Development and Validation of Sterility Systems for Trees

The overall goal of this project was to develop and validate sterility systems in poplar with the ultimate goal of fulfilling the basic requirements for commercial use. For this, sterility must be complete and stable over multiple growing seasons, cause no detrimental effects on vegetative growth, and successful transformation events must be identifiable via molecular tests when trees are still juvenile. Because of the inherent difficulties in achieving and demonstrating complete sterility in trees, our approach was to study alternate sterility systems in Arabidopsis and/or early-flowering tree systems. The public benefit from this work is the capacity for containment of genes or exotic forms of trees so they can be of benefit for industry for production of wood, energy, and renewable products, while having minimal impact on wild populations of trees. We tested three methods for engineering sterility: dominant negative mutant (DNM) proteins, floral tissue ablation, and RNA interference (RNAi) to suppress the expression of several floral regulatory genes. The ultimate goal of this work was to produce a number of transgenic poplars that could be outplanted to enable future assessments of the effectiveness of these transgenic sterility methods. Our attempts to produce ablation constructs that did not interfere with tree health were partially successful. Using the poplar LEAFY gene promoter and the barnase/barstar system, we were able to regenerate plants that grew well in the greenhouse, but they showed poor health in the field. Four of seven DNM genes tested were considered promising enough, based on results in Arabidopsis, to produce transgenic poplars. Single, double, and triple RNAi genes were produced and transformed into poplar. Over all, we produced 1,964 PCR-confirmed transgenic events with 19 different kinds of sterility genes and several kinds of control genes. We propagated 5,640, 6,820, and 7,055 trees for each of three test poplar genotypes, and field plantings were begun in Spring of 2003 and will be finished in Spring 2007. Continued field studies and monitoring will be required to establish if any of the approaches we have taken will prove to be safe for tree health, stable, and provide reliable containment

Genome Enabled Discovery of Carbon Sequestration Genes in Poplar

The goals of the S.H. Strauss laboratory portion of 'Genome-enabled discovery of carbon sequestration genes in poplar' are (1) to explore the functions of candidate genes using Populus transformation by inserting genes provided by Oakridge National Laboratory (ORNL) and the University of Florida (UF) into poplar; (2) to expand the poplar transformation toolkit by developing transformation methods for important genotypes; and (3) to allow induced expression, and efficient gene suppression, in roots and other tissues. As part of the transformation improvement effort, OSU developed transformation protocols for Populus trichocarpa 'Nisqually-1' clone and an early flowering P. alba clone, 6K10. Complete descriptions of the transformation systems were published (Ma et. al. 2004, Meilan et. al 2004). Twenty-one 'Nisqually-1' and 622 6K10 transgenic plants were generated. To identify root predominant promoters, a set of three promoters were tested for their tissue-specific expression patterns in poplar and in Arabidopsis as a model system. A novel gene, ET304, was identified by analyzing a collection of poplar enhancer trap lines generated at OSU (Filichkin et. al 2006a, 2006b). Other promoters include the pGgMT1 root-predominant promoter from Casuarina glauca and the pAtPIN2 promoter from Arabidopsis root specific PIN2 gene. OSU tested two induction systems, alcohol- and estrogen-inducible, in multiple poplar transgenics. Ethanol proved to be the more efficient when tested in tissue culture and greenhouse conditions. Two estrogen-inducible systems were evaluated in transgenic Populus, neither of which functioned reliably in tissue culture conditions. GATEWAY-compatible plant binary vectors were designed to compare the silencing efficiency of homologous (direct) RNAi vs. heterologous (transitive) RNAi inverted repeats. A set of genes was targeted for post transcriptional silencing in the model Arabidopsis system; these include the floral meristem identity gene (APETALA1 or AP1), auxin response factor gene (ETTIN), the gene encoding transcriptional factor of WD40 family (TRANSPARENTTESTAGLABRA1 or TTG1), and the auxin efflux carrier (PIN-FORMED2 or PIN2) gene. More than 220 transgenic lines of the 1st, 2nd and 3rd generations were analyzed for RNAi suppression phenotypes (Filichkin et. al., manuscript submitted). A total of 108 constructs were supplied by ORNL, UF and OSU and used to generate over 1,881 PCR verified transgenic Populus and over 300 PCR verified transgenic Arabidopsis events. The Populus transgenics alone required Agrobacterium co-cultivations of 124.406 explants

