2021 WILTON R. EARLE AWARD
Generating Cell Wall Deficient Plant Cells for Enhanced Recombinant Protein Production
Plant cell-based expression system have been established as a cost-effective alternative production platform for bioactive therapeutic proteins in industrial scale due to their intrinsic safety, low production and downstream cost and the capability of post-translational modification. Due to its central role in bio-production and fundamental research, tobacco BY-2 cell has been referred to as the “CHO-cell in molecular farming” and the “HeLa cell in the biology of higher plants”. However, major challenges exist for the BY-2 cell bioproduction system, such as low protein production and secretion, presence of large vacuoles, and difficulty in cryopreservation, which prevent the commercialization of this bioproduction platform. These problems can largely be attributed to the distinctive plant cell wall structure that is composed of a complex matrix of interconnected polysaccharides forming a thick semi-permeable rigid barrier which protects the cell. Understandably, plant cells devote substantial amounts of energy and resources to the synthesis of cell wall polymers, such as cellulose and pectins. However, complete cell wall structure may not be crucial for in vitro cultured plant cells because optimized culture conditions (medium composition, pH, temperature, etc.) are provided to the cells to support their rapid propagation. The long-term goal of our research is to create cell wall deficient (CWD) plant cells for improving production of recombinant proteins. This could be achieved by cellular engineering of plant cells using modern molecular biology tools, especially genome editing technology. In this research, to provide a quick and easy-access to CWD-like plant cells, pseudo-CWD BY-2 cells were created by providing a cellulose synthesis inhibitor, 6-dichlorobenzonitrile (DCB) to the culture media. The morphology, cell wall structural composition, cell growth, bioproduction properties and transcriptomics of the pseudo-CWD BY-2 were characterized. This study provides a conceptual proof for creating CWD plant cells by cellular engineering in the future.
Uddhab Karki, Arkansas Biosciences Institute and Molecular Biosciences Program, Arkansas State University, Jonesboro, AR 72401. In Vitro Cellular and Developmental Biology, 57:S37 2021
2021 JOHN S. SONG AWARD
Induction of Totipotency and Non-gametophytic Morphogenesis within the Soybean Microspore
Androgenesis-based doubled haploidy is a method used to achieve homozygosity in plant breeding programs. However, its application is limited within the Fabaceae, which has historically demonstrated morphogenic recalcitrance in comparison to other plant families (e.g., Brassicaceae and Poaceae). In this study, the immature microgametophyte, or microspore, was used to investigate in vitro androgenesis in soybean (Glycine max [L.] Merrill). An induction regimen was constructed to support non-gametophytic growth, and consisted of (i) donor plant temperature stress; (ii) a low-nitrogen, phytohormone-supplemented basal medium; and (iii) step-wise, post-isolation culture conditions. In addition, cytological characteristics in soybean microspores were compared to those reported in model androgenesis systems, revealing that totipotent induction/non-gametophytic morphogenesis within the soybean microspore was marked by (i) symmetrical mitotic division; (ii) vacuolar fragmentation; (iii) production and secretion of intrinsic arabinogalactan proteins; and (iv) stress-induced pollen dimorphism; among other features. Lastly, the transcriptomic landscape of stress-induced microspores was evaluated by mRNA Sequencing, which supported cytological findings and suggested that donor plant temperature stress induced totipotency by suppression of the microgametogenesis (pollen) developmental program. The findings herein provide insight into androgenesis-based doubled haploidy in vitro and may prove useful in legume breeding programs.
Brett Hale, Molecular Biosciences Graduate Program, Arkansas State University, State University, AR 72467. In Vitro Cellular and Developmental Biology, 57:S38 2021
2021 GORDON SATO AND WALLY MCKEEHAN AWARD
Assessing CCR1 Antagonists for Chemotaxis Inhibition in a Multiple Myeloma in Vitro Model
Stephanie Lourdes Echeverria
Multiple myeloma (MM) is a plasma B-cell malignancy characterized by osteolytic bone lesions. MM cells secrete and express CCL3/MIP1α which upregulates osteoclastogenesis. Elevated CCL3 levels display a chemotactic ability on isolated osteoclast precursors. Increased levels of CCL3 in MM patients correlates with a greater disease burden, due to the increase of bone resorption, and is indicative of a worse prognosis when compared to MM patients exhibiting lower CCL3 levels. CCR1, a GPCR chemokine receptor, is endogenously expressed on MM cells, and can bind with CCL3. In a previous study, the RPMI8226 MM cell line, CCL3-mediated CCR1 chemotaxis was inhibited in a dose-dependent manner by six CCR1 antagonists (AZD4818, BX471, CCX354, CP481715, MLN3897, PS031291). In this study, we assessed the MM cell line U266 as well as a transfected cell line, U266_CCR1. While U266 cells express lower levels of CCR1 than RPMI8226, U266_CCR1 express higher levels of CCR1. Cells were treated with a serial dilution of the same six CCR1 antagonists and chemotaxis to either supernatant from osteoclast precursor RAW 264.7 cells or Fetal Bovine Serum (FBS) in which a multitude of growth factors and chemokines are present was examined. We hypothesized the six CCR1 antagonists would result in a dose-dependent inhibition of chemotaxis similar to that seen with RPMI8226. Instead, we found only two of the compounds (AZD4818 and BX471) inhibited chemotaxis of the U266 and U266_CCR1 MM cell lines towards RAW 264.7 supernatant or FBS. For both AZD4818 and BX471 there were differences between the chemotactic response of the U266 and U266_CCR1 cell lines, with the U266_CCR1 cell line having a greater degree of inhibition, suggesting the inhibition is driven by CCR1.
