The effect of silicon microsegregation on the mechanical properties of high silicon alloyed ductile cast iron under monotonous loading.

High silicon alloyed ductile cast iron (Si-DCI) can show unpredictable brittle fracture which currently prevents a widespread application of this material. The brittleness is associated with local superstructure formation due to silicon segregation which influences the deformation mechanisms of the matrix phase. In order to understand the effect of silicon segregation on the mechanical properties of Si-DCI under monotonous loading, three alloys with different cooling conditions were examined and micromechanical simulations were carried out by using the phenomenological crystal plasticity model. Here, the segregation profiles were determined through multi phase field simulations. The influence of segregation on the mechanical properties was only evident from the model but not from the experimental results. The simulated results show that the toughness of Si-DCI decreases with stronger silicon segregation when ductile damage is considered.

Femur reconstruction in 3D ultrasound for orthopedic surgery planning.

PURPOSE: Derotation varisation osteotomy of the proximal femur in pediatric patients usually relies on 2-dimensional X-ray imaging, as CT and MRI still are disadvantageous when applied in small children either due to a high radiation exposure or the need of anesthesia. This work presents a radiation-free non-invasive tool to 3D-reconstruct the femur surface and measure relevant angles for orthopedic diagnosis and surgery planning from 3D ultrasound scans instead. METHODS: Multiple tracked ultrasound recordings are segmented, registered and reconstructed to a 3D femur model allowing for manual measurements of caput-collum-diaphyseal (CCD) and femoral anteversion (FA) angles. Novel contributions include the design of a dedicated phantom model to mimic the application ex vivo, an iterative registration scheme to overcome movements of a relative tracker only attached to the skin, and a technique to obtain the angle measurements. RESULTS: We obtained sub-millimetric surface reconstruction accuracy from 3D ultrasound on a custom 3D-printed phantom model. On a pre-clinical pediatric patient cohort, angular measurement errors were [Formula: see text] and eventually [Formula: see text] for CCD and FA angles, respectively, both within the clinically acceptable range. To obtain these results, multiple refinements of the acquisition protocol were necessary, ultimately reaching success rates of up to 67% for achieving sufficient surface coverage and femur reconstructions that allow for geometric measurements. CONCLUSION: Given sufficient surface coverage of the femur, clinically acceptable characterization of femoral anatomy is feasible from non-invasive 3D ultrasound. The acquisition protocol requires leg repositioning, which can be overcome using the presented algorithm. In the future, improvements of the image processing pipeline and more extensive surface reconstruction error assessments could enable more personalized orthopedic surgery planning using cutting templates.

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET.

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.

Characterization of Fluorescent Proteins with Intramolecular Photostabilization*.

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series*.

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.

Triggering Closure of a Sialic Acid TRAP Transporter Substrate Binding Protein through Binding of Natural or Artificial Substrates.

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.