2020 Poster Authors and Abstracts

2020 CSU Biotechnology Symposium
Posters with Author Listings and Abstracts

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Poster #: 1
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: biochemical methane potential, landfill, microbial enumeration
Project Title: Effects of waste diversion on biochemical methane potential and microbial population in landfill samples
Author List:
Ayoubi, Lynn; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Blair, Kylie; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Salvador, Brittany; Staff, Civil and Environmental Engineering, California Polytechnic State University, San Luis Obispo
Yesiller, Nazli; Staff; Global Waste Research Institute, Civil and Environmental Engineering, California Polytechnic State University, San Luis Obispo
Hanson, James; Civil and Environmental Engineering, California Polytechnic State University, San Luis Obispo
El Badawy, Amro; Civil and Environmental Engineering, California Polytechnic State University, San Luis Obispo
Yeung, Marie; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: An impending California waste policy AB-32 requires organics be diverted from landfills in hopes of reducing greenhouse gas emission such as methane; though this would also reduce revenue for landfills that convert methane into electricity. Therefore, it is important to estimate the quantitative effect of organics reduction on methane production. The objectives of this study were to 1) calculate the biochemical methane potential (BMP) of municipal solid waste (MSW) samples with varying organic loads, and 2) determine the extent of anaerobic microbes in the MSW samples. Samples were prepared in serum bottles by mixing 2-g synthetic solid waste containing varying amount of organic substance with 30-mL anaerobically digested sludge, and 120-mL defined medium. Organic content for each were designated as 0, 50, and 100% food waste removed (FWR). Serum bottles were purged with N2 gas, sealed with rubber stoppers and aluminum crimps, and incubated at 35°C until gas production ceased. Methane % was determined by injecting 1-mL headspace gas into a gas chromatograph equipped with a packed column and thermal conductivity detector. Adhering to analytical models developed by the US EPA, BMP data was analyzed using first-order decay approximation. A regression analysis was used to calculate k, the first order decay constant (yr-1) using the formula: L=L0(1 ─ e-kt) where: L is the cumulative methane yield at time t (m3/Mg); L0 is the ultimate methane yield. Controls (cellulose, blank, sludge alone) were tested to correct L0. Cumulative methane depreciated with 100% FWR (L0= 123.16, k =0.042), compared to 50% FWR (L0= 189.61, k =0.061) and 0% FWR (L0= 217.72, k =0.085). After the methane production peaked (14 days), most probable number (MPN) was used to enumerate total anaerobes via anaerobic incubation in thioglycolate broth for 48 hrs. The microbial count was 3.8-5.0 x106 MPN/g in 0% and 50% FWR samples, compared to 2.2×106 MPN/g in 100% FWR sample. DNA was extracted for downstream PCR analyses, including endpoint-PCR wherein all samples were positive for the presence of 16S rDNA. A primer set was designed to amplify the methyl coenzyme-M reductase (mcrA) gene, with quantitative PCR showing methanogens present in all samples; however, there was no distinct correlation between Ct values and level of organics removed. In conclusion, our study suggests that diverting 50% and 100% organics from landfill will reduce 13% and 44% methane production, respectively.

Poster #: 2
Campus: CSU San Marcos
Poster Category: Agriculture/Biofuels/Environment
Keywords: phytoplankton , lipids, light
Project Title: Red Light-Blue Light: Alkenone Production in Isochrysis galbana Is Enhanced by Red Light, while Growth is Optimized under Blue Light
Author List:
Huynh, Thi; Undergraduate, Biological Science, California State University San Marcos, Presenting Author, Nagel Award Nominee
Read, Nicole; Lowel High School, Presenting Author
Comstock, Stacey; Undergraduate, Biological Sciences, California State University San Marcos

Abstract: Isochrysis galbana commonly used as a food source in the aquaculture industry because of its nutritional content and the ease with which it can be cultured and harvested. In addition to being a rich source of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, Isochrysis galbana is one of five species of algae that produce alkenones. Alkenones are very long chains fatty acids that contain between 35 and 41 carbon atoms and between 1 and 5 trans double bonds. While the genes and proteins involved in the biosynthesis and metabolism of alkenones remain largely unknown, their ability to absorb UV radiation, and the antifungal and antibacterial properties they exhibit, suggests they may have some valuable commercial applications. For example, alkenones may be useful as an active ingredient in sunscreen, a novel drug delivery system, and/or as a protective coating for food products. Because of these potential commercial applications, the focus of our lab is to determine optimal conditions to maximize alkenone biosynthesis and growth of I. galbana. In this experiment, we determined the effects of different colored lights on the growth and alkenone production when I. galbana was cultured under different colored lights. Cells were grown in f/2 filtered seawater media in 250. ml batch cultures for 14 days under constant illumination at 580±50 lux with green, orange, yellow, blue, red, purple, and white lights at 19°C. Culture were monitored daily. Cell counts were performed using BD Accuri C6 flow cytometer. Lipid content was also assessed using Nile Red staining. After 14 days, cultures were harvested, and total lipids were extracted by using organic solvents. Neutral lipids were further purified using silica chromatography and profiled by gas chromatography-mass spectrometry (GC/MS). Alkenone productivity was highest under the red light while growth of I. galbana was maximal under the blue light. A total of seven alkenones species were identified in I. galbana across the different colored lights. Future research will be directed at fine-tuning conditions to maximize the production of individual alkenones, and the genes and proteins involved in their biosynthesis.

Poster #: 3
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: Nematicides, C. elegans, Plant parasitic nematodes
Project Title: Effects of Chalcones on the Life Span of Several C. elegans Strains
Author List:
Xiong, Daisy; Undergraduate, Biology, California State University, Fresno, Presenting Author
Tapia, Phillip; Undergraduate, Biology, California State University, Fresno, Presenting Author
Jiménez, Sophie; Undergraduate, Biology, California State University, Fresno
Echeveria, Dustin; Undergraduate, Biology, California State University, Fresno
Calderon-Urrea, Alejandro; Biology, California State University, Fresno

Abstract: Plant Parasitic Nematodes (PPNs) cause billions in monetary loss in the agricultural industry each year. Most nematicide agents are a type of pesticide that are harmful to humans and the environment. PPNs are also becoming more resistant to many nematicides. Thus, a new, more environmentally friendly form of pesticide is needed to control nematodes in agriculture to prevent economic losses. Previous research discovered that certain chalcones (17, 25, and 30), derived from the parent molecule 1,3-diphenyl-2-propen-1-one, and belonging to the flavonoid family of organic compounds, are 100% effective against the model nematode Caenorhabditis elegans and the PPN Meloidogyne incognita at concentrations of 10-5 M. In addition, in an attempt to understand the action mechanism of the chalcones, mutant nematodes were identified and isolated that are resistant to Chalcone 17 and Chalcone 30. In the present study, we conducted experiments to study the lifespan of four (4) different strains of C. elegans, including a GFP standard strain used to identify and isolate the mutants, the N2 Wild Type Bristol strain, and the two chalcone mutant strains (Ch17-m and Cha30-m), when treated with Chalcone 17 and Chalcone 30. The purpose of this research is ultimately to test whether Chalcone 17 and 30 mutant strains’ lifespan is the same as the GFP standard strain under normal conditions and under chalcone stress. For the lifespan studies, each strain was synchronized and the L4 stage adults were transferred into 96-well plates. Subsequently, FUDR (to prevent reproduction) and OP50 (food) were added, and after 24 hours, the chalcone treatments were added to each plate. Living, dead, and censored worms were counted on a regular schedule until all the worms were dead. Statistical analysis and curves were developed using the OASIS2 system. Our results indicated that the GFP Standard Strain used to generate the mutants has a similar lifespan as the N2 strain. The two control strains are highly susceptible to both Chalcones. The control groups of C. elegans lived significantly longer than the C. elegans exposed to the chalcones, as expected. When analyzing the GFP standard strain for this experiment, Chalcone 17 was more effective than Chalcone 30 in killing nematodes at the L4 stage. On the other hand, in the N2 Wild Type experiment, Chalcone 30 more effectively killed L4 stage nematodes than Chalcone 17. Finally, both mutant strains have longer lifespans than the control strains.

Poster #: 4
Campus: CSU San Marcos
Poster Category: Agriculture/Biofuels/Environment
Keywords: Brassica rapa, Fire Ecology, Hyrdoseeding
Project Title: The Effects of Fire and Post-Fire Management on Soil Nutrient Cycling in Southern California Chaparral Using Brassica rapa
Author List:
Cartmill, Ryan; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Mey, Ken; Undergraduate, Biological Sciences, California State University San Marcos
Wulf, Gwendalyn; Undergraduate, Biological Sciences, California State University San Marcos
Sethuraman, Arun; Biological Sciences, California State University San Marcos

Abstract: The effects of fire on chaparral soils as well as the effects of hydroseeding as a post-fire management solution are not well known. This study aims to investigate the effects of fire on the nitrogen and carbon cycling in chaparral soils in three different areas: an unburned site, a site that was burned five years prior and left to naturally recover, and a site that was burned and then hydroseeded. This was achieved through a two-week plant growth study using Brassica rapa as a model system grown in soil collected from the unburned, naturally recovering, and hydroseeded stands. The shoot height for each treatment was measured periodically along with their final shoot weight, dry-weight biomass, mineralization rates, total carbon, and total nitrogen. Results from burned stands showed a negative effect on nitrogen cycling in the form of lower rates of nitrification (ug*g/day; p < 0.05) and total extractable nitrate (ug/g; p < 0.05). In the hydroseeded treatment therewas a decrease in nitrogen mineralization (mg/plant; p < 0.05), indicating nutrient limitations and suggesting hydroseeding might not be an ideal method of post-fire management. Results suggest that while hydroseeding is beneficial in the prevention of excess soil erosion and quickened stand recovery, there remains a need for future post-fire management protocols to factor in the long-term negative effects of hydroseeding to native plants and ecosystem recovery

Poster #: 5
Campus: Stanislaus State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: depolymerase, compost, PHB
Project Title: Enumerating Depolymerase-producing Bacteria in Compost
Author List:
Marshall, Miranda; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Avalos, Angel; Undergraduate, Biological Sciences, California State University, Stanislaus, Presenting Author
Hanne, Larry; Biological Sciences, California State University, Chico
Kirk, Larry; Chemistry, California State University, Chico
Thao, My Lo; Biological Sciences, California State University, Stanislaus