Small sized EGFR1 and HER2 specific bifunctional antibody for targeted cancer therapy

licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2014.07.10; Accepted: 2015.01.05; Published: 2015.01.21 Targeting tumors using miniature antibodies is a novel and attractive therapeutic approach, as these biomolecules exhibit low immunogenicity, rapid clearance, and high targeting specificity. However, most of the small-sized antibodies in existence do not exhibit marked anti-tumor ef-fects, which limit their use in targeted cancer immunotherapy. To overcome this difficulty in targeting multiple biomarkers by combination therapies, we designed a new bifunctional antibody, named MaAbNA (multivalent antibody comprised of nanobody and affibody moieties), capable of targeting EGFR1 and HER2, which are widely overexpressed in a variety of tumor types. The small-sized (29 kDa) MaAbNA, which was expressed in E.coli, consists of one anti-EGFR1 nano-body and two anti-HER2 affibodies, and possesses high affinity (KD) for EGFR1 (~4.1 nM) and HER2 (~4.7 nM). In order to enhance its anti-tumor activity, MaAbNA was conjugated with adriamycin (ADM) using a PEG2000 linker, forming a new complex anticancer drug, MaAbNA-PEG2000-ADM. MaAbNA exhibited high inhibitory effects on tumor cell

Ethyne Reducing Metal-Organic Frameworks to Control Fabrications of Core/shell Nanoparticles as Catalysts

An approach using cobalt metal-organic frameworks (Co-MOF) as precursors is established for the fabrication of cobalt nanoparticles in porous carbon shells (core/shell Co@C). Chemical vapor deposition of ethyne is used for controlling the reduction of cobalt nanoclusters in the MOF and the spontaneous formation of the porous carbon shells. The metallic cobalt cores formed are up to 4 - 6 nm with the crystal phase varying between hexagonally-close-packed (hcp) and face-centre-packed (fcc). The porous carbon shells change from amorphous to graphene with the ethyne deposition temperature increasing from 400 to 600 oC. The core/shell Co@C nanoparticles exhibit high catalytic activity in selectively converting syngas (CTY: 254.1 - 312.1 μmolCO·gCo-1·s-1) into hydrocarbons (4.0 - 5.2 gHC·g-cat-1·h-1) at 260 oC. As well as the crystal size and phase, the coordination numbers of the cobalt to oxygen and to other cobalt atoms on the surface of the cobalt nanoparticles, and the permeability of the porous carbon shell have been related to the catalytic performance in FTS reactions

Single-Phase θ-Fe3C Derived from Prussian Blue and Its Catalytic Application in Fischer-Tropsch Synthesis

Elucidation of the intrinsic catalytic principle of iron carbides remains a substantial challenge in iron-catalyzed Fischer-Tropsch synthesis (FTS), due to possible interference from other Fe-containing species. Here, we propose a facile approach to synthesize single-phase θ-Fe3C via the pyrolysis of a molecularly defined Fe-C complex (Fe4[Fe(CN)6]3), thus affording close examination of its catalytic behavior during FTS. The crystal structure of prepared θ-Fe3C is unambiguously verified by combined XRD and MES measurement, demonstrating its single-phase nature. Strikingly, single-phase θ-Fe3C exhibited excellent selectivity to light olefins (77.8%) in the C2-C4 hydrocarbons with less than 10% CO2 formation in typical FTS conditions. This strategy further succeeds with promotion of Mn, evident for its wide-ranging compatibility for the promising industrial development of catalysts. This work offers a facile approach for oriented preparation of single-phase θ-Fe3C and provides an in-depth understanding of its intrinsic catalytic performance in FTS