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Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System
No full textThe classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation
Mechanistic Interplay between Light Switching and Guest Binding in Photochromic [PdDithienylethene] Coordination Cages
No full textPhotochromic [PdL] coordination cages based on dithienylethene (DTE) ligands L allow triggering guest uptake and release by irradiation with light of different wavelengths. The process involves four consecutive electrocyclic reactions to convert all chromophores between their open and closed photoisomeric forms. So far, guest affinity of the fully switched species was elucidated, but mechanistic details concerning the intermediate steps remained elusive. Now, a new member of the DTE cage family allows unprecedented insight into the interplay between photoisomerization steps and guest location inside/outside the cavity. Therefore, the intrinsic chirality of the DTE backbones was used as reporter for monitoring the fate of a chiral guest. In its “open” photoisomeric form (o-L, [Pd(o-L)] = o-C), the C-symmetric DTE chromophore quickly converts between energetically degenerate P and M helical conformations. After binding homochiral 1R-(−) or 1S-(+) camphor sulfonate (R-CSA or S-CSA), guest-to-host chirality transfer was observed via a circular dichroism (CD) signal for the cage-centered absorption. Irradiating the R/S-CSA@o-C host–guest complexes at 313 nm produced configurationally stable “closed” photoisomers, thus locking the induced chirality with an enantiomeric excess close to 25%. This value (corresponding to chiral induction for one out of four ligands), together with DOSY NMR, ion mobility mass spectrometry, and X-ray structure results, shows that closure of the first photoswitch is sufficient to expel the guest from the cavity
Investigation of geometrically necessary dislocation structures in compressed Cu micropillars by 3-dimensional HR-EBSD
No full textMechanical testing of micropillars is a field that involves new physics, as the behaviour of materials is non-deterministic at this scale. To better understand their deformation mechanisms we applied 3-dimensional high angular resolution electron backscatter diffraction (3D HR-EBSD) to reveal the dislocation distribution in deformed single crystal copper micropillars. Identical micropillars (6 μm μm μm in size) were fabricated by focused ion beam (FIB) and compressed at room temperature. The deformation process was stopped at different strain levels ( , and ) to study the evolution of geometrically necessary dislocations (GNDs). Serial slicing with FIB and consecutive HR-EBSD mapping on the (100) side was used to create and compare 3-dimensional maps of the deformed volumes. Average GND densities were calculated for each deformation step. Total dislocation density calculation based on X-ray synchrotron measurements were conducted on the pillar to compare dislocation densities determined by the two complementary methods. Scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM) images were captured on the pillar to visualize the actual dislocation structure. With the 3D HR-EBSD technique we have studied the geometrically necessary dislocations evolving during the deformation of micropillars. An intermediate behaviour was found at the studied sample size between bulk and nanoscale plasticity: A well-developed dislocation cell structure built up upon deformation but with significantly lower GND density than in bulk. This explains the simultaneous observation of strain hardening and size effect at this scale
PymePix : a python library for SPIDR readout of Timepix3
No full textPymePix is a new Python 3 library that provides control and acquisition for the Timepix3-SPIDR hardware. The rich set of data-structures and intuitive routines reduces time and coding effort to quickly configure, acquire, and visualize data from Timepix3. The highly extensible high-performance data-pipeline allows for alteration of the Timepix3 datastream into a form that is convinient for the user. This library is intended to be easily inserted into a standard scientific software stack as well as to allow for more direct interaction of Timepix3 with interactive flavors of Python. Included with the library are two example programs using PymePix: PymePix-acq is a command line control and acquisition program that can capture UDP packets and decode them into pixels and triggers. The second is pymepixviewer, an online control and data-acquisition program for general use, but with features geared toward mass-spectroscopy and ion imaging
The Hierarchy Problem and the Cosmological Constant Problem Revisited
No full textWe argue that the Standard Model (SM) in the Higgs phase does not suffer from a “hierarchy problem” and that similarly the “cosmological constant problem” resolves itself if we understand the SM as a low energy effective theory emerging from a cutoff-medium at the Planck scale. We actually take serious Veltman’s “The Infrared–Ultraviolet Connection” addressing the issue of quadratic divergences and the related huge radiative correction predicted by the SM in the relationship between the bare and the renormalized theory, usually called “the hierarchy problem” and claimed that this has to be cured. We discuss these issues under the condition of a stable Higgs vacuum, which allows extending the SM up to the Planck cutoff. The bare Higgs boson mass then changes sign below the Planck scale, such that the SM in the early universe is in the symmetric phase. The cutoff enhanced Higgs mass term as well as the quartically enhanced cosmological constant term provide a large positive dark energy that triggers the inflation of the early universe. Reheating follows via the decays of the four unstable heavy Higgs particles, predominantly into top–antitop pairs, which at this stage are massless. Preheating is suppressed in SM inflation since in the symmetric phase bosonic decay channels are absent at tree level. The coefficients of the shift between bare and renormalized Higgs mass as well as of the shift between bare and renormalized vacuum energy density exhibit close-by zeros at about 7.7×1014 GeV and 3.1×1015 GeV, respectively. The zero of the Higgs mass counter term triggers the electroweak phase transition, from the low energy Higgs phase and to the symmetric phase above the transition point. Since inflation tunes the total energy density to take the critical value of a flat universe and all contributing components are positive, it is obvious that the cosmological constant today is naturally a substantial fraction of the total critical density. Thus taking cutoff enhanced corrections seriously the Higgs system provides besides the masses of particles in the Higgs phase also dark energy, inflation and reheating in the early universe. The main unsolved problem in our context remains the origin of dark matter. Higgs inflation is possible and likely even unavoidable provided new physics does not disturb the known relevant SM properties substantially. The scenario highly favors to understand the SM and its main properties as a natural structure emerging at long distance. This in particular concerns the SM symmetry patterns and their consequences
Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1
No full textPro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1′ residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile
Integrin α6β4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes
No full textMechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6β4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of β4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of β4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-β4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in β4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation
Analysis of cyclic phase transformations during additive manufacturing of hardenable tool steel by in-situ X-ray diffraction experiments
No full textAppendix: Simulating CTA
No full textThe expectations for the scientific capabilities of CTA presented in this document are based on sensitivity and response functions derived from detailed Monte Carlo simulations of the CTA observatory. The simulations model extensive air showers initiated by high-energy (HE) primary particles (gamma rays or background cosmic-ray nuclei and electrons), the Cherenkov light production by the shower particles, and the measurement processes of imaging atmospheric Cherenkov telescopes