Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([¹³C₆, ¹⁵N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC₅₀ of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [¹³C₆, ¹⁵N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC₅₀ values of the inhibitors were in concordance with previously reported values. Furthermore, the [¹³C₆, ¹⁵N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([¹³C₆, ¹⁵N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.     

Theoretische Untersuchungen zum Zerfallsgleichgewicht des Harnstoffs in Gasphase und in wäßriger Lösung

Theoretical Investigations on the Decomposition Equilibrium of Urea in Gaseous State and Aqueous Solution There is empirical evidence that among the two possible decomposition equilibria of urea in the gaseous and aqueous states the desammonation process prevails. In the present work the thermodynamic and kinetic conditions of desammonation and dehydration based on intramolecular mechanisms were examined by means of quantum chemical methods. The application of MNDO method yields correct structural data, dipole moments and ionisation potentials; the solvation is taken into account using NDDO based solvation and reaction field models. Maximum deviations concerning enthalpies of formation in π-electron rich compounds reach up to 40kJ/mole. A detailed examination of the energy hyperfaces reveals that the dehydration is kinetically hindered by a significantly high activation barrier. This situation is not essentially altered by taking into account nonspecific solvent effects in aqueous solution.     

Anti-Inflammatory Characteristics of Local Anesthetics: Inhibition of TNF-α Secretion of Lipopolysaccharide-Stimulated Leucocytes in Human Blood Samples

Background. Local anesthetics (LAs) have potent anti-inflammatory properties. Inflammatory down-regulation is crucial in diseases with overactive immune reactions, such as acute respiratory distress syndrome (ARDS) and chronic inflammation. We investigated the influence of four LAs, procaine, lidocaine, mepivacaine, and bupivacaine, on the reduction of tumor necrosis factor-alpha (TNF-α) secretion in lipopolysaccharide (LPS)-activated human leucocytes. Methods. Blood samples of 28 individuals were stimulated with LPS. The reduction of TNF-α production by each of the four LAs added (0.5 mg/mL) was measured and correlated with biometric variables. A response was defined as reduction to <85% of initial levels. Results. All four LAs down-regulated the TNF-α secretion in 44–61%: Bupivacaine (44.4%), lidocaine (61.5%), mepivacaine (44.4%), and procaine (50% of the individuals, “responders”). The TNF-α secretion was reduced to 67.4, 68.0, 63.6, and 67.1% of the initial values in responders. The effects in both patients and healthy persons were the same. Interindividual responses to LAs were not correlated with the duration or type of complaints, basal TNF-α serum level, sex, BMI, or age of responders. Conclusions. Four clinically relevant LAs (amid-LA and ester-LA) attenuate the inflammatory response provoked by LPS. They are potential candidates for drug repositioning in treating overactive immune reactions and chronic inflammation.     

Validation of coupled codes using VVER plant measurements

A data set of five transients at different VVER type nuclear power plants was collected in order to validate neutron kinetics/thermal hydraulics codes. Two of these transients ‘drop of control rod at nominal power at Bohunice-3’ of VVER-440 type and ‘coast-down of 1 from 3 working MCPs at Kozloduy-6’ of VVER-1000 type, were then utilised for code validation. Eight institutes contributed to the validation with 10 calculations using 5 different combinations of coupled codes. The thermal hydraulic codes were ATHLET, SMABRE and RELAP5 and the neutron kinetic codes DYN3D, HEXTRAN, KIKO3D and BIPR8. The general behaviour of both the transients was quite well calculated with all the codes. Even an elementary modelling of coolant mixing in reactor pressure vessel under asymmetric transients improved correspondence to the measurements. Some differences between the calculations seem to indicate that fuel modelling and treatment of VVER-440 control rods need further consideration. The simultaneous validation interacted with the data collection effort and thus improved its quality. The complexity of data collection systems and sometimes conflicting data, however, called for compromises and interpretation guides that also taught the analysts balanced plant modelling.     

