rezeptorpopulationen existieren, welche von unterschiedlichen Ligandenkonzentrationen angesprochen werden, und dass die Induktion der Alkalischen Phosphatase stark vom zeitlichen Verlauf der SMAD- und MAPK-Aktivierung abhängig ist.Bone Morphogenetic Proteins (BMPs), together with Activins, Growth and Differentiation Factors (GDFs) and Transforming Growth Factor β (TGFβ), are secreted signalling proteins that belong to the Transforming Growth Factor β superfamily. They play an important role in regulating the development, maintenance and regeneration of tissues and organs. Signalling of TGFβ superfamily members occurs by binding to two types of serine-/threonine kinase receptors termed type I and type II. First, the high affinity receptor (in case of BMP2 the type I receptor) is bound, and then the low affinity receptor is recruited into the signalling complex. The fact that there are only seven type-I and five type-II receptors are known implies a limited promiscuity in ligand-receptor interaction. The architecture of both receptor subtypes is quite similar, with a small extracellular ligand-binding domain, a single transmembrane domain and an intracellular kinase domain. Subsequent transphosphorylation of the intracellular receptor domains leads to phosphorylation of SMAD proteins, which then act as downstream mediators and activate gene transcription. The main part of this work was to analyze the initial steps in receptor complex formation and the mobility of TGFβ-superfamily receptors with fluorescence microscopy techniques. It could be shown that complex formation requires a certain ligand threshold concentration and shows an all-or-nothing switch-like behaviour. Furthermore, differences between different ligands using the same receptors could be visualized. The other parts of this work deal with the functionality of the different receptor domains in signal transduction, the analysis of high- and low-affinity binding sites on whole cells and the influence of the SMAD- and MAPK-pathways on alkaline phosphatase induction. It could be shown that the type of SMAD phosphorylation ist solely dependent on the type of the kinase domain, that there exist different receptor populations on a cell that are addressed by different ligand concentrations and that alkaline phosphatase induction is highly dependent on the time course of SMAD- and MAPK-pathway activation

Pulseq-CEST : Towards multi-site multi-vendor compatibility and reproducibility of CEST experiments using an open-source sequence standard

PURPOSE: As the field of CEST grows, various novel preparation periods using different parameters are being introduced. At the same time, large, multisite clinical studies require clearly defined protocols, especially across different vendors. Here, we propose a CEST definition standard using the open Pulseq format for a shareable, simple, and exact definition of CEST protocols.METHODS: We present the benefits of such a standard in three ways: (1) an open database on GitHub, where fully defined, human-readable CEST protocols can be shared; (2) an open-source Bloch-McConnell simulation to test and optimize CEST preparation periods in silico; and (3) a hybrid MR sequence that plays out the CEST preparation period and can be combined with any existing readout module.RESULTS: The exact definition of the CEST preparation period, in combination with the flexible simulation, leads to a good match between simulations and measurements. The standard allowed finding consensus on three amide proton transfer-weighted protocols that could be compared in healthy subjects and a tumor patient. In addition, we could show coherent multisite results for a sophisticated CEST method, highlighting the benefits regarding protocol sharing and reproducibility.CONCLUSION: With Pulseq-CEST, we provide a straightforward approach to standardize, share, simulate, and measure different CEST preparation schemes, which are inherently completely defined

Screening of the ryanodine 1 gene for malignant hyperthermia causative mutations by high resolution melt curve analysis

BACKGROUND: A diagnosis of malignant hyperthermia (MH) can be determined by performing an in vitro (muscle) contracture test (IVCT) or by identifying a known MH causative mutation in the ryanodine receptor 1 gene (RYR1). Genetic diagnosis has an advantage over IVCT because it is less invasive. Direct sequencing of the very large RYR1 coding region (15.117 bases) is a laborious and expensive task. In this study, we applied the High Resolution Melting (HRM) curve analysis as a tool to screen the entire coding region of the gene. METHODS: Genomic DNA was extracted from peripheral blood samples in a cohort of 16 MH-susceptible patients diagnosed by the IVCT. The total coding region of RYR1 was divided and amplified by polymerase chain reaction in 131 DNA fragments and the melting profiles were compared with those of control samples. HRM curves were evaluated by Rotor-Gene Q software and visual inspection. Fragments showing aberrant melting profiles were sequenced to identify the underlying sequence variation. RESULTS: A subset of 520 of 2520 DNA fragments (21%) showed significantly aberrant melting profiles. Upon sequencing, 131 known polymorphisms and 17 known or suspected mutations were found in 13 of 16 MH-susceptible patients (81%). Thus, the workload of sequencing was reduced by 79%. CONCLUSION: HRM curve analysis is a sensitive and cost-effective tool for the identification of nucleotide sequence variants in complex genes such as the RYR1 gene

Vibrationsverdichtung von hartmagnetischen Pulvern im Magnetfeld Schlussbericht

The excitation of vibrations in bulk material can increase the mobility of the powder particles in a powder bed. This project was dedicated to investigations of the possibilities of improving the orientation of magnetic fields through vibratory compaction of magnetically hard powders in the magnetic field. The magnetic powders (Nd, Fe, B and Sr ferrite powders) were filled into the die with/out pressing aids while maintaining vibrations (f = 100 - 1000 Hz, a = 5 - 25 g) and an external magnetic field (H _m_a_x = 330 kA/m). After precompaction by filling (t = ca. 15 sec) a specific load was applied to the powder for vibratory compaction (p _m_a_x = 13.2 MPa). The results obtained for Nd, Fe, B powders showed that pressing aids such as natural rubber have a negative effect on the magnetic properties. Without pressing aids the dimensional stability of the green compact is insufficient. The vibratory compaction of Sr ferrite powders showed that moisture combined with PVA additives suppress the effect of orientation through agglomeration and that vibratory compaction in the absence of moisture results in a remarkable magnetic field orientation. (orig.)SIGLEAvailable from TIB Hannover: F94B1152+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman