Search Poster #: 1 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: Plant, glutaredoxin, nitrogen Project Title: The Effects of Glutaredoxins on Arabidopsis thaliana Nitrogen Signaling Author List: Joaquin Lopez, Undergraduate, Biological Sciences, San Marcos, Presenting Author; Abstract  Glutaredoxins (GRXs) are small disulfide oxidoreductase enzymes found in all kingdoms of life. Class III GRXs are exclusive to land plants, and many Class III GRXs are regulated by nitrogen availability in soil. The purpose of this study was to determine whether two clusters of nitrate-induced class III GRX genes in Arabidopsis thaliana (AtGRXS3/4/5/7/8 and AtGRXS6/AtGRXC11) are required for “normal” plant responses to nitrogen availability. We studied growth phenotypes and the expression of nitrogen-regulated genes in wild type (WT), a AtGRXS3/4/5/7/8 mutant (line 823), a AtGRXS6/AtGRXC11 mutant (line 3810), and a AtGRXS3/4/5/7/8 + AtGRXS6/AtGRXC11 mutant (line 15-7). WT and GRX mutant lines were grown using nitrogen-rich hydroponic media, nitrogen-rich agar media, and soil. The septuple GRX mutant line 15-7 displayed statistically significant increases in the expression of several root nitrate transporter genes, in shoot biomass, and in shoot:root ratio. This finding suggests that AtGRXS3/4/5/7/8 and AtGRXS6/AtGRXC11 work cooperatively to regulate multiple plant nitrogen responses. Notably, the observed phenotypes were mostly absent in the AtGRXS3/4/5/7/8 mutant and the AtGRXS6+AtGRXC11 mutant, which are the parents of the septuple mutant line. This suggests that nitrogen-regulated GRX genes are functionally redundant, and can only be functionally characterized in higher-order mutants. Given these findings, future research will focus on characterizing changes in the whole transcriptome of septuple GRX mutant line 15-7 under nitrogen replete conditions, using RNAseq. Overall, these studies will further elucidate the molecular pathways involved in plant nitrogen signaling, which can potentially be applied to improve crop nitrogen use efficiency, an agriculturally important trait.
| Poster #: 2 Campus: California State University, Los Angeles Poster Category: Agriculture/Biofuels/Environment Keywords: Lactic acid producing bacteria, Climate Change, Oenology Project Title: Antimicrobial Activity of Protein Hydrolysates from Grape Seeds Intended for Color Stabilization in Winemaking Author List: Destiny Guerrero, Undergraduate, Biological Sciences, Los Angeles, Presenting Author; Naim Cerkezi, Undergraduate, Biological Sciences, Los AngelesJanette Dzul, Graduate, Biological Sciences, Los AngelesAna Belen Mora-Garrido, External Partner, Facultad de Farmacia, Non-CSU, Universidad de SevillaMaria Luisa Escudero-Gilete, External Partner, Facultad de Farmacia, Non-CSU, Universidad de SevillaFrancisco Heredia, External Partner, Facultad de Farmacia, Non-CSU, Universidad de SevillaMaria Jesus Cejudo-Bastante, External Partner, Facultad de Farmacia, Non-CSU, Universidad de SevillaEdith Porter, Faculty, Biological Sciences, Los Angeles Abstract A warming climate leads to changes in wine quality because of differences in the time between the grape maturity and the seed maturity; at the time of harvest, the grape is ripe while the seed is not fully ripe, leading a decrease in compounds that would otherwise help to stabilize the color of finished wine. It has been proposed to add grape seed protein hydrolysates (GSPH) to stabilize the color of wines. However, their antimicrobial effects against lactic acid-producing bacteria used in wine making are unknown. Lactiplantibacillus plantarum (LP) and Oenocuccus oeni (OO) are a vital part of the winemaking process through their role in malolactic fermentation. We set out to assess the metabolic activity of LP and OO with the resazurin assay in which bacterial reducing metabolites convert the non-fluorescent resazurin to the fluorescent resorufin. In contrast to OO, LP metabolic activity could not be assessed because of a loss of fluorescence likely due to excessive acid production. The objective of this study was to optimize the method for quantifying the metabolic activity of LP and determine if GSPH exhibit antimicrobial activity against LP. Effect of PBS on pH and resorufin fluorescence production after bacterial incubation was assessed. Subsequently, LP adjusted to McF 0.5 or McF 0.5 diluted 1:100 in liver broth was incubated for up to 42 h with varying concentrations of four different GSPH (P1-4) or solvent control before adding resazurin dissolved in PBS and measuring resorufin fluorescence. Buffering with PBS allowed for quantification of metabolic activity of LB in a bacterial concentration dependent manner. GSPH did not inhibit the metabolic activity of LP and P4 appeared to be rather growth promoting. For example, fluorescence readings (RFU) after 42 h incubation of LP at McF 0.5 inoculum with 0.5 mg/mL GSPH were as follows for control: 40568±9072, P1: 46336±851, P2: 46542±7249; P3: 47453±5044, and P4 (p < 0.01): 58708±1764. Our data suggest that GSPH do not inhibit LP metabolic activity supporting their use for the purpose of stabilizing the color of wine made from grapes grown in warm climates. This is significant because wine is an important economic product. This study was supported by NSF/CSU-LSAMP Grant #HRD-2204753, PID2021-127126OB-C22 and PID2021-124964OB-C22 del Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033, and by “ERDF A way of making Europe"; Alvinesa Natural Ingredients, S.A. (Daimiel, Ciudad Real, Spain).
| Poster #: 3 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Agriculture/Biofuels/Environment Keywords: Spirulina, Broiler, Growth Performance Project Title: Effect of Spirulina Supplementation on Body Weight, Body Weight Gain, and Feed Conversion Rate Author List: Emily Beckerman, Graduate, Animal Science , San Luis Obispo, Presenting Author; Ike Kang, Faculty, Animal Science , San Luis ObispoShreyash Patel, Graduate, Animal Science, San Luis ObispoNatalie Thiessen, Undergraduate, Animal Science , San Luis ObispoSiroj Pokharel, Faculty, Animal Science , San Luis ObispoDarin Bennett, Faculty, Animal Science , San Luis Obispo Abstract The poultry industry is constantly searching for new nutritional strategies and better feed conversion rates in order to enhance broiler growth and productivity. Microalgae have previously been shown to improve production rates on broilers. The objective of this study was to determine the effect of dietary Spirulina on broiler production performance. A total of 270 one-day-old chicks (Ross 708) were obtained from a local hatchery and vaccinated against Newcastle, Marek’s disease, and Infectious Bronchitis. They were weighed and sorted to 18 floor pens (4’x4’ each, 15 birds/pen, 6 pens/treatment). Each pen was randomly allocated to one of three dietary treatments consisting of corn-soybean meal diets containing graded levels of Spirulina (0%, 2.5%, and 5%). Birds were phase fed a starter (0-2 weeks), grower (2-4 weeks), and finisher (4-6 weeks) diets ad libitum for 6 weeks. Each set of diets were isonitrogenous and isoenergetic. Body weight and feed intake were measured weekly. Body weight gain and feed conversion ratio were then calculated. Data were analyzed with repeated measures ANOVA. Diets were the main effects, weeks were the repeat, and pen was the experimental unit. There were no significant dietary treatment effects. Feeding broilers Spirulina up to 5% of the diet does not affect body weight, body weight gain, and feed conversion rate (P > 0.05). These results are consistent with previous findings. Additional research needs to be conducted in order to determine the effect of Spirulina on broiler health and carcass quality.
| Poster #: 4 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: Chalcones, Nematicide, Plant parasitic nematodes Project Title: Isolation of Chalcone Mutants in Caenorhabditis elegans and the Identification of Putative Genes for Chalcone Resistance Author List: Quynh Nguyen, Graduate, Biology, Fresno, Presenting Author; Arwa Alsubhi, Graduate, Biology, FresnoMelissa Jauregui, Undergraduate, Biology, Fresno, Presenting Author; Zoei Gavel, Undergraduate, Biology, FresnoAlejandro Calderón-Urrea, Faculty, Biology, Fresno Abstract Plant parasitic nematodes (PPNs) are among the biggest constraints on the world’s food production and agriculture. The current methods of controlling these parasitic nematodes can be toxic to humans and the environment. Chalcones are organic compounds identified to be a safe and effective method of controlling nematodes. Chalcone 17 and Chalcone 30 were found to have 100% lethality against Caenorhabditis elegans and the PPN Meloidogyne incognita at the concentration of 10-4 M. However, their modes of action remain unknown. This study hypothesizes that Chalcone targets a product of a gene, causing the breakdown of certain biochemical pathways in nematodes, leading to their death. Using C. elegans as the model, we aimed to isolate C. elegans that survived in Chalcones 17 and 30 at 10-4M, and to generate a list of putative genes for Chalcone 30 resistance. We induced mutations in the wild-type VC2010 strain of C. elegans using methanesulfonate and growing the putative mutants in the presence of Chalcones 17 and 30 separately. Four lines of mutants were isolated: Ch17.6.b, Ch17.7.b, Ch17.10.a, and Ch30.3.b. Using a traditional genetic crossing scheme with wild-type VC2010 C. elegans, we found Chalcone resistance to be a recessive trait for both Chalcone 17 and 30. When screening in opposite Chalcone (Chalcone 17 mutants in Chalcone 30 and vice versa), neither mutant survived, suggesting that Chalcone 17 and 30 use different molecular pathways. To generate a list of putative genes for Chalcone 30 resistance, we performed two backcrosses of hermaphrodites from Ch30.3.b with wild-type males from the Hawaiian (CB4856) strain and screen their F2 generations for Chalcone 30 resistance. The DNAs of the surviving F2 generations of the first and second backcrosses were extracted for Whole Genome Sequencing (WGS). Using the bowtie2 and MiModD toolsets on the Galaxy online server to analyze the WGS data, we obtained a list of putative genes that could be involved in the Chalcone 30 molecular pathway of killing nematodes. Overall, Chalcone was observed to affect a product of a gene and Chalcone 17 and Chalcone 30 have different nematocidal pathways. Future research should focus on using targeted mutagenesis on the putative genes to confirm their involvement in the Chalcone 30 biochemical pathway. Research in Dr. Calderón-Urrea’s lab is partially funded by a grant from NIH (1R16GM145429).
| Poster #: 5 Campus: California State University, Chico Poster Category: Agriculture/Biofuels/Environment Keywords: Indole Acetic Acid (IAA), Surfactin, Bacteria Project Title: Bacterial Numbers Indole Acetic Acid and Surfactin From Burn Lines at LIttle Grass Valley Reservoir Author List: Jannette Ortiz, Undergraduate, Biological Science, Chico, Presenting Author; Emily Regan, Undergraduate, Biological Sciences, Chico, Presenting Author; Abstract Many soil bacteria enjoy the benefits that plants provide them, including carbohydrates, vitamins, organic molecules, and oxygen. Therefore fire may have an impact on bacterial numbers in soil. We tested this hypothesis by counting bacteria recoverable on several media, from soil samples taken near burn lines at a single location. Soil samples were diluted in water, plated on solid media, and incubated at 28*C. Salkowski's reagent on filter lifts was used to quantify indole acetic acid-producing colonies. Production of surfactin was detected by clearing on sheep's blood agar. The total number of recoverable bacteria on King Medium B was 9.8*10^6 CFU per milligram of soil. In comparison, the average total number of recoverable bacteria on burned soil was 2.5*10^7 CFU per milligram of soil. However, on blood agar from unburned soil, the average was 3.7*10^7 CFU per milligram of soil versus 4.7*10^7 CFU per milligram of soil. We are continuing our work with the examination of surfactin and IAA production by the isolated bacterial strains, and in the development of bioassays to evaluate the efficacy of bacterial applications to modify root growth and water tension in soils.
| Poster #: 6 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Agriculture/Biofuels/Environment Keywords: Essential Oil, Blood Metabolites, Feed Additives Project Title: Effects of Feeding Essential Oils on Laying Hens Blood Metabolites Author List: jasmine Moallem, Graduate, Animal Science, San Luis Obispo, Presenting Author; Zachary Ferrenberg, Graduate, Animal Science, San Luis Obispo, Presenting Author; Darin Bennett, Faculty, Animal Science, San Luis ObispoRodrigo Manjarin, Faculty, Animal Science, San Luis ObispoMohammed Abo Ismail, Faculty, Animal Science, San Luis ObispoSiroj Pokharel, Faculty, Animal Science, San Luis Obispo Abstract This study evaluated the effect of feeding essential oils (EO) on late-laying hens and their impact on various blood metabolites. Essential oils are increasingly used in the poultry industry as beneficial feed supplements that may be utilized to maintain gut health, promote growth, balance intestinal microflora, and exhibit antimicrobial properties. In this study, a total of 60 commercial caged White Leghorn laying hens were randomly allocated to one of four dietary treatments and fed ad libitum for twelve weeks. These treatments included 0% control (corn–soybean meal-based basal diet), 0.5% (low EO-based basal diet), 1% (high EO-based basal diet) diets, and a 1% prebiotic-based basal diet. Weekly body mass and feed conversion ratio were measured and recorded, and a complete blood profile was performed at the end of the twelfth week. All data was analyzed using SAS 9.4. Body mass was significantly (P < 0.05) decreased in prebiotic-treated birds compared to birds fed a control diet. Similarly, metabolite panels were lowered in prebiotic-treated hens. There was a stepwise decrease in Phosphorus and Calcium levels from control to prebiotic diets. The 0.5% low EO-based basal diet treated birds showed a significant increase (P < 0.05) in the Lipemic Index compared to other diets. Other metabolites measured showed no significance between treatments (P > 0.05). Further research will be necessary to evaluate essential oils' role as a potential feed additive in the diets of laying hens. We want to acknowledge our sponsor and ARI for funding this project.
| Poster #: 7 Campus: California State University, Chico Poster Category: Agriculture/Biofuels/Environment Keywords: small molecule organic synthesis, pesticide development, drug assessment Project Title: Synthesis of Anthelmintic Drugs with Increased Efficacy for Enhanced Parasite Control Author List: Emmanuel Gallegos, Undergraduate, Chemistry and Biochemistry, Chico, Presenting Author; Rene Sabala, Undergraduate, Chemistry and Biochemistry, Chico, Presenting Author; John-Paul Quinones, Undergraduate, Chemistry and Biochemistry, ChicoCarolynn Arpin, Faculty, Chemistry and Biochemistry, Chico Abstract Parasitic infections pose a significant challenge to ruminant farming, affecting livestock feed intake, growth, carcass weight, milk production, wool growth, fertility, and mortality, thereby impacting farm cash income. Increasing resistance of nematode populations to available drugs necessitates the development of new anthelmintics. A class of drugs known as benzimidazoles has been effective against various helminths due to their unique pharmacological activity and transport properties. Among benzimidazoles, Albendazole stands out for its broad spectrum of efficacy against nematodes and tapeworms affecting cattle and dairy herds. However, enhancing the absorption of Albendazole in nematodes remains a challenge. We have thus designed and synthesized a library of Albendazole derivatives, leveraging Albendazole's framework with strategically introduced fluorine substitutions in its thio-alkyl group. These substitutions aim to enhance the drug's lipophilicity, ultimately improving its absorption by parasites. We were successful in synthesizing one of these derivatives in good yield: between 24 to 100% yield for each step. Efforts are underway to synthesize the remainder of the library and assess compound lipophilicity. Albendazole derivative design, synthesis, and lipophilicity assessment assays will be presented.
| Poster #: 8 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Agriculture/Biofuels/Environment Keywords: Essential Oils, Prebiotics, Egg Quality Project Title: Effects of Feeding Essential Oils and Prebiotics as Feed Additives on Production Parameters & Egg Quality in Laying Hens Author List: Zachary Ferrenberg, Graduate, Animal Science, San Luis Obispo, Presenting Author; Jasmine Moallem, Graduate, Animal Science, San Luis Obispo, Presenting Author; Darin Bennett, Faculty, Animal Science, San Luis ObispoRodrigo Manjarin, Faculty, Animal Science, San Luis ObispoMohammed Abo Ismail, Faculty, Animal Science, San Luis ObispoSiroj Pokharel, Faculty, Animal Science, San Luis Obispo Abstract Antibiotic feed additives in livestock production have seen a rise in regulations. Antibiotic growth promotors (AGP) have widely been used in non-therapeutic dosages to improve livestock production and health. However, it is necessary to explore feed additives that exhibit similar properties without the continued usage of anti-microbial. One area currently being examined is the usage of bioactive compounds found in plants. One such bioactive compound to explore is essential oils (EO). The EO has been shown to exhibit anti-microbial properties, improve livestock gut health, and are less likely to introduce new antibiotic-resistant pathogens when used as a supplementation in animal feed.
This research examines the feasibility of EO and prebiotic implementation in feedstuff as an AGP substitution and its impact on production parameters, egg production, and quality. In this research trial, we used sixty White Leghorn laying hens at their peak production period. Fifteen laying hens were randomly assigned to each treatment group consisting of – 1) control corn-soybean meal-based basal diet, 2) 0.5% low EO, 3) 1% high EO, and 4) 1% prebiotic mixed diets. The addition of EO and a prebiotics diet lasted for 12 weeks. Egg production was determined by eggs collected daily with an expected egg output of 7 eggs per week, egg quality was measured bi-weekly, and body mass was measured monthly. This feeding trial has shown varying results in the potential usage of EO and prebiotics as feed additives. Data was analyzed using repeated measures ANOVA in SAS 9.4Ò. Results showed that body mass was affected significantly (P < 0.05) by the prebiotic diet when compared with the control diet-fed hens. Likewise, prebiotic-fed hens have significantly lower (P < 0.05) feed intake values when compared with the rest of the groups. Interestingly enough, for the egg parameters, albumen height was significantly (P < 0.05) higher in prebiotic-fed hens when compared with the low EO-fed hens. However, low EO-fed hens had significantly lower (P < 0.05) eggshell thickness and strength values when compared with high EO-fed hens. Continuing research in this area could prove beneficial to reducing antibiotic usage, which could further help impede the emergence of antibiotic-resistant pathogens. Future research should continue to examine specific EO supplementation and their individual properties on livestock production. We want to acknowledge our sponsor and ARI for funding this project.
| Poster #: 9 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: Genomics, Microbiome, Agriculture Project Title: Metagenomics and metabolomics of soils associated with Salinas Valley tomato fields Author List: Adam Dean, Undergraduate, Marine Science, Monterey Bay, Presenting Author; William Andreopoulos, Faculty, Computer science, San JoséMatthew Escobar, Faculty, Biology, San MarcosJohn Gieng, Faculty, Nutritional science, San JoséJeroen Gillard, Faculty, Biology, BakersfieldGreig Guthey, Faculty, Environmental studies, San MarcosFranz Kurfess, Faculty, Computer science, San Luis ObispoJuleen Lam, Faculty, Public health, East BayJoseph Ross, Faculty, Biology, FresnoNathaniel Jue, Faculty, Biology and chemistry, Monterey Bay Abstract Soil microbiomes, a community of microorganisms that live in the soil, are incredibly diverse and important ecosystems. They can have significant effects on plant health and agricultural yields. One active area of research is looking into how natural variation in microbial communities affects crops. This study examines the relationship between soil microbial communities and the metabolites present in the soil. We are interested in determining if changes in soil microbe composition can be associated with changes in soil metabolite composition. The goal of this project is to eventually manipulate soil microbiomes to produce a healthier agricultural product. We tested this idea by comparing the structure of microbial communities and their metabolite content for soil samples associated with mature tomato plant roots taken from a farm in Greenfield, California. DNA was isolated from the soil rhizosphere of these plants, followed by whole metagenome shotgun sequencing. The KBase platform facilitated the assembly of metagenome-assembled genomes (MAGs), and we annotated the MAGs using the DRAM program. Using annotated MAGs, we created Principal Component Analysis (PCA) and Multidimensional Scaling (MDS) plots to determine how similar or different the functions in the genomes of our different samples were. The PCA and MDS were then compared to previously collected metabolomic data from GC-MS analysis of the same soil samples to determine if certain bacteria and specific functions affect the metabolite content. While informative on patterns of variation within the field, the relationship between soil metabolites and soil microbiome for our samples remains unclear. Deeper sampling from multiple sites is needed to better determine how soil microbiomes influence soil metabolite content and if these changes in soil metabolite content are, ultimately, significant enough to affect fruit characteristics and subsequent human health benefits.
| Poster #: 10 Campus: California State University, Sacramento Poster Category: Astrobiology Keywords: protocells, energy generation, proton gradient Project Title: Sulfite as a reducing agent capable of establishing proton gradients across lipid bilayers of model systems for primeval cells Author List: Stephanie Ramirez, Undergraduate, Chemistry, Sacramento, Presenting Author; Amaan Sandhu, Undergraduate, SacramentoNaiki Judge, Undergraduate, SacramentoDavid Deamer, Faculty, Non-CSU, University of California at Santa CruzStefan Paula, Faculty, Chemistry, Sacramento Abstract A frequently debated topic related to origin of life research is the question of how complex modern life forms present on today’s Earth may have evolved from simpler predecessors. This includes processes involving the generation of proton concentration gradients across cellular membranes which are at the core of ATP synthesis in all cells. Modern cells produce these gradients using enzymes and electron carriers that are part of the respiratory chain. Since it is highly unlikely that such a sophisticated machinery was present on early Earth, primitive cells must have generated their proton gradients by simpler means, relying on materials readily available in the environment back then. Recently, it has been shown that substantial proton gradients can be established in liposomes using energy derived from redox reactions between inorganic solutes, such as dithionite and ferricyanide. Here, we expand on this topic by evaluating other sulfur compounds as potential electron donors, using a fluorescent pH-sensitive dye in conjunction with liposomes as model systems for primeval cells. We found that sulfite can act as an electron source whose oxidation leads to the buildup of proton gradients in the order of two pH units. Our findings add to a growing body of evidence which shows that the generation of proton gradients in protocells can be accomplished by simple components that were likely present on the early Earth.
| Poster #: 11 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Extremophile, Microbiology, Thermophile Project Title: Isolation of novel Dictyoglomus strains from US Great Basin hot springs Author List: Andreas Vamvakas, Undergraduate, Biology, San Bernardino, Presenting Author; Santiago De La Cruz, Undergraduate, Biology, San Bernardino, Presenting Author; Alexis Ortega, Undergraduate, Biology, San BernardinoJacqueline Gomez, Undergraduate, Biology, San BernardinoAllison Bledsoe, Undergraduate, Biology, San BernardinoRandy Jimenez, Undergraduate, Biology, San BernardinoJeremy Dodsworth, Faculty, Biology, San Bernardino Abstract The genus Dictyoglomus currently consists of two described species, D. turgidum and D. thermophilum, which are the only cultivated members of the bacterial phylum Dictyoglomerota. These strictly anaerobic thermophiles can ferment a variety of sugars and plant carbohydrate polymers, and their thermotolerant enzymes responsible for this activity have potential biotechnological applications. Cultivation-independent 16S rRNA gene sequencing studies have detected microbes related to, but likely distinct from, described Dictyoglomus species in hot springs in the US Great Basin, including Great Boiling Spring (GBS), NV and Little Hot Creek (LHC), CA. Our goal was to attempt to isolate these Dictyoglomus relatives, characterize them, and compare them to known species in the genus. To accomplish this, samples from GBS were used to inoculate media containing xyloglucan as a sole carbon and energy source under anaerobic conditions. These cultures were incubated at 70 degrees C and transferred repeatedly in the laboratory. Solid media of similar composition with either xyloglucan, carboxymethylcellulose, or a mix of sugars as carbon sources were used to obtain isolates of a strain of Dictyoglomus. This isolate had 99.8% 16S rRNA gene identity to sequences obtained from GBS and neighboring springs, but less than 97.6% identity to D. turgidum and D. thermophilum. The GBS isolate was phenotypically similar to known Dictyoglomus species, but differed from one or both in utilization of starch and various sugars such as lactose, and could grow at a broader pH range (3.75-9.25). The whole genome sequence of the GBS Dictyoglomus isolate had less than 77% average nucleotide identity to other Dictyoglomus genomes, providing additional evidence that it represents a distinct, novel species. More recently, a strain of Dictyoglomus with 99.2% identity to the GBS strain was obtained from samples from hot spring LHC using a similar approach, except that dilution-to-extinction was used for isolation because of a lack of growth on solid media. Future work will further characterize these isolates, and describe them as representatives of at least one new species in the genus Dictyoglomus.
| Poster #: 12 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Course-Based Undergraduate Research Experience, Enzyme Inhibition, Structure-activity relationship studies Project Title: Course-Based Undergraduate Research Experience Lab: Design, Synthesis and Biological Evaluation of New Alzheimer’s disease Therapeutics Author List: Stevan Pecic, Faculty, Chemistry and Biochemistry, Fullerton, Presenting Author; Abstract The goal of this Course-Based Undergraduate Research Experience (CURE) Lab was to offer students at California State University Fullerton (CSUF) early opportunities to practice authentic scientific research in the cross-disciplinary fields of medicinal chemistry and biochemistry through revisions of an existing upper division Medicinal Chemistry lab course. The proposed modifications focus on introducing research experience in drug discovery into a lab course while still covering essential organic chemistry and biochemistry laboratory skills. This interdisciplinary program is composed of a 1-credit introductory chemistry lab seminar, followed by a 2-credit medicinal chemistry lab course.
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory loss, dementia and cognitive impairment. Acetylcholinesterase (AChE) is an important enzyme that hydrolyzes the neurotransmitter acetylcholine. According to the Cholinergic hypothesis, AChE inhibitors represent a promising therapeutic strategy for treating the symptoms of AD. In this CURE-based teaching labs 8 undergraduate CSUF students majoring in Chemistry and Biochemistry are introduced to a real-life research experience - discovery of new AD therapeutics. Students synthesized, purified and biologically evaluated potential new therapeutics, followed by molecular modeling studies. The integration of an authentic research project into teaching lab is designed to serve as a foundation for a future curriculum that will better prepare students in their careers in biomedical field and pharmaceutical industries.
Standardized, specifically designed surveys for CURE, students’ attitudes and learning styles was used to assess the impact of the course modifications and student impressions of research.
Research reported here was supported by the 2020 CSUPERB Curriculum Development Grant.
| Poster #: 13 Campus: San Francisco State University Poster Category: Biochemistry Keywords: Mycobacterium tuberculosis, Flavin-Dependent Thymidylate Synthase, antibiotic Project Title: Synthesis and evaluation of novel inhibitors of MtbFDTS Author List: Su Ozcan, Graduate, Chemistry and Biochemistry, San Francisco, Presenting Author; Eric Koehn, Faculty, Chemistry and Biochemistry, San Francisco Abstract Tuberculosis (TB) poses a global health challenge. Mycobacterium tuberculosis (M.tb), the bacterium that causes TB, evolved variants that are extensively and multiple drug resistant resulting in an emerging need for new antimycobacterial drugs. Thus, none of the TB antibiotics target M. tb's DNA biosynthesis. Our goal is to develop potential antimycobacterial agents that target DNA biosynthesis by directly interrupting the the synthesis of deoxythymidine monophosphate (dTMP), which is an essential DNA base. Unlike humans, where Classical Thymidylate Synthase (TSase) catalyzes dTMP synthesis, M.tb employs a Flavin-Dependent Thymidylate Synthase (FDTS) to produce this nucleotide. Our innovative approach centers on nucleotide-based antibiotics targeting MtbFDTS to interrupt DNA biosynthesis in M.tb., without affecting human cell dTMP synthesis through selective enzyme inhibition.
