In vitro 2-deoxy-2-[18F]fluoro-D-glucose uptake: practical considerations

In oncology 2-deoxy-2-[F-18]fluoro-D-glucose ([F-18]-FDG), a glucose analogue, is the most used positron emission tomography (PET) tracer. There are however some limitations due to low metabolic activity or high surrounding physiological uptake in several tumors or regions. Investigating new tracers or methods is expensive and elaborative when animal experiments or phase I clinical trials are used. In vitro experiments can overcome these limitations. We analyzed the influence of incubation time, cell medium conditions, administered activity, and cell density on [F-18]-FDG uptake in six different cell cultures. Glucose transporter 1 (GLUT1)- and hexokinase 2 (HK2)-expression at high and low cell density was analyzed using immunocytochemistry. FDG-uptake increases over time and absence of glucose in the incubation medium increases uptake. By increasing the administered activity, uptake per protein also increases and tracer uptake per protein is lower at higher cell densities. Immunocytochemical analysis reveals a lower expression of both GLUT1 and HK2 at higher cell concentrations. All investigated parameters influenced FDG uptake and therefore we can conclude it is of utmost importance to keep administered activity, incubation medium, and time constant and to correct uptake when cell density changes due to environmental conditions, such as therapy

LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line

Development of a stereovision-based technique to measure the spread patterns of granular fertilizer spreaders

Centrifugal fertilizer spreaders are by far the most commonly used granular fertilizer spreader type in Europe. Their spread pattern however is error-prone, potentially leading to an undesired distribution of particles in the field and losses out of the field, which is often caused by poor calibration of the spreader for the specific fertilizer used. Due to the large environmental impact of fertilizer use, it is important to optimize the spreading process and minimize these errors. Spreader calibrations can be performed by using collection trays to determine the (field) spread pattern, but this is very time-consuming and expensive for the farmer and hence not common practice. Therefore, we developed an innovative multi-camera system to predict the spread pattern in a fast and accurate way, independent of the spreader configuration. Using high-speed stereovision, ejection parameters of particles leaving the spreader vanes were determined relative to a coordinate system associated with the spreader. The landing positions and subsequent spread patterns were determined using a ballistic model incorporating the effect of tractor motion and wind. Experiments were conducted with a commercial spreader and showed a high repeatability. The results were transformed to one spatial dimension to enable comparison with transverse spread patterns determined in the field and showed similar results

Paradigm of biased PAR1 (protease-activated receptor-1) activation and inhibition in endothelial cells dissected by phosphoproteomics

Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology

Alien macroinvertebrates in Flanders (Belgium)

Biological invasions of aquatic macroinvertebrates are gaining interest because of their potential for significant ecological and socio-economic impacts (positive and negative). In the present study, an inventory was made of the alien macroinvertebrates occurring in Flanders (northern Belgium) based on extensive existing collections of biological samples and supplemented with our additional sampling programs. Fresh and brackish waters as well as the Belgian coastal harbours, situated at the interface of the marine environment, were investigated. Over 2,500 samples containing alien macroinvertebrates were identified to species level, which allowed us to accurately map their distribution in Flanders. Alien macroinvertebrates are widespread and abundant in many watercourses in Flanders. Four new macroinvertebrate species for Flanders were discovered: Procambarus clarkii (Girard, 1852), Echinogammarus trichiatus (Martynov, 1932), Synurella ambulans (F. Müller, 1846) and Laonome calida Capa, 2007. Fifty-two alien macroinvertebrates were encountered in fresh and slightly brackish surface waters, and 21 alien species were reported for the Belgian part of the North Sea and its adjacent estuaries. Most alien macroinvertebrates collected were crustaceans and molluscs. Alien species found in fresh and brackish water mainly originate from the Ponto-Caspian area and North America; fewer species originated from Asia and South- and East-Europe. The major pathways were probably shipping and dispersal through canals. Based on observations in neighbouring countries, several additional species are expected to arrive in the near future. Follow-up work is needed to assess the ecological and economic impacts of existing alien macroinvertebrates, and a monitoring program is needed to detect new incoming species

Regional accuracy of ZTE-based attenuation correction in static and dynamic brain PET/MR

Accurate MR-based attenuation correction (MRAC) is essential for quantitative PET/MR imaging of the brain. In this study, we analyze the regional bias caused by MRAC based on Zero-Echo-Time MR images (ZTEAC) compared to CT-based AC (CTAC) in static and dynamic PET imaging. In addition the results are compared to the performance of the current default Atlas-based AC (AtlasAC) implemented in the GE SIGNA PET/MR. Methods: Thirty static [18F]FDG and 11 dynamic [18}F]PE2I acquisitions from a GE SIGNA PET/MR were reconstructed using ZTEAC (using a research tool, GE Healthcare), single-subject AtlasAC (the current default AC in GE's SIGNA PET/MR) and CTAC (from a PET/CT acquisition of the same day). In the 30 static [18F]FDG reconstructions, the bias caused by ZTEAC and AtlasAC in the mean uptake of 85 anatomical volumes of interest (VOIs) of the Hammers' atlas was analyzed in PMOD. For the 11 dynamic [18}F]PE2I reconstructions, the bias caused by ZTEAC and AtlasAC in the non displaceable binding potential BPnd in the striatum was calculated with cerebellum as the reference region and a simplified reference tissue model. Results: The regional bias caused by ZTEAC in the static [18F]FDG reconstructions ranged from -8.0% to +7.7% (mean 0.1%, SD 2.0%). For AtlasAC this bias ranged from -31.6% to +16.6% (mean -0.4%, SD 4.3%). The bias caused by AtlasAC showed a clear gradient in the cranio-caudal direction (-4.2% in the cerebellum, +6.6% in the left superior frontal gyrus). The bias in the striatal BPnd for the [18F]PE2I reconstructions ranged from -0.8% to +4.8% (mean 1.5%, SD 1.4%) using ZTEAC and from -0.6% to +9.4% using AtlasAC (mean 4.2%, SD 2.6%). Conclusion: ZTEAC provides excellent quantitative accuracy for static and dynamic brain PET/MR, comparable to CTAC, and is clearly superior to the default AtlasAC currently implemented in the GE SIGNA PET/MR.Comment: 23 pages in total, 7 figures, 1 table, 3 supplementary figures, 5 supplementary table