The following student awards were presented at the 2016 World Congress on In Vitro Biology in San Diego, California. Information on additional awardees at the 2016 World Congress will be presented in the next issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website (www.sivb.org) or by contacting the SIVB Business Office at email@example.com.
2016 HONOR B. FELL AWARD AND 2016 STUDENT TRAVEL AWARD
Bioavailability of the Antimalarial Drug Artemisinin Delivered Orally as Dried Leaves of Artemisia annua: the Role of Solubility and Protein
Malaria treatment using orally consumed dried leaves of the artemisinin producing plant Artemisia annua has recently shown promise. Previously we showed, oral consumption ofA. annua dried leaves (DLA) yielded >40 times more artemisinin in the blood of mice than treatment with pure artemisinin. Using the Caco-2 cell culture model of the human intestinal epithelium, we also showed that compared to pure artemisinin, digested DLA doubled the permeability (Papp). Here, using simulated human digestion, we show that artemisinin solubility is about seven times higher in digestates of DLA than in digestates of pure artemisinin, likely contributing to its enhanced bioavailability. Digestion with pure artemisinin combined with levels of essential oils comparable to that in DLA increased the solubility of artemisinin 2.5 times indicating essential oils play a role in increasing artemisinin solubility. Interestingly, increasing the starting concentration of artemisinin in Caco-2 transport studies did not alter Papp. Considering malaria affects mostly young children and about 60% of the population experiences DLA as unpleasant tasting, we also tested several protein rich foods as potential flavor-masking agents for their effects on bioavailability. We showed that while taste was masked, peanuts and a peanut-based paste used to treat malnutrition, PlumpyNut, reduced artemisinin and flavonoid levels in simulated digestates, respectively, likely decreasing their bioavailability. Experiments to further investigate the role of several compounds such as camphor, a principle component of the essential oil fraction, and flavonoids on artemisinin solubility and bioavailability are ongoing. The results of these experiments are helping to explain the increased bioavailability afforded by DLA seen in mice.
Matt Desrosiers, Worcester Polytechnic Institute, Department of Biology and Biotechnology, 100 Institute Road, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 52:S45, 2016
2016 CELLULAR TOXICOLOGY AWARD
The Therapeutic Effects of per os Artemisinin Delivered as Dried Leaf Artemisia annua vs. Artesunate in Non-small Cell Lung Cancer
Artemisinin, the active component of Artemisia annua L. used to treat malaria, also has therapeutic efficacy against many types of cancer. Solubility issues led to development of more soluble semi-synthetic derivatives. Artesunate (ART), in particular, is a more soluble derivative of artemisinin and has profound cytotoxicity toward many types of tumor cells, but healthy cells are less sensitive. Artemisinin delivered per os as dried leaves, referred to as dried leaf artemisinin (DLA), was shown in rodent studies to improve bioavailability by more than 40-fold. ART has been widely studied for its anti-cancer properties, but it has yet to be shown if DLA also improves therapeutic efficacy. As A. annua produces a wide array of phytochemicals with anti-cancer activity other than artemisinin, it is reasonable to expect DLA may provide a more potent therapeutic. Using two non-small cell lung cancer cell lines, PC-9 and H1299, artemisinin delivered as DLA effectively reduced viability with 24h IC50 values of 56.3 and 77.5 µM for PC-9 and H1299, respectively, as determined by MTT assay. For PC-9 cells, this was a 2.5-fold improvement in the 24h IC50 value for ART at 142.9 µM. However, for the H1299 cells, ART at 60.6 µM was better than DLA by about 25%. Ongoing studies are comparing the mechanism of action of DLA and ART on these two cell lines and will analyze markers for apoptosis, proliferation and metastatic migration and invasion. Xenograft models also will be used to compare in vivo efficacy of DLA and ART on tumor reduction. These studies will help us further understand the anti-cancer effects of artemisinin when delivered per os as dried plant leaves.
Dina Rassias, Worcester Polytechnic Institute, Dept. of Biomedical Engineering, 100 Institute Road, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 52:S47-48, 2016
2016 JOSEPH F. MORGAN AWARD
Light Intensity Stress as the Limiting Factor in Micropropagation of Sugar Maple (Acer saccharum Marsh.)
