Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.     

Rietveld and pair distribution function study of Hägg carbide using synchrotron X‐ray diffraction

Fischer–Tropsch (FT) synthesis is an important process in the manufacturing of hydrocarbons and oxygenated hydrocarbons from mixtures of carbon monoxide and hydrogen (syngas). The reduced iron catalyst reacts with carbon monoxide and hydrogen to form bulk Fe₅C₂ Hägg carbide (χ‐HC) during FT synthesis. Arguably, χ‐HC is the predominant catalyst phase present in the working iron catalyst. Deactivation of the working catalyst can be due to oxidation of χ‐HC to iron oxide, a step‐wise decarburization to cementite (θ‐Fe₃C), carbon formation or sintering with accompanying loss of catalytic performance. It is therefore critical to determine the precise crystal structure of χ‐HC for the understanding of the synthesis process and for comparison with the first‐principles ab initio modelling. Here the results of high‐resolution synchrotron X‐ray powder diffraction data are reported. The atomic arrangement of χ‐HC was confirmed by Rietveld refinement and subsequent real‐space modelling of the pair distribution function (PDF) obtained from direct Fourier transformation. The Rietveld and PDF results of χ‐HC correspond well with that of a pseudo‐monoclinic phase of space group Pī [a = 11.5661 (6) Å, b = 4.5709 (1) Å, c = 5.0611 (2) Å, α = 89.990 (5)°, β = 97.753 (4)°, γ = 90.195 (4)°], where the Fe atoms are located in three distorted prismatic trigonal and one octahedral arrangement around the central C atoms. The Fe atoms are distorted from the prismatic trigonal arrangement in the monoclinic structure by the change in C atom location in the structure.     

Assessment of normal flow patterns in the pulmonary circulation by using 4D magnetic resonance velocity mapping

Objective

The purpose of this study was to analyze flow patterns in the pulmonary circulation of healthy volunteers by using 4D flow magnetic resonance imaging.

Materials and Methods

The study was approved by the local ethics committee and all subjects gave written informed consent. Eighteen volunteers underwent a 4D flow scan of the whole-heart. Two patients with congenital heart disease were also included to detect possible patterns of flow abnormalities (Patient 1: corrected transposition of great arteries (TGA); Patient 2: partial anomalous pulmonary venous return and atrial septal defect). To analyze flow patterns, 2D planes were placed on the main pulmonary artery (PA), left and right PA. Flow patterns were assessed manually by two independent viewers using vector fields, streamlines and particle traces, and semi-automatically by vorticity quantification.

Results

Two counter-rotating helices were found in the main PA of volunteers. Right-handed helical flow was detected in the right PA of 15 volunteers. Analysis of the helical flow by particles traces revealed that both helices contributed mainly to the flow in the right PA. In the patient with corrected TGA helical flow was not detected. Abnormal vortical flow was visualized in the main PA of patient 2, suggesting elevated mean PA pressure.

Conclusions

Helical flow is normally present in the main PA and right PA. 4D flow is an excellent tool to evaluate noninvasively complex blood flow patterns in the pulmonary circulation. Knowledge of normal and abnormal flow patterns might help to evaluate patients with congenital heart disease adding functional information undetectable with other imaging modalities.     

Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz ultrasound

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT₁, 5-LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT₁. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT₁. Taken together, the current study reports the feasibility of a non-invasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing.