Student Poster Competition

In Vitro Animal Cell Sciences

First Place

Fibrotic Responses Differ after Treatment with Artemisia sp., Artemisinin, and Its Derivatives

Fibrosis is a pathology linked to most diseases and prior studies indicated that various artemisinin (ART) derivatives (artesunate (AS), artemether (AM), dihydroartemisinin (DHA)) reduced fibrosis in vitro and in vivo. Artemisia annua L., is the natural source of ART and globally used as a tea infusion especially in underdeveloped countries to treat many diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART) for their effects on fibroblast growth and expression of key fibrotic marker genes and proteins after 1 and 4 d treatment. A resazurin assay showed that AS, DHA, and both Artemisia teas significantly reduced fibroblast proliferation after 1 d post-treatment, whereas ART and AM did not. After 4 d, all treatments showed less proliferation except AM and dART. Unlike AS and DHA, ART had no significant effect on fibrotic gene expression. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART. Using mouse anti-SMA and anti-mouse Alexa488 conjugated secondary antibodies an immunofluorescent stain for smooth muscle actin (α-SMA) correlated with PCR results of drug-treated fibroblasts. Together, the PCR and immunocytochemistry results showed that ART, AS, DHA, and the two Artemisia teas have different anti-fibrotic responses at both the level of transcription and translation of α-SMA. The fibroblast fibrotic phenotype is inducible by TGF-β. A resazurin assay showed that CRL-2097 cell proliferation decreased by 16% and 22%, 2 and 4 d, respectively, post TGF-β treatment suggesting faster induction to myofibroblasts. The effects of the two Artemisia teas, ART, and its derivatives are being studied further to determine their relative anti-fibrotic effects on the same target genes post TGF-β treatment. These results provide a foundation for studying possibly new artemisinin-based therapeutics

Trevor Bush, Worchester Polytechnic Institute, 100 Institute Rd., Worcester, MA 01609. Abstract presentation: A-2012

Second Place

Development of Polyethylene Glycol Hydrogel Drug Delivery Device to Study Intramuscular Adipose Tissue Signaling

Intramuscular adipose tissue (IMAT) can potentially be stimulated to secrete pro-myogenic adipokines, emulating brown adipose tissue and promoting muscle regeneration. We have previously shown that β-3 adrenergic receptor agonists can successfully brown white auxiliary adipose depots with intraperitoneal (IP) injection. However, systemic delivery of β-3 adrenergic receptor agonists is associated with undesirable off-target effects. Thus, the aim of this project is to develop a sustained-release, biodegradable system for the local delivery of IMAT stimulatory molecules. Here we developed a tunable biodegradable polyethylene glycol (PEG) hydrogel with embedded net negatively charged nanosilicates (NS) for the delivery of the net positively charged small molecule IMAT stimulatory drug Mirabegron. Electrostatic complexation between Mirabegron and NS particles refines drug release kinetics, mitigating burst release. Preliminary in vivo results show mild inflammation around the gels following subcutaneous and intramuscular implantation, which resolves within 2 weeks. This indicates that the system will be adaptable for the IMAT signaling study. We determined the affinity of Mirabegron to the NS surface and demonstrated that the drug electrostatically binds to the NS. Bulk release experiments were performed to show the effect of NS-drug complexation on slowing down Mirabegron release and minimizing burst release. The sustained drug release from PEG-NS gels was quantified via capillary electrophoresis. Future work will finalize the delivery system formulation and perform in vitro and in vivo studies to characterize the effects of the drug.

Rebekah Boos, Department of Biomedical Engineering at Saint Louis University, St. Louis, MO. Abstract presentation: A-2019

Plant Biotechnology

First Place

Characterization of Phenylalanine Ammonia-lyases (PALs) in Arabidopsis thaliana

The phenylpropanoid pathway generates a variety of specialized secondary metabolites that play key roles in defense against pathogens, oxidative stress, and UV light. The first step of the pathway is the deamination of phenylalanine to trans-cinnamic acid catalyzed by phenylalanine ammonia-lyase (PAL). In Arabidopsis thaliana, PAL is encoded by four genes, PAL1-PAL4. PAL has been extensively studied in a variety of species, but previous findings with PAL T-DNA mutants in Arabidopsis are difficult to interpret because some of the alleles used appear to be leaky. Previous studies have shown that mutations in the PAL1 and PAL2 genes lead to increased expression of other PAL genes as well as decreased expression of Kelch F-box Protein 39 which has been shown to directly interact with PALs and lead to their degradation. To characterize PAL enzyme function and its transcriptional and post-transcriptional regulation, we have generated exon-specific CRISPR-Cas9 mutants and have observed various differences that distinguish our CRISPR mutants from the previous T-DNA mutants. PAL assays performed on our CRISPR pal lines agree with previous findings that PAL1 is the major isoform contributing to total PAL activity. CRISPR pal1/2 and pal1/2/3 lines show decreased rates of germination and growth compared to Col-0. CRISPR pal1/2 and pal1/2/3 plants produce yellow seeds due to decreased seed condensed tannin accumulation. Analysis of sinapoylmalate, a UV protectant phenylpropanoid, accumulation in our CRISPR pal mutants reveals that PAL1 and PAL2 are similarly the major isoforms contributing toward its synthesis. Indeed, pal1/2 plants exhibit a reduced epidermal fluorescence phenotype in which the leaves fluoresce red instead of blue due to the absence of sinapoylmalate. Additionally, CRISPR pal1/2/3 lines exhibited stunted growth and sterility and pal1/2/4 lines are so strongly dwarfed that they do not produce stems. In the future, we plan to perform RNA sequencing and proteomics analysis to explore the impact of our pal mutants on PAL expression, PAL protein accumulation, and PAL activity.

Lizhi Cheng, Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907. Abstract presentation: P-2083

Second Place

Expanding CRISPR-combo’s Scope: Targeting A-T Rich Sites for Advanced Genome Modification in Plants

The recently reported CRISPR-Combo systems provide an orthogonal approach to improve genome engineering in crops by allowing simultaneous gene editing and activation. However, these systems rely on SpCas9, which recognizes NGG PAM sites. Considering that genomic promoter regions are typically AT-rich, the adoption of alternative Cas nucleases in CRISPR-Combo systems is warranted. In this regard, we sought to expand the scope of CRISPR-Combo by using iSpyMacCas9, iSpyMacCas9-D10A cytosine base editor, and AaCas12b, targeting NAAR-PAM, NAAR-PAM, and VTTV-PAM sites, respectively. As a demonstration, we aim to achieve hormone-free regeneration of the gene-edited lines facilitated by simultaneous editing of agriculturally important traits while activating the morphogenic BABY BOOM 1 gene in rice. We expect these additional CRISPR-Combo systems with expanded targeting scope will enable versatile applications in plant breeding and crop improvement.

Innocent Byiringiro, Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD.  Abstract presentation: P-2024