Sugar maple (Acer saccharum Marsh.) is an important hardwood of North America and is the basis for the $350 million maple syrup industry in Canada. The energy to concentrate sap accounts for about 40% of syrup production costs and has a significant carbon footprint (Huyler & Garret, 1979). Developing elite germplasm with higher sap sugar content (SSC) to reduce this cost/impact has been the focus of much research, and while SSC is under genetic control, selection and propagation of “sweet” trees has not been feasible due to difficulties in vegetative propagation. Attempts of clonal propagation through tissue culture have been largely unsuccessful and the basis for this longstanding problem has remained unknown (Hanus and Rohr,1984, Connolly et al.,1998, Brassard et al., 2003 and Sutano and Stasolla, 2010). In the present study, we report that the critical factor limiting sugar maple’s response to in vitro propagation is light intensity. We used custom made growth chambers equipped with adjustable intensity LED lights of two spectra to demonstrate low light is required for in vitro multiplication. The effect of light quality on the electron transport chain in the in vitro leaves and various other stress parameters based on chlorophyll fluorescence imaging are discussed. Development of an efficient propagation system will help improve yields and reduce the carbon footprint of maple syrup production. Further, while this study is limited to maples, requirement of specific light intensity is likely the cause of recalcitrance in other woody species with similar ecological roles.
Amritpal S. Singh, University of Guelph, Department of Plant Agriculture, Guelph, ON, N1G 2W1, CANADA. In Vitro Cellular and Developmental Biology, 52:S67, 2016
2016 PHILIP R. WHITE AWARD
Novel Designer Glycopeptides as a Molecular Carrier Boosts Secreted Protein Yields in Plant Cell Culture.
Plant cell culture in controlled environment has been demonstrated to be a safe and cost-effective bioproduction platform for therapeutic proteins. However, Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Expression of recombinant proteins as fusion with a designer hydroxyproline-O-glycosylated glycopeptide (HypGP) tag have consistently boosted secreted protein yields as high as 1500-fold, due to the function of the HypGP tag as a molecular carrier to promote efficient transport of tagged-proteins into culture media. This prompted us to study the process development of this technology. Transgenic tobacco BY-2 cells expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of “Ser-Pro” dipeptide, or (SP)32, was used as a model system to study cell growth and protein secretion, manipulation of cell culture media and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in SH medium but secreted high yields of (SP)32-tagged EGFP (125 mg/L). The basal salts of culture media, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen supply increased the secreted EGFP yields to 170 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day-1, generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer HypGP engineering technology for use in commercial production of valuable proteins with plant cell cultures.
Ningning Zhang, Arkansas Bioscience Institute, Arkansas State University, Jonesboro, AR. In Vitro Cellular and Developmental Biology, 52:S69, 2016
2016 SIVB STUDENT TRAVEL AWARD
Analyzing Somatic Embryogenesis Gene Expression in Response to Tissue Culture Enhancer PLA1 Protein
It has been known for nearly half a century that ‘conditioned media’ aids tissue culture regeneration of plants. Advances in molecular biology and bioinformatics are now permitting unraveling the basis of these observations. Extracellular Phytocyanin-Like-Arabinogalactan-1 or PLA1 protein has been shown to improve somatic embryogenesis (SE) in cotton (Gossypium hirsutum); Poon et al. (2012) applied GhPLA1 proteins to tissue culture media for a two-fold increase in embryogenic calli production. Our work focuses on (1) production of orthologous PLA proteins and (2) investigation of their respective efficacies as a media additive towards enhanced regeneration of recalcitrant plants through SE. Specifically, we are evaluating PLA1 protein based on 1) observed effects on embryo production in Theobroma cacao SE tissue culture and 2) the effects of protein treatment on SE marker genes such as BBM, LEC1, LEC2, AGL15, and FUS3. Understanding how PLA1 interacts with SE genes can give insight into the complex orchestration of gene expression during the development of plant embryos. The generality of many elements surrounding the SE tissue culture process should allow improvement across all plants. GhPla1 protein was applied to T. cacao by dripping protein solution as a way to use less protein than media addition, while achieving the same enhancement effects. This is consistent with our goal to implement exposure in temporary immersion bioreactor propagation systems. Preliminary results showed embryos were produced earlier in protein treated tissue compared to non-protein treated tissue and GhPla1 treatment induced expression of SE transcription factor: BBM. Gene expression of BBM and other SE genes were analyzed via qPCR. In addition to GhPla1, we are developing PLA1 proteins from a variety of plant species. E. coli will be used to recombinantly express PLA1 proteins fromTheobroma cacao, Oryza sativa, and Dioscorea rotundata. PLA proteins may prove to be a valuable addition to the in vitro toolbox (beyond plant hormones) for plant species that are difficult to transform or propagate.
Tina S. Lai, Penn State University, Department of Plant Biology, University Park PA 16802. In Vitro Cellular and Developmental Biology, 52:S34-35 2016