Abstract: Compost is thought of by most as a way to turn one’s old banana peels into stinky, nutritious fertilizer. However, the process of composting is a delicate balance of moisture, temperature, and bacterial activity that converts would-be waste into useful soil. Bacterial degradation is an integral part of compost activity because bacteria break down different polymers through the use of depolymerases. In our study, we sought to detect and enumerate the percentage of microbes that possess different depolymerases at different stages of composting. Specifically, we wanted to determine if there would be microbes with different depolymerases (proteases, amylases, and polyhydroxybutyrate (PHB) depolymerases) at different stages of composting. Of particular importance were PHB depolymerase-producers since PHB is a degradable bioplastic. Thus, the isolation and identification of PHB-degrading bacteria may help in the reduction of the volume of plastic in the environment. The UC Davis “hot” compost method was used and samples were obtained periodically to detect bacteria that produce these depolymerases through spread plating and replica plating onto differential media at two temperatures (28 or 45 °C). The data showed that protease producers were the most abundant followed by amylase-producers, then PHB depolymerase producers. This could be due to the substrate availability in the compost (i.e., more protein, less starch, and much less PHB). The results also showed that the total percentage of 45 °C depolymerase-producing bacteria increased as the temperature of the compost decreased over time. This suggests that the 45 °C depolymerase-producing bacteria started the degradation process but were outcompeted by cross-feeders and other depolymerase-producers that thrive at lower temperature as more simple nutrients became available and the compost temperature dropped.

Poster #: 6
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: Thermophile, genome, geothermal
Project Title: Isolation, characterization, and complete genome sequencing of a novel Thermanaeromonas species from Little Hot Creek, CA
Author List:
Manriquez, Alexander; Graduate, Biology, California State University, San Bernardino, Presenting Author
Dodsworth, Jeremy; Biology, California State University, San Bernardino

Abstract: Hot springs in Little Hot Creek, California contain many unidentified, novel microbes. Currently, the genus Thermanaeromonas consists of only two thiosulfate-reducing species, T. toyohensis and T. burensis, both isolated from deep subterranean environments. Enrichment cultures from Little Hot Creek contained a microbe related to Thermanaeromonas. Our research goal was to isolate this Thermanaeromonas from Little Hot Creek and characterize it using phenotypic tests and comparative genomic analysis, so that it can be formally described as a member of this genus. Initial enrichments were performed using xyloglucan as carbon source, with further enrichment in the presence of thiosulfate, acetate, and lactate. Pure cultures were obtained through spread- and streak-plating on this medium solidified with gelrite. The isolate was strictly anaerobic, thermophilic, Gram-positive, and rod-shaped, with terminal endospores observed on cells. Growth was observed between 60-82.5 °C and pH of 5.75 and 8.25, with optimal conditions being 73 °C and pH of 6.5. Thiosulfate and nitrate served as electron acceptors, but not nitrite, sulfate, or elemental sulfur. Lactate, pyruvate, yeast extract, casamino acids, and several different sugars such as arabinose, fructose, lactose, and sucrose served as sole carbon sources. The isolate’s 16S rRNA gene had only 97.48% identity with T. toyohensis and 92.74% with T. burensis, suggesting the isolate is a distinct species. For further comparison, the complete genome sequence of the isolate was obtained by hybrid assembly of short and long reads from the Illumina iSeq and Oxford Nanopore sequencing platforms, respectively, yielding a single, circular 3.19 Mbp chromosome containing 3507 predicted protein-coding genes. Whole-genome comparison with T. toyohensis show only 84.7% average nucleotide identity and 35.3% in silico DNA-DNA hybridization, below the respective 95% and 70% cutoffs for distinguishing distinct species. The isolate’s genome contained a putative bacterial microcompartment gene cluster, which was not present in T. toyohensis, that may be involved in catabolism of propanediol or other alcohols. Overall, the results of the phenotypic and genotypic tests demonstrated that the isolate represents a distinct species, for which the name T. inyoensis is proposed. T. inyoensis represents the first characterized member of this genus from a surface hot spring environment.

Poster #: 7
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: lipoprotein, structural biology, apolipoprotein
Project Title: Ionic Residues in Helix 10 of the C-terminal Domain of Human Apolipoprotein A-I Regulate Self-association
Author List:
Burdick, John; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Nominee
Basi, Rohin; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Burns, Kaitlyn; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Cardiovascular disease is currently the leading cause of death in the modern world. High-density lipoprotein (HDL), or “good cholesterol”, remains a key component in research seeking to development treatments for such diseases. Apolipoprotein A-I (apoA-I) is the central protein of HDL. No high-resolution structure of the protein is currently available, but biophysical characterizations indicate apoA-I is comprised of two domains; a highly ordered N-terminal domain and a less structured C-terminal domain. The C-terminal domain initiates lipid binding and formation of discoidal HDL. Lipid-free apoA-I exists in an oligomeric state, which is also facilitated by the C-terminal domain. Six lysines reside in this domain at positions 195, 206, and 208 in helix 8 (H8) and 226, 238, and 239 in helix 10 (H10). Substitution of all six lysine residues with glutamine resulted in a monomeric form of the protein, indicating that ionic bonds play a role in self-association. To identify the lysines critical for self-association, site-directed mutagenesis was implemented to engineer seven single and two triple mutants. Upon expression in E. coli and purification by N-affinity and gel filtration chromatography, circular dichroism and fluorescence spectroscopy revealed all mutants retained their structural integrity. Dimethyl suberimidate crosslinking analysis demonstrated a strongly reduced amount of self-association for the H10 triple mutant, while the H8 triple mutant was much less affected by the mutations. FPLC elution profiles of the H10 triple mutant suggests that the protein is primarily monomeric, and may be amenable for studies to obtain the high-resolution structure. All H10 single mutants displayed increased elution times, however, not to the extent of the triple mutant, suggesting the H10 lysines work collectively to contribute to self-association. The K226Q mutation produced the largest increase in elution volume of all single mutants, and when nearby glutamate 223 was substituted with glutamine the elution profile of this E223Q mutant matched that of K226Q-apoA-I. This suggests an ionic bond between these two residues. The results of this study indicate that intrahelical salt bridges in H10 play a prominent role in apoA-I oligomerization through stabilization of amphipathic α-helices. Assembly of the H10 helices from neighboring proteins likely results in an intermolecular helix bundle thereby promoting protein oligomerization.

Poster #: 8
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Cancer, Reactive carbonyl species, Aldo-keto reductase
Project Title: Aldo-keto reductases may be a compensatory detoxification pathway in response to Glyoxalase 1 inhibition in cancer cells
Author List:
Ahmad, Saadman; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Reque, Leticia; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Cordova, Benjamin; Graduate, Chemistry & Biochemistry, California State University, Northridge
Tamae, Daniel; Chemistry & Biochemistry, California State University, Northridge

Abstract: Breast and prostate cancers are two of the most frequently diagnosed cancers in the developed world. Glyoxalase 1 and 2 (Glo 1 and 2) have been found to be over-expressed in breast and prostate tumors relative to adjacent normal tissue. In a recent clinical trial of prostate cancer patients (n=11,000), high levels of Glo 1 expression correlated with poor prognosis. Inhibition of Glo 1 by pBrBzGSH(Cp)2 results in cell death in a panel of breast and prostate cancer cell lines. The Glo1/2 pathway detoxifies the reactive carbonyl species such as methylglyoxal (MG) and glyoxal. The reaction of MG with cellular nucleophiles leads to the formation of advanced glycation end products (AGEs), which have been implicated in diabetes, aging and cancer. We are interested in the aldo-keto reductase (AKR) superfamily of NAD(P)H-dependent oxidoreductases as a possible compensatory mechanism for MG detoxification. AKR1B1 and AKR1B10 are known to be overexpressed in hyperglycemic cells. Enzyme kinetic studies have shown that AKR1B1 and AKR1B10 have relatively high catalytic efficiency towards glyoxal and MG. AKR1C3 is over-expressed in castration-resistant prostate cancers. We subjected our panel of cell lines to GLO 1 silencing through siRNA knockdown. Western blots showed robust expression of Glo 1 in our siRNA scrambled control samples and between 65-98% GLO 1 knockdown in our siRNA treated samples. qRT-PCR with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) compliant gene specific primers showed up to a 3-fold increase in AKR1B10 expression in our Glo1 knockdown samples compared to our control samples. Using a pharmacological approach, we inhibited our cell lines with the IC50 values of pBrBzGSH(Cp)2. We observed 2 to 3-fold increase in AKR1B10 and AKR1C1 in the MDA-MB-231 cell line and approximately 2-fold increase in AKR1C3 in the 22Rv1 cell line, as well as increases in AKR1B1 expression in both cell lines compared to our control samples. We are currently investigating changes in expression in sub-IC50 treated cells and developing a method for absolute quantification of mRNA transcripts via qRT-PCR. [This work was supported by a CSUPERB New Investigator Award and is currently supported by an NIGMS SCORE SC2 to D.T.]

