MACUB (2021) Conference

Abstract: Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12-15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, targeting cancer-driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre-mRNA transcript. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapy targeted the 5’ Splice Site of exon 10 of the EGFR pre-mRNA transcript, downstream region of intron 10 of EGFR with a G-quadruplex tail, with a 4GQ tail, the wild type 5’Splice site with no tail, and the Exonic Splicing Enhancer (ESE) with a 4GQ tail in comparison to no treatment with no therapies. The retention for 10A retention for the downstream region of intron 10 with a 4GQ tail was 61.9 (p < 0.002). The retention for the Wild Type 5’Splice site with a 4GQ tail, had a shift of 15.9 (p < 0.001). The retention for the Wild Type 5’Splice site with no tail was 10.4 (p < 0.002). The retention for the Exonic Splicing Enhancer (ESE) was 5.9 (p < 0.04).

Abstract: Prenatal exposure to a high fat diet results in the neurogenesis of orexigenic peptide neurons in the hypothalamus of the offspring. This increase in the number of peptide neurons leads to hyperphagic behavior and predisposes offspring to obesity. However, the molecular mechanism in which a high fat diet affects neurogenesis is unknown. The primary composition of a high-fat diet consists of the monounsaturated fatty acid, oleic acid, and the saturated fatty acid, palmitic acid. This study examined the effects of palmitic and oleic acid on the hypothalamic neuronal transcriptome and on two processes of neurogenesis, proliferation, and migration. Immortalized embryonic day 18 hypothalamic neurons were treated with 1,10, and 100 μm of palmitic acid or oleic acid. The RNAseq results show significant gene enrichment in cellular pathways involved in proliferation, migration, and apoptosis. Further investigation shows that low concentrations of oleic acid stimulate proliferation while elevated levels of both fatty acids cause apoptosis. High concentration of oleic and palmitic acid was also shown to reduce hypothalamic neuronal migration. These results indicate a direct relationship between fatty acids and proliferation, migration, and pathways associated with neurogenesis.

Abstract: Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12-15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, targeting cancer-driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapies targeted the EGFR pre-mRNA transcript, specifically, regions of exon 10 and intron 10. Therapies were either designed to enhance recognition of the alternative intronic polyadenylation signal or block recognition of canonical exon 10/exon 11 splicing. We targeted the Exonic Splicing Enhancer (ESE) wild-type 5’ Splice Site (wt-5’SS) of exon 10, and the regions surrounding the alternative intronic polyadenylation signal. Each target showed a significant increase in intron retention. Targeting the ESE showed a 5-fold increase in intron retention (p < 0.04), the wt-5’SS was 15-fold (p < 0.001), and the region surrounding the alternative intronic polyadenylation signal showed the strongest effect, with greater than 60-fold increase in intron retention (p < 0.002). Currently, we are examining additional antisense therapies, as well as generation of the therapeutic soluble decoy protein using ELISA.

Abstract: The Eastern Oyster, Crassostrea virginica, is a mollusk that is native to eastern North America. Eastern Oysters are ecologically important since they filter water, removing chemical contaminants, and they also build reefs that serve as habitats for other marine organisms. Eastern Oysters typically begin life as males, however, due to a combination of environmental and genetic influences they can change to females later in life. We are interested in understanding the genetic pathway(s) that control sex determination in C. virginica. Sex determination has been studied extensively in humans and other mammals. Studies have identified several important sex determination genes including Sox9 for males and FoxL2 for females. These genes have been identified in a closely related mollusk, C. gigas, and we were therefore interested in detecting their presence in C. virginica. In this study, we decided to focus on FoxL2 (Forkhead box protein L2), a transcriptional activator that may play a role in ovarian development. Our hypothesis is that the FoxL2 gene is present in the Eastern Oyster genome. As a first step, we used the human FoxL2 protein to perform a BLAST search against the C. gigas genome database at NCBI. This search identified two potential C. gigas FoxL2 proteins, one with 47.8% identity to the human protein and the other with 70.3 % identity. We used the C. gigas protein with highest identity to the human protein to BLAST the C. virginica genome and found two proteins with 90.3% identity to the C. gigas FoxL2 protein. The C. virginica proteins only differ from each other by one amino acid and are encoded by separate genes located on chromosome 5. Multiple sequence alignments showed that the C. virginica proteins have 48%-52% identity to the FoxL2 proteins from fruit flies, zebra fish, chickens, mice, and humans and this identity falls predominantly within the forkhead box of these proteins. These results support our hypothesis that C. virginica contains a FoxL2 protein. This work was supported by grant 2R25GM06003 of the Bridge Program of NIGMS and grant #0537-22-1091 of the CSTEP Program of NYSED.