Novel nucleoside-based analogs that have antimicrobial activity against MtbFDTS were synthesized based on previous lead compounds demonstrating either enzyme inhibition or antimycobacterial activity. These analogs with large hydrophobic groups in the C5 position, selectively interact with MtbFDTS since its active site is larger and more solvent exposed than the TSase. Based on that, the novel nucleoside-based analogs will be converted to nucleotide form by enzymatic phosphorylation by double mutant deoxycytidine kinase (DM-dCK). We confirmed the activity of our purified DM-dCK enzyme using deoxythymidine, a control substrate, to test DM-dCK activity. We optimized the reaction conditions to include (100mM Tris-KCl buffer at pH 7.5, 10mM MgCl2 stock, 1mM ATP stock, 200μM nucleoside stock, and 0.33uM enzyme stock were reacted for 4 hours at room temperature). The deoxythymidine monophosphate obtained as a result of this reaction was observed in LCMS analysis and proved that our DM-dCK stocks showed 97% conversion from dT to the desired dTMP. We are currently running reactions using novel nucleoside analogs with DM-dCK for enzymatic phosphorylation. To assess the potency of these newly synthesized inhibitors, the binding constants of nucleotide-based inhibitors with MtbFDTS will be calculated by monitoring the change in FAD absorbance at 454 nm. We measured the binding constant of the substrate nucleotide, dUMP, as a control to validate this methodology. Thus, critical information regarding new nucleotide-based inhibitors and their ability to selectively inhibit MtbFDTS will be gained.
| Poster #: 14 Campus: California State University, Stanislaus Poster Category: Biochemistry Keywords: toluene monooxygenase, drug metabolism, protein engineering Project Title: Application of Engineered Toluene o-Xylene Monooxygenases as Biocatalysts to Generate Human Metabolite of Chlorzoxazone Author List: Noella Younan, Undergraduate, Chemistry, Stanislaus, Presenting Author; Angeline Dauz, Undergraduate, Chemistry, Stanislaus, Presenting Author; Gonul Schara, Faculty, Chemistry, Stanislaus Abstract The biocatalyst toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 belongs to the great family of bacterial multicomponent monooxygenases and has been shown to have a potential for biotechnological applications. Chlorzoxazone, which is a muscle relaxant and sold under the brand names Lorzone or Paraflex, is mostly metabolized into 6-hydroxychlorzoxazone (6-HC) in human liver by P450 monooxygenases. In this study, metabolism of chlorzoxazone was examined with Escherichia coli TG1 strain expressing native ToMO for the very first time. Oxidation activity levels and regiospecificities were determined using reverse-phase high-performance liquid chromatography on a C18 column using a water/acetonitrile solvent system. Compounds were identified by comparison of retention times to those of authentic standards and by co-elution with the standards. The negative controls used in these experiments contained TG1 cells expressing ToMO without chlorzoxazone as well as empty TG1 cells with chlorzoxazone (no-ToMO control). We found that native ToMO was capable of metabolizing chlorzoxazone to 6-HC, facilitating human P450-like hydroxylation, but only at a very low rate (less than 0.1% conversion after overnight incubation). After this reaction was discovered, selected engineered enzymes containing mutations in ToMO were examined in order to generate further improvements in ToMO activity. We found that activity on chlorzoxazone has been improved by up to 48.0-fold relative to the native ToMO and the key mutations perturbing ToMO’s active site allowed the generation of 6-HC in milligram quantities, which is usually the desired amount as reference compounds for metabolic studies. This study confirms the advantages of protein engineering approaches and adds to the list of research on probing this remarkable enzyme. Furthermore, this study also shows the potential of non-human ToMO and its variants in drug metabolism applications; hence, their applicability to metabolize selected drug molecules is currently being investigated. This project is supported by the National Institutes of Health Support of Competitive Research Program.
| Poster #: 15 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: structure-function, protein function, gene expresssion Project Title: Understanding the Role of Phosphorylation in RNA Binding Protein Function Author List: Abigail Anastasi, Undergraduate, Chemistry and Biochemistry, Fullerton, Presenting Author; Abstract Alternative pre-mRNA splicing is a critical process that allows for a single gene to encode for more than one protein isoform, increasing protein diversity. RNA Binding Proteins (RBPs) play a crucial role in regulating alternative pre-mRNA splicing and, in turn, dictating the cellular proteome. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RBPs that regulate alternative splicing of pre-mRNA gene transcripts. The paralogs share 74% primary structure identity and a similar domain organization of four RNA-recognition motifs (RRMs) connected by three linker regions and an N-terminal region. Yet, the two proteins have distinct tissue-specific expression patterns where PTBP1 is expressed near ubiquitously but absent in neurons, while PTBP2 is expressed in differentiating neurons. Moreover, the paralogs regulate different sets of target exons and this difference in activity plays a critical role in the process of neuronal development and maturation. How two related structurally similar proteins can regulate distinct sets of target exons is not understood. Previous studies discovered that PTBP2 is distinctly phosphorylated in the unstructured N-terminal and Linker regions to a greater extent than PTBP1. We determined that PTBP2 distinctly phosphorylated residues are conserved in lower species, indicating an important role in PTBP2 function. Thus, we hypothesize that linker region phosphorylation plays a role in the neuronal-specific splicing activity of PTBP2. Our proposed experiments are to test this hypothesis. We used recombinant DNA technology to create phosphate mimic aspartate mutants of PTBP2 phosphorylated residues. Alanine mutants that cannot undergo phosphorylation (of phosphorylated residues) were also created. We are currently assaying these mutants in vivo in mouse neuro-2A cells to test for protein expression (via Western Blotting) and splicing activity on PTBP2-regulated exons (via reverse-transcription PCR). Results from this study will determine the role of phosphate modifications in PTBP2 splicing activity and determine whether phosphorylation plays a role in dictating the distinct splicing activities of related RBPs in a gene family.
This work is supported by a NIH SCORE award (1SC3GM132036) to NMK and funds from the CSUF ORSP to AA and NMK. We thank members of the Keppetipola research laboratory for their support and CSUBIOTECH for the opportunity to present this work.
| Poster #: 16 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Structure-activity relationship studies, Enzyme inhibition, Molecular modeling Project Title: Designed Multiple Ligands as Non-opioid Analgesics for Treating Chronic Pain Author List: Faye Yun , Undergraduate, Chemistry and Biochemistry, Fullerton, Presenting Author; Leah Duong, Graduate, Chemistry and Biochemistry, Fullerton, Presenting Author; Ram Kandasamy, Faculty, Psychology, East BayStevan Pecic, Faculty, Chemistry and Biochemistry, Fullerton Abstract Chronic pain is the leading cause of disability worldwide, impacting around 20% of adults in the U.S. alone. The annual cost of treating chronic pain is more than $500 billion per year - higher than the cost of heart disease, cancer, and diabetes. Currently, opioids are the primary treatment option for pain, but can lead to addiction and severe health complications. Current non-opioid alternatives, while safer than opioids, offer limited relief and are typically suited for mild to moderate pain treatment. Therefore, there is a pressing need for the development of more effective non-opioid pain medications.
The sEH (soluble epoxide hydrolase) and FAAH (fatty acid amide hydrolase) enzymes play significant roles in pain modulation. sEH breaks down epoxyeicosatrienoic acids (EETs), which have anti-inflammatory properties, thus influencing pain perception. FAAH, on the other hand, degrades endocannabinoids, molecules involved in pain sensation, mood, and memory. Inhibition of these enzymes can lead to increased levels of EETs and endocannabinoids, respectively and potentially offering a therapeutic avenue for non-opioid pain relief. The goal of our lab is to develop a dual sEH/FAAH inhibitors, a single molecule that will inhibit both aforementioned enzymes simultaneously.
In this project, using a 4-step synthetic route, we designed and synthesized twelve new 4-phenylthiazole-based compounds with the aim to explore the structure-activity relationships of this moiety in inhibiting both FAAH and sEH enzymes. We successfully optimized a novel microwave-assisted Hantzsch thiazole synthetic reaction and were able to obtain intermediates in high yield, above 90%, while decreasing the time of the reaction from 2.5 hours (conventional, previously reported method) to 3 minutes. Our study showed that placement of various groups (e.g. fluoro, chloro, methyl and methoxy) at the left side of the pharmacophore can improve the inhibition at both enzymes producing dual inhibitors in low nanomolar range. After determining their metabolic stability in microsomal liver assay, the most promising compounds will be sent to our collaborator for in vivo testing at Cal State East Bay.
This research is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1 R16GM149204-01. Faye Yun is supported by the U-RISE at Cal State Fullerton grant from the National Institutes of Health 5T34 GM149493-01.
| Poster #: 17 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: heat-shock proteins, lipids, pharmacology Project Title: Characterizing the lipid-dependency of plasma membrane and extracellular HSPA1A Author List: Allen Badolian, Graduate, Fullerton, Presenting Author; Carolina Briseno, Graduate, Fullerton Abstract Heat shock protein A1A (HSPA1A) translocates to the plasma membrane (PM) of heat-shocked and cancer cells. HSPA1A’s PM movement depends on lipids like phosphatidylserine (PS) and phosphatidylinositol-monophosphates (PIPs). HSPA1A is also found extracellularly, leading to predictions that PM-bound HSPA1A is exported. However, the relationship between membrane-bound (mHSPA1A) and extracellular HSPA1A (eHSPA1A) remains undefined. We hypothesized that lipid depletion, known to prevent mHSPA1A localization, would consequently abolish eHSPA1A. To test this, we pharmacologically inhibited key enzymes involved in PS and PIPs biosynthesis. We also sequestered these lipids using biosensor proteins with high-affinity lipid-binding domains. These complementary approaches achieved potent lipid depletion and disruption. We quantified eHSPA1A by western blotting of conditioned media after heat shock and lipid depletion. We also adapted the HiBit® luciferase-based system to simultaneously quantify eHSPA1A, mHSPA1A, and cytosolic HSPA1A under these experimental conditions. In all conditions tested, lipid depletion consistently blocked mHSPA1A but not eHSPA1A. These novel findings contradict current predictions and demonstrate mHSPA1A and eHSPA1A are independently regulated. Our data reshape the model for HSPA1A trafficking, suggesting separate mechanisms govern PM localization versus extracellular export. This finding has broad implications for understanding HSPA1A's roles in cellular stress response and cancer progression, where both PM and extracellular HSPA1A are functionally relevant and have contradictory roles. Overall, this work challenges prevailing assumptions about HSPA1A trafficking through rigorous examination of the relationship between mHSPA1A and eHSPA1A using pharmacologic and biosensor tools. It further indicates the complex, multifaceted regulation of this essential chaperone protein at the PM and in the extracellular environment.
| Poster #: 18 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Multidrug efflux pump, Antibiotic resistance, Escherichia coli Project Title: Identification of Endogenous Metabolic Substrates of the AcrABZ-TolC Multidrug Efflux Pump of Escherichia coli Author List: Allea Cauilan, Faculty, Biology, Northridge, Presenting Author; Cristian Ruiz Rueda, Faculty, Biology, Northridge Abstract Introduction: Antibiotic resistance is a growing problem in the clinical and agricultural settings. One mode of resistance are bacterial multidrug efflux pumps which bind and expel multiple classes of antibiotics. One such pump is the AcrABZ-TolC pump found in Enterobacteriaceae. Previous studies have shown that AcrABZ-TolC plays a role beyond removing antibiotics. These functions include removal of cellular toxic metabolites and virulence. These other roles of the AcrABZ-TolC pump have not been well studied. Our previous research found several metabolic compounds that accumulate intracellularly when this pump is inactivated, implying that they may be substrates of this pump. Objective: To identify these substrates by using an ethidium bromide accumulation assay which detects increased intracellular ethidium bromide accumulation when the AcrABZ-Tolc pump is inhibited by competing substrates. Methods: The strains used in this study were Escherichia coli Wild-Type and gene-deletion mutants for the AcrB, AcrZ, and TolC components of the pump. The four strains were grown in Lysogeny Broth overnight then subcultured and grown to mid-exponential phase. The cells were pelleted and resuspended in Phosphate Buffered Saline. Cells were placed in a black clear-bottom microplate and treated with a potential pump substrate/inhibitor and ethidium bromide. The relative fluorescence units of each well were measured at 37 ˚C over the course of 75 minutes. Results: We found that sodium citrate caused a dose-dependent increase in ethidium bromide accumulation in the Wild-Type strain but not the efflux pump mutants. Sodium malate also caused increased accumulation in the Wild-Type but also caused some accumulation in the AcrB and TolC pump mutants. Conclusion: Sodium citrate and sodium malate have been shown to be potential inhibitors of the AcrABZ-TolC pump. Further experiments such as minimum inhibitory concentration assays and efflux assays will be used to determine the effect of these compounds on antibiotic susceptibility as well as determine whether, besides being inhibitors, they are also substrates of the AcrABZ-TolC pump.
| Poster #: 19 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: enzyme inhibitor, ligand docking, enzyme activity assay Project Title: Naphthoquinones as Inhibitors of the Sarco/Endoplasmic Reticulum Calcium ATPase Author List: Nick Johnson, Undergraduate, Chemistry, Sacramento, Presenting Author; Stefan Paula, Faculty, Chemistry, Sacramento Abstract The sarco/endoplasmic reticulum calcium ATPase (SERCA) is a large integral membrane protein that resides in the membrane of the sarco/endoplasmic reticulum. It is a transmembrane protein that charges intracellular calcium ion stores by transporting calcium ions from the cytosol into the SR/ER lumen by using chemical energy gained from the hydrolysis of ATP. Inhibition of SERCA has been proposed a strategy for the treatment of various diseases, such as prostate cancer. Even though a variety of SERCA inhibitors has been characterized, a need for novel compounds remains. Naphthoquinone derivatives have shown promise as potential SERCA inhibitors in earlier studies, but no systematic exploration of this compound class has been performed to date. Here, a selection of 15 naphthoquinone derivatives and of several structurally related anthroquinones was evaluated in enzyme inhibition assays. Three compounds with good potencies were identified that displayed IC50 values in the low to mid micromolar range. In parallel to the assays, molecular modeling and docking techniques based on the crystal structure of SERCA were employed to visualize inhibitor binding and to identify critical inhibitor/enzyme interactions in the binding pocket. The findings will serve as a platform for a broader search for novel SERCA inhibitors based on the naphthoquinone scaffold.
| Poster #: 20 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Biochemistry, Alzheimer’s disease, Lipid and lipoproteins Project Title: Role of Glycosylation in Maintaining the Structure and Function of Human Apolipoprotein E. Author List: Vanessa Garcia, Graduate, Chemistry and Biochemistry, Long Beach, Presenting Author; Emaela Valdez, Undergraduate, Chemistry and Biochemistry, Long BeachGeorge Celis, Graduate, Chemistry and Biochemistry, Long BeachJasmine Nguyen, Undergraduate, Chemistry and Biochemistry, Long BeachVasanthy Narayanaswami, Faculty, Chemistry and Biochemistry, Long Beach, California St University Long Beach Foundation Abstract Human apolipoprotein E (apoE) is a cholesterol transport protein that plays a critical role in cardiovascular disease (CVD) and Alzheimer’s disease (AD). The APOE gene is polymorphic giving rise to common alleles ε2, ε3 and ε4 corresponding to three isoforms: apoE2, apoE3, apoE4 that differ at their amino acid positions 112 and 158. ApoE2 bears Cys, while apoE4 bear Arg at these positions. ApoE3 bears a Cys at 112 and an Arg at 158. While apoE3 is considered cardioprotective, apoE4 is an established risk factor for AD and CVD. While the molecular basis of this is not understood, it has been reported that apoE4 is more susceptible to proteolytic degradation than apoE3. All three isoforms are composed of an N-terminal and a C-terminal domain linked via a protease-sensitive loop, which contains a glycosylation site at Thr194. We hypothesize that glycosylation confers an isoform-specific protection against proteolytic degradation in apoE. To test this, we compared recombinant apoE2, apoE3 and apoE4 expressed in Chinese hamster ovary (CHO) cells with those expressed in E. coli. While CHO cells have the capability to add glycosylated moiety following protein translation, the bacterial system can only synthesize non-glycosylated proteins. CHO cells were transfected with pcDNA 3.1 bearing the coding sequence for apoE2, apoE3 or apoE4 with a His-tag at the N-terminal end to facilitate purification. The synthesized proteins were harvested from the medium and purified by affinity chromatography and compared with those expressed in E. coli. SDS-PAGE analysis reveals a band ~35 kDa and ~36 kDa for apoE from E. coli and CHO cells, respectively. In an alternate approach, apoE from guinea pig (GP) was investigated since sequence alignment reveals that GP apoE is missing residues 193-197, a segment that encompasses the O-glycosylation site, Thr 194. SDS-PAGE analysis revealed a band ~33 kDa for GP apoE isolated either source. Western blot using anti-human monoclonal antibody showed cross reactivity between human and GP apoE. Future studies will include structural and functional characterization of the proteins to investigate the effect of glycosylation. Taken together, the data from our study will yield valuable insight into the role of glycosylation on apoE’s role in cholesterol metabolism and in CVD and AD.
Acknowledgments: This project is by NIH GM105561, T32GM138075, NSF HRD-1826490 and the 2023 Leslie K. Wynston Research Assistantship Award.
| Poster #: 21 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: transporter, conformational change, In-Gel fluorescence Project Title: Determine Substrate-Binding Induced Conformational Changes in MFS Transporter Using Site-Directed Alkylation and In-Gel Fluorescence Author List: Sayeeda Mostafa, Undergraduate, San Bernardino, Presenting Author; Cindy Mercado, Undergraduate, San Bernardino, Presenting Author; Abstract Secondary active transporters transport substrates using energy released from an ion gradient and are crucial for cells to acquire essential nutrients and exclude hazardous wastes and toxins. The Major Facilitator Superfamily (MFS) is the largest secondary active transporter family in nature and found in all forms of life. In human, there are more than 500 MFS transporters transporting a great variety of substrates in cells. Understanding the conformational change of MFS transporters during the transport cycle is crucial for the full explanation of their mechanism. In this study, we use CscB, the MFS transporter of sucrose in E. coli, as a model to investigate the substrate-binding induced conformational change. We propose that CscB undergoes an alternating access mechanism, in which the sugar binding and release make CscB alternatingly open and close to the periplasmic side and cytoplasmic side of the cytoplasmic membrane. A site-directed alkylation method with in-gel fluorescence is applied to test this notion: a cysteine-less CscB-GFP fusion was constructed and used as the template to construct single-cysteine replacements on the periplasmic side (helix I and helix VII) and cytoplasmic side (helix IV and helix X) of the proposed sugar pathway in CscB. E. coli C43(DE3) cells expressing a given single-Cys replacement were grown in LB broth and the accessibility/reactivity of the replacement was determined by alkylation in whole-cell using methoxypolyethylene glycol maleimide, a 5 KDa membrane impermeant thiol reactive reagent, in the presence and absence of sucrose. In-gel GFP fluorescence was used for detection of the band shift and quantitative determination of the level of alkylation. More than 20 single-Cys replacements on the periplasmic side of CscB have been tested and sugar-binding effects to alkylation have been determined. 16 single-Cys replacements on the cytoplasmic side are under construction and will be tested by alkylation with isolated cell membrane. The combined alkylation results on both sides will generate a delineation of the sugar-induced global conformational change in CscB and provide new insights to the mechanism of MFS transporters. This work is supported by CSUSB Mini Grant, CSUSB OSR Student Research Grant and CSUPERB COVID-19 Post Pandemic Faculty Support Program Grant.
| Poster #: 22 Campus: California State University, East Bay Poster Category: Biochemistry Keywords: Tritonia diomedea, protein purification, acetylcholinesterase Project Title: Purification of the enzyme acetylcholinesterase from the marine gastropod Tritonia diomedea Author List: Timothy Catbagan, Graduate, East Bay, Presenting Author; James Murray, Faculty, East BayMonika Sommerhalter, Faculty, East Bay Abstract The marine gastropod Tritonia diomedea serves as a model organism in neurobiology. We are characterizing the enzyme acetylcholinesterase (AChE) from this model. AChE cleaves the neurotransmitter acetylcholine and thereby terminates signal transmission between cholinergic neurons. Despite its role as a model organism very little is known about T. diomedea’s enzymes involved in neurotransmission. Our goal is to purify AChE from T. diomedea for an enzyme kinetic characterization. Without purification AChE is surrounded by thousands of other proteins expressed in the gastropod’s tissues. To determine how many acetylcholine molecules are cleaved by AChE per minute we need to isolate the enzyme.
Previous purification attempts based on affinity chromatography (AC) in which a bait molecule (procainamide) is attached to a column resulted in very low yields. The AChE enzyme from the marine gastropod had a low binding affinity and did not stick to the column. We are therefore seeking to develop a multi-step column chromatography procedure involving hydrophobic interaction (HIC), anion exchange (IEX), and gel filtration (GF). These techniques are less selective than AC and require a large amount of starting material. In addition to the central nervous system, AChE is also expressed in the hemolymph of the marine gastropod. By combining the circulatory fluid of three to five invertebrates we obtained a starting volume over 100 mL. After each column purification step we measured AChE activity and total protein content with biochemical assays and conducted protein electrophoresis on samples high in AChE activity. With each purification step, we learned more about the enzyme. We now know that AChE is expressed in different molecular forms in the gastropod’s hemolymph. HIC showed two AChE forms, both with hydrophobic patches on the surface. IEX conducted in a pH 8.0 buffer revealed that AChE has negative surface charges. GF displayed two AChE forms with apparent molecular weights of 530 kDa and 1050 kDa.
Our purification procedure still requires further optimization. For example, we used ammonium sulfate precipitation followed by solubilization in a small buffer volume to concentrate our sample before HIC but we lost more than 50% of the total AChE activity. Ultrafiltration devices used to concentrate AChE-rich samples resulted in minimal sample loss. In our next attempt we will use ultrafiltration devices for all concentration steps.
| Poster #: 23 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Protein structure, Lipoprotein, Cardiovascular disease Project Title: Elimination of an intra-molecular salt bridge in the C-terminal domain of apoA-I did not prevent self-association Author List: Uyen Vo, Graduate, Chemistry and Biochemistry, Long Beach, Presenting Author; Bryan Kang, Undergraduate, Chemistry and Biochemistry, Long Beach Abstract Cardiovascular disease is the world's leading cause of death, which results from the buildup of plaque in the arteries, gradually blocking blood flow. HDL possesses anti-atherosclerotic properties via a mechanism known as reverse cholesterol transport (RCT). In this process, HDL travels through the blood, collects cholesterol esters and transports them to the liver for excretion. The major protein component of HDL is apolipoprotein A-I (apoA-I), which is known to play a critical role in cholesterol metabolism and HDL synthesis. ApoA-I consists of two domains, a highly ordered N-terminal domain (residues 1 – 179) and a less structured C-terminal (CT) domain (residues 180 – 243). The CT domain is responsible for mediating self-association of apoA-I and comprises three helices (8 to 10) that adopt helical structure upon lipid binding. Previous studies showed that when all three lysines of helix 10 were changed into glutamine, the protein became monomeric, and identified lysine 226 as the most critical residue. Therefore, an intrahelical salt-bridge with neighboring glutamate 223 may stabilize the amphipathic α-helix, forming a hydrophobic face that promotes self-association. To test this hypothesis, a double mutant was engineered via site-directed mutagenesis by substituting glutamate 223 and lysine 226 with glutamine, eliminating the positive and negative charge on these residues. The protein was expressed in E. coli, purified by Ni-affinity and size-exclusion chromatography. Self-association was studied by chemical crosslinking with dimethyl suberimidate, followed by SDS-PAGE. Crosslinking of the double mutant resulted in the appearance of large multimers, evidence that the protein remained in a self-associated state. However, a measurable amount of apoA-I did not form intermolecular crosslinks. Size-exclusion chromatography FPLC analysis showed that the protein exhibited monomeric character, but large apoA-I complexes were also abundantly present, confirming the crosslinking analysis. Thus, while the mutations increased the amount of monomeric apoA-I, a large portion of the protein population remained in a multimeric state. Since disruption of the interhelical salt-bridge did not prevent formation of multimeric apoA-I complexes, lysine 226 may form intermolecular salt-bridge with a neighboring apoA-I. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number GM089564.
| Poster #: 24 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Protein kinase B, Small molecule inhibitors, Project Title: Investigating the Mechanism of Action of SC66 – A Small Molecule Modulator of Protein Kinase B Author List: Tuhina Bhattacharya, Graduate, Long Beach, Presenting Author; Deepali Bhandari, Faculty, Long Beach Abstract According to the World Health Organization, cancer is the major cause of death worldwide with approximately 10 million deaths annually, or every one in six deaths. About 40% of all tumor types have aberrations in the signaling pathway involving protein kinase B, more commonly referred to as Akt. Akt plays a pivotal role in regulating numerous critical cellular processes, including growth, metabolism, and survival, all of which are essential for the sustenance of cancer cells. Consequently, Akt inhibitors represent a vital category of anti-tumor agents in the realm of cancer therapy. One such inhibitor, SC66, was discovered from a library of small molecules and was shown to allosterically inactivate Akt as well as promote its degradation. The authors proposed that SC66 binds to the amino-terminal pleckstrin homology (PH) domain, however, this was not experimentally tested. The main objective of this study is to determine whether SC66 interacts with the PH domain of Akt. To do so, we cloned the PH domain of Akt1 (residues 1-113) with an N-terminal hexa-histidine tag in the pET28a bacterial expression vector and expressed it in codon optimized Rosetta Escherichia coli DE3 cells. Based on a previous study, to improve solubility of the recombinant protein, three codons for lysine residues were added to the carboxyl-terminal end of the construct. The protein was partially purified by a single step affinity chromatography using Co2+ beads and the fractions were analyzed via staining with instant blue dye and western blotting. Subsequently, we employed fluorescence spectroscopy for the preliminary analysis of the binding between SC66 and the purified PH domain. We observed a change in the fluorescence intensity of Akt after SC66 was added to the solution suggesting a potential interaction. Our current studies include further purifying the protein using ion-exchange chromatography followed by isothermal calorimetry studies to analyze the binding between the PH domain and SC66. The findings from this study will enable a reassessment of prior research findings and their implications regarding SC66 as a potential anti-cancer drug.
| Poster #: 25 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Cancer, missense mutations, X-ray crystallography Project Title: Analyzing Cancer-Associated Mutations in the Retinoblastoma Protein Based on Protein Crystal Structure and Thermostability Author List: Alfredo Ruiz-Rivera, Undergraduate, Chemistry and Biochemistry, San Bernardino, Presenting Author; Chad Moorman, Staff, Chemistry and Biochemistry, San BernardinoAnthony Castro, Undergraduate, Chemistry and Biochemistry, San BernardinoJason Burke, Faculty, Chemistry and Biochemistry, San Bernardino Abstract Cancer's complexity and high death rate have emphasized the urgency of furthering knowledge of this disease. Tumor suppressor proteins, such as the Retinoblastoma protein (Rb) act as checkpoints in the cell cycle to prevent unregulated cell growth. Rb was first discovered in a rare form of eye cancer affecting infants and has since been identified in other malignancies. The Rb protein negatively regulates the cell cycle through interactions with its binding partner, the E2F family of transcription factors. We postulate when the protein is mutated, this may result in lowered protein expression levels or weakened binding strength to its binding partner. To test this hypothesis, cancer-associated mutations were studied through parameters including protein thermostability, binding strength, and here we report that we have optimized protein crystals of some of the most interesting variants, which are awaiting data collection. Protein variants were produced using BL21 E. coli, isolated through a series of column chromatography techniques, and crystallized using hanging drop diffusion. Notable mutations that have been crystallized include H555Y, R668C, M695I, and R763T. These display significant changes in melting temperature (Tm) of -2.53(± 0.51) for H555Y, -1.98(± 0.77) for R668C, -3.08(± 0.53) for M695I, and -1.6(± 0.56) for R763T. The negative shifts in Tm indicate internal structural disruptions, leading to reduced stability, and diminished function. Overall, this data will elucidate the role of cancer missense mutations in the Rb tumor suppressor protein. Thank you to the National Institute of Health (NIH) (Grant #SC3GM135037), CSUPERB (2019 New Investigator Grant), and CSUSB for financial support.