Poster #: 9
Campus: Cal Poly San Luis Obispo
Poster Category: Biochemistry
Keywords: neurodevelopment, Western Diet, inflammation
Project Title: NON-ALCOHOLIC FATTY LIVER DISEASE IS ASSOCIATED WITH MEMORY IMPAIRMENT AND CHANGES IN METABOLOMIC PROFILE IN BRAIN CORTEX OF JUVENILE IBERIAN PIGS
Author List:
Zeltser, Nicole; Undergraduate, Animal Science, California Polytechnic State University, San Luis Obispo, Presenting Author
Trahan, Matthew; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Mayer, Isabell; Undergraduate, Animal Science, California Polytechnic State University, San Luis Obispo
Onat , Hannah; Undergraduate, Animal Science, California Polytechnic State University, San Luis Obispo
Nawar, Ishman; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Hernandez, Gabriella; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo
Smith, Victoria; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo
Burrin, Douglas; Baylor School of Medicine, Houston, TX
Strand, Christy; Biological Sciences, California Polytechnic State University, San Luis Obispo
La Frano, Michael; Food and Sciences, California Polytechnic State University, San Luis ObispoBurrin, Douglas; Baylor School of Medicine, Houston, TX
Manjarin, Rodrigo; Animal Science, California Polytechnic State University, San Luis Obispo
Maj, Magdalena; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Growing evidence supports a link between neurodegenerative conditions, such as Alzheimer’s, Parkinson’s, and non-alcoholic fatty liver disease (NAFLD), whereby neurotoxic lipids synthesized in excess by the liver may cross the blood-brain barrier, leading to inflammation, amyloid plaque accumulation, and other hallmarks of neurodegeneration. Improving our understanding of the mechanisms by which diet impacts the brain development early in life has the potential to translate into practices that may improve pediatric health.
Our hypothesis is that pediatric NAFLD in neonatal pigs is associated with changes in the metabolomic signature and increased expression of inflammatory markers in the frontal cortex, as well as impaired memory and decreased physical activity of animals.
In this study, 28 10-d old pigs received during 10 weeks either control (CON) or high-fructose high fat (HFF) diet. Half of the pigs in each group were treated daily with probiotics. Pigs’ memory was assessed using a Novel Object Recognition test (NOR) once per week, while the animal movement was recorded daily to assess overall activity levels. Both behaviors were quantified with BORIS video tracking software. On week 10, all animals were euthanized, and hepatic and brain tissue was collected for liver histopathology, and brain metabolomics, inflammatory markers, and protein expression level analyses.
Based on serum liver enzymes and histopathological analysis, HFF diet induced NAFLD. HFF pigs had impaired memory during NOR test (P<0.05), however, different treatments did not affect the activity level of animals. Metabolomics analysis showed a significant increase in bile acids (P<0.05) and a decrease in choline and betaine (P<0.05) in frontal cortex of HFF animals as compared to CON. Immunohistochemical analysis of glial fibrillary acidic protein, an astrocyte activation marker showed an increase in trend for HFF group (P<0.1). Protein expression analysis revealed no difference in insulin signaling pathway nor in microglia activation marker (Iba-1), however, it demonstrated that probiotics increased myelin level in neurons of HFF-fed animals (myelin basic protein, P<0.05). Levels of pro-inflammatory cytokines in the frontal cortex did not differ between treatments.
In conclusion, NAFLD-induced by 10 week feeding of high fructose, high fat “Western diet” caused a memory impairment and changes in metabolomics profile in brain cortex with no apparent inflammation. ARI#58873, RSCA Maj.

Poster #: 10
Campus: CSU Sacramento
Poster Category: Biochemistry
Keywords: Artemisia douglasiana, ethnopharmacology, cytotoxicity
Project Title: Isolation and Identification of Cytotoxic Constituents in Artemisia douglasiana
Author List:
Nguyen, Jonathan; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Clapp, Adam; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Roush, Jessica; Undergraduate, Chemistry, California State University, Sacramento
McCarthy Hintz, Mary; Chemistry, California State University, Sacramento

Abstract: California mugwort (Artemisia douglasiana Besser) is a plant found in the western regions of North America. Known locally by the Chumash Nation for its anti-viral and anti-inflammatory properties, A. douglasiana is brewed as teas to treat various pain symptoms, upper respiratory ailments, and for women’s medicine. When ingested in large doses, the leaves induce labor and correct dysmenorrhea. More recently, ethanolic extracts (tinctures) are used by non-Native peoples. Previous studies showed that ethanolic extracts were cytotoxic to cancerous and normal human cell lines. The identification of these cytotoxic compounds is important since it is widely used as medicine. Solvents of various polarities were tested to determine the most cytotoxic extract. Between ethanol, ethyl acetate, and hexane, ethyl acetate was determined to extract the most cytotoxic constituents from California mugwort leaves via EC50 analysis. Therefore, the extraction was scaled up using ethyl acetate. Then thin layer chromatography was performed, and a 1:2 ethyl acetate/hexane solvent system was determined for initial separation of compounds. Flash column chromatography was performed and 87 fractions were tested for cytotoxicity analysis. A bioassay-guided fractionation on MDA-MB-231 cells was done, and 10 cytotoxic peaks were identified. Active fractions will be assessed for purity by gas chromatography and will further purified if necessary. Once purity is confirmed with high performance liquid chromatography, cytotoxic compounds will be identified using mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy.

Poster #: 11
Campus: Cal Poly Pomona
Poster Category: Biochemistry
Keywords: Crystallography, Bioinorganic, CURE
Project Title: Effects of Cisplatin Concentration on Drug Interactions with Thaumatin
Author List:
Brannon, Jacob P.; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author, Eden Award Nominee
Stieber, S. Chantal E.; Chemistry & Biochemistry, California State Polytechnic University, Pomona
McCulloch, Kathryn M.; Chemistry & Biochemistry, California State Polytechnic University, Pomona

Abstract: Cisplatin is widely used in cancer treatment and targets DNA, however the many side-effects lead to questions about the resulting effects cisplatin has on proteins. Specifically, cisplatin is known to bind to free sulfur residues, but interactions with other amino acids are less widely studied. A recent publication demonstrated that cisplatin could bind to the thaumatin protein at 8 free nitrogen sites. The current study aimed to determine whether the concentration of cisplatin affected the number of free nitrogen sites that cisplatin bound to. Thaumatin was crystallized with cisplatin using two methods: a cisplatin soak and a cisplatin co-crystallization method. Samples were prepared by combining 7.2 pH tartaric acid, 40% PEG3350, and water to make a buffered well solution. The hanging drop crystallization method was used to grow thaumatin crystals by mixing the buffer with a 30 mg/mL thaumatin solution in a 1:1 ratio for the drop. Thaumatin crystals were observed after 3 days, after which 1 µL of 3.3, 2.0, 1.0, 0.5, 0.1, and 0.05 mmol/L cisplatin soaks were added to drops. The co-crystallization method used a 1 µL drop of thaumatin already incubated with the aforementioned concentrations of cisplatin when setting the hanging drop. All crystals were left for 10 days to ensure adequate binding of the cisplatin before being analyzed by single crystal X-ray diffraction. Using protein crystallography software and anomalous scattering data, it was determined that for both crystallization methods, cisplatin did not bind to thaumatin. This work indicates that thaumatin, which has no free sulfur residues, does not form partial occupancy bonds with low concentrations of the anticancer drug cisplatin.

Poster #: 12
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, Cloning
Project Title: Yeast strains to investigate cell cycle-dependent resolution of Holliday Junctions via Slx4 and Yen1 in S. cerevisiae
Author List:
Hadi, Hafridha; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abuhanna, Kevin; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge

Abstract: Double-strand breaks (DSBs) in DNA can be harmful if left unrepaired. Eukaryotes use repair mechanisms, some of which involve intact chromosomes that template repair via an intertwined intermediate known as a Holliday Junction (HJ). In S. cerevisiae, recruitment of the endonuclease enzymes that resolve HJs is not well understood. Slx4 is “partially” required to recruit the Rad1-Rad10 endonuclease to repair sites during cell division (S, G2 and M phases) but the basis for this “partial” requirement has not been elucidated. Another endonuclease, Yen1, resolves HJs, but it’s unclear whether it localizes to anaphase bridges, the final intertwined regions during chromosomal separation. Our aim is to clone specialized yeast strains with fluorescently labeled genes that will allow us to track the position and timing of Slx4 and Yen1 during HJ resolution.
First, to help us clearly distinguish between cell cycle phases, we have integrated a fluorescently labeled spindle pole body protein (Spc110-CFP) because of its morphology shifts during cell division. Yeast genetic crosses were done to combine Spc110-CFP, SLX4 mutant genes, and other features to perform fluorescence microscopy and flow cytometry analyses on one progeny strain. Following cell cycle arrest by alpha-factor, greater than 90% of cells showed a single Spc110-CFP focus, indicating that most cells were successfully arrested at the G1/S phase boundary. However, flow cytometry analysis showed that only about 60% of cells contained a DNA content consistent with G1/S arrest. These data indicate that our Spc110-CFP strain can effectively be cell cycle arrested, but further efforts are needed to optimize the protocol.
Second, toward investigation of Yen1 by fluorescence microscopy, using standard cloning methods, we cloned DNA plasmids containing yeast Enhanced Monomeric Red Fluorescence Protein (yEMRFP). Candidate clones were screened by Polymerase Chain Reaction (PCR) and DNA sequencing which showed that the candidate plasmids had the desired sequences with no mutations. The plasmids were co-transformed into yeast to make recombinant Yen1-yEMRFP. Transformants were screened for the integration of the Yen1-yEMRFP cassette by PCR and fluorescence microscopy. PCRs showed that DNA integration had been successful but that integration didn’t occur at the YEN1 locus within the genome. Microscopy showed no red fluorescent signal. We are redesigning the cloning vectors to target the YEN1 locus more specifically.

Poster #: 13
Campus: CSU San Marcos
Poster Category: Biochemistry
Keywords: Nanomaterial, Cancer , Chemotherapeutic
Project Title: Tunable Fluorescent Ionic Nanomaterials with Selective Toxicity Towards Cancer Cells
Author List:
Burch, Shalise; Graduate, Biology, California State University San Marcos, Presenting Author
Arrioja, Luis; Undergraduate, Chemistry, California State University San Marcos, Presenting Author
Bituin, Amanda; Undergraduate, Chemistry, California State University San Marcos
Torres, Dianna; Undergraduate, Chemistry, California State University San Marcos
Luna Lopez, Carlos; Biology, California State University San Marcos
Bwambok, David ; Chemistry, California State University San Marcos

Abstract: The development of chemotherapeutic agents that offer high efficacy against cancer cells and have low toxicity to non-tumor tissues are one of the main goals of cancer research. Our project aimed to investigate the effects of tunable ionic nanomaterials as novel chemotherapeutic agents against cancer cells. Our research focuses on rhodamine 6G and hexamethylindotricarbocyanine (HMT) -based fluorescent nanomaterials paired with various anions. By careful choice of anions paired with these cations it is possible to generate nanoparticles with selective toxicity towards tumor cells. Additionally, these selected anions have been shown to influence the uptake of these nanoparticles into specific cell organelles such as mitochondria. Our first objective was to design ionic nanomaterial with different chemical and fluorescent properties and test their diagnostic and anti-cancer properties. Rhodamine 6G and HMT-based fluorescent ionic salts were prepared using an anion exchange approach and demonstrated remarkable photostability with tunable chemotherapeutic properties. Fluorescence of these nanoparticles were monitored over a span of 25 days with ~ 99% retention of fluorescent emission. The fluorescent nanoparticles were incubated with cancer and stem cells (SC) at various concentrations for 72 hrs. Due to a highly polarized mitochondrial membrane, the ionic nanoparticles were internalized in mitochondria and were found to be selectively toxic toward cancer cell and nontoxic toward SC. These results inspired us to explore a unique way to deliver the ionic nanomaterials by taking advantage of the already present mitochondrial transfer between SCs and cancer cells. In a 2D system we show SCs can deliver mitochondria that are fluorescently labeled by rhodamine 6G and HMT-based ionic nanoparticles into co-cultured HEK293 tumorigenic cells. Our next step is to measure the rate and tumor toxicity of nanomaterials after transfer using a viability assay. The combined use of: i) SCs for transferring ionic nanomaterials to tumors and ii) chemotherapeutic effect of ionic nanomaterials can open a novel area of cancer and SC research that could lead to unexplored diagnostic and therapeutic applications. Additionally, HMT fluorophore emits in the NIR wavelength region of the spectrum, which is useful diagnostic window for biomedical imaging.

