Taspoglutide, a novel human once-weekly GLP-1 analogue, protects pancreatic ß-cells in vitro and preserves islet structure and function in the Zucker diabetic fatty rat in vivo.

AIM: Glucagon-like peptide-1 (GLP-1) has protective effects on pancreatic ß-cells. We evaluated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, on ß-cells in vitro and in vivo. METHODS: Proliferation of murine pancreatic ß (MIN6B1) cells and rat islets in culture was assessed by imaging of 5-ethynyl-2'-deoxyuridine-positive cells after culture with taspoglutide. Apoptosis was evaluated with the transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labelling assay in rat insulinoma (INS-1E) cells and isolated human islets exposed to cytokines (recombinant interleukin-1ß, interferon-γ, tumour necrosis factor-α) or lipotoxicity (palmitate) in the presence or absence of taspoglutide. Islet morphology and survival and glucose-stimulated insulin secretion in perfused pancreata were assessed 3-4 weeks after a single application of taspoglutide to prediabetic 6-week-old male Zucker diabetic fatty (ZDF) rats. RESULTS: Proliferation was increased in a concentration-dependent manner up to fourfold by taspoglutide in MIN6B1 cells and was significantly stimulated in isolated rat islets. Taspoglutide almost completely prevented cytokine- or lipotoxicity-induced apoptosis in INS-1E cells (control 0.5%, cytokines alone 2.2%, taspoglutide + cytokines 0.6%, p < 0.001; palmitate alone 8.1%, taspoglutide + palmitate 0.5%, p < 0.001) and reduced apoptosis in isolated human islets. Treatment of ZDF rats with taspoglutide significantly prevented ß-cell apoptosis and preserved healthy islet architecture and insulin staining intensity as shown in pancreatic islet cross sections. Basal and glucose-stimulated insulin secretion of in situ perfused ZDF rat pancreata was normalized after taspoglutide treatment. CONCLUSIONS: Taspoglutide promoted ß-cell proliferation, prevented apoptosis in vitro and exerted multiple ß-cell protective effects on islet architecture and function in vivo in ZDF rats.

Interventional 4D motion estimation and reconstruction of cardiac vasculature without motion periodicity assumption.

Anatomical and functional information of cardiac vasculature is a key component in the field of interventional cardiology. With the technology of C-arm CT it is possible to reconstruct static intraprocedural 3D images from angiographic projection data. Current approaches attempt to add the temporal dimension (4D). In the assumption of periodic heart motion, ECG-gating techniques can be used. However, arrhythmic heart signals and slight breathing motion are degrading image quality frequently. To overcome those problems, we present a reconstruction method based on a 4D time-continuous B-spline motion field. The temporal component of the motion field is parameterized by the acquisition time and does not assume a periodic heart motion. The analytic dynamic FDK-reconstruction formula is used directly for the motion estimation and image reconstruction. In a physical phantom experiment two vessels of size 3.1mm and 2.3mm were reconstructed using the proposed method and an algorithm with periodicity assumption. For a periodic motion both methods obtained an error of 0.1mm. For a non-periodic motion the proposed method was superior, obtaining an error of 0.3mm/0.2mm in comparison to 1.2mm/1.0mm for the algorithm with periodicity assumption. For a clinical test case of a left coronary artery it could be further shown that the method is capable to produce diameter measurements with an absolute error of 0.1mm compared to state-of-the-art measurement tools from orthogonal coronary angiography. Further, it is shown for three different clinical cases (left/right coronary artery, coronary sinus) that the proposed method is able to handle a large variability of vascular structures and motion patterns. The complete algorithm is hardware-accelerated using the GPU requiring a computation time of less than 3min for typical clinical scenarios.

Bulk fluorescence measurements cannot probe the survival-time distribution of single molecules.

Using a straightforward theoretical approach, we show that an ensemble of dyes with a simple first-order photobleaching kinetics yields a power-law decaying emission intensity in a heterogeneous excitation field in bulk fluorescence experiments. Our theoretical considerations are experimentally confirmed for two distinct classes of small organic fluorophores represented by the classical laser dye rhodamine-6G on glass in air and the cyanine dye DiI(C18) in a thin polymethylmethacrylate film. Our results provide evidence that the time course of bleaching in a bulk sample in general does not allow derivation of the properties of survival times of individual quantum systems.

Visualizing odorant receptor trafficking in living cells down to the single-molecule level.

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of approximately 190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

Single-molecule identification by spectrally and time-resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.

Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light.

A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.