| Poster #: 26 Campus: San Diego State University Poster Category: Biochemistry Keywords: RNA Structure, Viral RNA, RNA DNA Hybridization Project Title: Investigating RNA Molecule Clusters Using Complementary DNA Oligos Author List: Herman Dhaliwal, Graduate, San Diego, Presenting Author; Rees Garmann, Faculty, San Diego, San Diego State UniversityDaniel Villareal, Graduate, San DiegoFernando Vasquez, Graduate, San DiegoTimothy Chiang, External Partner, Non-CSUOfer Kimchi, External Partner, Non-CSUMichael Brenner, External Partner, Non-CSUVinothan Manoharan, External Partner, Non-CSU Abstract Understanding the intricate structure of RNA molecules is paramount, as RNA plays a central role in directing every facet of the viral lifecycle, from encoding essential proteins to modulating gene expression. These RNA secondary structures form due to connections established between complementary bases. These connections can occur between neighboring nucleotides or extend over vast distances within the molecule itself. This complex formation of RNA structure raises questions about what transpires during transcription. In the presence of long RNA molecules with extensive long-range connections, early-transcribed regions may seek to establish connections with other segments on the same RNA molecule or other RNA molecules. Remarkably, this phenomenon is observable even in vitro transcription, our most rudimentary model that includes only essential transcription materials, indicating prominent intermolecular connections forming. To deepen our comprehension, we subject these RNA clusters to heat, a critical approach for gauging the strength of different intermolecular connections. Furthermore, we employ complementary DNA oligos, an innovative approach that hybridizes with RNA molecules, deliberately perturbing their secondary structures. This technique unveils vital regions intricately involved in intermolecular and intramolecular connections and serves as a pioneering step in our quest to differentiate between these two connection types. By combining heat and complementary DNA oligos, we gain a comprehensive understanding of the strength and locations of various intermolecular connections. This innovative approach using DNA oligos to study RNA molecules is paving the way for a new method to investigate RNA structure and dynamics.
| Poster #: 27 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: Enzyme mechanism, Terpene synthase, Metal ion Project Title: Inhibition of Pinene Synthase by Mg2+ Author List: Devyn Nguyen, Undergraduate, Chemistry, Sacramento, Presenting Author; Jacob Johnsen, Undergraduate, Chemistry, Sacramento, Presenting Author; Abstract Monoterpene synthases catalyze the conversion of geranyl diphosphate to generate C10 monoterpene hydrocarbons, and have been used as models to better understand how enzymes catalyze electrophilic reaction sequences involving carbocation intermediates. Monoterpene synthases require a divalent metal ion to initiate the reaction sequence, and most can utilize either Mg2+ or Mn2+. Pinene synthase, a monoterpene synthase that catalyzes the conversion of geranyl diphosphate to (-)-α-pinene and (-)-β-pinene, is unusual in that Mn2+ but not Mg2+ supports catalysis. To better understand the specificity of the enzyme-divalent metal ion interaction we assessed the ability of Mg2+ to inhibit Mn2+-supported activity. After purifying His-tagged pinene synthase expressed in E. coli, we incubated the enzyme with saturating levels of geranyl diphosphate and increasing levels of Mn2+ in the absence and presence of 20 mM Mg2+. The enzyme had a high affinity for Mn2+, with half-maximal levels of activity at 50nM Mn2+ and maximal levels of activity at 100 nM Mn2+. Co-incubation with 20 mM Mg2+ decreased maximal activity levels by 75% but did not affect the concentrations of Mn2+ required to meet half-maximal (50 nM) or maximal (100 nM) activity. These results suggest that Mg2+ does not inhibit pinene synthase by competing for the same binding as Mn2+at the enzyme active site but instead imposes more global structural changes of the enzyme to reduce maximal activity. These results thus suggest that divalent metal ion specificity is established by amino acid side chains that exclusively bind Mn2+ at the enzyme active site. Site-directed mutagenesis studies are currently underway to identify the active site residues involved in divalent metal ion specificity, which will ultimately lead to a better understanding of how enzymes use metal-ion catalysis to facilitate the propagation of electrophilic reaction sequences.
| Poster #: 28 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: aging, aggression, Cytochrome P450 Project Title: Ubiquitous knockdown of a Cytochrome P450 family member in D.melanogaster and its effects on aggression behavior Author List: Trinity Foster, Undergraduate, Chemistry, Sacramento, Presenting Author; D’Yahna Baker, Undergraduate, Chemistry, SacramentoNang Noi, Undergraduate, Chemistry, SacramentoArwa al-Rakabi, Graduate, Chemistry, Sacramento Abstract The fruit fly Drosophila melanogaster (D.melanogaster) has been successfully used as a genetic model system, and has recently emerged as a powerful system to study complex behaviors. One such behavior is aggression, which in fruit flies is characterized by a series of stereotypical fighting moves. We aim to further understand the molecular basis of aggression behavior by investigating the function of the gene Cyp6a20.
Cyp6a20 is a member of the Cytochrome P450 superfamily of proteins, which are involved in the degradation of and breakdown of various compounds, such as hormones or lipids. As such, they play a critical role in the detoxification and clearance of drugs and xenobiotics, and are important in drug metabolism. The Drosophila P450 protein Cyp6a20 is an orphan member of this superfamily, i.e. its specific function or substrates are unknown. Cyp6a20 has been suspected to function in the degradation of pheromones, small molecules that regulate fruit fly behavior.
Here, we perform RNAi knockdown (KD) studies to investigate the tissue-specificity of Cyp6a20. Cyp6a20 KD was conducted ubiquitously, as well as in tissues related to cVA production. Our data show that flies lacking Cyp6a20 have increased levels of the aggression pheromone cVA and display hyper-aggressive behavior. These data suggest that Cyp6a20 modulates behavior through the breakdown of pheromones, particularly pheromones associated with aggression behavior.
| Poster #: 29 Campus: San José State University Poster Category: Biochemistry Keywords: intracellular pH, cell death, Drosophila Project Title: Increased intracellular pH promotes cell death in the developing Drosophila eye Author List: Alan Wong, Undergraduate, Biological Sciences, San José, Presenting Author; Rachel Ann Soriano, Undergraduate, Biological Sciences, San José, Presenting Author; Juan Manuel Reyna Pacheco, Graduate, Biological Sciences, San JoséJoanne Mendez, Undergraduate, Biological Sciences, San JoséBlake DuPriest, Graduate, Biological Sciences, San JoséJobelle Peralta, Graduate, Biological Sciences, San JoséBree Grillo-Hill, Faculty, Biological Sciences, San José Abstract Background: Regulated cell death is essential during development to precisely pattern tissues and avoid developmental errors. Dysregulation of cell death is associated with pathologies including cancer (reduced cell death) and neurodegeneration (increased cell death). Dysregulated intracellular pH (pHi) dynamics are also associated with these diseases, where cancer cells have constitutively higher pHi than normal cells while degenerating neurons have lower pHi. Supporting this, in vitro experiments show that apoptotic cell death caspase enzymes showed increased activity at low pH. Together, these observations led to the current view that cell death is enhanced at low pHi and inhibited at higher pHi. Our objective is to directly test this prediction in vivo.
Methods: We used transgenic Drosophila lines that overexpressed the Na-H exchanger DNhe2 specifically in the eye (DNhe2-OE), which increases pHi and results in a smaller, mispatterned adult eye. We used another transgenic line that overexpressed the proto-oncogene protein Myc (Myc-OE), which regulates cellular processes such as proliferation and cell growth. Using confocal microscopy, we imaged the eye discs, performed cell counts in pupal eyes, and analyzed our results for statistical significance.
Results: In pupal eyes, we found a significant decrease in cell number, from an average of 15 cells in control to 12.4 cells with DNhe2-OE. This suggested that cells overexpressing DNhe2 were dying inappropriately. We next tested for genetic interactions between DNhe2-OE and genes that regulated cell death. We found that the pH-dependent cell death is p53-dependent but caspase-independent. When we crossed a fly line deficient in a gene that promotes autophagic cell death, mutant Atg1, with Dnhe2, we saw an eye with wild type appearance. Together, these data are inconsistent with apoptosis but suggest that the cells are eliminated through autophagic cell death. We also identified a genetic interaction between Myc and DNhe2, where co-expression showed suppression of the rough eye phenotype and resembled control, with 15 cells, suggesting that Myc suppresses the pH-induced autophagy. We are currently testing whether molecular markers for autophagy are increased in DNhe2-OE eyes. Together, our findings will elucidate mechanisms for pH regulation of conserved, critical developmental processes and provide evidence for new paradigms in growth control. Funded by NIH SC3GM132049 and CSUPERB NI Award.
| Poster #: 30 Campus: San Diego State University Poster Category: Biochemistry Keywords: Single-particle measurements, Hepatitis B, Capsid disassembly Project Title: Single-Particle Studies of the Disassembly of Hepatitis B Virus Capsids in GuHCl Author List: Daniel Villarreal, Graduate, Chemistry, San Diego, Presenting Author; Rees Garmann, Faculty, Chemistry, San DiegoAdam Zlotnick, External Partner, Chemistry, Non-CSU, Indiana University Abstract Viral capsids are highly functional nanoscale structures. They can be self-assembled around various cargos—such as nucleic acids—to protect the cargo from degradation, and they can be disassembled by changing buffer conditions to deliver their contents back to the solution. While much work has focused on the assembly of viral capsids, less is understood about how they disassemble. To better understand the disassembly process, we use interferometric scattering microscopy to measure the mass of individual Hepatitis B virus capsids as they break apart in a buffer solution containing the denaturant GuHCl. We perform two types of experiments. In one experiment, we mix together the capsids and denaturant and then record changes in the mass distribution of the particles as a function of time, revealing the disassembly kinetics of the ensemble. In another experiment, we monitor changes in the mass of individual capsids as they break apart, revealing the disassembly kinetics of each particle. While the disassembly process is different for each particle, many of the particles lose mass in bursts punctuated by pauses. The average duration of the pauses is shorter at higher denaturant concentrations, and the bursts become faster and larger on average as more mass is lost from the particle. These results show that interferometric scattering microscopy can be used to measure the disassembly kinetics of viral capsids and also provide initial clues about the underlying disassembly pathways.
| Poster #: 31 Campus: California State University, Fresno Poster Category: Biochemistry Keywords: Drosophila melanogaster, Neurotoxin, Neurodegenerative diseases Project Title: Exploring the Effects of a Neurotoxic on Neurodegenerative Diseases: Investigating the Impact on Fruit Flie Author List: Prabhjot K. Gill, Undergraduate, Biology, Fresno, N/A, Presenting Author; Joy J. Goto, Faculty, Fresno Abstract Neurodegenerative disease such as amyotrophic lateral sclerosis (ALS), Parkinson's (PD), and Alzheimer's (AD) disease may be influenced by the presence of beta-methylamino-L-alanine (BMAA), a neurotoxic non-protein amino acid. Studies suggest that human chronic exposure to BMAA through diet or environmental exposure to cyanobacterial blooms can cause neurodegenerative disease. In vivo (D. melanogaster (fruit fly) experiments in our lab show chronic exposure to BMAA may be a risk factor for progressive neurodegenerative disease, while in vitro experiment shows deleterious results based on the toxin interfering with glutamate receptors. We have found the viability of Drosophila melanogaster is dose-dependent on the BMAA-fed fruit flies. Further, the circadian rhythms are disrupted by low doses (~5-7 mM) and high doses (36 mM) of BMAA over a period of 1-9 days; there is a level of hyperactivity measured using a Drosophila Activity Monitory (DAM). The BMAA effects can be reversed upon removal of the BMAA from the fly diet.
My hypothesis is BMAA effects the viability of Drosophila melanogaster in a dose-dependent manner and the circadian rhythms are disrupted based on the chemical reaction between BMAA and the glutamate receptors in Drosophila. Further, the locomotor behavior is negatively affected in a dose-dependent manner. My current research will also focus on which specific neurons are affected by BMAA, by using cholinergic neurons tagged with green fluorescent protein (GFP) to see how these are affected by BMAA in the fruit fly diet. I further hypothesize, the cholinergic neurons are a target for BMAA based on the receptors that are glutamate-drive in fruit flies.
| Poster #: 32 Campus: California State University, Los Angeles Poster Category: Biochemistry Keywords: cell thermal lysis, ImageJ analysis, protein purification Project Title: Determination of Thermolysis Efficacy via ImageJ Analysis Author List: Qianyang Ding, Undergraduate, Los Angeles, Presenting Author; Shanti Raminani, External Partner, Los AngelesXin Wen, Faculty, Los Angeles Abstract Antifreeze proteins (AFPs) are a class of proteins that possess versatile applications, a result of their ability to inhibit ice-crystal growth and depress the freezing point of fluids in cold-adapted organisms. Given their implications, improving the cost and efficacy of expression and purification of recombinant AFPs from Escherichia coli (E. coli) is of particular interest. We have demonstrated that thermolysis, a method of cell lysis utilizing heat for selective denaturation, is a viable alternative lysis method for the release of a beetle AFP from Tenebrio molitor (TmAFP). In this study, we aim to assess the optimal thermolysis conditions of TmAFP. Thermolysis was implemented by subjecting the cells expressed the AFPs to various temperatures from 50°C to 75°C for a range of duration (up to 30 min). The release of TmAFP was monitored using protein electrophoresis and the efficacy of the lysis was quantified based on the analysis of the region of interest (ROI) using ImageJ, a Java-based image processing program. Our results indicate that, among the tested temperatures, 65°C yielded the highest efficacy of the release of TmAFP. These results, when coupled with the protein stability and function studies, provide valuable insights into cost-effective biomanufacturing process of recombinant AFPs.
| Poster #: 33 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: Keywords are Brain-Computer Interface (BCI), Brain Waves, Electroencephalography (EEG) Project Title: Investigation of Brain Activity while Listening to Music using Brain Control Interface (BCI) Author List: Rachel Behan, Graduate, Biology, FresnoSukhraj Gill, Graduate, Biology, Fresno John Herrera, Undergraduate, Electrical and Computer Engineering, FresnoZachary O’Neil, Undergraduate, Electrical and Computer Engineering, FresnoJacob Perry, Graduate, Biology, FresnoDaniel Savala, Graduate, Biology, FresnoAnahit Hovhannisyan, Faculty, Biology, Fresno, Presenting Author; Hovannes Kulhandjian, Faculty, Electrical and Computer Engineering, Fresno, Presenting Author; Abstract “There are five main types of brain waves: alpha, beta, delta, gamma, and theta, which are associated with different states of the mind (Figure 1)1. Previous research has shown that music alters the ratio of brain waves in the brain and has significant effects on the brain and body in a clinical setting2. Figure 1. Brain Waves Bigliassi et al. (2015) study showed that calm music can lower vagal withdrawal through increased activation of the pre-frontal cortex. Additionally, Nawaz et al. (2018) explained that stimulating and calm music increase the beta and alpha waves in the frontal and parietal regions of the brain, respectively. Although the effects of music on the brain are well studied, the quantification of these effects is not well documented in the current literature. Therefore, our study focuses on the quantification of these effects. We have used BCI technology, which creates a communication pathway between neural activity and external devices, such as drones or prosthetic arms, via neural signals. BCI technology requires recording of brain activity, which can be done invasively or non-invasively with electrical conductors5. The neural activity required for BCI is measured through electroencephalograms (EEGs), which are thought to be generated by cortical pyramidal neurons6. For our research, we used our BCI technology to accumulate and quantify EEG data to address the effects of music on brain waves. Refferences Frey, St Louis, E. K., & Britton, J. W. (2016). Electroencephalography (EEG) : an introductory text and atlas of normal and abnormal findings in adults, children, and infants (Frey & E. K. St Louis, Eds.). American Epilepsy Society. Kučikienė, D., & Praninskienė, R. (2018). The impact of music on the bioelectrical oscillations of the brain. Acta Medica Lituanica, 25(2). https://doi.org/10.6001/actamedica.v25i2.3763 Bigliassi, M., Barreto-Silva, V., Altimari, L. R., Vandoni, M., Codrons, E., & Buzzachera, C. F. (2015). How Motivational and Calm Music May Affect the Prefrontal Cortex Area and Emotional Responses: A Functional Near-Infrared Spectroscopy (fNIRS) Study. Perceptual and Motor Skills, 120(1), 202–218. https://doi.org/10.2466/27.24.pms.120v12x5 Nawaz, R., Nisar, H., & Voon, Y. V. (2018). The Effect of Music on Human Brain; Frequency Domain and Time Series Analysis Using Electroencephalogram. IEEE Access, 6, 45191–45205. https://doi.org/10.1109/access.2018.2855194 Hinterberger, T., & Neumann, N. (2018). Invasive and non-invasive brain- computer interfaces. In S. Coyle, M. Prasad, & H. Lotze (Eds.), Brain-Computer Interfaces Handbook: Technological and Theoretical Advances (pp. 33-42). CRC Press. https://doi.org/10.1201/9781315371605-3 Frey, St Louis, E. K., & Britton, J. W. (2016). Electroencephalography (EEG) : an introductory text and atlas of normal and abnormal findings in adults, children, and infants (Frey & E. K. St Louis, Eds.). American Epilepsy Society. “ | Poster #: 34 Campus: California State University, Long Beach Poster Category: Bioengineering Keywords: Biological Hydrogen Gas, Anaerobic Digestion, Food Waste Project Title: Parameters Enhancing Biological Hydrogen Production from Food Waste Batch Anaerobic Digestors using Batch Experiments Author List: Thy Duong, Undergraduate, Civil Engineering and Contruction Engineering Management Department , Long Beach, Presenting Author; Pitiporn Asvapathanagul, Faculty, Civil Engineering and Contruction Engineering Management , Long Beach, Presenting Author; Abstract From existing publications, food waste (FW), an auspicious substrate, yields the highest bio-hydrogen gas from the anaerobic digestion process. To expand on this advantage, our project continued this research endeavor by investigating bio-hydrogen (bi H2) gas production through different operational treatments. Manipulating combined substrates between FW and cooking oil, treating different levels of NaHCO3 concentrations, and operating at various temperatures were included. To activate the anaerobic digestors, each batch bioreactor required of seed anaerobic digested sludge mixture. Consequently, the bioreactors were incubated at two different temperatures, 35oC or 55oC. Up to four days, the samples were analyzed by measuring H2 gas content, chemical oxygen demand (COD), volatile fatty acid (VFA), alkalinity, and ammonium (NH4+-N). The results show the bio-hydrogen gas measured at 55oC was remarkably lower than those from 35oC. Importantly, bio-hydrogen gas generation yielded the highest at 20% after 24 hours at 35oC for 4 mL FW with 1 mL cooking oil and 5 mL FW. This implies that FW can enhance bio-hydrogen gas recovery. A small fraction of cooking oil can promote H2 production, but not significantly compared to sole FW. As for the effects of having NaHCO3, high hydrogen gas was measured in low NaHCO3 concentrations for both temperatures. Combing all the discoveries, high gas production peaked early on the first two days and was highest at 35oC, low NaHCO3 content, and low oil content. The multi-variable analysis achieved using principal component analysis (PCA) confirmed a positive relationship between bi H2 gas content and alkalinity reduction and NH4+-N removal, but with negative correlation between bi H2 gas generation and temperature, NaHCO3 and COD removal.
| Poster #: 35 Campus: California State University, Long Beach Poster Category: Bioengineering Keywords: Epilepsy, Electroencephalograph, Time-frequency Project Title: Discovering Electroencephalogram Biomarkers of Lennox-Gastaut Syndrome through Unsupervised Time-Frequency Image Analysis Author List: Derek Hu, Faculty, Biomedical Engineering / Computer Engineering and Computer Science, Long Beach, Presenting Author; Marco Pinto-Orellana, External Partner, Biomedical Engineering, Non-CSU, University of California, IrvineMandeep Rana, External Partner, Neurology, Non-CSU, Children’s Hospital of Orange CountyDavid Adams, External Partner, Pediatric Neurology, Non-CSU, Children’s Hospital of Orange CountyLinda Do, External Partner, Neurology, Non-CSU, Children’s Hospital of Orange CountyDaniel Shrey, External Partner, Pediatric Neurology, Non-CSU, Children’s Hospital of Orange CountyShaun Hussain, External Partner, Pediatric Neurology, Non-CSU, University of California, Los AngelesBeth Lopour, External Partner, Biomedical Engineering, Non-CSU, University of California, Irvine Abstract The development of electroencephalogram (EEG) biomarkers for epilepsy relies on observations from visual review, which often vary by reviewer and can hinder the development of automated detection algorithms. Here, we developed a novel method for unsupervised time-frequency image analysis to automatically identify and characterize potential EEG biomarkers without relying on empirical clinical definitions.
A ten-minute clip of sleep EEG was retrospectively collected for twenty subjects diagnosed with Lennox-Gastaut Syndrome (LGS) and twenty age-matched healthy controls. Continuous time-frequency points with power exceeding the baseline were marked as events. Each event was characterized based on frequency range, duration, spatial spread, time-frequency density, frequency of peak power, and mean power in the delta, theta, alpha, sigma, beta, and gamma frequency bands. Events were grouped together using two different clustering methods.
A total of 11,708 events were identified across forty subjects, consisting of 4,964 LGS events and 6,744 control events. Six of the twelve clusters had a significantly greater number of LGS events than control events, making them candidate biomarkers of LGS. The visual review of these clusters revealed that four of these clusters primarily contained known LGS-associated waveforms. The last two candidate clusters consisted of short, high-frequency events that were labelled by raters as “nothing,” yet 81.6% of these events came from LGS subjects. The second clustering method corroborated this finding, showing that 81.3% of events with high beta/gamma power were from LGS subjects and were a potential novel biomarker of LGS.
Time-frequency image analysis is a novel method for EEG biomarker discovery that can objectively identify known biomarkers of LGS while also identifying novel candidate biomarkers that were unremarkable in visual review. This work can help improve the clinical diagnosis of LGS by providing a quantitative characterization of EEG waveforms, which can improve interrater reliability. Moreover, this method could be broadly applied to any pathology relying on EEG interpretation by quantifying known waveforms and identifying new potential biomarkers.
| Poster #: 36 Campus: California State University, Sacramento Poster Category: Bioengineering Keywords: Endothelial progenitor cells,, Normothermic machine perfusion, Expanded polytetrafluorethylene Project Title: Testing the Binding Affinity of the LXW7 Ligand-Modified Expanded Polytetrafluorethylene Author List: Grace Garcia, Graduate, Biological Sciences, Sacramento, Presenting Author; Dake Hao, Postdoc, Department of Surgery, School of Medicine; and Pediatric Regenerative Medicine, Non-CSU, UC DavisAijun Wang, Faculty, Biomedical Engineering; Pediatric Regenerative Medicine; and Department of Surgery, School of Medicine, Non-CSU, UC Davis Abstract Kidney failure requires patients to undergo hemodialysis; an arteriovenous graft (AVG) approach is the most common form of vascular access. Currently, synthetic AVGs are commonly used but they have high failure rates due to thrombosis, stenosis, and infection primarily because they possess no regenerative or growth potential and lack a functional endothelium. There is a significant unmet clinical need for long-lasting vascular grafts. Our lab recently identified a peptide ligand LXW7 with high affinity and specificity for endothelial progenitor cells (EPCs) and endothelial cells. We developed a technology to coat AVG grafts with LXW7. To further test the binding affinity of LXW7 with human EPCs under physiologically relevant condition, we will apply a normothermic machine perfusion (NMP) ex vivo model to test human EPC binding to LXW7 modified expanded polytetrafluoroethylene (ePTFE) AC|VG grafts. Steen solution, which is typically used for organ perfusion with the NMP model, was as the carry solution for EPCs. We first confirmed the viability of the circulating EPCs at three different timepoints (0.5, 1 and 2 hours). The cells were counted pre and post NMP circulation using the Luna Cell Counter. LXW7 modified ePTFE grafts were then attached to the NMP system with the untreated grafts as the control. The parameters included the 37°C temperature, 50mL Steen solution with EPCs per each graft, 9.5mL/min blood flow rate, with oxygenation. The grafts were fixed in formalin, blocked with BSA and cut into various 5mm x 5mm squares for staining. CD31 and CD62p primary antibodies were used with AlexaFluor647 and AlexaFluor555 secondary antibodies, and confocal images were captured. We found that circulating the cells in both solutions resulted in over 50% survival at every time point, except a low of 35% in Steen solution at the 30-minute point. We observed CD31 fluorescence scattered throughout the LXW7 treated graft after 2 hours of circulation. The untreated graft displayed very few puncta throughout the graft. No CD62p fluorescence was seen on either graft. The LXW7 treated graft showed promising results on capturing human EPCs in physiologically relevant conditions. Future studies will characterize the morphology and function of the attached EPCs to the graft luminal surface. Funding sources–This work was in part supported by the California Institute for Regenerative Medicine (CIRM) grant (TRAN3-13332) and the CIRM Bridges Training program (EDUC2).
| Poster #: 37 Campus: Sonoma State University Poster Category: Bioengineering Keywords: Sensors , Assistive Technology (AT), Emotions Project Title: EMOTE: A Wearable Device for Emotion Estimation and Classification Author List: Jorge Romero, Undergraduate, Department of Engineering , Sonoma, Presenting Author; Oscar Avendano , Undergraduate, Department of Engineering, Sonoma, Presenting Author; Gabe Esquibel, Undergraduate, Department of Engineering, SonomaFlor Luna, Undergraduate, Department of Engineering, Sonoma Abstract Non-verbal individuals on the autistic spectrum and others with similar disabilities, along with their caretakers, suffer from difficulties in communication on a daily basis. In this project, we designed a non-invasive wearable device to estimate and classify the emotional state of its user, with the aim of addressing the needs of those individuals with communication difficulties and their caretakers. The project goal is to determine the accuracy of its application in contrast to other existing methods such as facial recognition, and develop a system that interprets data trends by monitoring changes in vital signs recorded using various sensors (Heart Rate Monitor [HR], Skin Conductivity [GSR], Blood Oxygen Saturation [SpO2], and Body Temperature [SKT]) to detect one of four primary emotions. Experimental data are collected from volunteers, who were subjected to visual and auditory stimuli via VR and music, biographical recollection, and taste tests, to stimulate the different types of emotions. The collected data are processed to establish correlations between each emotion and deviations from baseline readings for all measured vital signs. A total of five individuals volunteered to participate in the data collection. Even with a small sample size, there were noticeable patterns; sensors like the GSR and HR proved to be much easier to analyze since they showcased the greatest change in values in contrast to the SKT and SpO2 sensors. The SKT provided reliable measurements but proved to be an ineffective method of indicating changes in emotion because temperature fluctuations were negligible. The opposite stands for the SpO2; the blood oxygen levels were not accurate and had large fluctuation. In conclusion, we found collecting GSR and HR data to be effective in emotion classification. As a result, we began synthesizing a non-invasive method that allows behavior specialists and caretakers to interpret the emotional state of the individual with communication difficulties and provide assistance to them when needed.
| Poster #: 38 Campus: San Diego State University Poster Category: Bioengineering Keywords: Cardiovascular Engineering, Medical Devices , Project Title: The Impact of LVAD Outflow Graft Diameter on Flow in the Aortic Arch Author List: Britton Mennie, Undergraduate, San Diego, Presenting Author; Karen May-Newman, Faculty, San DiegoHailey Harkness, Graduate, San Diego Abstract Introduction
Left Ventricular Assist Devices (LVAD) are implantable rotary pumps used to treat heart failure patients by redirecting blood flow from the Left ventricle to the aorta, bypassing the aortic valve. New LVAD models such as the HeartMate3 (Abbott Laboratories, Chicago IL) are equipped with an artificial pulse to restore flow pulsatility. However, the non-planar curvature and complex geometry of the aortic arch give rise to unique hemodynamic and flow features that may not be preserved during LVAD support. The LVAD flow outlet is attached to the ascending aorta by an outflow graft, with the aortic root perfused in a retrograde manner. Previous studies have found that LVAD outflow graft angle and position affect flow patterns in the aortic arch and can increase the risk of stroke. Less research has been performed to investigate the effect of outflow graft diameter, despite evidence that links small graft size to increased neurological event risk. Our goal in this study was to measure the effect of outflow graft diameter on 3-D flow and pulsatility in the aortic arch during support with a HeartMate3 LVAD.