Poster #: 14
Campus: San José State University
Poster Category: Biochemistry
Keywords: thermodynamics, calorimetry, water
Project Title: Test Study of New Binding Equation: Hydration Energies Deduced from Sulfocalixarene Host-Guest Binding Model
Author List:
Camat, Leslie; Undergraduate, Chemistry, San José State University, Presenting Author
Huynh, Emily; Undergraduate, Chemistry, San José State University, Presenting Author
Eggers, Daryl; Chemistry, San José State University

Abstract: The classic equation for binding equilibria, ∆G° = -RTlnK, is widely known and appears in nearly every science textbook. Our laboratory, however, believes this definition of ∆G° is only valid for an infinitely dilute solution. Furthermore, though it is well established that the hydrophobic effect is driven by energy differences between the water molecules residing at a nonpolar surface and the surrounding aqueous environment, the energy contribution of the water is not fully accounted for by the classic equation. The Eggers lab proposes an alternate equation that appears to remedy both of these issues. In order to test the validity of this equation, our team measured the change in binding affinity (K) between a ring-like host molecule, sulfocalix[4]arene, and a known guest molecule, tetramethylammonium ion. Isothermal titration calorimetry (ITC) was used to measure K at specific temperatures (10, 25, and 37 °C) and at reactant concentrations ranging from 0.10 – 5.0 mM. As predicted by our governing equation, the measured binding affinity was not constant with concentration, and, when analyzed as -RTlnK versus the corresponding product concentration, a linear relationship was observed that allows one to deduce the free energy change of the water. From such an analysis of our preliminary data, we conclude that ∆G(H2O) is approximately +310 kcal/mol at 25 °C for this binding system. This large energy value for the solvent contribution in the binding of two relatively small molecules is supported by previous work from our laboratory and by computational studies from other investigators. Insights into the energetic role of the solvent, as reported here, may be exceedingly useful for many biotechnologies, including the design of drugs by the pharmaceutical industry with stronger binding affinity for their target molecule.
This work was funded by a grant from the National Institutes of Health, R15 GM110654.

Poster #: 15
Campus: San José State University
Poster Category: Biochemistry
Keywords: thermodynamics, microscale thermophoresis, water
Project Title: Microscale Thermophoresis to Measure DNA-DNA Binding: Trials and Tribulations
Author List:
Kim, Joon; Undergraduate, Chemistry, San José State University, Presenting Author
Eggers, Daryl; Chemistry, San José State University

Abstract: In aqueous binding reactions, when two solutes bind, a small portion of water around each solute is displaced into the surrounding environment. This displacement of water coincides with a change in solvation energy. In the classical Gibbs free energy equation, the change in solvation energy is not considered explicitly. The Eggers lab proposes a modification to the classic equation by including the change in energy of the water, the magnitude of which depends on the concentration of the complex formed at equilibrium. With this new equation, our lab predicts the observed equilibrium will shift with increasing concentrations of the reactants. The current project uses microscale thermophoresis (MST) to measure the equilibrium quotient, K, and to test the equation with a model binding system, the hybridization of two complementary DNA oligomers. MST allows one to follow the change in response of a fluorescent molecule when it binds to another molecule in the presence of a temperature gradient. Initial measurements resulted in an unusual binding curve that displayed a maximum at high concentration ratios of the two oligomers. In attempts to understand and/or bypass this anomalous behavior, we made the following modifications to our experimental protocol: (1) changing the DNA counterion from Na+ to Cs+, (2) substituting a larger oligo that includes a short hairpin loop in addition to the complementary sequence, and (3) adding a non-reactive DNA oligo to maintain a constant extent of crowding. Unfortunately, none of these changes resolved our problem. We conclude that MST is not a reliable technique for measuring binding equilibria at the high DNA concentrations necessary to test our governing equation. Future work will shift the DNA measurements from MST to calorimetry techniques.
This work was funded by a grant from the National Institutes of Health, R15 GM110654.

Poster #: 16
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Molecular recognition of DNA, biophysical chemistry, antitumor agents
Project Title: DNA binding and cell viability studies of synthetic derivatives of the triphenylmethane dye crystal violet
Author List:
Shaktah, Ryan; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Chowdhury, Marvin ; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Vardanyan, Laura; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Manlutac, Joshua; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Gardon, Nicole; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Tamae, Daniel; Chemistry and Biochemistry, California State University, Northridge
Minehan, Thomas; Chemistry and Biochemistry, California State University, Northridge

Abstract: In an effort to develop sequence-selective major groove binding small molecules, we have prepared monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a – 1f) and evaluated their affinity and sequence selectivity for duplex DNA. Competitive ethidium displacement assays indicate that 1a – 1f have apparent association constants for CT DNA in the range of 1.80-16.2 x10^7 M^-1 and binding site sizes of 10-14 base pairs. Viscosity experiments performed on ligand 1f confirmed that these dyes associated with duplex DNA by a non-intercalative mode of binding. Circular Dichroism (CD) and competition binding studies of the tightest binding ligand, 1e, suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers. This suggests a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove. Finally, we performed cell viability MTT assays using MCF-7 breast cancer cell lines with doses of our most promising ligand, 1e. An IC(50) value range of 9.154×10^-6 M to 2.034×10^-5 M was obtained with a 95% confidence interval, indicating that this compound may be capable of disrupting the growth of tumor cells.
This project was supported by a CSUPERB Faculty-Student Collaborative Resarch Development Grant (2019, to T.M.) and by an NSF-RUI grant (CHE-1508070, to T.M.)

Poster #: 17
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Cancer, Glyoxalase, DNA damage
Project Title: Investigating the metabolic and biological effects of Glyoxalase 1 inhibition in cancer cells
Author List:
Cordova, Benjamin; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author, Eden Award Nominee
Anderson, Jennifer; Undergraduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Kenney, Sydney; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Shuck, Sarah; Beckman Research Institute at City of Hope
Termini, John; Beckman Research Institute at City of Hope
Tamae, Daniel; Chemistry & Biochemistry, California State University, Northridge

Abstract: The detoxification enzyme, Glyoxalase 1 (Glo 1) is over-expressed in breast and prostate tumors relative to adjacent tissue. In a clinical trial of over 11,000 patients, Glo 1 expression correlates with poor prognosis and the TMPRSS2:ERG gene fusion that is a clincal biomarker for castrate-resistant prostate cancer (CRPC). The glutathione-dependent Glo 1/2 enzyme system efficiently detoxifies reactive carbonyl species such as methylglyoxal and glyoxal. Additional cellular defenses include the de-glycase, DJ-1/PARK-7 and the aldo-keto reductase super-family of NAD(P)H oxidoreductases. The α-oxoaldehyde, methylglyoxal (MG) primarily results from the breakdown of glucose and the triose phosphates generated during glycolysis. MG forms adducts with cellular nucleophiles such as nucleic acids, amino acids and lipids, leading to diminished cellular function and cell death. The primary aim of our study is to investigate the metabolic and biological effects of Glo 1 inhibition by S-p-bromobenzylglutathione cyclopentyl diester (pBrBzGSH(Cp)2). We used a panel of breast, prostate and ovarian cancer cell lines for these studies. Using the glutathione analogue, pBrBzGSH(Cp)2, we have determined the IC50 values in our cancer cell lines. They are as follows in the breast cancer cells, 14.6 μM for MCF-7, 19.5 μM for MBA-MB-231, and 11.7 μM for MDA-MB-468; in the prostate cancer cells, 11.3 μM for LNCaP, 9.0 μM for DU 145, 14.5 μM for 22Rv1; and in the ovarian cancer cells, 12.7 μM for UWB1.289 and 29.2 μM for UWB1.289+BRCA1. We quantified total glutathione (GSH) during Glo I inhibition. Treatment with pBrBzGSH(Cp)2 at the IC50 concentration elevated intracellular GSH by 10-50% for 5 of the 8 cell lines tested. We quantified a well characterized protein adduct known as carboxyethyl lysine (CEL) to probe intracellular accumulation of methylglyoxal. We found that the intracellular levels of CEL increased 1.3-to-2-fold in pBrBzGSH(Cp)2 treated cells versus untreated cells. We quantified the MG DNA adduct, (R)-/(S)-N2-(1-carboxyethyl)-2’-deoxyguanosine ((R)-/(S)-CEdG) in both pBrBzGSH(Cp)2 treated and untreated cells. Preliminary results indicate that the MDA-MB-231 breast cancer, 22Rv1 prostate cancer and UWB1.289 ovarian cancer cells have a 6-to-10-fold increase in CEdG levels in pBrBzGSH(Cp)2 treated cells versus untreated cells. CEdG adducts have been well-established to lead to transversions and single strand breaks. We have commenced studies pairing the clinically approved poly(ADP-ribose) polymerase inhibitor, Olaparib, with our novel Glo1 inhibitor pBrBzGSH(Cp)2 in our ovarian cancer cells at an equimolar ratio of 1:1 and are using the Chou-Talalay method to quantify effects of this combinatorial treatment. We hypothesize that pairing the two inhibitors will have synergistic effects and amplify synthetic lethality in Glo 1 over-expressing, BRCA1/2 defective cells. Taken together, Glo 1 inhibition may be part of a viable therapeutic strategy in Glo 1 over-expressing tumors that are not responsive to conventional therapies. [This work was supported by a CSUPERB New Investigator Award and is currently supported by an NIGMS SCORE SC2 to D.T.]

