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.     

Sensorische Analyse, Malat- und Saccharosegehalt und Fruchtfleischfestigkeit verschiedener Apfelsorten

Zusammenfassung Neben einer ausführlichen sensorischen Beurteilung der Frühsorte Jamba, der mittelspäten Sorten Holsteiner Cox und Roter Holsteiner Cox sowie der späten Sorte Gloster wurden parallel dazu der Malat- und Saccharosegehalt und die Fruchtfleischfestigkeit bestimmt. Die Korrelation dieser chemisch-physikalischen Parameter mit der Bewertung des Geschmacks und der Fruchtfleischstruktur sollte die Frage klären, ob eine Qualitätsaussage unter Umgehung der sensorischen Beurteilung möglich ist. Durch Vergleich ergab sich, daß für alle untersuchten Sorten der Malat- und/oder Saccharosegehalt wenig über die geschmacklichen Qualität der Früchte aussagt. Engere Beziehungen traten zwischen den Parametern sensorische Fruchtfleischbewertung und Festigkeit auf, wo bei einem Festigkeitswert von 5–5,3 kp/cm² und mehr das entsprechende senorische Urteil nicht schlechter als mittelmäßig ausfiel.

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Sensory evaluation, content of malate and sucrose, and fruit firmness of different apple varieties

Summary Parallel to a detailed sensory evaluation of the apple varieties Jamba, Holsteiner Cox, Red Holsteiner Cox and Gloster the content of malate and sucrose and fruit flesh firmness were measured. Comparisons between chemical and physical parameters and the sensory evaluation of taste and fruit flesh structure were performed to see, if apple quality can be determined without sensory assessment. The results for all samples show that content of malate and sucrose means little in relation to fruit taste. Recommendations for an optimum storage period could not be made. There was a closer relationship between sensory fruit flesh assessment and flesh firmness with a firmness of 5–5,3 kp/cm² or higher sensory evaluation was not worse than fair.

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Teil der Dissertation Mast, Kiel 1982     

Probing molecular mobility during crosslinking process of commercial resins by NMR multiexponential relaxation data analysis

In this study, we present the experimental results for the crosslinking process of a commercial polyester resin based on measurements of the spin lattice relaxation time T₁ of protons, as function of the crosslinking time evolution. Multiexponential decomposition of the evolution of magnetization measured in inversion‐recovery experiments is performed. The population of “rigid” and “mobile” nuclear spin sites was estimated as function of time evolution. In analogy to the usual monomer conversion u, site conversion from “mobile” to “rigid” sites u_M were also estimated as a function of time evolution and initial concentrations of the reagents. The multiexponential decomposition approach of T₁ relaxation data allows one to follow crosslinking processes. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

New Perspectives to Improve Mesenchymal Stem Cell Therapies for Drug-Induced Liver Injury

Drug-induced liver injury (DILI) is one of the leading causes of acute liver injury. Many factors may contribute to the susceptibility of patients to this condition, making DILI a global medical problem that has an impact on public health and the pharmaceutical industry. The use of mesenchymal stem cells (MSCs) has been at the forefront of regenerative medicine therapies for many years, including MSCs for the treatment of liver diseases. However, there is currently a huge gap between these experimental approaches and their application in clinical practice. In this concise review, we focus on the pathophysiology of DILI and highlight new experimental approaches conceived to improve cell-based therapy by the in vitro preconditioning of MSCs and/or the use of cell-free products as treatment for this liver condition. Finally, we discuss the advantages of new approaches, but also the current challenges that must be addressed in order to develop safer and more effective procedures that will allow cell-based therapies to reach clinical practice, enhancing the quality of life and prolonging the survival time of patients with DILI.     

Physicochemical and Theoretical Characterization of a New Small Non-Metal Schiff Base with a Differential Antimicrobial Effect against Gram-Positive Bacteria

Searching for adequate and effective compounds displaying antimicrobial activities, especially against Gram-positive bacteria, is an important research area due to the high hospitalization and mortality rates of these bacterial infections in both the human and veterinary fields. In this work, we explored (E)-4-amino-3-((3,5-di-tert-butyl-2-hydroxybenzylidene)amino) benzoic acid (SB-1, harboring an intramolecular hydrogen bond) and (E)-2-((4-nitrobenzilidene)amino)aniline (SB-2), two Schiff bases derivatives. Results demonstrated that SB-1 showed an antibacterial activity determined by the minimal inhibitory concentration (MIC) against Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus (Gram-positive bacteria involved in human and animal diseases such as skin infections, pneumonia, diarrheal syndrome, and urinary tract infections, among others), which was similar to that shown by the classical antibiotic chloramphenicol. By contrast, this compound showed no effect against Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, and Salmonella enterica). Furthermore, we provide a comprehensive physicochemical and theoretical characterization of SB-1 (as well as several analyses for SB-2), including elemental analysis, ESMS, ¹H and ¹³C NMR (assigned by 1D and 2D techniques), DEPT, UV-Vis, FTIR, and cyclic voltammetry. We also performed a computational study through the DFT theory level, including geometry optimization, TD-DFT, NBO, and global and local reactivity analyses.     

Neural Crest-Derived Stem Cells (NCSCs) Obtained from Dental-Related Stem Cells (DRSCs): A Literature Review on Current Knowledge and Directions toward Translational Applications

Evidence from dental-related stem cells (DRSCs) suggests an enhanced potential for ectodermal lineage differentiation due to their neural crest origin. Growing evidence that DRSC cultures can produce cells with a neural crest-derived stem cell (NCSC)-like phenotype supports their potential for future therapeutic approaches for neurodegenerative diseases and nerve injuries. However, most of the evidence is limited to the characterization of DRSCs as NCSCs by detecting the expression of neural crest markers. Only a few studies have provided proof of concept of an improved neuro-glial differentiation or direct applicability in relevant models. In addition, a current problem is that several of the existing protocols do not meet manufacturing standards for transferability to a clinical scenario. This review describes the current protocols to obtain NCSCs from DRSCs and their characterization. Also, it provides important considerations from previous work where DRSCs were established and characterized as mesenchymal stromal cells but studied for their neuro-glial differentiation potential. The therapeutic advancement of DRSCs would depend on establishing protocols that can yield a neural crest-like phenotype efficiently, using appropriate manufacturing standards and testing them in relevant models of disease or injury. Achieving these conditions could then facilitate and validate the therapeutic potential of DRSC-NCSCs in regenerative therapies.     

Supporting inheritance hierarchy changes in model-based regression test selection

Software and Systems Modeling - Models can be used to ease and manage the development, evolution, and runtime adaptation of a software system. When models are adapted, the resulting models must be...