Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D ¹H-detected ¹H,¹⁵N and 3D ¹H,¹³C,¹⁵N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.     

Click-Chemistry (CuAAC) Trimerization of an αvβ6 Integrin Targeting Ga-68-Peptide: Enhanced Contrast for in-Vivo PET Imaging of Human Lung Adenocarcinoma Xenografts

α_vβ₆ Integrin is an epithelial transmembrane protein that recognizes latency-associated peptide (LAP) and primarily activates transforming growth factor beta (TGF-β). It is overexpressed in carcinomas (most notably, pancreatic) and other conditions associated with α_vβ₆ integrin-dependent TGF-β dysregulation, such as fibrosis. We have designed a trimeric Ga-68-labeled TRAP conjugate of the α_vβ₆-specific cyclic pentapeptide SDM17 (cyclo[RGD-Chg-E]-CONH₂) to enhance α_vβ₆ integrin affinity as well as target-specific in-vivo uptake. Ga-68-TRAP(SDM17)₃ showed a 28-fold higher α_vβ₆ affinity than the corresponding monomer Ga-68-NOTA-SDM17 (IC₅₀ of 0.26 vs. 7.4 nM, respectively), a 13-fold higher IC₅₀-based selectivity over the related integrin α_vβ₈ (factors of 662 vs. 49), and a threefold higher tumor uptake (2.1 vs. 0.66 %ID/g) in biodistribution experiments with H2009 tumor-bearing SCID mice. The remarkably high tumor/organ ratios (tumor-to-blood 11.2; -to-liver 8.7; -to-pancreas 29.7) enabled high-contrast tumor delineation in PET images. We conclude that Ga-68-TRAP(SDM17)₃ holds promise for improved clinical PET diagnostics of carcinomas and fibrosis.     

Effects of timing of formative feedback in computer-assisted learning environments

This study examines the effects of timing of corrective formative feedback on processing text information on question-answering. Undergraduate students read an expository text and answered questions in two attempts. Students were randomly assigned to a no feedback, immediate feedback and delayed feedback conditions. Students in the feedback conditions received feedback on the correctness of their answer after the first attempt and were informed about the right answer after the second attempt. Students were prompted to restudy the text after failing in their first attempt. However, students in the no feedback condition were just prompted to search the text. All students were tested on question-answering, corrective probability and a post-test cued-recall test. Results showed that: (a) feedback reduced the initial time reading the text; (b) feedback increased performance on question answering and cued-recall; (c) delayed feedback produced no advantages over immediate feedback. Theoretical and practical implications of these results are discussed.     

Impact of calcium and TOC on biological acidification assessment in Norwegian rivers

Acidification continues to be a major impact in freshwaters of northern Europe, and the biotic response to chemical recovery from acidification is often not a straightforward process. The focus on biological recovery is relevant within the context of the EU Water Framework Directive, where a biological monitoring system is needed that detects differences in fauna and flora compared to undisturbed reference conditions. In order to verify true reference sites for biological analyses, expected river pH is modeled based on Ca and TOC, and 94% of variability in pH at reference sites is explained by Ca alone, while 98% is explained by a combination of Ca and TOC. Based on 59 samples from 28 reference sites, compared to 547 samples from 285 non-reference sites, the impact of calcium and total organic carbon (TOC) on benthic algae species composition, expressed as acidification index periphyton (AIP), is analyzed. Rivers with a high Ca concentration have a naturally higher AIP, and TOC affects reference AIP only at low Ca concentrations. Four biological river types are needed for assessment of river acidification in Norway based on benthic algae: very calcium-poor, humic rivers (Ca < 1 mg/l and TOC > 2 mg/l); very calcium-poor, clear rivers (Ca < 1 mg/l and TOC < 2 mg/l); calcium-poor rivers (Ca between 1 and 4 mg/l); moderately calcium rich rivers (Ca > 4 mg/l). A biological assessment system for river acidification in Norway based on benthic algae is presented, following the demands of the Water Framework Directive.     

Vorkommen und Verhalten eines Arzneimittels (Clofibrinsäure) im Grundwasser

As to nowadays knowledge drugs in groundwater originate from anthropogenic input by urine and production residue. Clofibric acid, a blood lipid regulator is one of the human drugs already detected in groundwater and drinking water with concentrations of up to 7300 ng/l. Recent investigations indicate, that some drugs are insufficiently or not degraded by sewage treatment or by drinking water production. Groundwater contamination is likely to be due to leaky sewage systems, influent streams and bank infiltration. The distribution of clofibric acid beneath the sewage farms south of Berlin shows the high mobility of this contaminant. Nevertheless, results indicate that there is no correlation between the concentrations of clofibric acid and physico-chemical parameters, groundwater constituents nor sediment material. First results of a column experiment show that clofibric acid is transported with tracer velocity, although some variations in concentration of clofibric acid occur. Again, no correlation between clofibric acid and pH, oxygen content nor the concentration of groundwater constituents has been found whereas a possible dependence of the redox potential on clofibric acid was observed. During the column experiment no sorption processes could be identified, but there were hints for a degradation of clofibric acid.