Poster #: 18
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: metabolism, IDH1, catalysis
Project Title: The mechanisms of catalytic regulation of isocitrate dehydrogenase 1
Author List:
Strom, Alexandra; Undergraduate, Biology, San Diego State University, Presenting Author
Widjaja, Vinnie; Undergraduate, Chemistry, San Diego State University
Pucher, Dahra; University of Southern California, Chemistry
Duc Nguyen, Tin; University of California, San Diego, Biology
Luna, Lucas; Graduate, Chemistry, San Diego State University
Matteo Avellaneda, Diego; Graduate, Chemistry, San Diego State University
Hoang, An; Staff, Chemsitry, San Diego State University
Sohl, Christal; Chemistry, San Diego State University

Abstract: Isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to α-ketoglutarate in the presence of NADP+ as a coenzyme. Tumorigenic IDH1 mutants gain the ability to catalyze a NADPH-dependent neomorphic reaction of converting α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate, which drive brain cancer, acute myeloid leukemia, and chondrosarcoma. However, further studies of IDH1 are required to fully understand the mechanisms behind its regulation. Post-translational modifications (PTMs), which include acetylation, represent one effective means of protein regulation. Here we hypothesize that IDH1 acetylation results in decreased catalytic efficiency. To test this hypothesis, we performed kinetic studies on select lysine to glutamine IDH1 mutants (K81Q, K224Q, K3221Q) that mimic acetylation. We report a decreased rate of isocitrate to α-ketoglutarate conversion in this mutant when compared to wild type IDH1. We also show that treatment with wild type IDH1 with acetylCoA similarly decreases activity. These studies suggest that acetylation may be an important PTM for regulating IDH1 activity, helping us to understand pathways relevant to IDH1 activity.

Poster #: 19
Campus: CSU Sacramento
Poster Category: Biochemistry
Keywords: Drosophila, pheromones, GC/MS
Project Title: A method to quantitatively measure cuticular hydrocarbon content in individual Drosophila melanogaster.
Author List:
Brown, Samuel; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Bauer, Johannes; Chemistry, California State University, Sacramento

Abstract: Background
The insect cuticle carries a complex mix of hydrocarbons, which play a significant role in disease resistance, water-proofing and prevention of desiccation. These cuticular hydrocarbons (CHC) play an additional role as pheromones, and thus regulate diverse insect behaviors, such as courtship, mate choice and mating, aggression, aggregation as well as foraging and pathfinding. Recently, we have shown that pheromones are not only linked to behavior, but also to aging and longevity. Our own data shows that altering the CHC mix displayed by fruit flies significantly changes fly longevity, suggesting an important role for pheromones in the aging process.
Interestingly, even genetically identical Drosophila show a wide range of age at the time of death, suggesting that non-genetic factors may influence longevity. These non-genetic factors may include individual interactions with other flies, and thus may be reflected in the CHC profiles of individual flies. Here, we report on a method to measure pheromones in individual flies as a first step in addressing this question.
Method
Traditionally, CHC are measured using groups of flies, and little is known about how CHC profiles differ between individuals. In order to elucidate this CHC variability, we extracted CHC from individual flies using iso-octane. Extracts were then separated on an Agilent GC/MS, at 280˚C column temperature. We used a set of saturated, mono- and diunsaturated hydrocarbons as references to create standard curves to aid with identification of the CHC present in the extracts.
Results
Using hydrocarbon standards, we were able to positively identify ~80% of all CHC present on the insect cuticle. Comparing extracts prepared from individual flies to extracts prepared from groups of flies yielded no statistically significant differences, indicating that conducting CHC analysis on single flies is feasible without the danger of losing low quantity CHC. Importantly, using at least ten replicates of single-fly extracts, we were able to show that considerable differences exist between the CHC displayed by individual flies, especially with respect to high-abundance CHC. These differences are masked when using extracts prepared from groups of flies. Next, we will use the single-fly analysis to compare the CHC profiles of freshly-dead and still-living flies. We hypothesize that individual CHC variability is closely linked to longevity, and thus may be used as a predictor of remaining life span.

Poster #: 20
Campus: CSU Sacramento
Poster Category: Biochemistry
Keywords: Drosohila, sleep , aging
Project Title: Sleep-length differences are associated with altered body composition and longevity in the fruit fly D. melanogaster.
Author List:
Thompson, Jacqueline ; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Bauer, Johannes; Chemistry, California State University, Sacramento

Abstract: Background:
Sleep deprivation has been shown to negatively impact health, e.g. memory loss, raised stress levels, weight gain, loss of insulin sensitivity, including diabetes-like phenotypes, and even behavioral changes. These observations suggest that sleep deprivation interferes with important physiological functions, including metabolic pathways of energy utilization.
Little is known about how disruption of sleep affects long-term health outcomes, especially aging and longevity. Here, we investigate these outcomes, using the fruit fly D. melanogaster as our model system. We hypothesize that flies with shortened sleep cycles have health defects, accelerated aging and shortened longevity.
Methods:
We obtained several isogenic fruit fly lines with either long- or short-sleep periods. Sleep patterns were verified by using a Drosophila Activity Monitor that measures fly movement on a continuous basis. Flies that displayed expected long- or short-sleep patterns were then analyzed for effects on longevity by recording their age at the time of death. The resulting survivorship curves were transformed into Gompertz mortality curves to determine the rate of aging.
In addition, we evaluated the general health of flies with shortened sleep, by determining their stress resistance, defined as survival when exposed to high levels of external stresses, such as starvation stress or oxidative stress. Finally, we assessed the metabolic status of these flies by measuring important markers of metabolic homeostasis by performing enzymatic determination of whole-body glucose and triacylglyceride (fat) levels.
Results:
Surprisingly, our data show that that both male and female flies with short-sleep periods have ~16% longer life span, as well as reduced aging rate, compared to flies with long-sleep. This increased longevity is accompanied by a ~10% reduction in body weight for male and female short sleep animals, compared to the long sleepers. This weight reduction is accompanied by fat and glucose storage reduction in short- versus long-sleep flies.
Interestingly, short-sleep flies have ~10% shortened longevity when exposed to Calorie Restriction, which extends life span by ~10% in control flies. Together, these data indicate that the short-sleep flies experience a metabolic state similar to Calorie Restriction, with reduced body weight and energy storage. Our data suggest that sleep length influences metabolism, which in turn affects health and longevity outcomes.

Poster #: 21
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: chaperone protein, NMR spectroscopy, protein folding
Project Title: Examination of structural HdeA mutants to better elucidate the mechanism of acid chaperone activation
Author List:
Geddes-Buehre, Dane; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Aguirre Cardenas, Imex; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge

Abstract: The periplasmic protein HdeA is a known acid-stress chaperone, promoting survival of several species of bacteria in the low pH of the stomach, in turn allowing colonization of the intestines by the invasive bacteria. The colonization of bacteria in the intestines usually results in food poisoning and ultimately dysentery if left untreated. This is a potentially fatal result in third world countries.
HdeA is a pH sensitive chaperone that becomes activated only at low pH, preventing aggregation of the other periplasmic proteins that unfold in the acidic environment of the stomach. Previous work from our lab as well as others have shown HdeA becomes active near pH 2.4, dissociating from an inactive homodimer into active monomers. My research focuses on mutating key residues and identifying the resulting structural changes at different pHs using Nuclear Magnetic Resonance (NMR) spectroscopy. Improving our understanding of the mechanism by which charged and flexible residues of HdeA aid in protein stability and chaperone activation can also provide insight into their roles in intrinsically disordered proteins as a whole.
Three mutants were generated to examine the impact of critical residues in HdeA: two mutants investigate the importance of specific charged residues (HdeA-D20N-D51N and HdeA-E37Q), while the third (a deletion mutant, delta44-48) evaluates whether a long flexible loop in the dimer interface is a trigger point of unfolding and chaperone activation. A pH titration of each mutant from pH 6 down to 2 in 1-unit increments was employed to quantify the changes in NMR chemical shifts to track alterations in tertiary structure as a function of pH. Although each mutant unfolds in a similar manner to wild type HdeA, there is variation in the pH at which this process begins. Of particular interest is the deletion mutant: large chemical shift perturbations are observed near the mutation site, indicating that region is heavily destabilized as the pH is decreased. Chemical shift data were used to quantify secondary structure at each pH for each mutant. Some key results show that the proportion of beta strand character at the N-terminus varies widely despite the fact that none of the mutations are located nearby in sequence. In the future I will test if the mutations impact chaperone activity.
We gratefully acknowledge support from the NIH for research funds (SC3-GM116745), and the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).

Poster #: 22
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat shock proteins, lipid biosensors, cellular membrane
Project Title: Characterization of the lipid-driven translocation of HspA1A, a stress inducible 70-kDa heat shock protein, to the cell surface
Author List:
Smulders, Larissa; Graduate, Biological Science, California State University, Fullerton, Presenting Author, Eden Award Nominee
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton

Abstract: HspA1A is a stress-inducible seventy-kilodalton heat shock protein (Hsp70) that plays essential roles in cellular survival. This protein also localizes at the plasma membrane (PM) of 90% of stressed and tumor cells. In cancer cells, in particular, the presence of HspA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, inhibiting this protein from being at the surface of tumors is a promising therapeutic. However, the PM-translocation pathway of HspA1A to the cell surface remains enigmatic, because this protein lacks membrane localization signals. Taking into account that HspA1A interacts with lipids, including phosphatidylserine (PS) and several mono-phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HspA1A with these lipids allows the chaperone to localize at the PM. To test this hypothesis, we subjected different cell lines to mild heat-shock and the PM-localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that HspA1A’s membrane localization and embedding increased during recovery from non-apoptotic heat-shock. Next, we selectively reduced PS, PI(4)P, and PI(3)P targets by co-transfecting HspA1A with known PS-, PI(4)P-, and PI(3)P- biosensors, and determined that HspA1A’s membrane localization was greatly reduced in all three cases. In contrast, the reduction of PI(4,5)P2 availability by overexpression of the PLCδ-PH biosensor had minimal effects on HspA1A’s PM-localization. Additionally, we manipulated the cellular lipid content using ionomycin, a calcium ionophore that activates PLC, and phenylarsine oxide (PAO), a PI4-Kinase inhibitor, using several lipid-biosensors as positive and negative controls. These experiments revealed that the PM localization of HspA1A was unaffected by ionomycin, but was greatly reduced in the presence of PAO, corroborating the findings obtained by the lipid-biosensors. Collectively, these findings strongly support the notion that HspA1A’s PM localization and anchorage is a complex lipid-driven phenomenon controlled by the binding of the chaperone to endosomal and plasma membrane PS, PI(4)P, and PI(3)P. These findings provide the basis for future interventions to inhibit the lipid-driven translocation of HspA1A to the PM of tumor cells, making them sensitive to radiation therapy.

