Orthogonal Peptide-Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ET_AR and ET_BR), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ET_AR/ET_BR. Further, we demonstrate, that ET_AR expression regulates the signaling of co-expressed ET_BR. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.     

Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion

The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments. METHODS: The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique). RESULTS: The renal uptake in the amino acid group was significantly lower (p<0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group. CONCLUSION: The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.     

Arthroscintigraphy in suspected rotator cuff rupture

AIM: In order to evaluate the diagnostic efficiency of arthroscintigraphy in suspected rotator cuff ruptures this new imaging procedure was performed 20 times in 17 patients with clinical signs of a rotator cuff lesion. The scintigraphic results were compared with sonography (n = 20), contrast arthrography (n = 20) and arthroscopy (n = 10) of the shoulder joint. METHODS: After performing a standard bone scintigraphy with intravenous application of 300 MBq 99m-Tc-methylene diphosphonate (MDP) for landmarking of the shoulder region arthroscintigraphy was performed after an intraarticular injection of 99m-Tc microcolloid (ALBU-RES 400 MicroCi/5 ml). The application was performed either in direct combination with contrast arthrography (n = 10) or ultrasound conducted mixed with a local anesthetic (n = 10). Findings at arthroscopical surgery (n = 10) were used as the gold standard. RESULTS: In case of complete rotator cuff rupture (n = 5), arthroscintigraphy and radiographic arthrography were identical in 5/5. In one patient with advanced degenerative alterations of the shoulder joint radiographic arthrography incorrectly showed a complete rupture which was not seen by arthroscintigraphy and endoscopy. In 3 patients with incomplete rupture, 2/3 results were consistent. A difference was seen in one patient with a rotator cuff, that has been already revised in the past and that suffered of capsulitis and calcification. CONCLUSION: Arthroscintigraphy is a sensitive technique for detection of rotator cuff ruptures. Because of the lower viscosity of the active compound, small ruptures can be easily detected, offering additional value over radiographic arthrography and ultrasound, especially for evaluation of incomplete cuff ruptures.     

Development of a 60 Hz Power Standard Using SNS Programmable Josephson Voltage Standards

We are implementing a new standard for 60 Hz power measurements based on precision sinusoidal reference voltages from two independent programmable Josephson voltage standards (PJVS): one for voltage and one for current. The National Institute of Standards and Technology PJVS systems use series arrays of Josephson junctions to produce accurate quantum-based DC voltages. Using stepwise-approximation synthesis, the PJVS systems produce sinewaves with precisely calculable RMS voltage and spectral content. We present measurements and calculations that elucidate the sources of error in the RMS voltage that are intrinsic to the digital-synthesis technique and that are due to the finite rise times and transients that occur when switching between the discrete voltages. Our goal is to reduce all error sources and uncertainty contributions from the PJVS synthesized waveforms to a few parts in 10 ⁷ so that the overall uncertainty in the AC-power standard is a few parts in 10⁶     

BerlinMOD: a benchmark for moving object databases

This document presents a method to design scalable and representative moving object data (MOD) and two sets of queries for benchmarking spatio-temporal DBMS. Instead of programming a dedicated generator software, we use the existing Secondo DBMS to create benchmark data. The benchmark is based on a simulation scenario, where the positions of a sample of vehicles are observed for an arbitrary period of time within the street network of Berlin. We demonstrate the data generator’s extensibility by showing how to achieve more natural movement generation patterns, and how to disturb the vehicles’ positions to create noisy data. As an application and for reference, we also present first benchmarking results for the Secondo DBMS. Whereas the benchmark focuses on range queries, we demonstrate its ability to incorporate new future classes of queries by presenting a preliminary extension handling various nearest neighbour queries. Such a benchmark is useful in several ways: It provides well-defined data sets and queries for experimental evaluations; it simplifies experimental repeatability; it emphasizes the development of complete systems; it points out weaknesses in existing systems motivating further research. Moreover, the BerlinMOD benchmark allows one to compare different representations of the same moving objects.     

Influence of heat distribution and zone shape in the floating zone growth of selected oxide compounds

The effects of heat distribution, process parameters, and the ambient atmosphere on the characteristics of the molten zone are considered for floating zone (FZ) crystal growth of selected oxide compounds with special reference to cuprates. The outer shape of the FZ, which is stabilized only by capillary forces, and the interfaces between melt and growing crystal are decisive for process stability and crystal perfection. They do not only depend on external parameters such as zone heights, growth velocity, and rotation mode but are closely related to the phase diagram and the constitution of the individual substance. Therefore, the choice of the optimum process conditions is a subtle balance of parameters like appropriate focusing or defocusing of the light, the direction of growth and the ambient atmosphere, which are adapted to the individual substance. A pyrometric method of FZ temperature measurement was developed as an appropriate tool for process control.     

Posture and muscular behaviour in emergency braking: An experimental approach

In the field of numerical crash simulations in road safety research, there is a need to accurately define the initial conditions of a frontal impact for the car occupant. In particular, human models used to simulate such impacts barely take into account muscular contracting effects. This study aims to quantify drivers’ behaviour in terms of posture and muscular activity just before a frontal impact.Experiments on volunteers were performed in order to define these conditions, both on a driving simulator and on a real moving car. Brake pedal loads, lower limbs kinematics and muscle activation were recorded.Coupling instantaneous data from both experimental protocols (simulator versus Real car), a standard emergency braking configuration could be defined as (1) joint flexion angles of 96°, 56° and 13° for the right hip, knee and ankle respectively; (2) a maximum brake pedal load of 780 N; (3) a muscular activation of 55% for the anterior thigh, 26% for the posterior thigh, 18% for the anterior leg and 43% for the posterior leg.The first application of this research is the implementation of muscle tone in human models designed to evaluate new safety systems.