Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector

Ultra-broadband surface-normal coherent optical receiver with nanometallic polarizers

A coherent receiver that can demodulate high-speed in-phase and quadrature signals of light is an essential component for optical communication, interconnects, imaging, and computing. Conventional waveguide-based coherent receivers, however, exhibit large footprints, difficulty in coupling a large number of spatial channels efficiently, and limited operating bandwidth imposed by the waveguide-based optical hybrid. Here, we present a surface-normal coherent receiver with nanometallic-grating-based polarizers integrated directly on top of photodetectors without the need for an optical hybrid circuit. Using a fabricated device with the active section occupying a 70-{\mu}m-square footprint, we demonstrate demodulation of high-speed (up to 64 Gbaud) coherent signals in various formats. Moreover, ultra-broadband operation from 1260 nm to 1630 nm is demonstrated, thanks to the wavelength-insensitive nanometallic polarizers. To our knowledge, this is the first demonstration of a surface-normal homodyne optical receiver, which can easily be scaled to a compact two-dimensional arrayed device to receive highly parallelized coherent signals.Comment: 23 pages, 4 figures (main manuscript) + 4 pages, 2 figures (supporting info

Effects of KP-496, a Novel Dual Antagonist for Leukotriene D4 and Thromboxane A2 Receptors, on Contractions Induced by Various Agonists in the Guinea Pig Trachea

Background: A dry powder inhaler of KP-496 is currently in clinical development in Japan as an antiasthmatic agent. The aim of this study was to evaluate the in vitro pharmacological profile of KP-496. Methods: The antagonistic activities of KP-496 for leukotriene (Lt) D4 and thromboxane (TX) A2 receptors were examined using the LTD4 - and U46619-induced contractions of the isolated guinea pig trachea. The selectivity of KP-496 was examined using various agonist-induced contractions in the isolated guinea pig trachea. Results: KP-496 produced parallel rightward shifts of the LTD4 and U46619 concentration-response curves in a concentration-dependent manner. Schild plot analyses of the antagonistic activities of KP-496 demonstrated that it is a competitive antagonist for LTD4 and TXA2 receptors with pA2 values of 8.64 and 8.23, respectively. The LTD4 antagonistic activity of KP-496 was comparable to that of pranlukast and zafirlukast but was more potent than that of montelukast. The TXA2 antagonistic activity of KP-496 was comparable to that of seratrodast. KP-496 and seratrodast also inhibited the prostaglandin (PG) D2 - and PGF2α-induced contractions of the isolated guinea pig trachea. KP-496 had no effect on the histamine-, acetylcholine-, serotonin- and substance P-induced contractions of the isolated guinea pig trachea. Conclusions: These results indicate that KP-496 is a selective dual antagonist for LTD4 and TXA2 receptors. LTD4 and TXA2 play important roles in asthma, and antagonists for these mediators are being used for the treatment of asthma. Thus, KP-496 is expected to become a novel potent therapeutic agent for asthma

Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E−4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction. Keywords: Early afterdepolarization, hiPSC-CMs, Microelectrode array, Proarrhythmia, Safety assessmen