Materials/methods
A mock circulatory loop consisting of a silicone left ventricle, HeartMate3 LVAD, Tygon tubing, outflow graft and a glass aorta model with three branches representing the brachiocephalic (B), carotid (C), and subclavian (S) arteries. The aortic valve was closed with a silicone plug to ensure full bypass flow conditions, when the LVAD provides all flow into the aorta. The HeartMate3 artificial pulse consists of a speed change every two seconds that is ± 400rpm from the set speed. Flow and pressure sensors were placed on the vessels and aorta and measured at 200Hz using LabChart. Three-dimensional velocity in the aortic arch and branch vessels was measured with particle image velocimetry (PIV). 63-micron fluorescent particles were injected into the circulatory loop and imaged with the LaVision Mini-shaker camera and an LED light for 4 sec periods at four LVAD speeds (3900, 4000, 5000 and 5800 rpm) with no cardiac function (asystolic heart). Experiments were performed for two clinically relevant OGDs, 10mm and 14mm, at matched hemodynamic conditions. LaVision DaVis Shake-the-box and Tomo-PIV algorithms were used to track the particles as they entered the aortic arch. Average velocity in rectangular regions of interest (ROIs) were computed for the proximal and distal aorta, and the brachiocephalic, carotid, and subclavian blood vessels.
Results and discussion
Pressure and flow increased with LVAD speed as shown in Table 1, with the highest speed (5800 rpm) corresponding to clinical patient values. As the aortic valve was closed off in these studies, all flow entered the aorta via the LVAD outflow graft and 20-25% entered the arch branches with the remainder proceeding downstream. The 14mm outflow graft developed higher pressure and flow for the same LVAD speed, reflecting the lower resistance compared to the 10mm outflow graft.
For all four LVAD speeds, average velocity was highest at the proximal aorta and decreased as flow traveled downstream to the distal aorta. Overall, average velocity increased with increasing speed but was consistently higher for the 10mm outflow graft condition in the proximal, distal and branch vessels. Velocity measured in the individual branch vessels was also higher for the 10mm outflow graft but the ratio of flow entering the three branches increased slightly at higher speeds, especially for the 14mm outflow graft.
Conclusions
The results of these studies indicate that overall flow and pulsatility is higher with the 14mm outflow graft, but that the 10mm graft imparts higher velocity to the flow which increases flow velocity in the aorta and branch vessels.
| Poster #: 39 Campus: California State University, Northridge Poster Category: Bioengineering Keywords: prostheses, wearables, mechanical design and control Project Title: Foot controller and haptic feedback systems for below-the-elbow prosthetic arms Author List: Gerbert Funes Alfaro, Undergraduate, Northridge, Presenting Author; Matthew Hoff man, Undergraduate, Northridge, Presenting Author; Sara Ali, Undergraduate, NorthridgeAdrian Author 4 Last Name: * Lima, Undergraduate, NorthridgeKevin Amaya, Undergraduate, NorthridgeVincent Rebultan, Undergraduate, NorthridgeAlyssa Cancio, Undergraduate, NorthridgePeter Bishay, Faculty, Northridge Abstract Surveys with arm amputees revealed that there are several factors that lead to the high
rejection rates of prosthetic devices, despite the currently used technology. These factors include
impaired mobility, unreliable functionality, and lack of sense of touch. To address these factors, this work
presents two wirelessly controlled systems; a foot controller system that allows the user to have reliable
control of the prosthetic arm, and a haptic feedback system that allows the user to have a sense of
touch. The foot control system features push buttons integrated inside a toe-sleeve and a sensor
controller unit with integrated gyroscope and accelerometers. The user could activate predetermined
hand grips or gestures by pressing the push buttons with their toes. The user's arm would rotate and
bend as a result of the user’s foot rotating and bending. The haptic feedback system has force sensors
placed in critical hand areas that, upon contact with an object, generate vibrations in the user's residual
limb in the desired locations. To test the reliability of the foot controller, an algorithm was developed to
rate the participant’s performance in a series of tasks. Preliminary testing showed that after a few
minutes of initial training, users were able to perform all provided tasks with a reasonable accuracy. The
most identifiable locations for the vibration motors on the upper arm of the user were determined
through testing in a similar approach. Future work will focus on connecting the microcontrollers through
bluetooth technology, utilizing a wireless server, and introducing a new means of sensory feedback to
the haptic feedback system.
| Poster #: 40 Campus: San Francisco State University Poster Category: Bioengineering Keywords: Fallopian Tube, Morphology, micro CT Project Title: Detecting 3D morphological aspects of the fallopian tube from micro-CT images that are needed in Finite element modeling and analysis of the tissue for fertility research Author List: Aaron Thomas Nolasco, Undergraduate, Engineering , San Francisco, Presenting Author; Pedro Castro, Faculty, Gynecology, Non-CSU, the Federal University of Rio de JaneiroMojtaba Azadi, Faculty, Engineering , San Francisco Abstract Introduction: This research is part of main research that tries to improve the IVF success rate by detecting, quantifying, and uncovering some of the key biomechanical aspects of the fallopian tube (FT) including morphological aspects of the FT. The FT is a complex organ that plays a critical role in the reproduction process, providing an optimal biomechanical and biochemical environment for fertilization and for early stages of embryo development. To date, the biomechanical aspects of the FT have received little attention in scientific communities, despite their significance in understanding the fertilization process, mechanotransduction and mechanobiology during embryo development, and replication of the FT in lab environments for the purpose of infertility treatments. This work focuses on detecting complex 3D morphological aspects of the FT by analyzing micro-CT images captured by the Brazilian coauthor, Dr. Castro. The research is being done by students at San Francisco State University (SFSU) in collaboration with Dr. Pedro Teixeira Castro, a Gynecologist and faculty member at the Federal University of Rio de Janeiro (UFRJ) in Brazil.
Research Goals: To construct a representative 3D structural model of the Human fallopian tube at different length-scale (i.e., micro to Macro corresponding to cellular-scale, tissue-scale to organ-scale) by using key morphological of the FT associated with its function with a focus on fertilization and embryo development sites in human during fertilization (about one day after ovulation). The result of this research will enable the next phase of the research where the multi-scale biomechanical properties of the Human FT will be found by the Finite element Method (FEM) and studied by looking at the interaction of the FT with egg and embryo at different anatomical sections of the FT where eggs are collected (fimbria), fertilization site (Ampulla) and where the embryo grows and moves through before implantation (isthmus).
Background
Basic Anatomy of FT: The FT, which is also known as the uterine tube, oviduct, or salpinx, transports gametes (i.e., sperm and ovum) in opposite directions. The FT contains four main regions: the intramural, the isthmus, the ampulla, and the infundibulum. Each region of the FT has distinct functions and anatomical features. The infundibulum opens into the peritoneal cavity through the abdominal ostium. The fimbria surround and embrace the ovary during ovulation to aid oocyte pickup while the cilia beating helps the process. The fertilization, the union of the spermatozoon (male gamete) with the ovum (female gamete), regularly takes place in the ampulla close to its junction with the isthmus. The ampulla is the longest and widest region of the FT. The isthmus is the narrowest and the final portion of the extrauterine part of the FT. The isthmus plays a crucial role in controlling the spermatozoa while they are entering the ampulla and in controlling the embryo when it enters the uterus. In addition to acting as sperm reservoir, the isthmus can limit polyspermy, preserve the viability of sperm, and adjust the sperm's physiological state, particularly capacitation and motility hyperactivation. Intramural traverses the wall of the uterus and opens into the uterine cavity at the uterine horn. The intramural region differs from the other regions of the FT in which there is no peritoneal coating. Intramural functions as a sphincter by regulating the passage of gametes between the uterus and the FT.
Basic Functions of FT: FT is a complex organ and plays a critical role during ovulation, fertilization, and embryo development, including: 1) capturing the oocyte after ovulation, 2) timed transportation of the gametes and embryo towards the uterus, 3) controlling storage and migration of spermatozoa, 4) providing an optimized biomechanical and biochemical environment for fertilization, 5) nourishing the early stage of an embryo while being carried to the uterus, and 6) contributing to the early stages of embryo development before implantation. Tubal transport, one of the main functions of an FT, depends on several factors including complex morphology of the FT, cilia beating, contractile activity of the muscular layer, and tubal fluid. Biomedical researchers have looked at the anatomy, physiology, tubal transport and its related events such as gametes metabolism, and storage, selection, and capacitation of sperm. While many of these functions are related to FT’s biomechanical aspects, these aspects have received little attention in Biomedical Science.
Within recent years, new technologies relevant to reproductive science have made enormous advancements. But so far little research has been done on the biomechanical aspects of the FT. The biomechanical aspects refer to multi-scale mechanical properties of the FT (i.e., material models such as linear elastic, hyperelastic, viscoelastic, and poroelastic) considered with morphological and ultrastructural aspects at different length scales. When these biomechanical aspects are known, finite element method (FEM) can be used to study and understand the physical interaction (i.e., loads, stress, strain) between the FT and gametes or embryos while they are moving along the FT and are surrounded and loaded by the mucosal layer of the FT.
Summary of the accomplished works
The morphometric measurements done by other researchers are reviewed by Dr. Azadi and his former students/collaborators. We realized that the different measurement techniques and conditions as well as the lack of a measurement protocol are sources of large uncertainty in the measurements of the FT. Many of the measurements are not systematically and accurately captured We particularly aim to encourage researchers to use engineering practices and new measurement techniques such as high-resolution 3D measurement techniques and micro-CT to measure and reveal the three-dimensional anatomical structure of the FT.
Multi-scale structural models of the human FT at different locations of the FT using Micro-CT
Embryo length scale (up to 200 μm): To model the 3D structure and micron range contact of the FT at this length scale for 3D FEM the cross-section of the Micro CT 3D images provided by the collaborator (Dr. Castro) are being used to model the structure of the contact between egg and FT. We are analyzing the available images and are providing feedback so our collaborator can capture more targeted micro-CT images of the human FT with a higher resolution of about 2 μm.
Organ scale (200 μm-12 cm): Abundant histological images of the FT usually provide high-resolution image but only at a few cross sections of the FT. Unfortunately, so far we have not found any informative histological images along the FT’s length to explain the morphological changes along the FT which is crucial for the organ scale FEM studies here. Fortunately, in recent years the collaborator Dr. Castro and his team in Brazil has captured 3D images of human FT with reasonable resolution (~ 10 μm) using confocal microtomography (micro-CT). To enable Organ scale FEM analysis, we worked with Dr. Castro and we are reconstructing the needed topography of the internal surface of the FT mucosa along FT’s length using the digitized available files shared with us. The Brazilian collaborator has provided access to captured images to our team in the US. Multi-scale modeling to study mechanics of the FT at different length scale.
One of the main roadblocks that is being dealt with currently is the issue of file size. Because of the vast amount of information in each segment of the Micro-CT scan that the software and hardware must accommodate. This has led to a deep dive into computing hardware research as well as research on compatible software. Another issue that was addressed was “the initial thresholding” where we decide how much of the file information to exclude at the commencement of this process. The results have been promising. We have already been able to create a 3D “mesh file” as well as a .obj file compatible with Cad software packages and 3d printing software packages.
| Poster #: 41 Campus: California State University, Northridge Poster Category: Clinical Keywords: Antibiotic resistance, carbapenem, β-lactamase Project Title: Characterization and Whole Genome Sequencing a carbapenem-resistant Aeromonas veronii environmental isolate carrying three β-lactamase genes Author List: Rafael Estrada, Graduate, Biology, Northridge, Presenting Author; Cristian Ruiz Rueda, Faculty, Biology, Northridge Abstract Carbapenems are last-resort antibiotics used in clinical settings to treat antibiotic-resistant bacterial infections. Thus, the emergence and spread of resistance to carbapenems is a major public health concern. Here, we have studied a carbapenem-resistant Aeromonas veronii strain previously isolated from a water sample from Sam Simeon Creek (Hearst San Simeon State Park, CA). Analysis of this isolate using disk-diffusion, CarbaNP, eCIM, and mCIM assays revealed that it was resistant to amoxicillin-clavulanic acid and all carbapenems tested and that this isolate produced a potentially novel carbapenemase of the Metallo-β-lactamase family. Whole genome sequencing analysis revealed that this A. veronii isolate carries a novel variant of the blacphA class B carbapenemase gene that was closely related to the blacphA7 gene of Aeromonas jandaei. This isolate also carried a novel variant of the blaOXA class D carbapenemase gene that was most closely related to the blaOXA-912 gene found in other Aeromonas veronii isolates. Finally, we also identified a novel class C β-lactamase gene moderately related to the blaFOX-17 gene of Providencia stuartii and other blaFOX variants identified in Klebsiella pneumoniae, Escherichia coli, and other Enterobacteriaceae. Overall, our findings reveal that environmental isolates are an important reservoir of multiple carbapenemases and other β-lactamases of potential clinical significance.
| Poster #: 42 Campus: Sonoma State University Poster Category: Clinical Keywords: At home data collection, Breath based glucose monitoring, Smart biomedical sensor Project Title: At-Home Patient Self-Breath Data Collection with a Sensor Device for Diabetes Study Author List: Sokiyna Naser, Graduate, Engineering, Sonoma, Presenting Author; Natalie Liang, Undergraduate, SonomaJessie Lopez, Undergraduate, SonomaAnja Damazyn, Undergraduate, SonomaRubi Santiago Martinez, Undergraduate, SonomaDeborah Roberts, Faculty, SonomaSudhir Shrestha, Faculty, Sonoma, Sonoma State, Presenting Author; Abstract Our team is developing a smart breath glucose monitor (BGM), a hand-held device that uses volatile organic compound (VOC) sensors to predict blood-glucose levels from human breath. Our goal is to collect data from patients with type-2 diabetes (non-insulin-dependent) and train machine learning (ML) models with this dataset to enable real-time glycemia predictions.
We conducted a pilot at-home data collection over the past year using a sensor device developed at the Intelligent Systems Lab at Sonoma State University. This poster presents the methodology used for this pilot study and the lessons learned. The study design includes two major steps. The first step was recruiting patients with type-2 diabetes from a medical clinic in Santa Rosa, CA, and giving them sensor devices to take home. The patients were trained on the device at the clinic by healthcare practitioners. The patients were instructed to measure their blood glucose using a fingerstick glucometer and breathe into the sensor device. The sensor data were wirelessly received or retrieved from a memory card, while the finger-stick readings were noted on a separate chart that was collected by the clinic and shared with the research group after removing all identifying information. Six patients participated in this study. After the first step's completion, we received 176 clean data points. The second step involved analyzing the collected BGM data using statistical analysis techniques such as PCA to reduce the number of features in the dataset. Then, we employed the use of Support Vector Machine (SVM) algorithms to build a prediction model that could classify glucose levels as high, normal, or low. We used cross-validation processes to train the SVM model and determine the SVM parameters. This poster will present the study design lessons learned, and highlight important results from the study.
Our future research will include larger-scale data collection and testing, developing a user interface on the device to input finger-stick BG measurements and display BG states, and creating a companion smartphone application for monitoring and tracking BG readings by patients and medical professionals. Such a solution would enable type 2 diabetes patients to receive instant glucose status readings without the need for finger pricks, test as many times as they desire, and easily monitor BG history, revolutionizing patient care. Diabetes requirescontinual management of blood glucose levels, and failure to do so can lead to acute and long-term complications. The proposed solution has the potential to help reverse nonadherence to treatment plans, which is a significant problem among diabetes patients. Overall, the research into the BGM device has the potential to transform diabetes management and improve patient outcomes while also driving innovation in healthcare and beyond. By advancing our understanding of breath analysis using machine learning, this research has significant implications for the future of healthcare and the well-being of people around the world.
| Poster #: 43 Campus: California State University, Long Beach Poster Category: Clinical Keywords: adolescent, development, psychostimulant Project Title: Liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, does not affect the expression of methamphetamine preference in adolescent male and female rats. Author List: Tammy Nguyen, Graduate, Psychology, Long Beach, Presenting Author; Annie Lin, Graduate, Psychology, Long Beach, Presenting Author; Stephanie Vargas, Undergraduate, Psychology, Long BeachArturo Zavala, Faculty, Psychology, Long Beach Abstract Adolescent use of methamphetamine (METH) leads to poor treatment outcomes later in life, and currently, there are no FDA-approved medications for the treatment of psychostimulant use disorder. Preclinical studies have linked drugs that activate glucagon-like peptide-1 (GLP-1) receptors, such as Liraglutide (an FDA-approved medication for diabetes), to a decrease in the rewarding effects of cocaine, heroin, and oxycodone. However, the effects of activating GLP-1 receptors on METH reward in adolescent rats has not been examined. Therefore, the present study tested the effect of Liraglutide on METH preference in adolescent male and female Sprague-Dawley rats using conditioned place preference (CPP), an animal model of reward, which involves a 10-day procedure. On day 1, baseline, rats had free access to both sides of a two-chamber apparatus for 15 min. During days 2-9, conditioning, rats were injected on alternating days with METH (0.0, 0.3, or 1.0 mg/kg, intraperitoneally) or saline and were immediately confined to one side of the apparatus for 30 min. During day 10, testing, rats were pretreated with Liraglutide (0.0 or 0.1 mg/kg, subcutaneously) 60 min before having free access to both sides of the two-chamber apparatus for 15 min. Preference scores were calculated by subtracting the time spent on the drug-paired side on testing day from the time spent on that same side on baseline. Male rats showed a strong preference for the METH-paired side, but female rats did not show a preference for the METH-paired side. Liraglutide did not affect male rats’ preferences for the METH-paired side and female rats’ preferences. Future studies will examine higher doses of Liraglutide and the effects of Liraglutide on the acquisition of METH-induced CPP. Developmental differences in the effects of Liraglutide are evident from the differential effects of Liraglutide on METH-induced CPP in male and female adolescent rats.
| Poster #: 44 Campus: California State University, Long Beach Poster Category: Clinical Keywords: Adolescence, Development, Addiction Project Title: Activation of 5-HT1B Receptors with Zolmitriptan on the Acquisition of Ethanol Preference in Adolescent Female Sprague-Dawley Rats Author List: Sullivan Moore, Undergraduate, Psychology, Long Beach, Presenting Author; Emily Siu, Graduate, Psychology, Long Beach, Presenting Author; Ana Garcia, Undergraduate, Psychology, Long BeachArturo Zavala, Faculty, Psychology, Long Beach Abstract Background. Initiation of alcohol consumption typically begins in adolescence and has become a highly abused drug among youth. In preclinical studies, serotonin (5-HT)1B receptors play a role in mediating the effects of ethanol, given that activation of these receptors decreases ethanol intake and the reinforcing effects of ethanol in adult male rats. However, the effects of activating 5-HT1B receptors have not been examined in adolescent female rats. Thus, the present study investigated whether administering Zolmitriptan, a 5-HT1B agonist, attenuates the preference for ethanol in adolescent Sprague-Dawley female rats. Ethanol preference was examined using the conditioned place preference (CPP) paradigm, a validated animal model of drug reward. Methods. Rats were given free access to both sides of a two-chambered CPP device for 15 minutes on postnatal day (PD) 31 to determine their initial preferences. During the following eight days of conditioning, rats were administered an injection of Zolmitriptan or vehicle (s.c.) prior to receiving 0, 0.625, or 2.0 g/kg (i.p.) of ethanol on alternating days. Preference for the ethanol-paired side was assessed on PD 40, during which rats again had access to both sides of the CPP apparatus for 15 min. Results. Rats exhibited a preference for the 2.0 g/kg dose of ethanol but not when administered the 0.625 g/kg of ethanol, evident as a significant increase in time spent on the ethanol-paired side by rats administered the 2.0 g/kg dose of ethanol compared to rats in the vehicle-saline group. The results of Zolmitriptan pretreatment are intriguing, considering that Zolmitriptan pretreatment alone resulted in a substantial preference for the Zolmitriptan-paired side (i.e., in the absence of any ethanol). Furthermore, Zolmitriptan appears to have enhanced the rewarding effects of ethanol, as pretreatment with Zolmitriptan and 0.625 g/kg ethanol resulted in a CPP. Lastly, Zolmitriptan pretreatment attenuated the preference induced by the 2.0 g/kg dose of ethanol. Conclusion. Our findings indicate that activating 5-HT1B receptors through administering Zolmitriptan may have dose-dependent effects on the rewarding effects of ethanol. , as well as rewarding effects of its own in female adolescent rats. These findings are partially consistent with previous studies that have examined the effectiveness of 5-HT1B agonists in reducing the reinforcing effects of ethanol in male adult rats, but also highlight sex- and age-dependent effects of Zolmitriptan. Future research will focus on replicating these effects using a wide range of ethanol doses in male and female adolescent rats.
| Poster #: 45 Campus: California State University, Fullerton Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: orthotic devices design, natural walking gait trajectory, physical rehabilitation Project Title: Hip-Knee-Ankle-Foot Orthoses (H-KAFO) for Physical Rehabilitation of Patients with Reduced Limb Mobility Author List: Severino Hernandez, Graduate, Fullerton, Presenting Author; Vincent Le, Undergraduate, Fullerton, Presenting Author; Vanessa Bustos, External Partner, Non-CSUJames Lopez, External Partner, Non-CSUAnvi Murarka, External Partner, Non-CSUZilan Patel , External Partner, Non-CSUKausthubh Veldanda , External Partner, Non-CSUNina Robson, Faculty, Fullerton Abstract The goal of this project is to explore the creation of a novel reconfigurable Hip-Knee-Ankle-Foot Orthosis (H-KAFO) for physical rehabilitation of patients, that coordinates the motion of all three leg joints in a natural manner, guiding the foot smoothly throughout the human walking gait trajectory.
During the mechanical design phase the leg of the subject is approximated as a kinematic chain, sized according to the wearer’s anthropometric data. Eight-bar linkage is then synthesized using GeoGebra software to coordinate the hip, knee and ankle movements. The procedure yields number of design candidates, followed by final design selection and SolidWorks CAD drawing steps. Finally, a reduced scale actuated H-KAFO preliminary prototype, sized according to the limb lengths of a wooden mannequin (with a femur length of 65.0 mm and tibia length of 70.1 mm) is 3D printed and assembled. While comparing the natural human knee joint angle trajectories for one cycle with those obtained from the GeoGebra design model, our analysis results show that both curves were characterized by two peaks with a 9 degree difference in the amplitude of the first peak. The obtained hip angle trajectories for one gait cycle, also characterized by two peaks, were very similar with less than 1 degree difference.
To assess the performance, the preliminary reduced scale prototype was secured to the hip, femur and tibia of the wooden mannequin through number of straps. The foot trajectory produced by the mannequin wearing H-KAFO were recorded using Adobe After effects and were compared to the GeoGebra simulations. Overall, the preliminary experimental tests showed that H-KAFO was able to successfully synchronize the leg joint motion and follow a slightly different from the desired natural walking foot trajectory shape, due to the 9 degrees difference within the designed knee angle.
| Poster #: 46 Campus: California State University, Fullerton Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: wearable hand devices for physical therapy, anthropomorphic , control-oriented naturalistic kinematic models Project Title: Creating Non-Anthropomorphic Hands for Human-Like Tasks Using Control-Oriented Naturalistic Kinematic Models Author List: Severino Hernandez, Graduate, Fullerton, Presenting Author; Sebastian Ledesma, Undergraduate, Fullerton, Presenting Author; Nina Robson, Faculty, Fullerton Abstract The goal of this research is to explore the possibility of creating future grasping devices, such as robotic hands and wearable hand devices for physical rehabilitation, based on a recently developed and experimentally validated control-oriented planar kinematic model for natural human hand configuration.
The model, developed using MATLAB software, describes the geometry of a thumb-finger(s) system assumed to be softly enclosing the surface of a virtual cylindrical object and calculates independently the natural joint rotation angles, as well as joint velocities and accelerations of the system by a single parameter, the radius of the virtual cylinder R. Based on the MATLAB model, we explore the design of a three-fingered robotic hand for multiple anthropomorphic tasks and assess how closely the motion of the pilot prototype resembles natural grasping motion using thumb, index and middle fingers.
The motion analysis of inter-finger coordination of the proposed design, performed in GeoGebra software, shows that the base joints for the index finger and the thumb move together and have their peak velocity at approximately 50% movement in its excursion. Similar trends are observed in the rest of the joints with their peak velocity at about 60% of the whole movement. In the assessment of the inter-joint coordination of the 3D printed preliminary prototype, related to the sequence of movement in the phalanges of a finger, it is observed that for the extension movement (opening), a proximal-to-distal sequence is evident in the index finger and distal-to-proximal sequence in the thumb, which is consistent with existing literature. The latter proofs the applicability of the proposed naturalistic model.
Future research includes detailed motion analysis and assessment of the preliminary prototype as well as extending the proposed techniques to the synthesis and design of enhanced wearable hand devices and articulated robotic hands for natural motion.
| Poster #: 47 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Machine Learning, Pneumonia detection, Chest X-ray Project Title: Using Convolutional Neural Networks for the Early Detection and Classification of Pneumonia in Chest X-Ray Images Author List: Shauryan Ratan, Undergraduate, Computer Science and Software Engineering, San Luis Obispo, Presenting Author; Franz Kurfess, Faculty, Computer Science and Software Engineering, San Luis Obispo, Presenting Author; Abstract Pneumonia is a common respiratory infection affecting millions worldwide each year. It is typically diagnosed through a chest x-ray, which can reveal the presence of inflammation or uid in the lungs. However, accurately detecting and classifying pneumonia from chest X-rays can be a challenging task for radiologists and healthcare providers. In recent years, deep learning techniques such as convolutional neural networks (CNNs) have shown great promise in assisting with image analysis, object detection and object classification. This project aims to improve the accuracy and efficiency of pneumonia diagnosis, ultimately leading to better patient outcomes. We trained a modified version of the VGG-16 convolutional neural network on 5861 chest X-ray images (1583 normal, 2781 bacterial pneumonia, 1497 viral pneumonia) with an 8:1:1 ratio between randomly assembled training, testing and validation sets. To adjust the imbalance between the three categories, we doubled the number of normal chest X-ray images and those depicting viral pneumonia. For this three-way classification problem, we obtained an accuracy of over 75% on the validation set, with F1-scores of 0.87 for normal, 0.72 for bacterial and 0.66 for viral pneumonia X-rays. The distinction between normal and pneumonia is very good, but differentiating between viral and bacterial is more challenging as evident in confusion matrices: For the normal images, 234 were correctly identified, 7 incorrectly labeled as bacteria, and 18 incorrectly as viral. Bacterial images were correctly classified in 216 cases, and incorrectly as normal in 23 and viral in 46. Viral images were correctly labeled 173 times, 94 times as bacterial and 21 times as normal.
The experiments for this work were run on the Google Colab platform for Jupyter notebooks. With our relatively small data set the resources provided there were sufficient, but for larger datasets and more demanding computational models resources beyond the free tier of Colab are needed.
We are planning to explore different architectures, training methods, data augmentation and hyperparameter tuning approaches to further improve the performance of our model. Using a large dataset of over 100,000 chest X-ray images for 14 common lung diseases, we will also expand the scope of the diagnosis significantly.
| Poster #: 48 Campus: California State University, Long Beach Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Nanomotor, Product quantum yield, Molecular dynamics Project Title: First-principles prediction of wavelength-dependent product quantum yields of a second-generation molecular nanomotor. Author List: Michelle Menkel-Lantz, Graduate, Chemistry and Biochemistry, Long Beach, Presenting Author; Abstract First-principles prediction of wavelength-dependent product quantum yields of a second-generation molecular nanomotor.
Michelle Menkel-Lantz
California State University Long Beach
Synthetic light-driven motors have promising potential for biomedical applications due to their temporal and spatial control. Applications include light-induced drug delivery and the permeabilization of biological membranes. Nanomotors are comprised of a polycyclic rotator and stator connected by a carbon-carbon double bond that isomerizes upon light irradiation, producing a rotary motion.
To optimize functionality of nanomotors, it is necessary to gain a detailed understanding of the mechanism of action on an atomic level. To this end, we study the dynamics of the photoinduced Z-E isomerization of a light-driven, second-generation molecular nanomotor. We use non-adiabatic molecular dynamics based on time-dependent density functional theory (TDDFT) to predict the wavelength-dependent product quantum yields of Z-E isomerization. Using replica exchange molecular dynamics (REMD) we obtain four different ground state conformers, labeled as aMsE, sMsE, aPuE and sPuE. From the ground state ensemble of structures, 375 were chosen for excited state non-adiabatic dynamics. The wavelength dependent product quantum yield was obtained by dividing the absorption spectrum averaged over the initial structures of the successful trajectories by the average absorption spectrum of the initial structures of all trajectories. Spectra were computed using TDDFT as well as second-order approximate coupled cluster theory (CC2).
