The following student awards were presented at the 2017 In Vitro Biology Meeting in Raleigh, NC. Information on additional awardees at the 2017 Meeting 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 firstname.lastname@example.org.
2017 WILTON R. EARLE and SIVB TRAVEL AWARDS
Scavenging for Bacteria: Identification and Characterization of Rainbow Trout MARCO
Class A scavenger receptors (SR-As) are a family of key innate immune receptors, which bind to a wide range of polyanionic ligands including bacterial components and nucleic acids. Macrophage receptor with collagenous structure (MARCO) is a SR-A that has been studied in mammals largely for its role in binding bacteria. To date there is little information about SRAs in fish, and what ligands specific SR-A family members bind remains largely unknown. In this present study a novel rainbow trout MARCO transcript has been identified and its sequence and putative protein domains have been analyzed. While there is only moderate sequence similarity to mammalian MARCO sequences there are notable protein domain similarities and MARCO clusters with MARCO sequences from other species more closely than other fish scavenger receptors. MARCO transcript was found in rainbow trout gonadal cell line RTG-2 and the macrophage/monocyte splenic cell line RTS-11; presence was not detected in the gut or gill cell lines RTgutGC or RTgill-W1. When overexpressed in CHSE-214, a cell line that lacks functional scavenger receptors, rainbow trout MARCO is able to bind the class SR-A ligand, acLDL as well as gram-positive, and gram-negative bacteria (of both mammalian and aquatic sources). MARCO did not show any binding to the yeast cell wall component zymosan. When the MARCO sequence was truncated to remove a domain necessary for bacterial binding in mammals, the scavenger receptor cysteine-rich domain, MARCO no longer bound bacteria or acLDL. This is the first time rainbow trout MARCO has been identified, and the first in-depth study exploring a fish class A scavenger receptor ligand binding profile. This study provides novel insight into the role of rainbow trout MARCO in bacterial innate immunity.
Sarah J. Poynter, Department of Biology, University of Waterloo, Waterloo CANADA. In Vitro Cellular and Developmental Biology, 53:S25, 2017
2017 HOPE E. HOPPS AND SIVB STUDENT TRAVEL AWARDS
Overexpression of AhCuZnSOD and Pyramiding of AhCuZnSOD with the AhcAPX Gene Enhance Salinity and Dehydration Stress in Brassica juncea
Antioxidant enzymes play an important role in conferring abiotic stress tolerance. The antioxidant metabolism protects cells from oxidative damage caused by ROS, such as peroxidation of membrane compounds, polysaccharide degradation, enzyme denaturation and DNA lesions Several enzymes act jointly to maintain redox status homeostasis. The antioxidative defense is imitated by SOD, which converts superoxide radicals to H2O2. The H2O2 that is also potentially harmful is converted to non-toxic water and monodehydroascorbate by the APX enzyme utilizing ascorbate as the electron donor. In the present study, genes for two different cytosolic antioxidant enzymes, superoxide dismutase (AhCuZnSOD) and ascorbate peroxidase (AhcAPX) were isolated from salt tolerant cell lines of Arachis hypogaea and overexpressed the AhCuZnSOD gene alone or with AhcAPX gene in highly important oilseed crop, Brassica juncea (Indian Mustard). Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. The enhanced levels of antioxidant enzymes in the transgenic plants correlated with higher relative water content, improved photosynthetic efficiency, less electrolyte damage, elevated accumulation of compatible osmolytes, less malondialdehyde as well as less lower level of ROS accumulation in the transgenic plants expressing AhCuZnSOD and AhcAPX as compared to the UC plants or AhCuZnSOD gene alone under stress conditions. Compared with UC plants, several independent AhCuZnSOD transgenic lines showed improved capability for tolerating exposure to high mannitol and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity and drought stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and untransformed control plants under salinity and drought stress. The present study seems to suggest that, for combining drought and salinity tolerance together, co- transformation is a better approach.
