RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants.

A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector

UPLC-MS-ELSD-PDA as a Powerful Dereplication Tool to Facilitate Compound Identification from Small-Molecule Natural Product Libraries

The generation of natural product libraries containing column fractions, each with only a few small molecules, using a high-throughput, automated fractionation system, has made it possible to implement an improved dereplication strategy for selection and prioritization of leads in a natural product discovery program. Analysis of databased UPLC-MS-ELSD-PDA information of three leads from a biological screen employing the ependymoma cell line EphB2-EPD generated details on the possible structures of active compounds present. The procedure allows the rapid identification of known compounds and guides the isolation of unknown compounds of interest. Three previously known flavanone-type compounds, homoeriodictyol (<b>1</b>), hesperetin (<b>2</b>), and sterubin (<b>3</b>), were identified in a selected fraction derived from the leaves of <i>Eriodictyon angustifolium</i>. The lignan compound deoxypodophyllotoxin (<b>8</b>) was confirmed to be an active constituent in two lead fractions derived from the bark and leaves of <i>Thuja occidentalis</i>. In addition, two new but inactive labdane-type diterpenoids with an uncommon triol side chain were also identified as coexisting with deoxypodophyllotoxin in a lead fraction from the bark of <i>T. occidentalis.</i> Both diterpenoids were isolated in acetylated form, and their structures were determined as 14<i>S</i>,15-diacetoxy-13<i>R</i>-hydroxylabd-8­(17)-en-19-oic acid (<b>9</b>) and 14<i>R</i>,15-diacetoxy-13<i>S</i>-hydroxylabd-8­(17)-en-19-oic acid (<b>10</b>), respectively, by spectroscopic data interpretation and X-ray crystallography. This work demonstrates that a UPLC-MS-ELSD-PDA database produced during fractionation may be used as a powerful dereplication tool to facilitate compound identification from chromatographically tractable small-molecule natural product libraries

A Theory of Natural Addiction

Economic theories of rational addiction aim to describe consumer behavior in the presence of habit-forming goods. We provide a biological foundation for this body of work by formally specifying conditions under which it is optimal to form a habit. We demonstrate the empirical validity of our thesis with an in-depth review and synthesis of the biomedical literature concerning the action of opiates in the mammalian brain and their effects on behavior. Our results lend credence to many of the unconventional behavioral assumptions employed by theories of rational addiction, including adjacent complementarity and the importance of cues, attention, and self-control in determining the behavior of addicts. Our approach suggests, however, that addiction is “harmful” only when the addict fails to implement the optimal solution. We offer evidence for the special case of the opiates that harmful addiction is the manifestation of a mismatch between behavioral algorithms encoded in the human genome and the expanded menu of choices--generated for example, by advances in drug delivery technology--faced by consumers in the modern world