Poster #: 23
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: butyrylcholinesterase, coumarins, Alzheimer’s Disease
Project Title: Synthesis, biochemical evaluation, and molecular modeling of organophosphate-coumarin hybrids as potent and selective butyrylcholinesterase inhibitors
Author List:
Macklin, Lee; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Alzheimer’s Disease (AD) is a multi-factorial brain disorder characterized by formation of deleterious plaques, and concomitant degeneration of nearby neurons. The most effective treatments to date have targeted a family of enzymes known as cholinesterases (which function to terminate synaptic activity by degradation of the neurotransmitter acetylcholine). The cholinergic hypothesis suggests that an increased synaptic concentration of acetylcholine, as a result of brain acetylcholinesterase (AChE) inhibition, provides some relief to AD-related cognitive deficits. An important isoform of AChE, known as butyrylcholinesterase (BChE) is significantly upregulated in AD brains, while AChE levels are unchanged. Biochemical work with organophosphorous (OP) inhibitors has highlighted the importance of BChE-selective inhibitors that do not alter AChE activity. On the other hand, coumarin derivatives are a widely appreciated class of bioflavinoids which have proven beneficial in medicine. Coumarins have been implicated as inhibitors of several key proteins involved in AD, including such cholinesterases. Purpose: To combine the structural features of two different fields of inhibitor study by the design and synthesis of OP-coumarins and evaluate them for inhibition of BChE and AChE in vitro. Methods: The OP-coumarin library was synthesized, purified, and evaluated spectrophotometrically using a chemically coupled enzyme assay. Relative inhibition, specific dose-response, and time-dependent inhibition curves were generated from readout of initial enzymatic velocities. SwissDock software was used to obtain top-ranked docking poses. Conclusion: Nearly all of the inhibitors were found to be selective for BChE and did not inhibit AChE at 34 μM. The most potent inhibitor, 0bi (IC50 = 0.363 μM), differed from its constitutional isomer by 100-fold, indicating that the position of the phosphate is critical. Docking of 0bi revealed that it binds as a transition state mimic, leading to a much more rapid phosphorylation rate (k >> 100 L/mmol*s). However, when the non-bridging oxygen atom of the phosphate was changed to a sulfur, this obliterated the rate of covalent modification by 100-fold (k = 8.4 L/mmol*s). Future work will aim to replace the OP group with an organosulfonyl which will preserve the inhibition type and prevent unwanted neuropathic effects often associated with OP compounds.
This work has been funded by the Graduate Research Fellowship awarded by CSULB.

Poster #: 24
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Protein Design, TIM, In Silico
Project Title: Forbidden Protein Angles: Exploitation of Unfavorable Dihedrals for the Enhancement of Human-Designed Enzymes
Author List:
Allen, Patrick; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Nominee
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Over the past 3.5 billion years of evolution, enzymes have adopted a seemingly infinite number of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be forbidden to all amino acids, save glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site Lysine 13 (K13) with a dihedral within the forbidden zone (phi=69/psi=-139). Both the amino acid and the dihedral angle are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted beta-sheet dihedral values of phi=-135/psi=170 but experiments show a 1,000-fold loss in activity. Based on these results, we hypothesized that adopting the forbidden torsion angle for K13 contributes to catalysis. Using a combination of computational and experimental approaches, four residues that interact with K13 (N11, M14, Q64, and E97) were mutated to alanine from single to quadruple mutants. In silico Molecular Dynamic (MD) simulations of 5x200ns per mutant species were made using 2JK2 unliganded human TIM as a starting structure. Heat maps, containing 400,000 K13 dihedral values per run reveal full or partial loss of forbidden zone angles depending on mutant combination. Initial experimental results with E97A single mutant showed a three-fold decrease in kcat with little change in the KM value suggesting a modest catalytic role for this residue. For this mutant the heat maps revealed the K13 angle spends less than 30% of its time in the forbidden zone compared to over 50% occupation in the wildtype suggesting the rate decrease may be due to the mutation affecting the positioning of K13. In addition to ongoing in vivo characterization of the remaining mutants, double mutant cycles will be used to evaluate their energetic connections with K13. As modern protein design relies on naturally occurring protein folds, understanding how to recreate forbidden angles may enhance catalytic rates in unnatural enzymes and achieve chemical reactions not yet envisioned by nature nor organic chemistry.
We would like to thank California State University Long Beach and the Carl E. Riley Endowed STEM scholarship for their generous funding.

Poster #: 25
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Tunneling nanotubes, CD7, Intercellular communication
Project Title: The Role of CD7 Protein in Tunneling Nanotube Formation
Author List:
Azami, Paria; Graduate, Biology, California State University, Fresno, Presenting Author
Gousset, Karine; Biology, California State University, Fresno

Abstract: Intercellular communication is crucial for the function, development, and survival of cells within an organism. Tunneling nanotubes (TNTs) are a newly discovered mode of cell-to-cell communication. They are long actin containing structures that connect distant cells, allowing the transfer of signals, molecules, organelles, and pathogens between them. Due to their recent characterization, there is still a significant gap in knowledge about their composition and possible mechanism(s) of formation. Recently, the transmembrane glycoprotein CD7 was identified in TNT samples analyzed using our newly developed LCM/MS method, hence it became a protein of interest for TNT formation/function. It was previously established that Succinylated Concanavalin A (Succ Con-A) and Phorbol 12 Myristate 13 Acetate (PMA) oppositely regulate CD7 expression in T cells. Interestingly, these two drugs were also shown to affect formation of cellular protrusions such as filopodia. Therefore, we hypothesize that CD7 could play an important role in TNT formation. As the first aim of this project, we wanted to verify that Succ Con-A up-regulates CD7 expression in the neuronal Cath.-a-differentiated (CAD) cells and that PMA down-regulates CD7 expression. Western blots were carried out to visualize the protein levels. Densitometric analysis showed a clear increase in CD7 expression upon treatment of cells with Succ Con-A and a significant decrease following PMA treatment, compared to untreated control cells. As a second aim, we wanted to verify that Succ Con-A and PMA treatments affect filopodia formation and more importantly establish if there is a correlation between the level of CD7 expression and TNT formation in CAD cells treated with Succ Con-A and/or PMA. Using a membrane dye and fluorescence microscopy to identify TNTs, we observed a dramatic increase in the number of filopodia and TNTs in cells treated with Succ Con-A and a significant decrease in the cells treated with PMA. Thus, we show here for the first time that CD7 might be an important TNT inducing protein. The next steps will be to further breakdown this pathway and specifically determine if these TNTs are Myo10-dependent or a different subset of TNTs. Overall, our results can be valuable to the medical and scientific fields providing scientists with the groundwork to develop selective and targeted tools and therapies for numerous diseases that utilize TNTs to spread and proliferate.

Poster #: 26
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Tunneling nanotubes, Myosin-X, glutamate
Project Title: The Effect of Hydrogen Peroxide and Glutamate on Myosin-X expression and TNT Formation
Author List:
Xiong, Zoua; Undergraduate, Biology, California State University, Fresno, Presenting Author
Pham, Alan; Undergraduate, Biology, California State University, Fresno, Presenting Author
Ajanel, Abigail; Graduate, Biology, California State University, Fresno
Gousset, Karine; Biology, California State University, Fresno

Abstract: Discovered in 2004, Tunneling nanotubes (TNTs) are long, actin based cellular protrusions, whose main function is to transfer subcellular components between cells. Often called “intercellular highways”, TNTs have been observed in many different cell types, including cancer cells. Recent studies have suggested that cancer cells can form TNTs with healthy cells as a way of transferring mitochondria and redistributing chemotherapy drug among cells. Despite the enormous interest in TNTs, the exact mechanisms that lead to TNT formation remain elusive. In the past, our lab has demonstrated that the actin molecular motor protein Myosin-X (Myo10) plays an important role in both the formation and functionality of TNTs in neuronal CAD cells, macrophages, and HeLa cells. It was also found to play a key role in HIV-1 intercellular transfer. In addition, we have recently shown that physiological concentrations of hydrogen peroxide (H2O2) and glutamate, two substances abundantly secreted by cancer cells, increase TNT formation in both HeLa and CAD cells. Here, our goal is to determine whether treatment with H2O2 and glutamate affect the expression levels of Myo10. Preliminary data in CAD cells showed a 3.85 fold and 7 fold increase in Myo10 in CAD cells treated with 6.4 uM glutamate and 0.5 nM H2O2 respectively. These data once more highlight the importance of Myo10 as a critical TNT inducer, independently of the cell type or the type of stimuli. Overall, it might provide valuable insight on what may be a potential pathway for infectious and cancerous cells.