Neelam Negi, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, INDIA. In Vitro Cellular and Developmental Biology, 53:S47, 2017
2017 SIVB STUDENT TRAVEL AWARD
RNAi Knockdown of Potato Genes Crucial for Potato Virus Y Replication
Potato virus Y (PVY) continues to cause potato producers enormous economic losses worldwide. PVY can cause significant damage in several species of Solanaceae, but the greatest economic impact is on potato (Solanum tuberosum L.), reducing crop yields from 10 to 75% and causing internal tuber defects. PVY is transmitted by many aphid species in a non-persistent manner and because of this, insecticides are considered an ineffective means of control. The difficulty of potato breeding, along with emerging PVY strains and recombinants has made breeding for PVY resistant cultivars problematic. PVY is a single-stranded positive sense RNA virus belonging to the family Potyviridae. A biotechnological approach could offer solutions to combating PVY in potato. In this study, two highly conserved host genes in potato, referred to as Chloe and Thor that have an essential role in potyviral replication have been targeted by RNA interference (RNAi). RNAi is a gene knockdown technology that is successfully being used as a method of viral control in many crops. Primers were specifically designed to amplify a fragment of the two different host genes. Gene fragments were independently cloned into the entry vector pENTR/D-TOPO (Invitrogen), then transferred into the Gateway-compatible binary vector pANDA35HK (Shimamoto Lab). This vector is used in Agrobacterium-mediated transformation to deliver a hairpin RNA containing the Chloe and Thor gene fragments into potato plants, thus triggering RNAi, and potentially resulting in PVY-resistant potato lines.
Whitney Harchenko, Montana State University, 119 Plant BioScience, Bozeman, MT 59717. In Vitro Cellular and Developmental Biology, 53:S47-48, 2017
2017 SIVB STUDENT TRAVEL AWARD
Enhancement of Halotolerance in Fremyella diplosiphon by Electroporation-mediated Overexpression of Malate Dehydrogenase Gene
Fremyella diplosiphon is a freshwater cyanobacterium that has great potential as a biofuel agent due to its ability to grow in low light intensity and acclimation to different wavelengths. Prior efforts to enhance salt tolerance in F. diplosiphon via heat mutagenesis have led to a mutant that thrives in 20 g L-1 NaCl. Further augmentation of halotolerance to 35 g L-1 NaCl which is the average salinity of seawater would be a desirable trait to exploit the organism for biofuel production in naturally available salt water systems. In the present study, we identified a homolog of the malate dehydrogenase (mdh)genein F. diplosiphon using PCR screening and sequence analysis. The plasmid containing the mdh gene, designated pGEM-7Zf-MDH, was cloned in an expression vector and transformed into the wild type. Electroporation-mediated overexpression of the gene resulted in a transformant designated HSF33 with a 20-fold increase in mdh transcript level. Physiological evaluation in BG11/HEPES medium and seawater adjusted to 35 g L-1 NaCl revealed that the transformant could thrive in high salinity, while a complete cessation of growth was observed in the wild type. Additionally, decrease in 630: 680 nm phycobiliprotein to chlorophyll a absorption peak ratio was observed solely in the wild type indicating that HSF33 maintains its photosynthetic pigment accumulation in seawater. Our results provide new insights into the role of the mdh gene in F. diplosiphon salt stress response enabling its cultivation in marine waters for large-scale biofuel production.
Benham Tabatabai, Morgan State, Spencer G6, 1700 East Cold Spring Lane, Baltimore, MD 21251. In Vitro Cellular and Developmental Biology, 53:S31-32, 2017
2017 JOHN S. SONG AWARD
A New Balancing Act: Melatonin and Serotonin as Mediators of Plant Morphogenesis
Melatonin (Mel) and serotonin (5HT) are indoleamines first identified as neurotransmitters in vertebrates; they have now been found to be ubiquitously present across all forms of life. Though Mel and 5HT possess important roles in plant growth and development, their roles in morphogenesis are still poorly defined. We hypothesize that Mel and 5HT function as a novel class of plant growth regulators (PGRs). To investigate this, we used a dual approach: phytochemical analysis and in vitro culture experiments. First, a simple and efficient method for the phytochemical analysis of Mel, 5HT and several established classes of PGRs, was developed for in vitro grown plants. Second, we examined the morphogenetic effects of Mel and 5HT in several plant culture systems including breadfruit (Artocarpus altilis) and St. John’s wort (Hypericum perforatum: SJW). In breadfruit, though Mel had a minimal effect on growth, 5HT appeared to act as both an anti-browning agent and to possess cytokinin-like effects in culture. Particularly, it was found that 5HT (100 µM) could replace kinetin supplementation, in multiplication medium. Our lab possesses unique lines of SJW, a model for the study of Mel and 5HT, with high (L4) and low (L112) endogenous Mel levels, in comparison to wild-type plants. Neither root, nor shoot explants of the three lines showed a significant difference in growth. Grown on media supplemented with Mel, 5HT or their precursors (5, 10 or 30 µM), both root and shoot cultures showed a dose-dependent morphogenetic response, particularly with respect to shoot and root initiation. High levels (10 – 30 µM) of these compounds induced a generally inhibitory effect, while low concentrations (5-10 µM) showed improved growth and regeneration. Additionally, L4 showed inhibition at lower levels than did L112, supporting the dose dependent nature of Mel and 5HT, a defining characteristic of PGRs. Together this research presents a) a platform for the investigation of Mel and 5HT in morphogenesis, and b) suggests Mel and 5HT should be classified as a novel class of PGRs.
