Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

Which factors influence the ability of a computational model to predict the in vivo deformation behaviour of skeletal muscle?

Deep tissue injury (DTI) is a severe form of pressure ulcer where tissue damage starts in deep tissues underneath intact skin. Tissue deformation may play an important role in the aetiology, which can be investigated using an experimental–numerical approach. Recently, an animal-specific finite element model has been developed to simulate experiments in which muscle tissue was compressed with an indenter. In this study, the material behaviour and boundary conditions were adapted to improve the agreement between model and experiment and to investigate the influence of these adaptations on the predicted strain distribution. The use of a highly nonlinear material law and including friction between the indenter and the muscle both improved the quality of the model and considerably influenced the estimated strain distribution. With the improved model, the required sample size to detect significant differences between loading conditions can be diminished, which is clearly relevant in experiments involving animals.     

How does muscle stiffness affect the internal deformations within the soft tissue layers of the buttocks under constant loading?

Mechanical loading of soft tissues covering bony prominences can cause skeletal muscle damage, ultimately resulting in a severe pressure ulcer termed deep tissue injury (DTI). Deformation plays an important role in the aetiology of DTI. Therefore, it is essential to minimise internal muscle deformations in subjects at risk of DTI. As an example, spinal cord-injured (SCI) individuals exhibit structural changes leading to a decrease in muscle thickness and stiffness, which subsequently increase the tissue deformations. In the present study, an animal-specific finite element model, where the geometry and boundary conditions were derived from magnetic resonance images, was developed. It was used to investigate the internal deformations in the muscle, fat and skin layers of the porcine buttocks during loading. The model indicated the presence of large deformations in both the muscle and the fat layers, with maximum shear strains up to 0.65 in muscle tissue and 0.63 in fat. Furthermore, a sensitivity analysis showed that the tissue deformations depend considerably on the relative stiffness values of the different tissues. For example, a change in muscle stiffness had a large effect on the muscle deformations. A 50% decrease in stiffness caused an increase in maximum shear strain from 0.65 to 0.99, whereas a 50% increase in stiffness resulted in a decrease in maximum shear strain from 0.65 to 0.49. These results indicate the importance of restoring tissue properties after SCI, with the use of, for example, electrical stimulation, to prevent the development of DTI.     

Results of automatic image registration are dependent on initial manual registration

Measurement of static alignment of articulating joints is of clinical benefit and can be determined using image-based registration. We propose a method that could potentially improve the outcome of image-based registration by using initial manual registration. Magnetic resonance images of two wrist specimens were acquired in the relaxed position and during simulated grasp. Transformations were determined from voxel-based image registration between the two volumes. The volumes were manually aligned to match as closely as possible before auto-registration, from which standard transformations were obtained. Then, translation/rotation perturbations were applied to the manual registration to obtain altered initial positions, from which altered auto-registration transformations were obtained. Models of the radiolunate joint were also constructed from the images to simulate joint contact mechanics. We compared the sensitivity of transformations (translations and rotations) and contact mechanics to altering the initial registration condition from the defined standard. We observed that with increasing perturbation, transformation errors appeared to increase and values for contact force and contact area appeared to decrease. Based on these preliminary findings, it appears that the final registration outcome is sensitive to the initial registration.     

Comparison of superoxide detection abilities of newly developed spin traps in the living cells

This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 µM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Detection of Free Radicals and Cholesterol Hydroperoxides in Blood Taken from the Coronary Sinus of Man During Percutaneous Transluminal Coronary Angioplasty

Patients undergoing percutaneous transluminal coronary angioplasty (PTCA) were investigated for the production of free radicals and cholesterol hydroperoxides during reperfusion. Fifteen patients were studied. Ischaemia during balloon inflation was assessed by serial coronary sinus lactate analysis (mean maximal increase in anterior descending artery diltation was 130%), and by the demonstration of reperfusion hyperaemia (mean increase of coronary sinus oxygen saturation 74%).

Free radicals were detected by electron spin resonance (ESR) spin trapping using the spin trap PBN (N-t-Butyl-α-phenylnitrone). Radical adducts were detected in up to 50% of samples taken during reperfusion after anterior descending lesion angioplasty. No radicals were detected in control samples or during the ischaemic phase. Radical detection was positively correlated with the change in coronary sinus lactate (p<0.025).

Coronary sinus cholesterol hydroperoxide analysis did not show a significant increase over control during reperfusion, due in part to unexpectedly high pre angioplasty levels.

This study provides clear evidence for the production of a burst of free radicals and evidence for lipid peroxidation in the minutes following myocardial reperfusion during angioplasty. A relationship between the severity of the ischaemic insult and the detection of radical adducts has also been found.     

Spin Trapping of Free Radicals Produced in vivo in Heart and Liver During Endotoxemia

Studies using free radical scavengers and measurements of lipid peroxidation have suggested that free radicals are generated during endotoxemia. Conclusions from these studies have implied that free radicals may participate in the sequence of pathologic events following endotoxin challenge in the experimental animal. Current inferences of free radical generation and involvement have been derived from indirect evidence and are therefore inconclusive. To quantitate the generation of free radicals in vivo during endotoxemia this study employed the use of electron paramagnetic resonance spectroscopy (EPR) combined with spin trapping techniques. Five minutes before intraperitoneal endotoxin administration, trimethoxy-a-phenyl-t-butyl-nitrone [(MeO), PBN] was administered intraperitoneally. Experimental animals were always matched with control animals receiving no endotoxin. At either five minutes or twenty-five minutes following endotoxin administration animals were decapitated and hearts and livers were rapidly taken for lipid extraction and EPR evaluation. Analysis of the EPR spectra revealed hyperfine splitting constants that indicated the presence of carbon-centered radical spin adducts in both organ tissues from animals exposed to endotoxin for twenty-five minutes. No signals were present in hearts and livers taken five minutes after endotoxin administration. EPR evaluation did not indicate spin adduct formation in control tissue. These data directly demonstrate that activation of processes in vivo involving free radical generation occur early during endotoxemia, but are not detectable immediately after the endotoxin challenge.