Poster #: 27
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: breast cancer, chemoresistance, surface plasmon resonance microscopy
Project Title: Measuring the effect of t-Darpp phosphorylation on trastuzumab drug binding to breast cancer cells via surface plasmon resonance microscopy
Author List:
Nguyen, Lisa; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Chang, Megan; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Avanes, Arabo; Staff, Chemistry and Biochemistry, California State University, Los Angeles
Wang, Yixian; Chemistry and Biochemistry, California State University, Los Angeles
Momand, Jamil; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: Trastuzumab (Herceptin) is a well-tolerated biologic drug often used to treat HER2+ breast cancers. HER2 is a receptor-tyrosine kinase type receptor on the surface of breast cancers. Several reports show that the majority of late stage metastatic HER2+ breast cancer patients acquire resistance to trastuzumab. HER2+ breast cancer cells that are resistant to the chemotherapeutic drug trastuzumab frequently overexpress the gene PPP1R1B. One protein product of PPP1R1B, t-Darpp, has been reported to form a complex with HER2 and block trastuzumab-induced HER2 dephosphorylation. We hypothesize that t-Darpp decreases the binding affinity between trastuzumab and HER2, thus causing a block to dephosphorylation. In this study we determine the effect of t-Darpp on the binding affinity (KD) of trastuzumab to HER2 using surface plasmon resonance microscopy (SPRM). SPRM combines an optical microscope with traditional surface plasmon resonance spectroscopy, an optical sensing technique that utilizes the collective excitation of electrons in a thin metal film to probe small changes occurring at the solid/solution interface. SPRM was used to measure the molecular interaction between trastuzumab and HER2 in isogenic t-Darpp-expressing breast cancer cell lines. Wild-type t-Darpp is phosphorylated at Thr39 by cyclin dependent kinases 1 and 5 (CDK1/5). Thr39 is critical for mediating t-Darpp chemoresistance activity. In breast cancer cell lines expressing wild-type t-Darpp, Ala39 mutant t-Darpp, and Asp39 mutant t-Darpp (phosphomimetic residue) the binding affinities were 5.103 ± 1.80 nM, 32.09 ± 14.2 nM, and 6.203 ± 0.43 nM respectively. Our study suggests that non-phosphorylated t-Darpp lowers the affinity of trastuzumab to HER2 by approximately 5-fold. Phosphorylated t-Darpp appears to cause chemoresistance through a mechanism other than trastuzumab binding to HER2.

Poster #: 28
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: small molecule inhibitor, botulinum neurotoxin, amino acid
Project Title: Structural-Based Optimization of Small Molecule Inhibitors for the Botulinum Neurotoxin Serotype A
Author List:
Thompson, Jordan; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Dao, Wendy; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton

Abstract: The botulinum neurotoxin (BoNT) is the most toxic protein known to man, causing the neuroparalytic disease, botulism. The neurotoxin consists of a heavy (HC) and light chain (LC) that work together to cause fatal paralysis in humans. The HC attaches to nerve cells and injects the LC into the cytosol. The LC is a zinc metalloprotease that cleaves SNARE proteins required for neurotransmission. Once the SNARE protein is cleaved, neurotransmission is halted, resulting in flaccid paralysis that can lead to death or long recovery times. The BoNT potency and ease of production perpetuate BoNT as a possible bioterrorism weapon, in which countermeasures are urgently needed. A therapeutic avenue for treatment of botulism is inhibition of the BoNT LC by small molecule inhibition.
The goal of this research is to design novel therapeutics to inhibit the BoNT LC. This goal was accomplished by synthesizing a novel scaffold that mimics peptide binding to the active site. The scaffold contains an amino acid with a hydroxamic acid at the C-terminus and a sulfonamide linked biphenyl at the N-terminus. Compounds were synthesized through a multistep synthesis, to yield molecules with various amino acids and substituted biphenyl rings. Furthermore, two amino acids were incorporated into the scaffold to produce dipeptides.
The compounds were evaluated for inhibition by enzymatic assay and computational docking. The enzymatic assay indicates that the sulfonamide is essential for inhibition as substituting the sulfonamide for a methylene resulted in no inhibition. The sulfonamide is proposed to hydrogen bond with residues in the active site and also contributes to a molecular shape that encourages binding. The stereochemistry of several amino acids was evaluated for inhibition. The assay results indicate D-isoleucine induces the highest increase in inhibition, with a 30% increase in inhibition compared to L-isoleucine. Computational docking additionally revealed that electronegative atoms, such as oxygen and chlorine at the 2’ position of the biphenyl make key polar contacts. Residues that were identified interacting with oxygen and chlorine are TYR366 and HIS223, respectively. Dipeptide analogs were synthesized to assess the inhibitor length within the BoNT/A LC pocket. The dipeptides displayed a 10-fold increase in inhibition compared to the amino acid scaffold. The results indicate that the amino acid and dipeptide scaffolds are promising leads for BoNT therapeutics.

Poster #: 29
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Lipoprotein Lipase, Nuclear Receptors, Cholesterol Efflux
Project Title: A role for PPAR in LPL-associated regulation of ABCA1 in THP-1 macrophages
Author List:
Ohana, David; Undergraduate, Biology, California State University, Northridge, Presenting Author
Pashanyan, Davit; Undergraduate, Biology, California State University, Northridge, Presenting Author
Christian, Bryan; Graduate, Chemistry and Biochemistry, California State University, Northridge
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge

Abstract: Cholesterol accumulation by macrophage cells of the vascular wall is a key contributor to atherogenesis. ATP binding cassette transporter A1 (ABCA1), a protein found on the membrane of foam cells, mediates the efflux of excess cellular cholesterol to extracellular lipid-deficient apolipoprotein A-I. ABCA1 is transcriptionally up-regulated via nuclear receptor transcription factors PPAR (peroxisome proliferator activated receptor) and LXR (Liver X Receptor). These receptors are activated by derivatives of fatty acids and cholesterol, and their activation is implicated in cholesterol homeostasis. Prior research from our lab has shown an inverse relationship between lipoprotein lipase (LPL, an enzyme responsible for triglyceride hydrolysis) and ABCA1 expression; however, the mechanism of this regulation is unclear.
We hypothesized that the reason for this LPL-associated downregulation of ABCA1may be the availability of abundant fatty acid products of LPL-mediated hydrolysis, which may interfere with ligand binding to PPAR and LXR. In this study, we determined the effect of LPL-down-regulation on the level of PPAR and LXR transcripts in THP-1 macrophages. We directly determined the effect of fatty acids on the binding of PPAR to the PPAR response element (PPRE).
Short hairpin RNA was used to specifically silence LPL in THP-1 macrophages. End-point RT-PCR was used to compare gene-specific transcript levels in wild-type (WT) and LPL-KD THP-1 macrophages. PPARγ binding to its response element was measured by first immobilizing the PPRE oligonucleotides to microtiter wells. Activated PPARγ from samples were allowed to bind in the presence of specific fatty acids. A PPAR-specific antibody was used to detect the amount of PPARγ bound.
LPL transcript levels in LPL-KD macrophage cells were 63% of that in WT cells. This decrease in LPL transcript resulted in a slight decrease in the transcript levels of PPAR and LXRα. Quantitative real-time PCR confirmed repression of PPARγ transcript levels in LPL-KD THP-1 cells. The PPRE binding assay showed that stearic acid and oleic acid decreased PPARγ binding to its response element, but linoleic acid increased it compared to the vehicle alone. Our data show that LPL silencing results in a down-regulation of nuclear receptors in THP-1 macrophages. Also, fatty acids were found to inhibit the activation of PPAR. Thus, ABCA1 up-regulation in LPL-KD cells may be due to abolition of PPAR repression by fatty acids.

Poster #: 30
Campus: CSU Chico
Poster Category: Biochemistry
Keywords: Medicinal chemistry, hemoglobin binding assay, undergraduate experiment
Project Title: Hemoglobin Drug Synthesis and Assessment – An Undergraduate Laboratory Experiment
Author List:
Maldonado, Letzi; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Becker, Casey; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Barnett, Nicholas; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Chavez, Dahlia; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Diaz, Juan; Undergraduate, Chemistry and Biochemistry, California State University, Chico
McDonald, Matthew; Undergraduate, Chemistry and Biochemistry, California State University, Chico
DuBose, Austin; Undergraduate, Chemistry and Biochemistry, California State University, Chico
Arpin, Carolynn; Chemistry and Biochemistry, California State University, Chico

Abstract: As students continue to pursue careers in the medicinal chemistry field, it becomes increasingly important to provide them with relevant experiences in the academic laboratory. Our goal is to provide such an experience as an undergraduate organic chemistry experiment that is an informative and enjoyable introduction to the medicinal chemistry field. In the experiment, students carry out tasks similar to those assigned to medicinal chemists in the field: drug synthesis, assessment of biological activity, and data analysis. The experiment begins with the synthesis of a drug, which students choose from one of four scaffolds. The second step is to assess how well the drug binds to the active site of apohemoglobin, a variation of the protein hemoglobin with its heme group removed. Drug binding is assessed via a novel fluorescence competitive displacement assay developed by our lab. The final step of the experiment for students is to analyze their fluorescence data, estimate their drug’s potency of binding, and compare their results with those of others to deduce strong and weak binders. Our results of this work include the development and optimization of the apohemoglobin binding assay, identification of drug scaffolds that bind to the target protein, and feedback from the experiment’s initial launch in the fall of 2018. Feedback was generally positive, but included comments on confusing synthesis instructions, and the fluorometer being a bottleneck. Thus, our most recent results include optimization of the drug syntheses and creating filler activities in programs such as Microsoft Excel and the molecule modeling software Chimera for students to complete while waiting for time on the fluorometer.