Neutron-kinetic code validation against measurements in the Moscow V-1000 zero-power facility

Measurements carried out in an original-size VVER-1000 mock-up (V-1000 facility, Kurchatov Institute, Moscow) were used for the validation of three-dimensional neutron-kinetic codes, designed for VVER safety calculations. The significant neutron flux tilt measured in the V-1000 core, which is caused only by radial-reflector asymmetries, was successfully modeled. A good agreement between calculated and measured steady-state powers has been achieved, for relative assembly powers and inner-assembly pin power distributions. Calculated effective multiplication factors exceed unity in all cases. The time behaviour of local powers, measured during two transients that were initiated by control rod moving in a slightly super-critical core, has been well simulated by the neutron-kinetic codes.     

Experiments at the mixing test facility ROCOM for benchmarking of CFD codes

For the validation of computational fluid dynamics (CFD) codes, experimental data on fluid flow parameters with high resolution in time and space are needed.Rossendorf Coolant Mixing Model (ROCOM) is a test facility for the investigation of coolant mixing in the primary circuit of pressurized water reactors. This facility reproduces the primary circuit of a German KONVOI-type reactor. All important details of the reactor pressure vessel are modelled at a linear scale of 1:5. The facility is characterized by flexible possibilities of operation in a wide variety of flow regimes and boundary conditions. The flow path of the coolant from the cold legs through the downcomer until the inlet into the core is equipped with high-resolution detectors, in particular, wire mesh sensors in the downcomer of the vessel with a mesh of 64 × 32 measurement positions and in the core inlet plane with one measurement position for the entry into each fuel assembly, to enable high-level CFD code validation. Two different types of experiments at the ROCOM test facility have been proposed for this purpose. The first proposal concerns the transport of a slug of hot, under-borated condensate, which has formed in the cold leg after a small break LOCA, towards the reactor core under natural circulation. The propagation of the emergency core cooling water in the test facility under natural circulation or even stagnant flow conditions should be investigated in the second type of experiment. The measured data can contribute significantly to the validation of CFD codes for complex mixing processes with high relevance for nuclear safety.     

Bandwidth performance of 16-element thinned phased array withtapped delay-line filter

The bandwidth performance of a thinned phased adaptive array with and without a delay-line processor are compared. Five- and nine-jammer scenarios are considered for two EHF bands, 0.125 and 2.0 GHz. The null-depth which was used as the measure of performance was computed over the nulling band for three different delay-line configurations. The simulation results give some insight into the way in which delay-line compensation provides improved performance as the interference bandwidth increases     

Expression level of a transgenic lambda2 chain results in isotype exclusion and commitment to B1 cells

Two new lambda2 chain-transgenic mouse lines were established, both of which showed stable transgene expression during aging of the mice. The line L23, which expressed the transgene at low levels, exhibited normal B cell development, antibody responses and serum Ig levels. Most of the B cells in this mouse line co-expressed the transgenic lambda2 chain together with an endogenous kappa chain, thus showing poor allelic exclusion of endogenous L chains. On the other hand, high expression of the transgenic lambda2 chain in the other mouse line, L2, resulted in nearly complete exclusion of endogenous L chain isotypes. In this line, the lambda2 transgene was already detectable in the cytoplasm of all preB-II cells and some pro/preB-I cells. Its expression during these early phases obviously inhibited development of conventional B2 cells, since the B cells in the periphery of these mice were almost exclusively of the B1 type. This finding was confirmed by adoptive transfer of transgenic bone marrow into lethally irradiated recipients. Very few B cells were present in the spleen of such recipients. The serum IgM levels of L2 mice were close to normal and the majority of these IgM were associated with the transgenic lambda2 chain. Antibody responses to thymus-dependent antigens in such mice were almost exclusively found to be of IgM class. Together, these findings indicate a developmental bias leading to a predominance of B1 cells in the L2 line.