Research reported in this paper was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award number 1 R16GM149410-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We acknowledge technical support from the Division of Information Technology of CSULB.
| Poster #: 49 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Knowledge graph, Language Model, Chronic Pain Project Title: A Large Language Model Powered Pipeline for Knowledge Graph Creation from Chronic Pain Medical Text Author List: Damon Lin, Undergraduate, Computer Science, San Luis Obispo, Presenting Author; Paul Anderson, Faculty, Computer Science, San Luis ObispoJean Davidson, Faculty, Biological Sciences, San Luis Obispo Abstract Within chronic pain literature, areas of research are frequently independent and siloed, hindering efforts to translate ideas and approaches across subfields. In addition, medical texts within these fields of chronic pain are often dense with different systems of technical terminology, further inhibiting collaboration. In order to bridge the gaps between sub-fields, our team proposes a large-language model approach to digest and distill the dense technical literature of chronic pain into a manageable, queryable knowledge graph. Knowledge graphs are flexible representations of data where information is divided into entities that are connected by labeled relations, otherwise known as “triplets”. With knowledge graphs in hand, researchers will be able to bypass the dense technical jargon and utilize the underlying data structure for graph algorithms and analysis. In order to streamline knowledge graph creation, our team introduces a large-language model powered pipeline for distilling chronic pain literature into Knowledge Graphs. First, the pipeline performs coreference resolution by utilizing SpanBERT to identify words in the text that refer to the same identity, and replacing the terms with a unified mapping. Second, the pipeline simplifies each sentence within the text by prompting GPT-3.5 to transform each sentence into a simpler, more straight-forward copy. Afterwards, the pipeline will extract the Knowledge Graph triplets by prompting GPT-3.5 to split each modified sentence into a subject, verb, and object. Third, the pipeline uses the triplets generated by GPT-3.5 to create the Knowledge Graph. Afterwards, the pipeline registers entities and relations that refer to the same meaning to a unified mapping, tuning the Knowledge Graph ahead of further analysis. To evaluate the performance of the pipeline, we compared the Knowledge Graph generated by our automated pipeline with a Knowledge Graph developed by a biology expert. Through manual inspection, we have observed general knowledge preservation and major similarities between Knowledge Graphs, although the automated Knowledge Graph is less precise and more cluttered. Overall, we believe that our pipeline for automated KG construction can serve as a stepping stone for greater knowledge analysis over chronic pain literature.
| Poster #: 50 Campus: San Diego State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Tissue Engineering, Multiscale Modeling, Elastin-Like Polypeptide Project Title: Investigating the Role of Elastin-Like Polypeptide (ELP) and ELP-PEI Copolymer in Scaffold Design for Enhanced Tissue Engineering Author List: Praneel Singla, Undergraduate, Mechanical Engineering, San Diego, Presenting Author; Sara Adibi, Faculty, Mechanical Engineering, San Diego Abstract Tissue engineering is a rapidly advancing field, harnessing the potential of various biomaterials to craft tissue constructs for medical applications. Among these, elastin-like polymers (ELPs) have gained significant attention due to their biocompatibility, biodegradability, and especially their unique adhesive ability, which facilitates binding to tissues and cells. Despite their promise, the mechanisms driving ELPs' adhesive properties remain elusive.
To bridge this knowledge gap, our research employs multiscale material modeling, a potent tool that offers insights into biomaterial behavior across different length scales. We're particularly interested in understanding the role of ELPs and their copolymers in designing advanced scaffolds for tissue engineering. Traditional collagen scaffolds, while foundational in regenerative medicine, often fall short in mechanical and physical properties. Infusing these scaffolds with ELPs has been shown to enhance their mechanical attributes and promote osteogenic differentiation.
Investigating the adhesive property of ELPs in tissue engineering using multiscale material modeling is an important area of research. Molecular dynamic (MD) simulations, a cornerstone of our research, delve into the intricate dynamics of these copolymer arrangements and their impact on tissue culture surface morphology. Employing software tools like GROMACS and LAMMPS, we can visualize and comprehend cellular interactions with the copolymers, offering a microscopic perspective of the forces and interactions in action. The results obtained from this study will contribute to the development of novel biomaterials for tissue engineering applications. By understanding the molecular mechanisms underpinning the adhesive behavior of ELPs, we can design ELP-based tissue constructs with enhanced adhesive properties. The insights gleaned will be pivotal in steering the development of innovative material designs, with far-reaching applications spanning biomedicine, diagnostic devices, and advanced tissue engineering tools.
| Poster #: 51 Campus: California State University, Northridge Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Solvation, Protein folding, ribosome Project Title: Computational Modeling of Magnesium Ions around Escherichia Coli 70S Using 3D Reference Interaction Site Model of Molecular Solvation Author List: Tiannah York Van Elslande, Graduate, Physics and Astronomy, Northridge, Presenting Author; Felipe Silva Carvalho, Postdoc, Physics and Astronomy, NorthridgeTyler Luchko, Faculty, Physics and Astronomy, Northridge Abstract The ionic environment around the ribosome affects protein synthesis and folding and may be critical to understanding deleterious aggregation-based diseases. However, no existing experimental methods can provide a detailed description of the ionic environment, and molecular simulations are too expensive to be practical. Instead, we have computed the density distribution of ions around the Escherichia Coli 70S ribosome in atomistic detail using the 3D reference interaction site model (3D-RISM) of molecular solvation. 3D-RISM uses the same molecular models as used in simulations but computes the distribution of the solvent using statistical physics, rather than brute-force simulation. To compute the distribution of water and ions, we first parameterized the crystal structure of Escherichia Coli 70S ribosome (PDBID 4YBB) with the Amber force field. Then we used 3D-RISM to calculate the equilibrium distribution of water with 100 mM NaCl and 1 mM MgCl2. The density distributions show tight binding of magnesium throughout the structure, as well as diffuse distributions of sodium and magnesium that could impact protein folding.
This material is based upon work supported by the National Science Foundation under Grants 2102668, 2320846, and 2018427
| Poster #: 52 Campus: San José State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: protein switch, sequence descriptors, model ensemble Project Title: Optimizing Characterization of Switch-Like Regions in SIRT1 Author List: Britney Nguyen, Undergraduate, Chemistry, San José, Presenting Author; Brooke Bellinghausen, Undergraduate, Chemistry, San José, Presenting Author; Richard Pearson, Graduate, Chemistry, San JoséBenjamin Strauss, Graduate, Computer Science, San JoséJonathan Oribello, External Partner, San José, NASA AmesBrooke Lustig, Faculty, Chemistry, San José Abstract Proteins can display conformational changes associated with allosteric regulation, with some residues consistent with a switch-like region. Here we refine identification of such regions, including in the N-terminal domain of SIRT1, a lysine deacylase that plays important roles in regulating cellular pathways. Initially applying sequence-based computational screening to SIRT1, a region between residues 185-194 was determined to exhibit switch-like behavior. Sequence-based descriptors, 6-term sequence entropy (E6), Lobanov-Galzitzskaya disorder propensity (IsUnstruct) and variability in predicted secondary structure (Vkabat) were previously found to be inclusive in the best performing logistic regression models for switch residue propensity. These entailed a learning set of 48 N-acetyltransferases, where the simpler application of these descriptors as overlays for SIRT! sequences indicated patches consistent with low E6, moderate IsUnstruct and high Vkabat.
Additional analysis of these regions, exploring secondary structure variability of an ensemble of 3D-model structures, was used to filter out less-likely regions and rank the high-probability regions. A total of twenty model structures were generated via I-TASSER, C-I-TASSER, AlphaFold2 and RoseTTAFold for Q96EB6, the complete hSIRT1 sequence. Vkabat values were calculated from the resulting conformational ensembles. The regions of interest ranked highest-to-lowest in the following order: 185-194, 295-298, 447-452, and 279-286. This methodology for ranking the switch-like regions seems to be reasonably robust to changes in the structure prediction software used, as exchanging the models generated from RaptorX for those generated by AlphaFold2 did not change the regions’ ranked order. Interestingly, regions ranked 2 and 3 are directly involved in ligand binding and 1 and 4 are adjacent to this binding region. The ensemble of 3D models indicates for 153-165 (described as disordered by X-ray methods) some conformations with an additional helix in proximity to a nearby three-helix structure that includes a switch. Our findings suggest integrating such computational approaches to experiment may facilitate the identification of key residues in enzymes and other proteins for therapeutic drug targeting and other applications.
Acknowledgements: This work was supported by NSF MRI 1626645.
| Poster #: 53 Campus: California State University, Sacramento Poster Category: Diagnostics/Imaging/Analytical Keywords: metal-organic framework, course-based undergraduate research experience, chemical analysis Project Title: Development of Advanced Analytical Laboratory Term Projects Focused on Metal Organic Framework Characterization and Applications Author List: Roy Dixon, Faculty, Chemistry, Sacramento, Presenting Author; Jordan Sharp, Undergraduate, Chemistry, SacramentoJastina Makeyenko, Undergraduate, Chemistry, SacramentoSimone Baker, Undergraduate, Chemistry, SacramentoRituparna Ivatury, External Partner, Sacramento, Vista Del Lago High SchoolYujuan Liu, Faculty, Chemistry, Sacramento Abstract A series of term projects on analysis related to metal-organic frameworks (MOFs) were developed for an advanced analytical chemistry laboratory to help students develop more critical thinking skills. These projects were tied to a general chemistry course-based undergraduate research experience in which general chemistry students synthesized HKUST-1, a copper and benzene tricarboxylic acid containing MOF. Term projects were oriented toward characterization of the synthesized MOF, investigations of MOF stability in water, and applications of the MOF for trapping molecules utilizing the high surface area of HKUST-1. Six teams of two students each performed the term projects over a three-week period.
MOF characterization was performed by powder x-ray diffraction and FTIR analysis on MOF samples. MOF water stability was investigated by measuring released copper and benzene tricarboxylic acid in water, using atomic absorption spectroscopy and HPLC, respectively, in two separate projects. For the applications, synthesized MOF was used to trap carbon dioxide, as a potential method to remove caffeine from beverages and to pack a GC column for permanent gas analysis. For the carbon dioxide trapping application, students prepared flow systems, used dry ice to prepare standards, and set up a data acquisition system to record carbon dioxide from a chip sensor. Caffeine was analyzed by HPLC in three beverages with and without addition of MOF for caffeine trapping. Students packed a short 1.6 mm diameter column with small MOF particles and used it for separation of permanent gases in three air/gas mixtures using a GC with thermal conductivity detection.
Because the experiments performed did not come with a detailed description of the work needed, students needed to review literature, to make decisions on the analysis and to trouble-shoot problems to complete projects. Each project was successful, at least to the extent of being able to make the relevant analyses. Students presented their results through both video-recorded and in-person poster presentations. The video recorded presentations also were shared with the general chemistry students involved in the MOF synthesis. Students appeared to be both more challenged (through the more independent nature of the projects) and more motivated by the term projects.
This project was funded by a CSUPERB Curriculum Development grant.
| Poster #: 54 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: neurotransmitters , imaging, electrochemical Project Title: Development of a Plasmonic Electrochemical Microscopy System for the Enhanced Sensing of Neurotransmitters Author List: Samuel Groysman, Undergraduate, Chemistry and Biochemistry , Los Angeles, Presenting Author; Yixian Wang, Faculty, Chemistry and Biochemistry , Los Angeles Abstract Detecting vesicles and their internal components, such as neurotransmitters, is essential in several biological, biomedical, and clinical contexts due to the pivotal roles vesicles play in cellular function and disease pathogenesis. Most conventional analytical methods conduct ensemble measurements of samples, so much remains unknown about the heterogeneity of vesicles. Plasmonic electrochemical microscopy (PEM) is a high throughput and highly temporally sensitive optical imaging technique that utilizes surface plasmon resonance to map refractive index changes near a sensor surface. However, applying the PEM setup to vesicle detection and electrochemical sensing of neurotransmitters would likely result in a lack of signal due to the minuscule quantities of chemicals released during exocytosis, an excessive amount of noise generated when cells and vesicles are in direct contact with the electrode, and damage to cells caused by potentials applied during PEM. To that end, this study develops the technique of non-contact plasmonic electrochemical microscopy (NC-PEM), in which the gold electrode surface of the PEM setup is coated with a mesoporous silica film via electrochemically assisted self-assembly to prevent direct contact between cellular components and the sensing surface while allowing exocytosed neurotransmitters to travel through the pores and reach the sensing surface. A model redox species, 1,1’-ferrocenedimethanol, was used to investigate the performance of the NC-PEM system. Our results demonstrate that the mesoporous silica film modification in NC-PEM allows redox species to access the sensing surface through the mesopores. More importantly, we observed at least two orders of magnitude enhancement of the sensing signal, allowing semi-quantitative detection of concentrations as low as 10 μM, while the PEM setup without modification was unable to detect concentrations as high as 1 mM. Signal enhancement is hypothesized to arise from the attraction of oxidized cations to the negatively charged silica films, which is thought to increase the local concentration change, thereby magnifying the NC-PEM signal. Future work will attempt to demonstrate the feasibility of this setup for sensing neurotransmitter release from vesicles during exocytosis.
This research has been funded by the CSUPERB Presidents’ Commission Scholars Program, NSF Major Research Instrumentation Program (CHE 1828334), and NSF CAREER award (CHE 2045839).
| Poster #: 55 Campus: California State University, Fresno Poster Category: Diagnostics/Imaging/Analytical Keywords: primary tooth, x-ray fluorescence, enamel Project Title: X-ray fluorescence and x-ray attenuation measurements of a primary incisor tooth slice Author List: Benjamin Awad, Undergraduate, Physics, Fresno, Presenting Author; Mihai Gherase, Faculty, Physics, Fresno, Fresno State University Abstract Primary teeth are easily accessible human tissues. Their analysis can reveal important issues of child development. Measurements of bulk and trace elemental composition can assess exposure to toxic elements such as lead or assist in diagnosis of malnutrition and other medical conditions. Modern x-ray fluorescence (XRF) elemental analysis is nondestructive, fast, and inexpensive. This study focused on concentration measurements of chemical elements in a primary incisor tooth slice using XRF. Such measurements require accounting for intrinsic variations of thickness, density, and elemental composition of teeth. Measured x-ray linear attenuation coefficient (μ) can account for these variations without additional calibration. A volunteer’s incisor primary tooth was sonicated in distilled water for 3 hours. The tooth was then embedded in resin and cut into thin slices with a diamond saw blade. The selected slice thickness, length, and width were 0.63 mm, 5 mm, and 1 to 3 mm, respectively. A 3D positioning stage assembly placed the sample perpendicular to the microbeam (~0.1 mm) from an integrated x-ray lens and x-ray tube unit. X-ray spectra for μ and XRF analysis were acquired by an x-ray detector in transmission and backscatter (135 degrees scattering angle) geometries, respectively. Sequential 30-s transmission spectra at positions separated by 0.1 mm for the dentin layer and 0.05 mm steps for the enamel layer were acquired. Three 300-s spectra for XRF analysis were also acquired at two locations in the dentin and enamel layers, respectively. The μ values were found to be uniformly distributed across the dentin and enamel layers. On average, the μ values of enamel were more than twice larger than those of dentin. For the photon energy range of 9.6 keV to 15.5 keV, the average μ of dentin and enamel were 5.3 mm-1 down to 0.59 mm-1, and 9.8 mm-1 down to 2.0 mm-1, respectively. The Kα peak area measurements in counts keV units for phosphorus (P), calcium (Ca), copper (Cu), zinc (Zn), and strontium (Sr) were 0.31(9), 23.8(4), 21.6(8), 3.3(3), 2.7(3) for dentin, respectively, and 0.15(6), 20.7(4), 6.1(4), 7.7(4), 1.5(2) for enamel, respectively. Uncertainties in the last significant figures were provided in the round parentheses. Sample attenuation corrections and external contamination for Cu and Zn will be performed and results will be compared to reported data.
| Poster #: 56 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: nanoplastics, cytotoxicity , scanning ion conductance microscopy Project Title: Cytotoxicity of Nanoplastics in SH-SY5Y Neuroblastoma Cells Author List: Tammy Pham, Undergraduate, Chemistry and Biochemistry , Los Angeles, Presenting Author; Tony Tran , Graduate, Chemistry and Biochemistry, Los Angeles, Presenting Author; Kevin Diego-Perez, Graduate, Chemistry and Biochemistry , Los AngelesArya Patel, Undergraduate, Chemistry and Biochemistry , Los AngelesTiffany Smith , Undergraduate, Chemistry and Biochemistry , Los AngelesYixian Wang, Faculty, Chemistry and Biochemistry , Los Angeles Abstract Since 1950, the surge in plastic production and waste mismanagement has heavily polluted the environment, with items like plastic bags, bottles, and tires breaking down over time due to UV exposure and natural processes into microplastics (<5 mm) and subsequently, nanoplastics (<1000 nm). These tiny particles, prevalent in marine ecosystems, cause DNA damage, oxidative stress, and reduce the quality of life for marine species. In a study using the SH-SY5Y human neuroblastoma cell line (ATCC CRL-2266) as an in vitro neurotoxicity model, we assessed the cytotoxic effects of commercially available polystyrene nanoplastics and house-made polyethylene terephthalate nanoparticles via ball milling. Cell health and death were gauged using the XTT assay, while scanning ion-conductance microscopy (SICM) revealed cell membrane changes post nanoplastic exposure. Our results highlight an increased cytotoxicity with rising nanoplastic concentration and show that treated cells exhibit higher membrane roughness, indicative of deteriorated cell health. Future studies aim to explore the effects of other nanoplastic varieties. This work has been partially supported by NIH R15 (Grant 1R15NS120157-01) and NSF HRD-2112554.
| Poster #: 57 Campus: San José State University Poster Category: Diagnostics/Imaging/Analytical Keywords: CRISPR/Cas9, Oral Microbiome, Next Generation Sequencing Project Title: Novel bacterial lineages discovered when a Human Oral Microbiome is treated with CRISPR/Cas9 Author List: Marianna Velasco, Undergraduate, Justice Studies, San José, Presenting Author; Leif Greene, Undergraduate, Biological Sciences, San JoséMichael McFarlin, Graduate, Biological Sciences, San JoséWilliam Huang, Undergraduate, Biological Sciences, San JoséKaori McDaniel, Graduate, Biological Sciences, San JoséCleber Ouverney, Faculty, Biological Sciences, San José Abstract A deeper understanding of the human oral microbiome is crucial as many diseases have been associated with oral pathogens. Despite advances in high-throughput sequencing, challenges remain to fully characterize the complexity of this microbial community due to the overwhelming abundance of six prevalent bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria, Fusobacteria, Spirochaetes, Bacteroidetes) which account for nearly 96% of the total bacteria. We propose a modification of the standard metagenomics approach to detect rare and significant microbes. Due to PCR bias common bacteria are preferentially amplified, making rare and novel bacteria difficult to detect. The goal was to use the highly precise CRISPR-Cas9 nuclease in vitro paired with a single guide RNA to target and digest the 16S rDNA gene of the highly abundant phyla. We hypothesized that by diminishing the most abundant 16S genes from a sample prior to PCR amplification, the DNA sample would be enriched for rare bacteria placing them at detectable levels. Validation of this concept started by digesting 16S rDNA amplified from pure cultures of known bacteria. One to four sgRNA guides were combined in the same Cas9 reaction to maximize the enrichment desired. Additionally, one broad-range and one reverse-complement guide were used as positive and negative controls respectively. Once preliminary results confirmed satisfactory, we treated genomic DNA purified from human oral plaque with our Cas9-sgRNA cocktail. The remaining DNA was PCR amplified using barcoded primers targeting the full length of the 16S gene. The PCR amplicon was cleaned using AMPure XP magnetic beads, and DNA was quantified using a Bioanalyzer prior to pooling samples and sequencing using Oxford Nanopore. Results showed a decrease of abundant bacterial phyla when paired with the respective sgRNA. Firmicutes decreased by 37.2%, Proteobacteria by 25%, Bacteroidetes by 9.8%, and Actinobacteria by 99% in comparison to the untreated control at statistically significant levels. The experiment was replicated with similar outcomes. Furthermore lesser-known bacterial phyla were revealed only in Cas9-treated samples including Campylobacter, Agrobacterium and Parvimonas associated with immune deficiencies and chronic renal insufficiency. CRISPR-Cas9 enrichment of the human oral microbiome may provide an alternative way to increase detectability of bacterial phyla that were previously unaccounted, validating our hypothesis.
| Poster #: 58 Campus: California State University, Fresno Poster Category: Diagnostics/Imaging/Analytical Keywords: bone, strontium, x-ray fluorescence Project Title: A two-dimensional K-shell x-ray fluorescence model applied to strontium measurements in a lamb bone sample Author List: Nikhil Hematillake, Undergraduate, Physics, Fresno, Presenting Author; Mihai Gherase, Faculty, Physics, Fresno Abstract Human bones store elements such as calcium, phosphorus, and strontium, and accumulate toxic elements such as lead. In vivo measurements of these elements are important for bone and environmental health assessments. X-ray fluorescence (XRF) is a non-destructive analytical technique based on detection of characteristic x-rays emitted following a photoelectric absorption event. Characteristic x-rays identify atoms based on their energies and relative intensities uniquely identifying an atomic electron transition. A calibration method is required to link in vivo or in vitro XRF measurements to elemental concentrations by accounting for several varying experimental factors such as sample size, sample x-ray attenuation, and excitation-detection geometry. A two-dimensional (2D) model was developed to compute the K-shell XRF (KXRF) signal of trace and bulk elements in bone with an overlying soft tissue. The model is a fast computational alternative to Monte Carlo methods and could guide future bone XRF studies. The boundaries of the bone and soft tissue transversal cross sections were approximated as elliptical curves and only KXRF photons generated by absorption of primary x-rays were considered. The model’s predictions for bone Sr were compared to Sr KXRF measurements using a bare lamb bone (LB) and the LB with overlying leather (LBOL) samples. The XRF experimental setup included a microbeam from an integrated x-ray tube and polycapillary x-ray lens unit, a silicon x-ray detector, and a linear positioning stage. Linear attenuation coefficients, required as input data in the 2D KXRF model, were measured to be in the 0.60 cm-1 to 3.4 cm-1 range for leather and 9.0 cm-1 to 40 cm-1 for cortical bone (using a 0.78 mm bone slice), for 17.5 keV down to 9.9 keV x-ray photon energies. The ratio between LBOL and LB Sr Kα (14.1 keV) peak area values was measured to be 0.55 ± 0.01 and its model-derived value was 0.535. The ratio between LBOL and LB Sr Kβ (15.8 keV) peak area values was measured to be 0.66 ± 0.07 and its model-derived value was 0.574. For LB and LBOL, the measured Sr Kβ/Kα ratios, corrected for detector efficiency values at 14.1 keV and 15. 8 keV photon energies, were 0.20 ± 0.01 and 0.24 ± 0.02, respectively, while their corresponding model-derived values were 0.225 and 0.240. Employing the 2D KXRF model and experimental data, the unknown LB Sr concentration was estimated to be ~0.5 mg/g which is lower than the reported human cortical bone Sr concentrations in the 0.02 to 0.07 mg/g range. In conclusion, the model correctly predicted bone and soft tissue attenuation of Sr x-rays, but likely overestimated LB Sr concentration.
| Poster #: 59 Campus: California State University, San Bernardino Poster Category: Disease (Pathogens) Keywords: Feline Coronavirus, Macrophage, Immortalization Project Title: Characterization of a New Domestic Feline Macrophage Cell Line to Study Feline Coronavirus Author List: Adam Espinoza, Graduate, Biology , San Bernardino, Presenting Author; Novalee Erickson, Undergraduate, Biology, San Bernardino, Presenting Author; Cameron Fraser, Undergraduate, Biology, San BernardinoLaura Newcomb, Faculty, Biology, San Bernardino Abstract Feline Coronavirus infects epithelial cells of the intestine, leading to a mild or subclinical infection termed feline enteric coronavirus (FECV). However, the virus can switch target cells and infect macrophages, causing Feline Infectious Peritonitis (FIPV), a systemic and often lethal disease. The factors responsible for conversion from FECV to FIPV are not well understood. In domestic cats, the switch from FECV to FIPV occurs in 5-10% of cases, whereas exposure of cheetahs to FCoV in the 1980s at U.S. Zoos resulted in 100% infected and 90% exhibiting FIPV. While domestic cats are genetically very diverse, cheetahs lack genetic diversity, especially at immune loci. We aim to investigate the increased rate of FECV to FIPV conversion in cheetahs compared to domestic cats. However, tools to study FCoV infection in vitro are lacking, with just one feline macrophage cell line available from the American Tissue Culture Collection (ATCC), Fcwf-4, a domestic feline cell line difficult to propagate. Using lentivirus integration of the immortalizing gene SV40 Large T-antigen, we generated a new domestic feline macrophage cell line, termed FMAC. Here we show this cell line is easy to propagate, displays macrophage characteristics, and is susceptible to infection with FIPV isolates of FCoV.
Cell counts at 24-hour intervals demonstrate FMAC doubles in less than 24 hours. We found FMAC can be grown to confluency, passaged over 30 times, and resuscitated from cryopreservation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirms mRNA expression of SV40-Large T-antigen (immortalizing gene), feline aminopeptidase (cellular receptor for FCoV), and CD4 (macrophage specific marker). FMAC are phagocytotic, demonstrated by uptake of bacteria expressing green fluorescent protein. Although FMAC show little cytopathic effect when infected with FIPV isolates of FCoV, RT-qPCR confirm viral gene expression and tissue culture infectious dose assays (TCID50) reveal production of infectious virus, demonstrating FMAC are susceptible. We will next infect FMAC with FECV isolates of FCoV to compare. We intend to deposit FMAC with ATCC to allow researchers access and advance studies of feline pathogens. Our laboratory intends to generate additional domestic feline macrophage cell lines and at least one cheetah macrophage cell line for continued studies.
