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 email@example.com.
2017 JOSEPH F. MORGAN AND SIVB STUDENT TRAVEL AWARDS
The First Wave of Inflammation: Elucidating the Ability of AF and NP Cells to Respond to IVD Herniation Triggered Damage
Lower back pain affects up to 80% of the population at some point during their life, with an estimated associated cost of $100 billion per year in North America alone. This results in considerable strains on our healthcare system despite having limited treatment options. Lower back pain can be attributed to mechanical compression of the nerve roots and inflammation caused by the innate immune response triggered by intervertebral disk (IVD) herniation, where a tear in the outer annulus fibrosus (AF) causes the migration of the inner nucleus pulposus (NP) through the AF. Macrophages are known to play a role in initiating inflammation at the site of herniation; however, very little is known of the role of resident IVD cells in initiating the first wave of inflammation and recruiting macrophages to the site of herniation. The present research aims to fill this void by studying resident AF and NP cells derived from rat trail IVDs; first developing primary cell cultures for each cell type, then characterizing their innate immune sensor repertoire and finally studying how these cells respond to inflammatory stimuli, both pathogen and host derived. Understanding the progression of the innate immune response and its role in lower back pain will allow treatments targeting IVD cell sensors in order to stop pain and inflammation before an innate immune response can be mounted.
Matthew Guerriero, Wilfrid Laurier University, 2-589 Beechwood Drive. Waterloo, ON N2T 2K9, Canada. In Vitro Cellular and Developmental Biology, 53:S42, 2017
2017 HONOR B. FELL AND SIVB STUDENT TRAVEL AWARDS
The Influence of Extracellular Topography on Mammalian Transgene Expression
Regeneration after wounding or traumatic injury can be a slow, complicated process that does not guarantee complete recovery. The extracellular matrix (ECM) plays a key role in mediating wound healing by providing structural and biochemical support to the surrounding cells. Importantly, the ECM consists of distinct chemical and physical cues. Previous studies have shown that biomimetic, physical ECM properties and cues can alter cell behavior and endogenous gene expression. Therefore, we hypothesized that transgene expression could also be influenced by the extracellular environment. Developing an implantable biomaterial that mimics the extracellular matrix, supplemented with therapeutic gene delivery could help promote controlled regeneration. The aim of this study is to examine how biophysical cues influence transgene expression. Our hypothesis is that transfected cells cultured on topographic surfaces with a repeating ridge and groove pattern of various pitch sizes will show significant changes in transgene expression compared to a chemically identical flat control. For our study, we used several cell types, including PC12 (pheochromocytoma), HEK293 (embryonic kidney), and C3H10 (mesenchymal) cells. Cells were transfected with a plasmid that contains a CMV promoter driving green fluorescent protein (GFP) and then plated onto surfaces with 800nm, 1400nm, and 4000nm pitches (1:1 ridge: groove ratio) as well as a flat control. The cells were imaged with a microscope and macro- scale imager at 24 and 48 hours after being plated on the surfaces and macro- scale changes in GFP intensity were quantified. The differences in GFP expression on the different topographic substrates have led us to conclude that physical cues alter transgene expression. In particular, we observed increases in transgene expression when the cells were growing on sub-micron scale feature sizes. These results may be translationally applied to the development of novel treatments for controlled wound healing or nerve regeneration in the form of gene therapy with implantable biomaterials.
Amanda Youssef, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 53:S28-29, 2017
2017 SIVB STUDENT TRAVEL AWARD
Micropropagation of Miscanthus x giganteus ‘Illinois’: Improved Regeneration Capacity of Callus Cultures and Optimized Microrhizome Development of in Vitro Plantlets
Miscanthus x giganteus exhibits favourable characteristics for cultivation as an advanced biofuel feedstock; however, because of its innate sterility, it must be propagated using vegetative means. Previous studies have successfully cultured Miscanthus calli on modified Murashige and Skoog (MS) mediums, though regeneration of shoots have been hindered after four months of culture. This has resulted in insufficient conservation of germplasm and an unreliable propagation system. In addition to this, induction of microrhizomes for propagule development have not been previously investigated in this species. The current study aimed to improve regeneration capacity of calli cultured for longer than four months by inhibiting the first step of the phenylpropanoid biosynthetic pathway with 2-aminoindan-2-phosphonic acid (AIP) supplemented in modified MS mediums, and optimize microrhizome development of in vitro plantlets using various concentrations of sucrose, benzylaminopurine (BAP), and naphthaleneacetic acid (NAA). We identified that calli cultured on MS media supplemented with 2.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 10 μM AIP exhibited higher frequencies of shoot regeneration (71.59±5.7 %) than the control lacking AIP (26.67±5.7 %) up to nine months of culture. AIP effectiveness was determined by measuring soluble phenolic content of calli with a modified Folin-Ciocalteu colorimetric assay. Significant differences were only observed in mediums with 2.0 mg l-1 2,4-D, with the lowest concentrations detected in mediums with 100 and 1000 μM AIP (1310.84 and 1365.45±0.7071 ug g-1 DW, respectively). Microrhizome induction of tillered plantlets was tested with liquid MS medium supplemented with sucrose (3, 8, and 10 %) with or without BAP (2.5 and 26.5 μM) and NAA (0.6 and 50 μM). Microrhizomes from the optimal medium were then tested for cold storage and growth in greenhouse and in vitro conditions. Findings from this study will help in establishing effective conservation of germplasm and alternative propagation systems.
Cassandra Downey, University of Guelph, 50 Stone Road East, Guielph, ON N1G 2WE1, Canada. In Vitro Cellular and Developmental Biology, 53:S50-51, 2017
2017 SIVB STUDENT TRAVEL AWARD
Identifying Cytoplasmic DNA Sensors, DHX9 and DDX3, in Rainbow Trout
Innate immunity constitutes the first line of defense during viral infections. Viruses produce nucleic acids, both RNA and DNA, during genome replication and transcript synthesis. These nucleic acids are foreign to the cell and are sensed by pattern recognition receptors, based on their type (RNA or DNA), their strandedness (ss or ds) and their location (endosomal, extracellular or cytoplasmic). When a viral nucleic acid is in the wrong compartment (ex. dsDNA in the cytoplasm), pattern recognition receptors (PRRs) detect it and activate signalling cascades, which culminate in the production of type I interferons (IFNs) and the induction of an antiviral state. Cytoplasmic RNA sensors, such as RIG-I and MDA5, have begun to be characterized in several fish species, but almost nothing is known of cytoplasmic DNA sensors (CDSs) in fish. To this end, two CDSs were cloned from the rainbow trout macrophage-like cell line RTS11. Both CDSs are ATP dependent RNA helicases that unwind DNA and RNA in the 3′ to 5′ direction as well as functioning as innate immune sensors to initiate an antiviral state via the IFN pathway during a virus infection. In this study the novel rainbow trout DHX9 and DDX3 sequences were compared to known vertebrates sequences to identify conserved protein domains, intron/exon structures and phylogeny. Knowledge of CDSs in rainbow trout will aid in a better understanding of innate antiviral immunity in this commercially and economically important fish species.
Shannee Herrington-Krause, Wilfrid Laurier University, 9 Elgin Place, Waterloo, Ontario N2J3X5, Canada. In Vitro Cellular and Developmental Biology, 53:S42-43, 2017