Abstract: Telomeres are the natural end of chromosomes and work to maintain chromosome stability. Telomerase functions to extend telomere length in eukaryotic species. In comparison, the telomeres of Drosophila melanogaster are composed of and extended by non-LTR retrotransposons including HeT-A, TART, and TAHRE. Previous research identified two mutations, called Tel and E(tc), that are capable of regulating telomere length in fruit flies. Both mutations are located within a short region on the third chromosome. For this study, we set out to identify additional telomere-length regulating genes from the same region on the third chromosome. We hypothesized that the genes capable of regulating telomere length may function in modulating chromosome structure, like a previously identified telomere-regulating gene Su(var)2-5. We used bioinformatics to identify candidate genes in the above-mentioned chromosome region that are predicted to remodel chromosome structures. After the identification process, we extracted genomic DNA from fruit flies with mutated versions of the candidate genes. Using real-time PCR, we compared telomere length between the fly mutants and the wild-type controls. We have found a new gene called CG6026 whose mutation can result in elongated telomeres. To investigate whether the mutation of CG6026 also affects the telomere structure, we are performing polytene chromosome staining and analyzing telomere fusion. This research will further our understanding of the molecular regulatory mechanisms of telomere elongation and structure in fruit flies. Because of the importance of alternative telomere lengthening in cancer development, this study of unraveling non-telomerase mechanisms may also contribute to human cancer research.

Abstract: Widespread anthropogenic noise acts as a sensory pollutant that presents a significant impediment to effective communication between birds. Noise from sources such as roadway traffic, airplanes, construction equipment, and landscaping equipment interrupts the flow of information between a sender and receiver while presenting both survival and fitness consequences. A songbird’s vocalizations (songs, calls) are essential for purposes such as courtship, the establishment of territories, warning others of predators, and maintaining contact between family members. Previous studies have presented evidence that birds in noisy environments alter the frequency (pitch) of their songs and calls to rise above the generally lower pitch of man-made noise sources. The goal of this study was to investigate whether songbirds changed their vocal behavior in environments with high levels of anthropogenic noise. Song Sparrow (Melospiza melodia) recordings were made at three sites with varying levels of background noise: two sites, a suburban and rural site, in East Brunswick (Middlesex County), New Jersey, and one site, an urban site, in Jersey City (Hudson County), NJ. Between June 8, 2020, and July 2, 2020, songs were recorded in .wav format using a Marantz Professional PMD-661 MKIII Handheld Solid-State Recorder with a sampling rate of 48 kHz and a Sennheiser MKE 600 Shotgun Microphone. A Tenma 72-945 digital sound level meter was used to measure real-time background noise at the locations where recordings were made. Bioacoustic measurements were made using Raven Pro 1.6 acoustic analysis software. Preliminary results suggest that Song Sparrows (Melospiza melodia) increased the minimum frequency of their songs and also sang shorter songs in areas that had higher levels of background anthropogenic noise. The soundscapes at many locations during the study period were altered due to widespread reductions in both human activity and noise due to mandated COVID-19 shutdowns. The next steps in this study include analysis and comparison of songbird vocalizations as soundscapes revert to more typical levels of varying anthropogenic noise.

Abstract: Because increased drought and rainfall variability are affecting natural environments in the Midwest and across the globe, it is imperative to study how plant interactions with other organisms influence drought tolerance. Microbial organisms, such as fungi and nematodes, can have both symbiotic and pathogenic interactions with plants. Nevertheless, studies have shown there is a gap of information and understanding of how these microbial interactions are beneficial to plants. For this REU project, I leveraged a new large-scale rainout shelter experiment with manipulations of fungal and nematode abundance to see 1. how fungicide and nematicide treatments alter plant growth and flowering and 2. how fungi and nematodes alter plant growth during drought and rainfall variability. Also, for this new experiment setup, I wanted to see how existing variation in plant communities differs across treatments. I measured plant growth in control, fungicide, and nematicide treatments in irrigated, drought, and variable rainfall shelters on early successional vegetation. I recorded plant height, specific leaf area, and leaf dry matter content (LDMC) on Red Clover (Trifolium pratense) and Goldenrod (Solidago canadensis). Finally, I measured percent ground cover and percent flower cover for the whole plant community. I found that LDMC for red clover was lower in nematicide subplots than in fungicide subplots( p=0.02). For the community measurements, I found that between the two sample times percent flower cover decreased in fungicide subplots more than nematicide subplots (p=0.04). Finally, I found that drought footprints had significantly lower initial ground cover than the other rainfall treatments. I showed that fungi and nematode abundance altered plant phenology and physiology in ways that could influence plant drought tolerance. With continued sampling, I will be able to test the impact of fungi and nematode abundance on plant drought tolerance.