Funding for this project was provided by Edison STEM-NET to CF and CSUSB-Office of Student Research to AE.
| Poster #: 60 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Disease (Pathogens) Keywords: iron acquisition, uropathogenic Escherichia coli, microbial pathogenesis Project Title: Inhibiting iron acquisition in uropathogenic Escherichia coli Author List: Chris Athens, Undergraduate, Biological Sciences, San Luis Obispo, Presenting Author; Adam Marin, Undergraduate, Biological Sciences, San Luis Obispo, Presenting Author; Spenser Platt, Undergraduate, Chemistry and Biochemistry, San Luis ObispoCole Detels, Undergraduate, Biological Sciences, San Luis ObispoMary Rose Ronquillo, Undergraduate, Biological Sciences, San Luis ObispoAbby Kaplan, Undergraduate, Biological Sciences, San Luis ObispoBella Oldenburg, Undergraduate, Biological Scienes, San Luis ObispoMadison Irons, Undergraduate, Chemistry and Biochemistry, San Luis ObispoScott Eagon, Faculty, Chemistry and Biochemistry, San Luis ObispoAlejandra Yep, Faculty, Biological Sciences, San Luis Obispo, Cal Poly Abstract Urinary tract infections (UTIs) are the second most common infections in humans and are primarily caused by strains of uropathogenic Escherichia coli (UPEC). UTIs are typically treated with antibiotics, however the steady increase in antibiotic and multidrug resistance has made the development of novel therapeutic strategies an urgent need. Iron acquisition is a promising target for developing new therapeutic agents, as it is both essential for bacterial growth and a limiting nutrient in urine. In order to successfully colonize the urinary tract, UPEC strains need to employ a variety of high-affinity iron acquisition systems. We describe a structure-activity relationship (SAR) study of small molecule inhibitors that are predicted to target and inhibit the high-affinity iron acquisition systems of UPEC. We tested the ability of a family of 11 compounds to inhibit iron acquisition in a whole-cell, growth-based assay in MOPS minimal medium. Five of the compounds inhibited growth of UPEC strain CFT073 under iron-limiting conditions. Iron supplementation abolished growth inhibition, suggesting that the compounds target iron acquisition mechanisms. Dose-response experiments show improved IC50 values for two of the newly synthesized compounds (1.93 ± 0.05 and 1.7 ± 0.1 μM, respectively) compared to previously published data for the parent scaffold (8 ± 2 μM). Based on patterns emerging from this first generation of molecules, we will design, synthesize, and test a second generation of compounds. This work further validates iron acquisition as an attractive target for antibacterial strategies against UTI pathogens.
| Poster #: 61 Campus: California State University, Fullerton Poster Category: Disease (Pathogens) Keywords: carbapenem-resistant Acinetobacter baumannii , lactic acid bacteria, antibiotic resistant bacterial infections Project Title: Chemical and Functional Characterization of postbiotics from Lacticaseibacillus rhamnosus CRL 2244 with Antimicrobial Activity against Carbapenem-Resistant Acinetobacter baumannii Author List: Kirsten Mcmanus, Graduate, Chemistry and Biochemistry, Fullerton, Presenting Author; Briea Gasca, Undergraduate, Biological Sciences, Fullerton, Presenting Author; Adiba Aziz, Undergraduate, Biological Sciences, FullertonCecilia Rodriguez, External Partner, Non-CSU, Centro de Referencia para Lactobacilos (CERELA), CONICET, Tucumán, ArgentinaNicholas Salzameda, Faculty, Chemistry and Biochemistry, FullertonMaria Soledad Ramirez, Faculty, Biological Sciences, Fullerton Abstract The increase in bacterial resistance to antibiotics is a major health concern throughout the world. As the number of antibiotic resistant bacterial infections rises, there is a need for new therapeutics to treat this health crisis. Specifically, carbapenem-resistant Acinetobacter baumannii (CRAB) is a dangerous pathogen that is resistant to multiple classes of antibiotics. With limited therapeutics for the treatment of CARB infections, there is an urgent need for new therapeutics to treat infections caused by CRAB and other antibiotic resistant pathogens.
A potential source of new therapeutics to treat antibiotic resistant bacterial infections is lactic acid bacteria (LAB). LAB are beneficial bacteria that have numerous health benefits and have been reported to have antimicrobial activities. LAB has been reported to inhibit a variety of pathogens, including CRAB, but these have been very limited studies and the mechanism and compound(s) responsible for the antagonist effects is still unknown.
In this study, we report the LAB strain, Lacticaseibacillus rhamnosus CRL 224 demonstrated significant inhibition of CRAB. The extracellular products of Lcb. Rhamnosus CRL 2244 caused CRAB cellular stress and ultimately cell death. The cell-free culture medium of Lcb. Rhamnosus CRL 2244 was subjected to extraction with organic solvents to isolate compound(s) of interest for further analysis. The crude organic extract was found to have inhibitor activity against CRAB with a preliminary minimum inhibitory concentration (MIC) to be 3.75 mg/ml. HPLC and GC/MS analysis suggest that a single small molecule (M.W. > 500) is responsible for the inhibitor activity. Additional studies will focus on the structure elucidation of the compound of interest and mechanism of action.
| Poster #: 62 Campus: California State University, Sacramento Poster Category: Disease (Pathogens) Keywords: Paraclostridium bifermentans, oxygen, spore Project Title: Oxygens impacts sporulation of the gut commensal bacterium Paraclostridium bifermentans Author List: Cassidy Fontaine, Undergraduate, Biological Sciences, Sacramento, Presenting Author; Christopher Lopez, Faculty, Biological Sciences, Sacramento Abstract Paraclostridium bifermentans is an opportunistic pathogenic bacterium that is part of the Class Clostridia. Bacteria in this Class have two main forms: vegetative cells and spores. Vegetative cells are cells that grow actively and reproduce in an environment that has favorable conditions. When a vegetative cell encounters an unfavorable environment, the vegetative cell turns into a dormant, highly resilient spore. These spores can stay metabolically dormant for an exceptionally long time until they are moved to an environment with favorable conditions. P. bifermentans has pathogenic potential but can also serve as a competitor to the gut pathogen Clostridioides difficile.
There is little known about how oxygen affects sporulation in P. bifermentans and the consequences in microbe-microbe interactions. Since P. bifermentans is a bacterium that grows in an environment with no oxygen (anoxic environment), our hypothesis was higher percentages of oxygen would be unfavorable for the bacteria thus forcing it to create a higher spore to vegetative cell ratio. Surprisingly, upon completing trials for this experiment our hypothesis has been challenged.
To investigate how oxygen affects the rate of sporulation in P. bifermentans, bacteria were plated onto BHIS agar and incubated under anoxic (0% oxygen) and oxic (2.5% oxygen) for 24 hours to allow a lawn to grow. Following incubation, samples of bacteria were taken from each environmental condition (anoxic and oxic) and either transferred to a glass slide or moved to 1 mL of phosphate buffered saline (PBS) for dilutions in a 96-well plate. The glass slide samples were stained with malachite green for the spores and safranin for the vegetative cells. Liquid samples were added to 1 mL of PBS and heat treated at 65 °C to kill vegetative cells, followed by dilution and spot plating for spore enumeration.
Results collected from these trials have shown that oxygen increases the vegetative cell to spore ratio, with more spores when oxygen is absent. Overall, this data shows how oxygen affects the rate of sporulation in P. bifermentans by lowering its sporulation in oxic conditions. These results can be used to formulate a new hypothesis on how oxygen causes changes in sporulation and how this can affect interactions with other microbes. This work was supported by the Summer Undergraduate Research Experience at California State University, Sacramento.
| Poster #: 63 Campus: California State University, Los Angeles Poster Category: Disease (Pathogens) Keywords: vulvuvaginal candidiasis, blue light therapy, Candida albicans Project Title: Efficacy of Antimicrobial Blue Light for Inhibiting Candida albicans Growth in Vulvovaginal Candidiasis Model Author List: Aaron Cruz, Graduate, Biological Sciences, Los Angeles, Presenting Author; Rikka Tagayuna, Undergraduate, Biological Sciences, Los Angeles, Presenting Author; Hyunsook Park, Faculty, Biological Sciences, Los Angeles Abstract Candida albicans, a fungus inhabiting human mucosal surfaces, is a primary cause of vulvovaginal candidiasis (VVC), affecting 75% of women at least once in their lives. Traditional antifungal treatments with oral or topical medications can be ineffective or cause adverse effects. Antimicrobial blue light (ABL) has emerged as an alternative therapy for microbial infections, leveraging its microbicidal properties through oxidative stress and DNA damage. While ABL has shown promise against various microbial pathogens in vitro and in vivo, limited research has explored its efficacy against fungal infections. ABL operates at wavelengths between 400-470 nm, inducing intracellular reactive oxygen species production, primarily in bacteria, to impede microbial growth. This study aims to assess ABL's effectiveness in inhibiting C. albicans growth and determine optimal parameters for VVC treatment, including wavelengths, energy levels, irradiance times, and photosensitizers. We also compared the viability of blue light-treated C. albicans with Gardnerella vaginalis, a common bacterial pathogen linked to bacterial vaginosis, and Lactobacillus crispatus, a vital bacterium for vaginal homeostasis. Microorganisms in log-phase growth were exposed to 400-450 nm blue lights at various durations and energy levels. Subsequently, quantitative culture assessments determined cell viability. This study revealed that 405 nm blue light treatment is the most effective for inhibiting C. albicans growth. C. albicans exhibits higher resistance to blue light treatment than G. vaginalis. A ninety percent reduction in C. albicans viability required 400 J/cm2 of blue light, compared to 200 J/cm2 for G. vaginalis. The energy level used for C. albicans did not affect Lactobacillus viability, suggesting blue light can be selective for vaginal pathogens while preserving beneficial Lactobacillus populations. This study highlights the potential of blue light treatment as a safe and effective approach for treating vaginal infection without disturbing normal commensal flora.
| Poster #: 64 Campus: California State University, Long Beach Poster Category: Disease (Pathogens) Keywords: SARS-CoV-2, Viral pathogenesis, CD4+ T cells Project Title: Determining whether the Spike/CD4 interaction correlates with enhanced pathogenesis of SARS-CoV-2 Author List: Michael Anderson, Graduate, Department of biological sciences, Long Beach, Presenting Author; Abstract Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the Coronavirus Disease 2019 (COVID-19) global pandemic and has accounted for nearly 7 million deaths worldwide. SARS-CoV-2 is an enveloped positive-sense single-stranded RNA virus (ssRNA) that mainly infects cells of the upper and lower respiratory tract. In some cases, SARS-CoV-2 causes lymphocytopenia and cytokine storm, though the exact causes for this immune dysfunction remained unknown. Recently, a study found that SARS-CoV-2 infects human CD4+ T helper cells, an important component of the adaptive immune system. Interestingly, the SARS-CoV-2 Spike glycoprotein (S), which binds ACE2 to mediate entry into host cells, directly bound to the CD4 molecule. However, whether other Spike variants of concern (VOC) also bind CD4 remains unclear. Here, we aimed to determine how conserved the Spike/CD4 interaction is among diverse Spike VOC. While more transmissible, infection with the SARS-CoV-2 Omicron variant displayed decreased pathogenesis than the SARS-CoV-2 ancestral strain. We therefore hypothesized that the Spike(Ancestral)/CD4 interaction may display increased binding than that of Spike(Omicron)/CD4. To test this, we employed bi-molecular fluorescence complementation (BiFC), an assay that quantifies protein-protein interactions within living cells. We constructed plasmids expressing SARS-CoV-2 Spike(Ancestral) or SARS-CoV-2 Spike(Omicron) fused to the N-terminus of a yellow fluorescent protein (YFP) and another set of plasmids expressing CD4 or ACE2 fused to the C-terminus of YFP. We transfected these plasmids into cells (HEK293T) and measured fluorescence via flow cytometry. Interestingly, our preliminary results detected a weaker interaction/signal between Spike(Omicron)/CD4 relative to Spike(Ancestral)/CD4 despite both proteins being reasonably well expressed. We are currently expanding our study to test more Spike VOC and conducting immunofluorescence and mutagenesis to both visualize and map regions that may be important for differential binding. Additionally, using SARS-CoV-2 Virus Like Particles (VLP), we are determining whether there are differences in the ability of Spike VOC to infect CD4+ T cells. Conducting this research will contribute toward our understanding of COVID-19 pathogenesis and virulence and the evolutionary history of SARS-CoV-2, which may inform the development of novel Spike peptide inhibitors.
| Poster #: 65 Campus: California State Polytechnic University, Pomona Poster Category: Disease (Pathogens) Keywords: Blocking Antibodies, Neutrophils, Trichomonas vaginalis Project Title: The role of Complement Receptors in neutrophil like cells’ killing of Trichomonas vaginalis. Author List: Isabel Romero, Graduate, Pomona, Presenting Author; Abstract Isabel Veronica Romero1, Emma N Trujillo1, Ashley D Ramirez1, & Frances Mercer1
1 Department of Biological Sciences, California State Polytechnic University Pomona
Trichomonas vaginalis is a protozoan parasite known to cause trichomoniasis, which is the third most common sexually transmitted infection in the United States. Once T. vaginalis infects its host, the parasite will live within the tissue of the prostate, urethra or vagina. To survive, T. vaginalis must adhere onto the vaginal or prostate epithelium since it is too large to enter cells. Neutrophils are crucial for our body’s immune response to T. vaginalis. A novel mode of killing that has recently been discovered, known as trogocytosis, involves neutrophils acquiring material from the pathogen. Neutrophils have been seen utilizing this mode of killing to defend against T. vaginalis. It also has been found that there must be serum present for the neutrophils to successfully kill the parasite. Thus, we hypothesize complement factors, abundantly found in serum, are playing a role in the function of trogocytosis. However, the receptor-ligand interaction between the neutrophils and parasite for trogocytosis to occur is still unknown. Using a cytotoxicity assay, we found that knocking out complement receptor CR3 and blocking CR3 with antibodies reduced killing of the parasite by neutrophil-like cells (NLCs).
| Poster #: 66 Campus: California State University, Fresno Poster Category: Disease (Pathogens) Keywords: Salmonella, Bacteria-killing assay, Finch Project Title: Variation in Cardueline finches’ constitutive innate immune response against Salmonella Author List: Lindsey Biehler, Graduate, Biology, Fresno, Presenting Author; Tricia Van Laar, Faculty, Biology, StanislausJoel Slade, Faculty, Biology, Fresno Abstract A recent outbreak of Salmonella enterica serovar Typhimurium in 2020-21 resulted in a large die-off of a wild Cardueline finch species in the Pacific Northwest and California. Studies predominantly focus on the presence of Salmonella in humans and agricultural animals, but studies on the relationship between this pathogen and wild songbirds are lacking. Cardueline finches occupy bird feeders, however Pine Siskins (Spinus pinus) seem to die during Salmonella outbreaks whereas other finch species, such as Lesser Goldfinches (Spinus psaltria), American Goldfinches (Spinus tristis), and House Finches (Haemorhous mexicanus) appear resistant. The aims of our research is to (1) quantify constitutive innate immune function by measuring bactericidal activity in the four wild finches against a locally cultured avian Salmonella strain in vitro, and (2) analyze genetic variation at Toll-like receptor 4 (TLR4), which is involved in immunity against this bacterium. We found that there was significant variation in their constitutive innate immunity against Salmonella, with Spinus spp. having low bactericidal ability in comparison to the Haemorhous sp. (House Finch). TLR4 analysis is underway. By comparing the results of both the BKAs and TLR4 variation, we will get a better understanding as to why certain Cardueline finches may be more susceptible to Salmonella infections. Overall, this research will provide data that helps with the conservation of local bird populations that are susceptible to bacterial infections and help with mitigating avian diseases.
| Poster #: 67 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Zostera marina, Population genomics, Triple restriction-enzyme associated DNA sequencing Project Title: Comparison of Eelgrass Population Origin and Genomic Variation Using Triple Restriction-Enzyme Associated DNA Sequencing Author List: Keezean Paguio, Undergraduate, Department of Biology, San Francisco, Presenting Author; Taylor Pantiga, Graduate, Interdisciplinary Marine and Estuarine Sciences, San FranciscoAnnie Chu, Graduate, Interdisciplinary Marine and Estuarine Sciences, San FranciscoC Sarah Cohen, Faculty, Biology and Interdisciplinary Marine and Estuarine Sciences, San Francisco Abstract Eelgrass, Zostera marina, is a coastal foundation species that provides environmental benefits such as blue carbon storage and wave attenuation. Conservation initiatives for these ecologically important species could benefit by understanding genetic diversity, structure, and connectivity between urbanized/anthropogenically influenced sites, such as those from San Francisco Bay, and those less impacted, such as those from Point Reyes National Seashore (PRNS). We used genome wide triple restriction-enzyme associated DNA (3RAD) sequencing to compare samples from central California within San Francisco Bay (3 sites), outside the bay in PRNS (2 sites), and two outlier locations in Washington state. We extracted DNA using a liquid nitrogen and CTAB protocol, then quantified and evaluated the quality of the DNA using gel electrophoresis and Qubit measures. Next, samples were normalized via DNA precipitation to a concentration of 20ng/uL. Finally, we performed the 3RAD protocol to create individual libraries to be sequenced. This was done in a genetics workshop, RADCamp2023, at Columbia University, New York, where participants did additional benchwork as well as bioinformatic analysis of RAD data. This process selectively sequenced the genomes of our samples and we compared genetic diversity across sites. We found that samples from geographically distant beds showed greater genetic differentiation with distance. Distinction across regions is evident, while individual sites within a region show mixing. We infer that genetic distance increases with geographic distance at the regional scale due to limited gene flow.
| Poster #: 68 Campus: California State University, Sacramento Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Gene amplification, genetic engineering, Acinetobacter baylyi Project Title: Uncovering the Mechanisms of Gene Amplification by Investigating the Roles of Various DNA Double-strand Break Repair Elements in Acinetobacter baylyi Author List: Hanna Franklin, Graduate, Biological Sciences, Sacramento, Presenting Author; Andrew Reams, Faculty, Biological Sciences, Sacramento Abstract Gene amplification mutations, where a genomic segment is abnormally copied many times, occur spontaneously in all life forms and are important drivers in cancer, pathogens, and evolution. Despite this, the detailed mechanisms and precise factors involved in their formation and collapse remain largely unknown in all organisms. Here, we investigate gene amplification formation using a bacterial model system in Acinetobacter baylyi, where spontaneous mutants carrying de novo high-copy gene amplification encompassing cat genes are exclusively selected on minimum benzoate agar plates. Using this system, this project analyzes three possible influences of DNA breaks and repair on the ability to form gene amplification mutations: 1) transposable elements, 2) R-loops, and the 3) RecBCD enzyme. To investigate the role of transposable elements, A. baylyi mutant strains were engineered lacking all six copies of the sole transposable element in its genome, IS1236, and tested by the minimum benzoate assay. Preliminary results indicate removal of transposable elements decreased cat gene amplification mutant frequencies by over 200-fold. To explore whether R-loops influence gene amplification formation, the A. baylyi parent strain was grown with protocatechuate as the sole carbon source. These growth conditions aimed to stimulate R-loop formation within a recombinational hot spot, the pca operon region, downstream of the cat genes targeted for amplification. Preliminary results show protocatechuate-grown cells increased cat gene amplification mutant frequencies by over 100-fold. Third, the role of the main enzyme complex involved in repairing double-stranded DNA breaks, RecBCD, was analyzed by engineering recB and recC null mutant strains and testing their ability to form cat gene amplification mutants. Results show both null mutants were unable to form cat gene amplification mutants on minimum benzoate plates. Together, these three experimental findings provide consistent evidence that DNA breaks and their repair mechanisms are key factors involved in forming gene amplification mutations.
| Poster #: 69 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: voltage gated sodium channels, muscle spindle afferents, electrophysiology Project Title: NaV1.1 and NaV1.6 play unique and essential roles in muscle spindle afferent function in adult mice Author List: Serena Ortiz, Graduate, San José, Presenting Author; Cyrrus Espino, External Partner, San José, UC DavisTheanne Griffith, External Partner, San José, UC DavisKatherine Wilkinson, Faculty, Biological Sciences, San José Abstract Voltage gated sodium channels (NaV) are crucial for the generation and propagation of action potentials. There are 9 different isoforms found in mammals (NaV1.1-1.9), and NaV1.1, 1.6 and 1.7 are found in the mechanoreceptors that sense muscle stretch, the muscle spindle afferents. This sensory information is critical for motor reflexes and proprioception, or the sense of body and limb position in space. We hypothesized that each of these NaV subunits play a unique role in the generation of stretch sensitive firing in muscle spindle afferents. Mice with a deletion of the NaV 1.1 or 1.6 genes (scn1a or scn8a) in peripheral sensory neurons were generated. We used an ex vivo muscle nerve preparation to record the response of muscle spindle afferents to ramp-and-hold stretch and vibration. The extensor digitorum longus muscle and the sciatic nerve were isolated from 33 adult mice (NaV1.1(WT), n=10; NaV1.1+/- (Het), n=12; NaV1.1-/- (cKO), n=11). We observed inconsistent firing in afferents with the loss of one or both copies of NaV1.1, which was almost never observed in WT afferents. We quantified this inconsistent firing by determining the coefficient of variation of the interspike interval (ISI CV) during the plateau phase of stretch (1.5–3.5 s into the hold phase) and found a significant effect of genotype, with the Het and cKO afferents both having higher ISI CV than the WT afferents (two-way ANOVA, main effect of genotype, p=0.015). In contrast, dynamic sensitivity and the response to sinusoidal vibration was not significantly different between genotypes. Mice lacking both copies of NaV1.6 also displayed motor deficits, however; these deficits appeared much more severe than those seen in NaV1.1 cKO mice. Preliminary results include 10 cKO animals, and 3 WT. Of the 10 cKO mice, only 3 displayed low sensitivity to stretch, and of these 3 only 1 reacted to sinusoidal vibrations. Seven of the ten mice did not fire in response to stretch at all.The 3 WT mice displayed normal firing patterns for both stretches and vibrations. These differences support our hypothesis that NaV1.1 and NaV1.6 play unique roles in MSA firing, where NaV1.6 is likely a primary generator of AP transmission and NaV1.1 plays a role in static mechanosensation. Future studies will determine whether these channels have different subcellular localizations that can explain our results.
This work was supported by NIH Grant 5SC3GM127195 (KAW) and a RISE Fellowship 5R25GM71381 (SO).
| Poster #: 70 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: antibiotic resistance, aminoglycoside, acetyltransferase Project Title: Reversal of plazomicin resistance by silver ions: a new hope against multidrug resistant infections Author List: David Ngo, Graduate, Biological Science, Fullerton, Presenting Author; Angel Magaña, Graduate, Biological Science, FullertonTung Tran, External Partner, Non-CSUJan Sklenicka, Graduate, FullertonKimberly Phan, Graduate, FullertonBrian Eykholt, Graduate, FullertonVeronica Jimenez, Faculty, FullertonMaria S. Ramirez, Faculty, FullertonMarcelo Tolmasky, Faculty, Fullerton Abstract Background: Plazomicin is a semisynthetic aminoglycoside recently approved by the U.S. Food and Drug Administration (FDA). Its structure is based on the sisomicin scaffold, with modifications that render the molecule resistant to inactivation by most aminoglycoside-modifying enzymes. However, the aminoglycoside 2′-N-acetyltransferase type Ia [AAC(2′)-Ia] recognizes plazomicin as a substrate conferring resistance to hosting bacteria. To overcome the action of this enzyme, we are designing formulations that combine the antimicrobial agent with an inhibitor of AAC(2′)-Ia. Methods: Acetylating activity was measured by the phosphocellulose binding assay. Silver acetate as an inhibitor of plazomicin resistance was assessed on Escherichia coli harboring recombinant clones that include aac(2′)-Ia. Bactericidal activity was confirmed using time-kill assays, and cytotoxicity of plazomicin/silver acetate mixtures was measured on HEK293 cells. Results: Addition of silver acetate inhibted acetylation of plazomicin in vitro. Silver acetate at 2 μM was sufficient to inhibit the growth of resistant E. coli cells in the presence of plazomicin (8 μg/ml). Sodium acetate did not influence resistance. Resistance was reversed when the gene was expressed from the natural or a constitutive promoter. Time-kill assays confirmed the bactericidal effect of plazomicin in these conditions. The mix of plazomicin with silver acetate did not exhibit toxicity toward HEK298 cells at concentrations several-fold higher than those needed to overcome resistance. Conclusions: We demonstrated that Ag1+ ions interfere with resistance to plazomicin mediated by AAC(2′)-Ia. This finding shows that this ion is an excellent candidate as a plazomicin adjuvant to eliminate this enzyme as a threat to the effectiveness of this antibiotic. The low cytotoxicity observed at the active concentrations makes the combination plazomicin/Ag1+ a viable option for treating multidrug resistant infections.
| Poster #: 71 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: symbiosis, bacteria, antibiotics Project Title: An ABC transporter confers resistance to beta-lactams, phenazines, and sodium dodecyl sulfate (SDS) in the alpha-proteobacterium Sinorizobium meliloti Author List: Nima Pendar, Undergraduate, Biology, San Francisco, Presenting Author; Klara Christensen, Undergraduate, Biology, San Francisco, Presenting Author; Joseph Chen, Faculty, Biology, San Francisco Abstract Sinorhizobium meliloti is a Gram-negative bacterium most known for its ability to form beneficial endosymbiosis with compatible legume plants, by colonizing root nodules and producing fixed nitrogen. It serves as a prominent model for elucidating molecular mechanisms that govern microbe-host interactions, ranging from mutualistic to pathogenic. Examination of various laboratory strains of S. meliloti serendipitously revealed that some exhibit increased sensitivity to the detergent sodium dodecyl sulfate (SDS) and the antibiotic faropenem, suggesting a possible defect in the cell envelope. Understanding the defect can provide insights into the physiology of this key symbiont and related species. Whole-genome sequencing and subsequent analysis, including verification via Sanger sequencing, identified three variations exclusive to the resistant or sensitive strains. One of the variations is a single nucleotide insertion/deletion that leads to a frameshift in a gene encoding a putative ATP-binding cassette (ABC) transporter. Deletion of the gene replicated the sensitivity to SDS and faropenem, and complementation with the ortholog from S. medicae alleviated the defect in the null mutants. These and additional results indicated that the ABC transporter confers resistance to various antimicrobial compounds, including specific detergents, beta-lactams, and phenazines. The molecular basis of this resistance is currently under investigation. Revealing the cellular mechanisms involved can shed light on antibiotic resistance in related alpha-proteobacteria, including human pathogens of concern.
| Poster #: 72 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Mastitis, Genome Wide Association Study, Dairy Cattle Project Title: Molecular mechanisms associated with clinical mastitis in dairy cattle Author List: Kevin Chen, Graduate, Animal Science, San Luis Obispo, Presenting Author; Fernando Campos-Chillon, Faculty, Animal Science, San Luis ObispoDaniel Peterson, Faculty, Animal Science, San Luis ObispoSiroj Pokharel, Faculty, Animal Science, San Luis ObispoKim Sprayberry, Faculty, Animal Science, San Luis ObispoPaul Anderson, Faculty, Computer Science & Software Engineering, San Luis ObispoJulie Huzzey, Faculty, Animal Science, San Luis ObispoJoy Altermatt, Faculty, Animal Science, San Luis ObispoMehdi Sargolzaei, External Partner, Animal BioSciences, Non-CSU, University of GuelphMohammed Abo Ismail, Faculty, Animal Science, San Luis Obispo Abstract Mastitis, the inflammation of the mammary gland caused by both gram-positive and gram-negative bacteria, is estimated to cost the global dairy industry about $20 billion annually due to changes in milk yield and quality. The goal of this study was to identify genetic variants, genes, and biological pathways that are associated with clinical mastitis. We performed genome-wide association studies (GWAS) on 2,121 cows with medium density single nucleotide polymorphisms (SNP; n=87,492) using a single SNP mixed linear model. The top one hundred significant (at P<0.0012) SNPs were identified on chromosomes 4, 14, 17, and 29. These SNPs were mapped to the bovine genome to corresponding genes. Then, gene enrichment analyses were performed using DAVID software. These genes were involved in nitric oxide regulation, apoptotic processes, and cell differentiation. Nitric oxide and apoptosis were found in previous studies to be involved in the immune response which directly impacts the susceptibility to mastitis. The identified genomic regions via GWAS for mastitis will be fine mapped and validated before implementation in genomic selection programs.
| Poster #: 73 Campus: Sonoma State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Chrysomela aeneicollis, Gene Duplication, Coleoptera Project Title: Duplication of and variation in succinate dehydrogenase affects response to stress in willow beetles Author List: Justin Brasil, Graduate, Biology, Sonoma, Presenting Author; Nathan Rank, Faculty, Biology, SonomaElizabeth Dahlhoff , Faculty, Biology, Non-CSU, Santa Clara University Abstract Montane environments pose unique challenges to animals living there, including elevation-induced hypoxia and variable temperatures, which exert strong selective pressures on metabolic genes. Succinate dehydrogenase (SDH) is a multisubunit enzyme complex that plays a central role in aerobic cellular metabolism and a secondary role in hypoxia signaling. In the willow leaf beetle Chrysomela aeneicollis, we discovered a duplication in the gene coding for the SDH-b subunit and characterized polymorphisms at introns and exons for both loci. A phylogenetic analysis of close BLAST matches to these SDH-b loci indicates that the duplication occurred within Insecta. Preliminary evidence suggests that interactions among the two SDH-b gene loci affect recovery of running speed after heat exposure. To further examine the role of duplicated SDH-b gene products in response to stress, we performed an RNA-seq experiment in which beetle hatchlings collected from an introgressed population were reared at high and low elevation in the laboratory before heat treatment. Analysis of resulting RNA-seq transcripts, including 16 genes involved in the hypoxia signaling pathway, revealed effects of rearing elevation, temperature and mitonuclear genotype on transcript expression, including those of the duplicated SDH-b loci. These results contribute to our understanding of the relationship between nuclear and mitochondrial genetic variation and ability to cope with a challenging and changing climate.