Lauren Erland, University of Guelph, 50 Stone Road W, Guelph, ON N1G 2W1, Canada. In Vitro Cellular and Developmental Biology, 53:S13-14, 2017
2017 CELLULAR TOXICOLOGY AWARD
Absorption and Distribution of the Antimalarial Drug Artemisinin Delivered Orally as Dried Leaves of Artemisia annua
The medicinal plant Artemisia annua is a promising option for the treatment of malaria. A. annua produces the antimalarial drug artemisinin (AN) whose derivatives make up the major component of artemisinin combination therapies, the frontline global treatment for malaria. Previously we showed that AN delivered as dried leaves of A. annua (DLA) is >40 fold more bioavailable in mice, 5 fold better at reducing parasitemia, and 3 fold better at slowing the development of AN resistance when compared to pure AN. Recently we showed that AN delivered as DLA is about 4 times more soluble in intestinal fluid and 37% more permeable to the intestinal membrane than pure AN. Here, using the Caco-2 model of the intestinal epithelium we investigate the effects of the essential oil fraction of A. annua on intestinal permeability of AN. We show that digested essential oil at two concentrations equivalent to 0.3 and 4.0% of that known in the plant, and from two sources decreased the intestinal permeability of AN. This result was surprising considering digested DLA increased AN permeability. Interestingly, when essential oil was added undigested, there was no change in the intestinal permeability of AN compared to pure drug. It appeared that the presence of bile changed essential oil permeability. To further characterize differences in bioavailability of AN delivered as DLA vs. pure drug, we performed tissue distribution (ADME) studies in rats. Groups of rats were orally dosed with either pure AN or a slurry of DLA containing an equal amount of AN. One hour after dosing, rats were euthanized, tissues harvested, and analyzed for AN content. AN delivered as DLA was distributed in significantly higher quantities, up to 6 fold greater than from pure AN throughout all the tissues tested. Together these studies begin to explain how bioavailability improves when AN is delivered via the plant than as pure drug.
Matt Desrosiers , Worcester Polytechnic Institute, Dept. of Biology and Biotechnology, 100 Institute Road, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 53:S39, 2017
2017 PHILIP R. WHITE AWARD
A Novel Approach to Cell Selection from Taxus Plant Cell Culture Via an Engineered Mammalian Caspase
Plant specialized metabolites provide a great diversity of valuable chemicals that can be used as flavorings, fragrances, pesticides and pharmaceuticals. One notable natural product is the chemotherapeutic agent paclitaxel (Taxol®), a drug commercially synthesized using Taxus plant cell culture. This production process is not optimal due to the inherent heterogeneity of plant culture which includes a large population of cells that do not produce and accumulate paclitaxel. Individual cells vary significantly in metabolic capacity, and there is not an effective method to cull cells due to their aggregated growth and inability to survive separately. We are investigating a new and exciting approach to select and propagate only those cells that synthesize paclitaxel. Our goal is to engineer a mammalian caspase protein to induce death in metabolically deficient cells by designing a caspase binding site which inhibits protease activity when bound by paclitaxel. This poster will highlight progress in several areas: (1) characterization of programmed cell death and necrosis in Taxus plant cell culture, (2) successful transient expression of mammalian caspase proteins in plant cells using particle bombardment and PEG mediated transformation, (3) quantification of caspase functionality within Taxus plant cells, and (4) robust assessment of protoplast transformation and viability via flow cytometry. This work serves as the foundation for future efforts towards caspase engineering and stable transformation to develop an efficient selection system and propagate superior cultures for use in paclitaxel synthesis bioprocesses.
Michelle McKee, Worcester Polytechnic Institute, 50 Prescott St., 4th Fl, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 53:S36, 2017