Stephanie Lourdes Echeverria, College of Graduate Studies, Midwestern University, Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 57:S32 2021
2021 HOPE E. HOPPS AWARD
Enhanced Production of Bioactive Protoberberine Alkaloids from In Vitro Cultures of Tinospora cordifolia (Willd.) Miers ex Hook F. & Thoms.
The inclination towards natural way of curing ailments has been existing dynamically from traditional to modern era. The present study is an endeavour towards highlighting the best alternative for prevention of over-exploitation of wild habitat and at the same time for their germplasm conservation. This is achieved by employing plant tissue culture technology to propagate large number of true-to-type, disease-free plantations as well as production of bioactive secondary metabolites in short duration and without any seasonal and regional constraints. In the present study, the callus cultures were obtained from leaf explants of Tinospora cordifolia. The explants were grown on MS basal medium fortified with plant growth regulators, 6-benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA), at specific concentrations. Further, analytical studies, such as High Performance Liquid Chromatography (HPLC) and Mass spectrometry were performed for the estimation of palmatine and jatrorrhizine, the primary nitrogen-containing alkaloids of interest, in the plant. Higher productivity of jatrorrhizine (10.86-fold) and palmatine (143.04-fold) was obtained from in vitro cell lines compared to that from the field grown leaves. Additionally, anti-staphylococcal (pertaining to Staphylococcus aureus) activity of purified alkaloids obtained from the cell culture extracts was also tested. Palmatine exhibited 87% inhibition while jatrorrhizine unveiled 78% inhibition in 48 hours, at their respective MBIC (minimum biofilm inhibition concentration), indicating feasible natural alternatives for future therapeutics.
Vartika Srivastava, Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, INDIA. In Vitro Cellular and Developmental Biology, 57:S32 2021
2021 CELLULAR TOXICOLOGY AWARD
Zero-valent Iron Nanoparticle-induced Reactive Oxygen Species in Fremyella diplosiphon, a Biodiesel-Producing Cyanobacterium.
Iron is important for various physiological processes in cyanobacteria; however, its bioavailability is a growth limiting factor. Nanofer 25s zerovalent iron nanoparticles (nZVIs) are proposed as “a new miracle” due to their bioavailability leading to enhanced growth, and unsaturated fatty acid methyl esters in F. diplosiphon, thus improving its biodiesel capability. In this study, reactive oxygen species (ROS) induced by nZVIs in F. diplosiphon strains, B481-WT (wild type) and B481-SD (engineered for enhanced lipids) was quantified using malondialdehyde (MDA) and 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA) assays. We used Energy Dispersive X-ray Spectroscopy (EDS) combined with Transmission Electron Microscope (TEM) to determine the localization of nZVIs in F. diplosiphon. Cultures treated with 1.6mg/L, 3.2 mg/L, 6.4 mg/L, 12.8 mg/L and 25.6 mg/L nZVIs were grown under continuous growth light for 15 days at 70 rpm. Culture not exposed to nZVIs served as the control. Three replicated treatments were maintained. On day 15, ROS was quantified using MDA and DCFH-DA assays, and data analyzed using one-way ANOVA and Tukey’s honest significant difference test. Our results indicated that cultures treated with 3.2 mg/L, 12.8 mg/L and 51.2 mg/L nZVIs exhibited significantly higher MDA concentration compared to the untreated control and 1.6 mg/L nZVI-treated cultures. Results from DCFH-DA assay showed that ROS in all nZVI-treated cultures was significantly higher than the untreated control. TEM images demonstrated aggregation of variously sized nZVIs outside F. diplosiphon cells and EDS analysis indicated the presence of iron within cells. Future studies will be aimed to investigate the effect of nZVIs on gene expression via. next generation sequencing. This study was supported by the National Science Foundation’s Division of Engineering [CBET-1900966] and National Institute of General Medical Sciences [RL5GM118972] grants awarded to Morgan State University.
Samson Gichuki, Department of Biology, Morgan State University, 1700 East Cold Spring Lane, Baltimore. In Vitro Cellular and Developmental Biology, 57:S49-50 2021