| Poster #: 74 Campus: California State University San Marcos Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Mismatch Repair, CCTG DNA Repeats, Myotonic Dystrophy Project Title: Investigating the Role of Mismatch Repair on CCTG DNA Repeat Instability Author List: Maya Qaddourah, Undergraduate, San Marcos, Presenting Author; Jane Kim, Faculty, Biology, San Marcos Abstract Myotonic Dystrophy type two (DM2) is a genetic disease in which muscles progressively grow weaker due to long tandem CCTG repeats that occur in the first intron of the CNBP gene. An affected individual can have 75 CCTG repeats to over 10,000. The process, or mechanism, behind the repeated shortening and lengthening of DNA, instability, remains unknown. One pathway of interest is the Mismatch Repair (MMR) system due to its role in DNA repair. MMR has its own set of proteins that work together to recognize small base-base mismatches, Msh2 and Msh6, and to loops and larger secondary structures, Msh2 and Msh3. Using the budding yeast Saccharomyces cerevisiae as the model organism, repeat lengths were monitored via Polymerase Chain Reactions to investigate the role of the MMR genes and their contribution to DNA repair on these CCTG repeats. We investigated the role of the DNA repair genes by knocking one out to see its effect on instability frequency. When msh2Δ was knocked out, it produced the most significant instability frequency among the samples: 3.6% compared to 0.44% for wild type. The msh3Δ demonstrated a similar instability level as the wildtype, both having the least. The msh6Δ demonstrated intermediate results as it was in the middle between the tested respective genes, which can leave select questions about its role in MMR open-ended. Through a Fisher’s Exact Test, the msh2Δ was the only gene to be deemed statistically significant among all the genes tested. Overall, these results demonstrate that specific genes might be essential in protecting against repeat instability which can provide insight into the unknown mechanisms of how the repeat instability of CCTG nucleotides are produced.
| Poster #: 75 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Caenorhabditis briggsae, Genetics, Fitness Project Title: Assessing temperature dependence of developmental delay frequencies within intra-species hybrids of Caenorhabditis briggsae Author List: Adrianna Grieco, Undergraduate, Biology, Fresno, Presenting Author; Joseph Ross, Faculty, Biology, Fresno Abstract Studying how genetic differences between individuals can cause their offspring to be less healthy is important for understanding human disease as well as fundamental biological processes like species formation. Offspring between genetically diverse parents can suffer from genetic incompatibilities that manifest as developmental deficits. A classical example is that mating of a horse with a donkey creates hybrid offspring (mules) that are sterile. Beyond this example, offspring of genetically divergent parents often suffer from developmental delay, which is evident as a protracted period of development in which the hybrid takes more time to reach adulthood than wild-type individuals. Developmental delay is commonly seen in the F2 generations of Caenorhabditis briggsae temperate x tropical hybrid crosses. Specifically, about 20% of F2 hybrid offspring of tropical AF16 (from India) and temperate HK104 (from Japan) exhibit developmental delay. The identity of the AF16 and HK104 alleles that negatively interact in F2 hybrids to cause them to be developmentally delayed is unknown. Prior studies suggest the possibility that this genetic interaction is temperature-dependent. With the ultimate goal of identifying the genetic differences that cause developmental delay, I sought to determine whether the temperature at which the worms are grown does influence the frequency of F2 hybrids that become delayed. I measured the frequency of developmentally delayed individuals in an AF16 x HK104 hybrid cross at 20°C and compared the results to an AF16 x HK104 hybrid cross at 25°C. Although developmental delay was significant when compared to the development rate of wild-type siblings (ANOVA, p=0.002), there was no significant interaction between the frequency of developmentally delayed F2 offspring and temperature (ANOVA, p=0.670). Although these results suggest that developmental delay is not affected by temperature, repeating this experiment to increase replication could produce a more accurate statistically significant value. Ultimately, studying the potential environmental influence on hybrid genetic developmental delay in C. briggsae improves our understanding of how changes in temperature, for example global warming, can impact the fitness of numerous organisms.
| Poster #: 76 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Mycobacteria, Mechanical lysis, Gene expression Project Title: High-quality RNA Extraction from the Acid-fast Bacterium Mycolicibacterium smegmatis, a model organism for Mycobacterium tuberculosis Author List: Janette Dzul, Graduate, Biological Sciences, Los Angeles, Presenting Author; Naim Cerkezi, Undergraduate, Biological Sciences, Los AngelesEdith Porter, Faculty, Biological Sciences, Los Angeles Abstract Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), continues to be the second leading cause of infectious death worldwide. Mtb primarily spreads through the air via droplets when an infected individual coughs, sneezes, or spits. About 5 % of new infections lead to the development of active TB which accounts for about 10 million new cases and over 1 million deaths yearly. One of the challenges encountered when treating TB is the rise in multidrug-resistant and extreme drug-resistant strains. This necessitates novel drug development, including approaches based on the immune defenses in the human body, which protect 95% of the population from TB development. Antimicrobial peptides, which are natural protein-based antibiotics, may be important players in fighting against this disease. Previously, our lab demonstrated that the antimicrobial peptide Human Beta Defensin 2 (HBD2) inhibits the metabolic activity of Mycolicibacterium smegmatis (Ms), a model organism for Mtb, consistent with the induction of dormancy, a temporary state of inactivity. To test the hypothesis that HBD2 induces dormancy in Ms our lab wishes to assess the gene expression of dormancy-regulated genes in Ms in the presence and absence of HBD2. This requires the extraction of high-quality RNA from these bacteria that are surrounded by a lipid-rich cell wall and are notoriously hard to lyse. The objective of this study was to establish a protocol to extract high-quality RNA from Ms. For this we used different methods to mechanically lyse the cells, and different RNA purification kits. We found that chemically stabilizing the RNA, mechanically lysing the bacterial cells through tip sonication, followed by a column-based RNA extraction combined with DNAse treatment and final RNA purification and concentration yielded high purity and quality RNA, as verified with NanoDrop and 2.5% ethidium bromide - stained agarose gel. We were able to extract from about 3 x 107 Ms cells, 1.5 mg of RNA with A260/A280 and A260/A230 values of 1.99 and 2.28 respectively. In addition, 16S rRNA and 23S rRNA bands were clearly distinguishable in the agarose gel. This protocol will be used to obtain mRNA for determining the expression of dormancy-induced genes by quantitative PCR. This work may assist in understanding the role that HBD2 plays in inducing dormancy in Ms, which can further lead to developing better treatments for Mtb. This work was funded by NSF/CSU-LSAMP Grant #HRD-2204753.
| Poster #: 77 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Multiple antibiotic resistance, Escherichia coli, gene regulation Project Title: Repression of multiple antibiotic resistance (mar) operon of Escherichia coli by cellular metabolites Author List: Pablo Segundo, Undergraduate, Biology, Northridge, Presenting Author; Rafael Estrada, Graduate, Biology, NorthridgeCristian Ruiz Rueda, Faculty, Biology, Northridge Abstract The spread of antibiotic resistance is a major threat to public health. Multidrug resistance (MDR) is a concerning growing issue and multidrug efflux pumps are one of the main modes of MDR, especially in Gram-negative bacteria. This project studied the multiple antibiotic resistance (mar) operon which regulates the main multidrug efflux pump of Escherichia coli, the AcrAB-TolC efflux pump, as well as outer membrane permeability. Previous research has suggested that other cellular functions including metabolism play a role in regulating MDR. Here, we used a mar promoter-lacZ gene fusion and β-galactosidase assays to determine whether central metabolism intermediates modify the expression of mar operon. Of the compounds tested, we found that the TCA intermediate succinate significantly represses the expression of the mar operon. This finding indicate that cellular metabolites play a role in regulating genes involved in antibiotic resistance, and suggest that succinate and its derivatives could be used to enhance antibiotic efficacy.
| Poster #: 78 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, alcohol, neurodegeneration Project Title: Ethanol interacts with mutations in parkin to damage the central nervous system during Drosophila development Author List: Navneet Sanghera, Graduate, Biological Sciences, San Jose, Presenting Author; Reza Almassi, Undergraduate, Biological Sciences, San Jose, Presenting Author; Rachael French, Faculty, Biological Sciences, San Jose Abstract Fetal Alcohol Spectrum Disorder (FASD) results from developmental exposure to alcohol in mammals, and causes a variety of developmental deficits, including developmental delays, low body weight, intellectual disabilities, behavioral changes, and reduced brain size. In fact, FASD is the leading non-genetic cause of intellectual disabilities worldwide. We have established a Drosophila model of Fetal Alcohol Spectrum Disorder (FASD), a highly variable disorder that results from the consumption of alcohol during pregnancy. Exposing fetuses to alcohol during development can lead to effects including but not limited to developmental delays, low body weight, intellectual disabilities, behavioral changes, and reduced brain size. We have replicated most of these effects in Drosophila exposed to alcohol during development. We also carried out a large-scale genetic screen for mutations that alter the sensitivity of flies to developmental alcohol exposure (DAE). Through this screen, we found that mutations in the gene Dementin (Dmtn), which encodes the Drosophila ortholog of the Alzheimer Disease associated protein TMCC2, result in sensitivity to DAE. Using quantitative RT-PCR and locomotion assays, we showed that DAE results in changes in Dmtn expression, and that adult flies, after exposure to DAE, show climbing defects indicative of CNS dysfunction. Such climbing defects are also seen in fly models of neurodegenerative diseases (including Alzheimer’s Disease). Neurodegenerative diseases attack the central nervous system which can cause issues with movement, power, senses, and cognition. We hypothesize that DAE causes neurodegeneration (and may target known pathways involved in neurodegeneration). Currently, we are surveying of the interaction between DAE and Drosophila models of neurodegeneration specifically focused on mutants associated with proteins involved in Parkinson’s Disease. We will present the results of rearing Pink and park mutant flies in ethanol. These two proteins work together in mitochondrial quality control by tagging damaged mitochondria with ubiquitin. Interestingly, our results suggested an inverse relationship between these two mutants when exposed to alcohol during development. The park mutants were highly sensitive, while Pink1 mutants were resistant to the effects of alcohol exposure. In addition, we have performed climbing assays to test the health of the CNS system and our analysis indicates that DAE exacerbates climbing defects in young park mutants, but this effect does not worsen over time (suggesting an effect on CNS development). We will also present data examining the interaction between DAE and climbing ability in Pink1 mutants and results from survival assays on other Parkinson’s disease associated mutants. | Poster #: 79 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: small RNA, antibiotic resistance, genetic circuit Project Title: Using a Genetic Circuit to Understand the Antibiotic Resistance Mechanism of small RNA MicF Author List: Nikita Opel, Undergraduate, Biology, Northridge, Presenting Author; Amy Sariles, Undergraduate, Biology, Northridge, Presenting Author; Abstract Antibiotic resistance has been an increasing issue in treating bacterial infections. Small RNAs (sRNAs) can quickly regulate various biosynthetic pathways within bacteria based on changes in their environment. Our research focus lies with the sRNA MicF, and its contribution to configuring antibiotic resistance in Escherichia coli. In the presence of antibiotics, E. coli produces MicF as a stress response. MicF then decreases the expression of the outer membrane protein OmpF, preventing the passage of antibiotics into the cell. The objective of this project is to understand how sRNAs, like MicF, influence antibiotic resistance in E. coli by being able to accurately detect its change in concentration when exposed to different environmental conditions. To accomplish this, we constructed a synthetic gene circuit that detects MicF. The proposed circuit is composed of three parts: MicF sensing, amplification, and detection. The circuit components are encoded on plasmids, which are then inserted and expressed in E. coli. Direct complementary binding of MicF to the sensing component of the circuit causes downstream events that lead to expression of the reporter gene, green fluorescent protein (GFP). GFP is produced in the presence of MicF whereas in the absence of MicF, the opposite is true. Circuit function is assessed based on fluorescence and optical density readings of the cells. After testing different combinations of the MicF sensing and amplification portion, we found two that expressed GFP in the presence of MicF with high dynamic range. We performed a quantitative analysis of the detection of MicF by testing two circuits with different concentrations of MicF to determine their sensitivity. Our next step is to add the detection component of the circuit to further increase the circuit sensitivity. With increased sensitivity in detecting MicF, the circuit will allow us to investigate various environmental triggers of MicF and their influence on the antibiotic resistance mechanism.
| Poster #: 80 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: epigenetics, neurodegenerative, Alzheimers Project Title: Identifying Potential Alzheimer’s Disease Biomarkers Beyond Amyloid-Beta and Tau Author List: Frank Cai, Graduate, Computer Science, San José, Presenting Author; Shamika Majmudar , Graduate, Computer Science, San José, Presenting Author; Rheya Mirani, Graduate, CS Department, San José Abstract Project Category: Bioinformatics
Motivation: Alzheimer's Disease (AD), as well as other forms of Mild Cognitive Impairment (MCI), affects more than 50 million people globally, incurring costs exceeding one trillion US dollars annually. The buildup of Amyloid-Beta (Aβ) and Tau proteins in the brain produced by the APP gene has been identified as an important cofactor in the onset and progression of AD and MCI. However, although patients diagnosed with AD or MCI exhibit Aβ and Tau buildup, approximately 40% of the subjects with Aβ and Tau buildup are not diagnosed with AD or MCI. While Aβ and Tau build up contribute towards AD or MCI, it is not sufficient. Therefore, we hypothesize the involvement of several epigenetic interactions between APP and related genes in addition to the buildup of Aβ and Tau proteins that might explain the onset and progression of AD and MCI.
Project Goal: To identify potential epigenetic biomarkers of AD and MCI, beyond the buildup of Aβ and Tau proteins, with a novel Python-based epigenetic computational pipeline.
Methods used: The developed pipeline has the ability to discern and predict structural and functional alterations in SNP-mutated proteins from epigenetically associated genes, such as APP. It also has the ability to perform statistical analysis between the predicted SNPs and their associated diseases. Multiple molecular biometrics have been employed resulting in nearly 70% accuracy in predicting structural and functional modifications. Hypothesis tests namely the two-tail F-test, t-test and the chi-square test have been conducted to statistically analyze 202,827 SNPs in 219 genes that are epigenetically related to APP. Finally, a comparison was made between the statistically analyzed SNPs and the AD and MCI patient mutation data from the AD Neuroimaging Initiative (ADNI) database.
Results: Out of the 202,827 SNPs analyzed, the Python-based epigenetic pipeline predicted 29,523 SNPs to be significant. Among the significant SNPs, 77 SNPs associated with 54 genes are contained in the ADNI database consisting of SNP data from healthy individuals (control group), AD, and MCI patient groups. The hypothesis test results from the F-test, t-test, and chi-square test carried out between the control, AD, and MCI groups predicted certain functional changes thereby confirming the significance of these 77 SNPs.
Conclusions: Using an integrated and automated epigenetic pipeline, we were able to identify a significant number of SNPs that are epigenetically associated with AD and MCI. These significant SNPs warrant further investigation as potential biomarkers linked to the onset or progression of AD or MCI.
| Poster #: 81 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Sinorhizobium meliloti, two-component system, sRNA Project Title: Investigating if the small RNA gene SmelC647 is transcriptionally regulated by ExoS/ChvI two-component signaling in Sinorhizobium meliloti Author List: Jackie Cruz, Undergraduate, Biological Science, Fullerton, Presenting Author; Megan Schreiber, Undergraduate, Biological Science, FullertonEsther Chen, Faculty, Biological Science, Fullerton Abstract Nitrogen is an essential element for plant growth, but the most abundant form of nitrogen, dinitrogen, is inaccessible to most organisms. Many agricultural fertilizers are produced by fixing nitrogen industrially, which causes air and water pollution. In contrast, legume plants like Medicago sativa can have a symbiotic relationship with nitrogen-fixing bacteria, such as Sinorhizobium meliloti. In this symbiosis, the plant host receives usable forms of nitrogen from the bacteria, while the bacteria receive carbon nutrients in return. This symbiosis can improve crop growth and reduce the use of environmentally harmful fertilizers. The two-component system ExoS/ChvI in S. meliloti plays a vital role in the symbiosis. The histidine kinase, ExoS, phosphorylates the response regulator, ChvI, which regulates gene expression important in the symbiosis process. From previous studies, small RNA (sRNA) encoding genes have been found to be candidates for ExoS/ChvI regulation. sRNAs are short non-protein-encoding RNAs that base-pair with specific mRNA transcripts, thereby affecting post-transcriptional gene expression. Using transcriptional fusion experiments, we investigated if the ExoS/ChvI signaling pathway transcriptionally regulates the expression of the candidate sRNA gene SmelC647. The upstream region of SmelC647 was ligated into a plasmid containing the GUS reporter gene to create a transcriptional fusion allele. The transcriptional fusion plasmid was conjugated into S. meliloti wildtype, then transduced into the following recipient S. meliloti strains: ChvI wildtype, ChvI gain-of-function (GOF) mutant, and ChvI partial-loss-of-function (LOF) mutant. GUS activity assays were used to compare SmelC647 gene expression in each strain. We found that the SmelC647 transcriptional fusion showed increased GUS activity in both the ChvI GOF and ChvI LOF mutants compared to the wild-type strain. This change in SmelC647 expression in the ChvI mutants compared to wild-type indicates that SmelC647 is regulated by the ExoS/ChvI pathway. However, since the two ChvI mutant strains did not show a reciprocal change in SmelC647 expression, additional transcriptional regulators may also be involved in controlling SmelC647 expression. Investigating the genes regulated by the ExoS/ChvI pathway can create foundational knowledge to assist in agricultural applications of sustainable models of nitrogen fixation. This research was supported by NIGMS NIH Award SC3GM144065 to E.J.C.
| Poster #: 82 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Cell cycle, Cardiac, Genes Project Title: Interferon regulatory factor-1: a candidate inhibitor of mammalian cardiomyocyte proliferation Author List: Tianna Young, Undergraduate, Biological Sciences, San José, Presenting Author; Maia Eugenio Quan, Undergraduate, Biological Sciences, San José, Presenting Author; Herman Huang, Graduate, Biological Sciences, San JoséAlexander Payumo, Faculty, Biological Sciences, San José Abstract After injury, the adult human heart fails to completely regenerate resulting in a fibrotic scar and lost contractile tissue. In comparison, other species possess the ability to fully regenerate their hearts. This is dependent on the robust proliferation of heart muscle cells called cardiomyocytes (CMs). CMs in adult mammals are stuck in a state of cell cycle arrest and lack the ability to proliferate. In contrast, neonatal rodents possess a temporary ability to fully regenerate their hearts for the first seven days after birth when their CMs can still divide. Recent studies identified that hormonal signaling from the thyroid gland and sympathetic nerves drive the loss of proliferative potential in mammalian CMs after birth. However, the molecular mechanisms acting downstream of these hormones are unknown. Based on RNA-sequencing data obtained from treating CMs directly with thyroid and sympathetic hormones in vitro, we hypothesize that interferon regulatory factor-1 (Irf1) acts as an inhibitor of CM proliferation. Irf1 is upregulated 2.8-fold after hormone treatment. It is a transcription factor known to induce cell cycle arrest in a variety of human cancer cell lines through a p21-mediated mechanism. The specific goal of this project is to generate an adenovirus-based tool that would allow us to overexpress Irf1 in primary CMs so we can test if it inhibits CM proliferation in vitro. Polymerase chain reaction (PCR) was first performed to clone the Irf1 coding sequence from a rat cDNA library. The product was then recombined into a Multi-Site Gateway cloning entry vector and sequence verified. This entry clone was then further recombined with clones containing the CM-specific cTnT promoter and an internal ribosomal entry site (IRES) driving an enhanced green fluorescent protein EGFP reporter into a destination vector with the adenovirus genome. The final assembled construct was validated by colony PCR and restriction enzyme digestion. Verified constructs were then transfected into AD293 cells to generate adenovirus, which were then harvested as a crude lysate. In future work, we will infect primary CMs with the virus and determine the role of Irf1 in CM proliferation. Defining the molecular mechanisms that limit mammalian CM proliferation may help identify new therapeutic targets in cardiac regenerative medicine. This work was supported by a CSUPERB Howell fellowship awarded to T.Y. and an NIH R16GM146643 grant awarded to A.P.
| Poster #: 83 Campus: Sonoma State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Chrysomela aeneicollis, Geneious , Molecular Biology Project Title: Evolution of heat shock 70 proteins in Willow leaf beetles Author List: Jacob Lapier , Undergraduate, Biology , Sonoma, Presenting Author; Abstract Heat shock proteins are widespread in the Metazoa and they are evolutionarily derived from molecular chaperones found in eubacteria, mitochondria and chloroplasts, and eukaryotic lineages. Heat shock proteins are some of the most conserved proteins among metazoans. The HSP70 family of proteins are induced in responses to temperature and other abiotic stress factors. I investigated the evolution of heat shock proteins in leaf beetles (family Chrysomelidae), focusing on the willow leaf beetle Chrysomela aeneicollis. My approach is to compare the diversification of these genes in beetles to a published paper that investigated the evolution of this gene family in butterflies. I used the program Geneious to upload the sequences of several leaf beetles to a phylogenetic tree from including the HSP70 lineages of other insects. Close homology was found between HSP70-5 proteins from the Glanville fritillary and leaf beetles. I will present the results of further work investigating additional genes in this family.
| Poster #: 84 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, nuclear envelope, stem cells Project Title: Regulation of Intestinal Stem Cells and longevity by the nuclear envelope protein Klaroid (Koi) Author List: Ithan Cano, Undergraduate, Biology, Northridge, Presenting Author; Carlos Asturias, Graduate, Biology, Northridge, Presenting Author; Abstract Multicellular organisms often rely on resident adult stem cells (ASCs) to maintain tissue homeostasis. These multipotent ASCs undergo self-renewing asymmetric divisions, by which they produce an identical copy of themselves and a differentiating sister cell that will replace cells lost to injury, disease or normal tissue turnover. The activity of these cells is governed by pleiotropic master regulator (MR) genes, which exert direct or indirect control over numerous downstream effector genes, most of which remain poorly understood.
In our lab, we use the fruit fly Drosophila melanogaster as a genetic model organism to study the function of MR genes in controlling a specific type of ASC, the intestinal stem cells (ISCs). Here we report our preliminary findings related to the nuclear membrane protein Klaroid (Koi), an experimentally validated target of two important MR genes in Drosophila ISCs. Previous work by others established Koi’s role in repairing double-strand DNA breaks in Drosophila cells in culture, and its requirement for attachment of the nucleus to the cytoskeleton in the Drosophila eye. No previous studies have specifically addressed its role in ISCs.
Here we used two complementary ISC-specific genetic manipulations of Koi (up- and down-regulation), coupled with immunofluorescence microscopy to determine their effect on the number, morphology and relative abundance of different cell types of the intestinal epithelium, both in young flies and during aging. Since our own preliminary observations indicated that Koi overexpression might be protective against aging-induced intestinal dysplasia, we also conducted lifespan assays. Flies with lower Koi expression in ISCs showed a significant shortening in lifespan compared to controls. Surprisingly, flies overexpressing Koi in ISCs had comparable survivorship to controls for approximately 40 days, but experienced a sudden and sharp mortality afterwards. We are currently trying to more carefully characterize the effect of Koi manipulations in ISC regulation, and thus potentially identify the link to their effect on survivability.
| Poster #: 85 Campus: California State Polytechnic University, Pomona Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: archaeal virus, lysogeny, Turriviridae Project Title: Investigating the lysogenic cycle of STIV viruses Author List: Kimberly Aguilar, Graduate, Biological Sciences, Pomona, Presenting Author; Abstract Viruses are the most abundant biological entities found in all domains of life. Archaeal viruses display similar characteristics to viruses found in the eukaryotic and bacterial domains. However, our knowledge of archaeal viruses is limited compared to our knowledge of viruses infecting the other two domains of life. Recent work on a particular virus family has resulted in the emergence of a model system for studying archaeal viruses. Members of the Turriviridae family of viruses infect Sulfolobus species that inhabit acidic (pH 1-4) hot springs (70-80°C) within Yellowstone National Park. STIV1 (Sulfolobus turreted icosahedral virus 1) was the first isolated member of this growing virus family and was the first lytic virus isolated from Sulfolobus. More recently, a new variant (STIV3) was discovered as a lysogen of Sulfolobus. Viruses can undergo two replication strategies: a lytic and a lysogenic cycle. In the lytic cycle, a virus will attach to the host cell, release its genetic material, hijack the host’s machinery to replicate, assemble virions, and egress the host cell. In the lysogenic cycle, the virus integrates into the host’s genome and persists in the host until the conditions are no longer favorable. Subsequently, as the cell replicates, the viral genome replicates as well; the viral DNA is passed onto progeny cells. Despite the abundance of archaeal viruses, little is known about viral integration. Integration of viral DNA is a form of site-specific recombination, but integrases can serve other functions. Even though STIV1 engages in a lytic replication cycle, it encodes an integrase-like protein (A510). However, there is no evidence demonstrating STIV1 integration into its host. With the recent discovery of an STIV variant that integrates, we decided to investigate the integrase proteins in both STIV1 and STIV3. The overall goal of this project is to learn more about the lysogenic cycle in STIV viruses. To learn more about integration, viral mutants will be created in both STIV1 and STIV3. Virus replication and integration will be assayed using qPCR, western blots, and Southern blots. Thus far, two integrase mutants have been generated. The first mutant was created by site-directed mutagenesis of the catalytic residue in the active site. The second mutant is a knockout created by filling-in a unique restriction site that resulted in a frameshift. Both viral mutants will be transfected into the host and tested for virus replication and integration. Understanding the role of the integrase protein in these two related viruses will provide more information about this unique virus family and provide further insight into virus evolution and replication.
| Poster #: 86 Campus: California State Polytechnic University, Pomona Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Delphinium, Complex floral organs, RNA-sequencing, Transcriptome Project Title: Understanding the genetic underpinnings of complex flowers of Delphinium Author List: Ana Alcaraz Echeveste, Graduate, Biological Sciences, Pomona, Presenting Author; Mankirat Pandher, Undergraduate, Biological Sciences, Pomona, Presenting Author; Bharti Sharma, Faculty, Biological Sciences, Pomona, Presenting Author; Rakesh KAundal, Faculty, Non-CSU, Utah State University, Logan Abstract Most information on the genetic networks underlying floral organ identity establishment is from studies conducted on species within the core eudicots or in grasses, primarily on model systems such as Arabidopsis thaliana and Oryza sativa. While this information provides a great foundation to understand the genetic pathways broadly, it is imperative to dig deeper and sample across angiosperm phylogeny to understand the genetic underpinnings of complex floral organs. To this end, we use Delphinium X "Bellamosum," a basal eudicot, a member of the Ranunculaceae family, as our model system. The zygomorphic flowers of Delphinium are composed of two distinct types of sepals and two types of petals. Sepals and petals with spurs and without spurs. The intricacies of these flowers are further exhibited by the paired dorsal petal spurs, which collectively grow into the outer sepal spur's pocket. The unique floral architecture of Delphinium makes it an interesting model system for evolutionary, genetic, and developmental studies. The aims of our study are 1) To understand the development, cell type, and expansion of perianth organs using light and Scanning Electron Micrography. 2) Use RNA-sequencing to understand the gene expression dynamics of sepals and petals. The comprehensive developmental analysis results for aim one show strikingly distinct cell types in the perianth organs. The evolution of such a complexity might be associated with pollination syndrome. For aim two, the extraction of RNA from sampled floral organs was standardized, and RNA libraries for 6-9 biological replicates were sequenced. We generated a reference transcriptome. The differential gene analysis being conducted on floral organs identifies the potential candidate genes underlying the complex floral organ identity of Delphinium.
