Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 μM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.     

Interleukin-8 expression associated with canine mammary tumors

The use of prognostic markers for mammary cancer is important for routine diagnosis and research. Interleukin-8 (IL-8) is a chemotactic cytokine, produced by several cell types in response to inflammation. The expression, regulation and function of IL-8 in dogs are little known. Recent studies have associated angiogenesis and inflammatory processes with tumor malignancy. We investigated a possible correlation between IL-8 expression and mammary tumor prognosis in female dogs. IL-8 expression was measured in 50 dogs with mammary neoplasia by immunohistochemistry and real-time PCR. Immunohistochemical staining was done with anti-IL-8 antibodies and PCR amplifications were performed in a 7500 Fast Real-Time PCR system. Gene expression stability was analyzed by the geNorm software. Quantitative real-time PCR showed that IL-8 expression decreased in malignant mammary cells compared to normal mammary tissue, while weak immunostaining was associated with a diagnosis of carcinoma. Complementing earlier studies on IL-8 expression in several types of cancer, including mammary cancer, we conclude that IL-8 has potential for use as a prognostic marker for canine mammary neoplasia.     

Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate

Objectives: We have investigated foetal mesenchymal stem cells (MSCs) obtained from first‐trimester chorionic villi (CV) and second‐trimester amniotic fluid (AF), comparing them to adult bone marrow‐derived MSCs. Materials and methods: We report on cell population growth in human allogeneic serum (HS) and platelet lysate (PL), immunophenotype, cytokine expression profile and immunoregulatory activity, of these foetal MSCs on stimulated peripheral blood mononuclear and lymphocyte subpopulations. Results: Chorionic villi cells grow rapidly in HS, with 20 populations doublings (PDs) after 59 days (six passages), and also in animal serum, with 27 PDs after 65 days (seven passages). PL allowed for expansion in 60% of the samples tested, although it was lower than in HS. HS supported an average of 40 PDs of expansion in 20% of AF cells after 90 days, whereas animal serum supported 28.5 PDs in 66 days. CV and AF cells inhibited proliferation of stimulated T lymphocytes, suppressing population growth of both CD4+ and CD8+ T subpopulations and sometimes also, CD19+ cells. Conclusions: Our results indicate that CV would be an optimal source of MSCs with high expansion potential in a HS propagation system and immunoregulatory capacity of T and B lymphocytes. More than 90% of CV samples achieved large‐scale expansion in HS, which is encouraging for potential clinical applications of these cells.     

Beeswax as Dental Filling on a Neolithic Human Tooth

Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individual’s death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling.     

Binding of an Indenoisoquinoline to the Topoisomerase-DNA Complex Induces Reduction of Linker Mobility and Strengthening of Protein-DNA Interaction

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect.