Convex Relaxation of a Class of Vertex Penalizing Functionals

We investigate a class of variational problems that incorporate in some sense curvature information of the level lines. The functionals we consider incorporate metrics defined on the orientations of pairs of line segments that meet in the vertices of the level lines. We discuss two particular instances: One instance that minimizes the total number of vertices of the level lines and another instance that minimizes the total sum of the absolute exterior angles between the line segments. In case of smooth level lines, the latter corresponds to the total absolute curvature. We show that these problems can be solved approximately by means of a tractable convex relaxation in higher dimensions. In our numerical experiments we present preliminary results for image segmentation, image denoising and image inpainting.     

The Role of Plasma Extracellular Vesicles in Remote Ischemic Conditioning and Exercise-Induced Ischemic Tolerance

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.     

Sixth graders’ evaluation strategies when reading Internet search results: an eye-tracking study

Eye-tracking technology was used to examine Internet search result evaluation strategies adopted by sixth-grade students (N?=?36) during ten experimental information search tasks. The relevancy of the search result’s title, URL, and snippet components was manipulated and selection of search results as well as looking into probabilities on the search result components was analysed. The results revealed that during first-pass inspection, students read the search engine page by first looking at the title of a search result. If the title was relevant, the probability of looking at the snippet of the search result increased. During second-pass inspection, there was a high probability of students focusing on the most promising search result by inspecting all of its components before making their selection. A cluster analysis revealed three viewing strategies: half of the students looked mainly at the titles and snippets; one-third with high probability examined all components; and one-sixth mainly focused on titles, leading to more frequent errors in search result selection. The results indicate that students generally made a flexible use of both eliminative and confirmatory evaluation strategies when reading Internet search results, while some seemed to not pay attention to snippet and URL components of the search results.     

Matrix models of discretely bending, stiff polymers

Polymer models which make use of the Ising model and transfer matrix techniques remind us, for example, of the work of Flory [Statistical mechanics of chain molecules, 1969] and Zimm and Bragg [J Chem Phys, 31 (1959) 526]. We investigate the properties of some such polymer models where the chain conformation can be described solely by an Ising-like parameterization and a set of independent, predetermined bond direction vectors or by a Potts-like model for directions of bond vectors on a lattice, with the specific aim of understanding more closely the connection of constraints and forces on the chain ends for polymers which, in general, are of arc length corresponding to their persistence lengths. Instances of these models are directed helical walks, random sequential walks, bimodally distributed in direction walks or relatively short, stiff chains fixed into a network. The behavior of this model under deformation in statistical mechanics and its dynamical properties under Glauber dynamics are discussed.     

Dissolution,Transport and Reaction at a DICY/DGEBA Interface

The curing of an epoxy consisting of the solid hardener dicyandiamide (DICY) and the resin diglycidyl ether of bisphenol A (DGEBA) is studied in a system consisting of a tablet of DICY embedded in liquid DGEBA. Dissolution of DICY within the liquid DGEBA in combination with the transport of dissolved DICY from the tablet border into DGEBA and the chemical reaction of both reactants is studied by scanning Brillouin microscopy and infrared spectroscopy. Scanning Brillouin microscopy demonstrates the spatial and temporal evolution of the static and dynamic hypersonic properties in the course of curing in the vicinity of the DICY tablet. Infrared spectroscopy performed on epoxy pieces extracted from the final sample at different distances from the tablet surface give information about the spatial evolution of the curing process. The results achieved by both techniques are finally combined to yield a better understanding of the curing of DICY-based epoxies, which transform upon curing from strongly heterogeneous systems towards increasingly homogeneous systems.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).     

Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.