| Poster #: 87 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: , , Project Title: Roles of the Androgen Receptor and Age on Locomotor Activity and Anxiety-Like Behaviors in Mice Author List: Adren Blanco, Graduate, Long Beach, Presenting Author; Darren Leung, Graduate, Long BeachJunho Lee, Undergraduate, Long BeachYada Treesukosol, Faculty, Long BeachHoung-Wei Tsai, Faculty, Long Beach Abstract While androgen receptor (AR) is necessary for the display of male-typical sexual and aggressive behaviors in mice, increasing evidence suggests that AR may also play an important role in non-reproductive behaviors, such as anxiety-like and locomotor behaviors. Using an open field maze to measure anxiety-like and locomotor behaviors, a large number of studies comparing young and aged animals have reported behavioral changes from young adulthood to middle age. However, little is known about the role of AR in mediating the effect of sex and age on those behaviors. In addition, systems for automated behavior analysis from videos have been increasingly used to provide the more reliable and objective investigation of specific behavioral parameters. To address these, we conducted the open field test to measure exploratory and locomotor activity in young and middle-aged, male mice carrying the testicular feminization mutation (Tfm) in the Ar gene as well as their wild-type littermates. Behavioral testing was recorded by a video camera mounted above the arena (L69 cm × W52 cm), and subsequently analyzed by EthoVision XT, a computer-assisted automatic tracking software. During young adulthood (5.26 ± 0.22 months old), young Tfm mice (n=17) spent more time in the center zone compared to wild-type males (n=22) (p=0.012). On the other hand, wild-type females (n=21) entered the center zone more frequently than both wild-type (p=0.014) and Tfm males (p=0.026). During middle age (12.04 ± 0.28 months old), wild-type males spent more time in the center than wild-type females (p=0.017), however both wild-type male (p=0.002) and wild-type female mice (p=0.038) entered the center zone more frequently than Tfm males. In addition, regarding locomotion, young and middle-aged Tfm mice both traveled a shorter distance than their age matching wild-type littermates of (p<0.001). In summary, our findings indicate that in mice, AR distinctly regulates specific components of anxiety-like behavior expressed in the open field paradigm in an age-dependent manner. Furthermore, we have also observed the critical role of AR in augmenting locomotor activity independent of sex, age, or androgen levels.
| Poster #: 88 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: HER2 positive , breast cancer , metabolomics Project Title: Investigating one carbon metabolic pathway in HER2-positive breast cancer Author List: Karina Hernandez-Hernandez, Graduate, Biology , Fresno, NIH Bridges to doctorate program , Presenting Author; Abstract Breast cancer is one of the most common cancers for women in the United States affecting nearly 300,000 women annually (American Cancer Society). Of that, 15-20% are diagnosed with overexpression of Human epidermal growth factor 2 (HER2). Studies have shown that patients diagnosed with HER2 overexpression are also associated with poor survivability due to its aggressive growth and recurrence. Although there are current therapies targeted for women with HER2 breast cancer, they often develop drug resistance after multiple exposures to anti-HER2 therapies. Because of this continued resistance and growth, understanding the molecular mechanisms of breast cancer metabolism is key in identifying new strategies to target the disease. Previous research in our lab pointed towards a positive role of the glycine/serine/threonine metabolic pathway or one-carbon metabolism. It is used by cancer cells for the biosynthesis of nucleic acids, proteins and lipids allowing for high capacity cell growth and survivability. For this project, we use a series of eight breast cancer cell lines with low, medium and high HER2 expression. Adapting each cell line for growth in two dimensional(2D) and three dimensional (3D) tissue culture to a common medium will facilitate evaluation of their HER2 expression by Western blot. In combination with high resolution one dimensional (1D) and two dimensional (2D) nuclear resonance spectroscopy (NMR) to further analyze metabolite changes to validate the role of one-carbon metabolism. This approach allows for better understanding of the global metabolic changes associated with HER2 positivity. We are currently re-adapting our cell lines to a common growth medium in two dimensional tissue culture and results show that extracted metabolites are still stable and can be re-evaluated on different instruments over time. These results are key in developing our understanding for potential biomarkers for metabolomic blockade that can be used in conjunction with current anti HER2 therapeutic strategies.
| Poster #: 89 Campus: California State University, East Bay Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: sleep, neural circuits, drosophila Project Title: Octopamine regulates sleep and context-dependent arousal via distinct neural pathways Author List: Preeti Sundaramurthi, Graduate, Biology, East Bay, Presenting Author; Martin Reyes, Staff, Psychology, East BayYi Shen Lee, Graduate, Biology, East BayDivya Sitaraman, Faculty, Psychology, East Bay Abstract Sleep is a fundamental behavioral state important for survival, and highly conserved across species. Although the physiological function of sleep is unknown, sleep deprivation leads to a range of cognitive, motor, attentional, and emotional deficits. An essential aspect of sleep-wake behavior is the ability to rapidly transition from one state to another, and persist in that state to carry out biologically important functions (for example- waking up and escaping upon receipt of sensory stimuli signaling danger, foraging, or staying awake while engaging in social behaviors).
Although goal-directed motivational processes and sleep expression have to be balanced and co-regulated for survival and reproduction the circuit mechanisms by which mutual exclusivity is enforced remain unknown. We recently identified a class of octopamine neurons (OA) that suppress sleep and promote courtship in a context-dependent manner. Using genetic, physiological, and behavioral approaches we find that molecular signaling and distinct circuit connectivity with sleep and arousal-regulating neurons within the central complex and mushroom body, OA mediates sleep suppression and courtship in a sex-specific manner. Here we will present these results and discuss potential mechanisms by which OA regulates sleep with a focus on tools we have developed to investigate key central complex neurons downstream of OA.
| Poster #: 90 Campus: California State University, Monterey Bay Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: muscle, Drosophila, anoikis Project Title: Investigating the role of bantam microRNA in promoting Drosophila muscle precursor homeostasis Author List: Riley Moulton, Undergraduate, Monterey Bay, Presenting Author; Paola Cabezas, Undergraduate, Monterey Bay, Presenting Author; Allysa Marie Domingo, Undergraduate, Monterey Bay Abstract In order for muscles to develop and function appropriately, precursor cells need to undergo stereotyped collective migration while also eliminating cells that behave abnormally. In Drosophila embryonic development caudal visceral mesoderm (CVM) cells undergo collective migration along a trunk visceral mesoderm (TVM) track. The TVM expresses ligands for various signaling pathways including the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) pathways. Previous research has uncovered a quality control system that utilizes both FGF and BMP signals to promote the elimination of abnormally migrating muscle precursor cells. However, this system does not account for the elimination of all mis-migrating cells, suggesting that a supporting quality control system is likely in place. We hypothesize that the microRNA bantam (mir-ban) is likely an additional component of this quality control system. mir-ban is a known downstream target of BMP signaling and is a negative regulator of the cell death protein Head involution defective (Hid). To investigate the effect mir-ban has on CVM cells we collected and fixed Drosophila embryos, focusing on wildtype, gain of function, and loss of function mutants. By performing fixations and immunohistochemistry stainings we were able to use both light and confocal microscopy to see how the CVM cells react in response to mir-ban overexpression or mir-ban loss of function. Loss of mir-ban results in decreased CVM cell number, suggesting a requirement for either CVM cell proliferation or survival. Additionally, preliminary results suggest that mir-ban expression supports survival of a small population of CVM cells even in FGF mutants, suggesting that it functions as a secondary quality control system prior to muscle assembly. We believe the insights from this study may provide insights into how microRNAs control homeostasis in organ systems, which has implications for both cancer biology and ex vivo organ generation.
| Poster #: 91 Campus: California State Polytechnic University, Pomona Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Cell Cycle, Genistein , CDC7 Project Title: Studying the Effects of Genistein on the Saccharomyces cerevisiae Cell Cycle Author List: Joseph Alas, Graduate, Biological Sciences Department, Pomona, Presenting Author; Wendy Dixon, Faculty, Biological Sciences Department, PomonaKarine Le Roch, Faculty, Department of Molecular, Cell and Systems Biology, Non-CSU, University of California, Riverside Abstract One of the leading causes of morbidity and mortality in the world is cancer. The most frequently diagnosed cancer types worldwide are lung, breast, and colorectal cancer. While many chemotherapeutics and anticancer drugs have been developed, they do not work on all types of cancers and can have unwanted side effects. Therefore, studying alternative agents is important. Genistein, a phytoestrogen found in soybeans, is known to lower the risk of cancer in patients, though limited research has been done. In some in vitro cancer cells, genistein has been shown to induce apoptosis. It has also been shown to have an effect on cell cycle progression including arrest in cell cycle checkpoints. In other cancer cells, genistein has been shown to override the G1/S phase arrest and increase cell division. The goal of our research is to develop a model to identify at the molecular level the effects of Genistein on various cancer cells. In particular we use the yeast Saccharomyces cerevisiae with different genetic backgrounds to mimic some of the phenotypes observed in cancer cells. We also use hydroxyurea to induce cell cycle arrest and analyze the effect of genistein and its potential on overriding the G1/S phase arrest. Using microscopy technique and FACS analysis as well as proteomic and transcriptomic studies, we aim to identify all molecular pathways affected by genistein. Preliminary results indicate that genistein can only overrides the cell cycle arrest in S. cerevisiae overexpressing CDC7 (Cell Division Cycle 7) gene. Several experiments are ongoing to validate our results and identify additional proteins affected by genistein.
| Poster #: 92 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, neurobiology, sleep Project Title: The molecular clock is necessary for downregulation but not upregulation of sleep drive in the Drosophila model Author List: Cynthia Hsu, Faculty, Bioloty, Fresno, Presenting Author; Abstract Homeostatic sleep centers promote sleep in response to prolonged wakefulness, but their contribution to circadian-regulated daily sleep is still unclear. We observed that silencing a small population of neurons that drive homeostatic sleep, those labeled by 23E10-Gal4, reduces sleep but only during the day, and show through long-term in vivo imaging that these sleep promoting neurons also show their peak activity during the day. We show that the 23E10+ neurons contribute to the maintenance of daytime siesta, and that both sleep and daytime neuronal activity remain high even in the absence of a molecular clock (a functioning period gene). However, clock signaling through the neuropeptide pigment dispersing factor (PDF) is necessary for downregulating 23E10+ neuronal activity and preventing mistimed sleep onset following the light-dark transition. Without functional PDF signaling, calcium activity in 23E10+ neurons remains elevated throughout the latter half of the day, resulting in earlier onset of night sleep. Silencing 23E10+ neurons rescues the timing of night sleep onset in pdfr mutants. Thus, the circadian clock suppresses, rather than recruits, homeostatic sleep neurons, in order to appropriately time daily wake and sleep.
| Poster #: 93 Campus: California State University, Sacramento Poster Category: Other Keywords: general chemistry, project-based learning, materials science Project Title: Incorporating a research-based laboratory project into the General Chemistry II Course Author List: Joshua Jaison, Undergraduate, Department of Chemistry, Sacramento, Presenting Author; Kaeryn Cruz, Undergraduate, Department of Chemistry, Sacramento, Presenting Author; Okeyiwobi Nnedimma, Undergraduate, Department of Chemistry, SacramentoXisen Wang, Faculty, Department of Chemistry, SacramentoYujuan Liu, Faculty, Department of Chemistry, Sacramento Abstract Project-based learning is an effective practice that has been used to improve student learning and engagement in chemistry laboratory courses. In a project-based activity, students effectively apply acquired knowledge and develop solutions to solve real-world problems. Therefore, students develop deep content knowledge as well as critical thinking, collaboration, creativity, and communication skills. The 2015 ACS guidelines encourage programs to integrate modern topics in chemistry into the undergraduate curriculum, such as materials science, environmental chemistry, and so on.
The project aims to introduce research experience to the General Chemistry II students by incorporating a research-based laboratory project. A materials science project has been developed based on project-based learning. The project includes synthesis, characterization, and application of a porous material. In particular, only water and ethanol were used during the synthesis of the material under room temperature and atmospheric pressure, which makes the project suitable for introductory-level laboratory courses. Moreover, the porous material was applied to remove organic pollutants from water, which integrates chemistry into real-world contexts. The project has been implemented in the General Chemistry II laboratory course for two semesters. Through this project, students learned about modern solid-state analytical techniques and the environmental impact of chemistry, as well as gained laboratory experience analogous to that of a researcher working in the field of materials science. After the project, students’ comments were collected. Students’ responses showed that they liked the experiment, in particular the easy synthesis and application of chemistry to solve real-world problems.
This work is supported by CSUPERB Curriculum Development Grant Program and Provost’s Progress to Promotion (P3) Program.
| Poster #: 94 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Other Keywords: cross resistance, food preservatives, antibiotic resistance Project Title: Antibiotic and Food Preservative Cross-Resistance in Bacteria Author List: Libby Hodge, Undergraduate, Biological Sciences, San Luis Obispo, Presenting Author; Haley Russell, Undergraduate, Biological Sciences, San Luis Obispo, Presenting Author; Jennifer VanderKelen, Ph.D., Faculty, Biological Sciences, San Luis Obispo Abstract Repeated or consistent exposure of bacteria to biocides can select for individual resistant cells giving rise to a population of biocide-adapted bacteria. We are investigating whether adaptation of Pseudomonas fluorescens and Escherichia coli to a food preservative, diacetyl, is correlated with increased resistance to antibiotics, a phenomenon called cross-resistance. Bacteria were adapted to diacetyl by sequential exposure to the biocide using a disk diffusion method. Cultured media was spread on an agar plate and a filter disk with the biocide was placed in the center. Biocide diffusion from the disk inhibits bacterial growth creating a halo of no growth around it. Cells were collected from the halo edge and re-exposed to diacetyl. As bacteria adapted to the biocide, the halo diameter decreased until the bacteria grew up to the edge of the biocide-impregnated disk. Single isolates from the adapted population were obtained and reassessed for adaptation. The resulting diacetyl-adapted strains from both species were analyzed using minimal inhibitory concentration (MIC) assays for cross-resistance to various antibiotics. Cells were exposed to different concentrations of antibiotics to determine the lowest concentration (MIC) that inhibits cell growth. Resistant cells can grow in higher concentrations of antibiotics. Cell density at the end of each assay assessed improved growth at sub-MIC levels. P. fluorescens replicates of the MIC were performed in a Curriculum Undergraduate Research Experience (CURE) class for first-year undergraduates. Three of the P. fluorescens diacetyl-adapted strains consistently showed cross-resistance to both chloramphenicol and ciprofloxacin antibiotics. Compared to the wild-type response to antibiotics, 5 of the E. coli diacetyl-adapted strains showed a slight decrease in sensitivity to gentamicin. Both results indicate that adaptation to diacetyl provides a competitive advantage when exposed to antibiotics. The development of antibiotic resistance poses a huge challenge to modern healthcare so it is vital we understand all mechanisms under which it can develop. In the future, we will explore what bacterial resistance mechanisms, such as increasing efflux pumps to remove excess biocide and antibiotics, result in cross-resistance. This research was funded by the Bailey College of Science and Mathematics at Cal Poly. A special thank you to Dr. Christopher Kitts for his contributions to this project.
| Poster #: 95 Campus: San José State University Poster Category: Other Keywords: catalysis, autoxidation trials, catecholase-like activity Project Title: Investigating autoxidation contributions to the catecholase-like activity of copper-containing enzyme mimics Author List: Dalia Cruz, Undergraduate, Chemistry, San José, Presenting Author; Nicole Caparros, Undergraduate, Chemistry, San José, Presenting Author; Dakota Balcer, Undergraduate, Chemistry, San JoséBrenda Lu, Graduate, Chemistry, San JoséMadalyn Radlauer, Faculty, Chemistry, San José Abstract Enzymes are important in facilitating chemical reactions because of their selective nature and ability to lower the activation energy barrier. Catechol oxidase is a dicopper enzyme that catalyzes the transformation of ortho-diphenols to ortho-quinones in the presence of molecular oxygen, a reaction we commonly see in the enzymatic browning of damaged fruits. Many scientists have synthesized their own catechol oxidase mimics, aiming to reproduce its oxidation activity, using the less-toxic, less-reactive 3,5-di-tert-butylcatechol (3,5-DTBC) as a model substrate. Running control reactions without catalyst when testing the catalytic activity of copper complexes for catecholase-like activity, we observed significant amounts of autoxidation. Surprisingly, previous studies typically do not mention autoxidation of 3,5-DTBC and instead solely focus on catalytic oxidation. We decided to study the autoxidation of 3,5-DTBC to further our understanding of previously published work. We use UV-vis spectroscopy to track the oxidation of 3,5-DTBC to 3,5-di-tert-butyl-quinone (3,5-DTBQ) via atmospheric oxygen. We are able to use this method because of the prominent peak at 400 nm that represents the production of 3,5-DTBQ. The extent of autoxidation appears larger than suggested by the literature, which, when it was mentioned, described it as “negligible” or “nonexistent,” so we decided to quantify autoxidation of 3,5-DTBC in different solvents and at a range of concentrations. We hypothesized that there is a direct relationship between the concentration of 3,5-DTBC and its absorbance, which may explain the discrepancy of results in the literature. To begin, we determined the molar absorptivity of 3,5-DTBQ in methanol, THF, and acetonitrile. Next, we conducted autoxidation trials of 3,5-DTBC in methanol and acetonitrile starting with 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, and 1.0 mM concentrations of 3,5-DTBC, under temperature controlled conditions, monitoring by UV-vis spectroscopy. We are developing a standard operating procedure for the purification of 3,5-DTBC and study of its catalytic oxidation that effectively accounts for autoxidation and will test it out with our own catalytic trials using pyridyl-bisimine-mono-copper complexes. This research was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) (R15GM132857-01A1) and by a CSU BIOTECH Presidents' Commission Scholarship (Dalia Cruz).
| Poster #: 96 Campus: California State University, East Bay Poster Category: Other Keywords: migraine, rat, enzyme Project Title: Effects of simultaneous inhibition of fatty acid amide hydrolase and soluble epoxide hydrolase on migraine pain in rats Author List: Siena Gunari, Undergraduate, Psychology, East Bay, Presenting Author; Daniel Carr, Undergraduate, Psychology, East Bay, Presenting Author; Christopher Chin, Staff, Psychology, East BayStephanie Sanchez, Staff, Psychology, East BayStevan Pecic, Faculty, Chemistry and Biochemistry, FullertonRam Kandasamy, Faculty, Psychology, East Bay Abstract Migraine is the most common neurological disorder in the world, and is characterized by severe headaches, nausea, and light sensitivity. While current migraine treatments, including opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), effectively address mild pain, they have dangerous and uncomfortable side effects. Moreover, modern anti-migraine drugs lack novel mechanisms of action, often resulting in side effects similar to traditional approaches. Our aim is to revolutionize migraine pain management by finding new medications with novel mechanisms that can provide relief from migraine without causing any negative side effects. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) are two pain-related enzymes. The potential benefits of dual-enzyme inhibitors include fewer drug-to-drug interactions and improved drug synergism compared to the administration of just one enzyme inhibitor. These dual inhibitors may require lower doses than the traditional anti-migraine agent, sumatriptan. We hypothesize that simultaneous inhibition of FAAH and sEH will alleviate migraine pain in rats. We investigated the effects of various doses of dual inhibitors of sEH and FAAH in a rat model of migraine to assess its efficacy. We used the von Frey test, which involves a thin, calibrated, plastic filament that is applied to the plantar surface of the hind paw to measure grams of force and the Hargreaves test, which uses an infrared heat source to measure the latency of hind paw withdrawal to assess pain thresholds. To induce migraine pain, we injected 10 mg/kg nitroglycerin, a vasodilator that induces migraine-like pain, every other day for five days. Thirty minutes after the last injection of nitroglycerin, we administered an intraperitoneal injection of either the dual-enzyme inhibitor in varying doses (0.1, 0.3, 1, 10 mg/kg) or sumatriptan. We tested von Frey and Hargreaves at 30, 60, and 120 minutes after drug injection. Repeated nitroglycerin injections lowered sensory thresholds in male rats indicating migraine-like pain. However, the effects of the dual inhibitor on migraine may be dose-dependent indicating that further studies are needed to determine the full efficacy of FAAH and sEH inhibition in alleviating migraine-like pain in rats. Funded by NIH grant R16 GM150781 (RK).
| Poster #: 97 Campus: California State University, Fresno Poster Category: Other Keywords: plasma, surface analysis, biomedicine Project Title: Functionalizing the Surface of PTFE with Primary Amine Groups: Can it Be Done Using Plasma Surface Modification? Author List: Sannad Jawad, Undergraduate, Chemistry and Biochemistry, Fresno, Presenting Author; Abstract The use of synthetic polymers has grown in many disciplines, especially in the medical field. In recent years, polytetrafluoroethylene (PTFE) has become widely used in cardiovascular patients for its favorable characteristics. This polymer has inert properties, is highly resistant to wear and tear, and has been used to treat several cardiovascular conditions such as congenital heart defects. PTFE, however, can present some challenges when introduced into biological environments. One challenge is that it can invoke a negative immune response which can be detrimental to treatment integrity. To combat this issue, researchers have explored methods to attach primary amine groups to the surface of PTFE by way of plasma surface modification. Plasma, the fourth state of matter, is a common method used to modify the surface of polymers without altering their bulk properties. Many studies have shown success in detecting nitrogen on the surface but concrete evidence of primary amine implantation is limited.
The goal of this study is to determine if primary amine group incorporation can be achieved on the surface of PTFE by a two-step plasma surface modification. In the first, water vapor plasma was used to implant active species, mainly hydroxyl groups. In the second step, the surface was treated with ammonia plasma, systematically varying both feedgas gas pressure and applied power. After treatments, a series of surface characterization techniques were performed to investigate the presence of amine groups. To begin, Fourier transform infrared spectroscopy (FTIR) was employed to determine if the IR active N-H bonds of amine groups were present. Subsequently, X-ray photoelectron spectroscopy (XPS) was used to determine the elemental composition of the surface, particularly using high-resolution fluorine, nitrogen, and oxygen spectra. Contact angle goniometry was also conducted to assess the hydrophilicity of the polymer surface. Finally, the orange II colorimetric assay was carried out to discern definitively the quantity of primary amine groups on the surface. Collectively, the results of this study will aid in the quest to determine if primary amine group functionalization of PTFE is achievable, extending the use of PTFE in medical contexts.
| Poster #: 98 Campus: California State University, Sacramento Poster Category: Other Keywords: Plastics, Rheology, Project Title: Rheological characterization of environmental plastics degradation Author List: Wesley Eby, Undergraduate, Physics and Astronomy, Sacramento, Presenting Author; Mikkel Herholdt Jensen, Faculty, Physics and Astronomy, Sacramento Abstract An estimated 22% of waste from the roughly 300 million tons of plastic materials, produced globally each year, ends up in our oceans, rivers, and creeks. Varied environmental conditions such as exposure to UV light and salinity, degrade these plastics into microplastics which are then taken up by organisms in the ecosystem, and subsequently become incorporated into the food chain. The adverse effects these molecules have on biological systems is well studied, but how plastics become microplastics and which combination of environmental conditions contribute to this breakdown, is less understood. Rheological techniques that can investigate the degradation of plastics into microplastics are needed. Our goal was to develop an instrument to collect data and have a semi-automated analysis that allows us to characterize these factors for plastics samples. We present the results of our development which consists of a force transducer and length controller, as well as code developed in Matlab used to analyze and characterize the collected force-extension data. We also present example uses of the system along with the data and analysis from those preliminary investigations.
| Poster #: 99 Campus: San Francisco State University Poster Category: Other Keywords: ImageJ, allometry, tissue regeneration Project Title: The effects of larval wing disc tissue damage in the hornworm, Manduca sexta, on body allometry in the adult Author List: Andrea Silva, Undergraduate, Biology, San Francisco, Presenting Author; Leslie Flores, Graduate, Biology, San FranciscoJoana Navarro, Undergraduate, Biology, San Francisco, Presenting Author; Megumi Fuse, Faculty, BIOLOGY, San Francisco Abstract Maintaining body allometry is a mechanism to ensure an organism’s survival in its specific environmental niche but is also important for proper physiological functioning. For instance, many holometabolous insects, those that go through complete developmental changes, depend on proper wing development to reproduce and feed properly. Proper development of organs and limbs in all animals is likewise important for proper communication and maintenance of physiology. External stresses may disrupt tissues within an organism sufficiently to alter development during the healing process. This has been noted in a few holometabolous insects (Manduca sexta, the hornworm, and Drosophila melanogaster, the fruit fly), where delays in development may putatively provide time for tissue regeneration to maintain body allometry. Thus, this organism is a useful model for understanding cell regeneration and the importance of body allometry. Whether body proportions are maintained during this regeneration, however, is unclear. We have previously found that cells within damaged wings appear normal, in that the cell count and size are not affected after regeneration. However, whether the wings develop appropriately has not yet been assessed. To test this, M. sexta larval wing imaginal tissues were damaged via irradiation and animals were then analyzed after becoming adults and compared to non-irradiated controls. Wing socket cells were stained with Phalloidin stain, and the size and cell count were determined using ImageJ software. Complete wings and other appendages along with the body length were also dried, weighed, and similarly measured. We did not find significant differences in cell count or size, and there did not appear to be differences between cells of either left or right wings, suggesting regeneration was complete and symmetry was not compromised during regeneration. We assessed whole body parts in a similar manner, and data will be discussed.
| Poster #: 100 Campus: California State University, San Bernardino Poster Category: Other Keywords: Psychopharmacology, Methamphetamine, rats Project Title: Impact of adolescent nicotine exposure on methamphetamine reward Author List: Johnna Richie, Undergraduate, psychology, San Bernardino, Presenting Author; Paola Uribe, Graduate, psychology, San Bernardino, Presenting Author; Alejandra Lujan, Graduate, psychology, San BernardinoAlexandra Mora, Undergraduate, psychology, San Bernardino Abstract Cigarette smoking and vaping are very common during adolescence and the majority of individuals who smoke begin during this developmental period. In the United States, one in three adolescents report experimenting with a tobacco product and approximately a third to one half of these adolescents will eventually become daily smokers, with the peak of experimentation occurring between the ages of 13 and 16. Tobacco use during this age developmental is not solely a result of social influences, such as peer pressure; recent research suggests that biological factors play a role during this period of increased vulnerability. In general, tobacco has a positive effect on mood and behavior, but the initial smoking engagement tends to be aversive. Interestingly, adolescents experience more positive and fewer aversive effects than adults during their first smoke. Importantly, there is a correlation between adolescent nicotine use and adult drug use and nicotine has been suggested to act as a “gateway” to other drug use. For example, adolescent nicotine exposure is associated with later psychostimulant and marijuana use. There are also clear preclinical sex differences with regard to the reinforcing properties of both nicotine and MA. During nicotine self-administration, female rats maintain a higher motivation to obtain nicotine than males, while no sex differences are observed during nicotine-induced reinstatement. Similar to the sex differences observed within nicotine self-administration, female rats are more susceptible to the reinforcing properties of MA than male rats.
this study, we assessed whether early adolescent nicotine treatment differentially affects the rewarding value of methamphetamine (MA) using the conditioned place preference procedure. To this end we exposed male and female rats with nicotine during the adolescent period and later assessed MA-induced conditioned place preference (CPP) in adulthood. Adolescent Sprague-Dawley rats were injected with nicotine (0.16, 0.32, or 0.64, sc) daily for 20 consecutive days beginning on postnatal day (PD) 25. On PD 45, MA-induced CPP was assessed using a 10-day biased CPP procedure, consisting of one preconditioning day, eight conditioning days and one test day. On the preconditioning day, rats received no injection and will be put in the gray placement chamber of the apparatus. After leaving the placement chamber, rats will be allowed 15 min access to the equal size conditioning chambers (access to the placement chamber will be blocked). On the conditioning days, rats received an injection of MA (0 or 1.0 mg/kg, ip) and be placed in their non-preferred compartment or they will receive an injection of saline and were placed in their preferred compartment. intial drug order was counterbalanced between groups. Conditioning sessions lasted 30 min. On the test day, rats were left uninjected and given free access to the two end compartments for 15 min. Test days were recorded, and time spent in the non-preferred compartment was measured. Experimenters were blind to both nicotine and MA treatment conditions. As expected, rats given MA showed a significant increase in time spend in the previously non-preferred chamber. This preference however was altered by sex and nicotine exposure. Specifically, female rats had higher preference scores than similarly treated male rats. addition, rats treated with the high dose of nicotine (0.64 mg/kg) had greater preference scores than saline and rats treated with 0.16 nicotine. These result support the theory that nicotine exposure enhances vulnerability to later drug use and that females are more susceptible than males.
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