Poster #: 31
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Lipoproteins, Oxidative stress, Alzheimer’s disease
Project Title: Lipid peroxidation product 4-hydroxynonenal alters tertiary structure and fold of apolipoprotein E in an isoform-dependent manner
Author List:
Abeer, Muhammad; Graduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Nominee
Abdulhasan, Abbas; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author

Abstract: Oxidative stress is a characteristic feature of aging and neurodegenerative diseases such as Alzheimer disease (AD). It causes extensive cellular damage leading to increased lipid peroxidation such as 4-hydroxynonenal (HNE), and protein oxidization. HNE is found in high levels in post mortem brains of AD patients. The overall goal of our study is to understand the effect of HNE modification on the structure and function of apolipoprotein E3 (apoE3) and apoE4, the latter known to be a major risk factor for AD. Western blot with HNE specific antibody confirms the modifications of apoE3 and apoE4 as appearance of major bands at ~36 kDa. Preliminary mass spectrometric analysis reveals that Lys75 is a hot spot for both modified proteins. Modified apoE3 and apoE4 were highly helical (60.5 ± 3.1 and 61.0 ± 2.7 %, respectively, n=3) comparable to that of unmodified proteins (56.4 ± 1.5 and 57.9 ± 3.3 %, respectively, n=3) as revealed by circular dichroism spectroscopy. A significant decrease in the intrinsic fluorescence emission was noted for both HNE-apoE3 and HNE-apoE4, compared to the corresponding unmodified proteins. GdnHCl (guanidine hydrochloride)-induced denaturation revealed a notable difference in terms of increased susceptibility to unfolding for HNE-apoE4, but not HNE-apoE3. Further, ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence emission spectra revealed a 10 nm red shift in the wavelength of maximal fluorescence emission for HNE-apoE4 (but not for HNE-apoE3) compared to their corresponding unmodified forms. HNE modification leads to a significant reduction in the ability of apoE3 and apoE4 to bind lipid vesicles and cause transformation to discoidal bilayer particles; the rate constant K for apoE3 and HNE-apoE3 were 1.01 x 10-4 s-1 and 3.22 x 10-5 s-1, respectively, while that for apoE4 and HNE-apoE4 were 3.37 x 10-3 s-1 and 1.02 x 10-3 s-1, respectively. Incubation of HNE modified proteins with brain microvasculature endothelial cells revealed punctate, perinuclear vesicles by suggesting cellular uptake as noted by confocal microscopy. Taken together, our data indicate that there are isoform-specific differences in protein conformation and tertiary fold, and in the functional ability as a consequence of modification of apoE by HNE. Further studies are needed to understand the mechanism of cellular clearance of HNE modified apoE and the role of oxidatively modified apoE4 as a risk factor for AD and amyloid pathology.

Poster #: 32
Campus: San José State University
Poster Category: Biochemistry
Keywords: Aedes aegypti, Serine Proteases, Blood-borne disease vectors
Project Title: Recombinant Expression and Purification of Aedes aegypti Midgut Juvenile Hormone-Regulated Chymotrypsin-like Serine Protease (JHA15)
Author List:
Aguilar, Eric; Graduate, Biology, California State University, Fresno, Presenting Author
Houghton, Kim; Undergraduate, Chemistry, San José State University
Rascon, Alberto; Chemistry, San José State University

Abstract: The Aedes aegypti female mosquito requires a blood-meal to successfully complete the gonotrophic cycle and successfully lay eggs. Transmission of blood-borne pathogens like Zika virus to humans can occur through the blood feeding process. Once imbibed, the blood-meal must then be digested into necessary amino acids by midgut proteases. One of these digestive enzymes is JHA15, which is suggested to be present before and slightly after blood meal digestion, and although the protease has been suggested to be involved in this process, no direct evidence is available to definitively indicate such. The purpose of this project is to recombinantly express and purify JHA15 serine protease in its inactive form in order to investigate the activation, proteolytic activity, and determine the role in the blood meal digestion process. The JHA15 gene was cloned into both the pET28a and pET29b expression vectors to produce an N-term and C-term his-tagged (no leader sequence) JHA15 protease, respectively. Initial bacterial growth experiments led to low yield expression of recombinant JHA15, but adding an osmolyte, betaine, enhanced bacterial expression. Protease expression was conducted in SHuffle T7 express competent E. coli cells due to their oxidative cytoplasm that allow for disulfide bridge formation in JHA15. Cells were induced with 0.05 mM isopropyl β-D-1-thiogalactopyranoside for 96 hours at 10°C, then his-tagged JHA15 was purified by affinity chromatography using a HisTrap Fast Flow column. Once purified, an Ala-Ala-Pro-Phe-Nitroanilide activity assay confirmed the presence of the inactive form of JHA15. Furthermore, western blot analysis using JHA15 specific antibodies confirmed that the purified protease is indeed JHA15. We are currently optimizing protocols to prevent proteolysis during the purification process for JHA15 (no leader) constructs and working with a pseudo propeptide construct with an enterokinase (EK) sequence in place of the natural propeptide region. The EK site serves to reduce any autoactivation since mosquitoes do not have the enterokinase enzyme needed to recognize the DDDDK (EK) sequence. Future work will consist of activation and kinetic experiments for both the pseudo EK and wildtype JHA15. This work establishes a successful approach to recombinantly expressing JHA15 and provides a starting point for the purification of JHA15, which can aid in future proteolytic studies to help determine the exact role in the blood meal digestion process.

Poster #: 33
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: West Nile Virus, Protease inhibitors, zafirlukast
Project Title: Investigating Zafirlukast as a novel allosteric inhibitor against the West Nile Virus NS2B-NS3 Serine Protease
Author List:
Nguyen, Shaina; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton

Abstract: The West Nile Virus (WNV) is a neurovirulent mosquito borne pathogen that is prevalent worldwide. 20 % of WNV infections will result in severe symptoms that include: encephalitis, seizures, tremors, and death. There is no known cure for WNV infections. Upon infection, the WNV genome is translated by host ribosomes into a genomic polyprotein containing all proteins necessary for viral replication. The viral NS2B-NS3 serine protease cleaves the genomic polyprotein at multiple positions to allow viral replication in the cell. Therefore, the protease is an integral component of the WNV replication cycle and is an attractive therapeutic target.
Zafirlukast has shown promising inhibition against the NS2B-NS3 protease with an IC50 value of 32 µM. The Zafirlukast scaffold consists of four major components: a cyclopental carbamate, a methylated indole core, an aromatic linking group, and an o-toluic sulfonamide. This research explores the synthesis of Zafirlukast analogs as potential inhibitors against the WNV NS2B-NS3 protease using an enzymatic inhibition assay. Initial enzymatic kinetic measurements and computational docking analysis suggest Zafirlukast binds to the NS3 protease at an allosteric site that disrupts the NS2B cofactor from binding, resulting in protease inhibition. We investigated the Zafirlukast scaffold by synthesizing analogs that substituted the indole core with quinoline, phthalimide and triazole scaffolds. The aromatic linker and o-toluic sulfonamide was substituted with a biphenyl. Also, the methylene connecting the indole with the aromatic linking group was replaced with a direct carbon bond. The results of the enzymatic assay, revealed analogs containing a modified indole core did not show any inhibition. This indicates the indole core is integral for Zafirlukast analogs to remain effective against the protease. Replacing the aromatic linking group and sulfonamide group with a biphenyl group resulted in 44% inhibition against the protease at a concentration of 60 µM. Additionally, replacing the methylene linker between the indole core and the aromatic linking group with a direct carbon bond displayed 63% inhibition at a concentration of 60 µM. Computational docking also predicts that the new analogs bind to the same allosteric site as Zafirlukast. These results reveal a novel class of inhibitors with a unique mode of inhibition for the NS2B-NS3 protease.

Poster #: 34
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Vesicular transport, Organelle, Cell signaling
Project Title: Msp1 regulates localization of the peroxisomal transmembrane Rab GTPase accelerating protein Gyp8
Author List:
Quinn, Monique; Graduate, Biology, California State University, San Bernardino, Presenting Author, Eden Award Nominee
Lin, Ariel; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Biology, California State University, San Bernardino

Abstract: Eukaryotic cells maintain integrity and biochemical functions of discrete organelles using a network of vesicular trafficking pathways regulated by lipid-anchored Rab GTPase signaling proteins (Rabs). Rabs control where and when vesicles fuse with target membranes, ensuring appropriate cargo (proteins, lipids, etc.) delivery. When bound to GTP (active) Rab proteins interact with effector proteins that control membrane molecular identity, docking and fusion, but Rabs are inactive when bound to GDP. Controlled cycling between active and inactive conformations is important for Rabs to properly regulate traffic. Rabs require Rab GTPase accelerating proteins (GAPs), to trigger GTP hydrolysis and cycle into an inactive state in which the Rab can be removed from a membrane. We report that the evolutionarily conserved yeast Rab GAP Gyp8 is a transmembrane protein whose localization is regulated by the ATPase Msp1, a chaperone that functions to remove tail-anchored proteins from peroxisomes and mitochondria. Fluoresence microscopy indicates that GFP-Gyp8 co-localizes with peroxisomal markers in wild type cells. Computational analysis of Gyp8 hydrophobicity predicts a single-pass transmembrane domain near the carboxy terminus, characteristic of a tail-anchored transmembrane protein. Biochemical experiments, including subcellular fractionation and salt-washing on membrane fractions, indicate that Gyp8 is a integral membrane protein that localizes to a membrane fraction that includes peroxisomes. Loss of Msp1 chaperone function resulted in localization of GFP-Gyp8 to both peroxisomal and mitochondrial membranes, indicating that Msp1 surveils the mitochondrion and tends to remove mitochondrial Gyp8. In the absence of peroxisomes or pre-peroxisomal vesicle formation at the ER, GFP-Gyp8 localizes to the endoplasmic reticulum (ER), consistent with the manner in which most tail-anchored proteins are first inserted at the ER. Truncation analysis of GFP-Gyp8 indicated that the C-terminal 90 amino acids, including the transmembrane and luminal domain, are necessary and sufficient to direct localization to peroxisomes. These data represent the first report in any experimental system of a Rab GAP that localizes to peroxisomes. Further study will explore the presumptive physiological role of Gyp8 in regulating peroxisome dynamics. This work was supported by Summer Undergraduate Research Program grants and two ASI Research Supplies grants from CSUSB Office of Student Research.

Poster #: 35
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: lower division undergraduate research , cross-disciplinary program, online STEM summer research outreach
Project Title: Engaging Middle, High School and Lower Division Undergraduate Students in Cross-Disciplinary Research
Author List:
Serrano, Allison ; Undergraduate, Biology, California State University, Fullerton, Presenting Author
Alvarez, Axel; Undergraduate, Presenting Author
Robson, Simone; Fountain Valley High School
Rasche, Madeline; Biochemistry, California State University, Fullerton
;

Abstract: For the past two years, the authors have integrated research-based high-impact practices into a lower division undergraduate cross-disciplinary program on Early Experiences in Biotechnology in the area of DNA Nano-robots. The program involves freshmen CSUF students from the College of Natural Science and Mathematics, as well as the College of Engineering and Computer Science and spans the Spring and Fall semesters during sophomore and freshmen year. The curriculum activities are designed to strengthen the foundation for authentic cross-disciplinary research productivity and graduate school/job preparation in the biotechnology workforce. Our direct pre- and post-course self assessment survey results show that the program enhanced CSUF students’ (i) readiness for cross-disciplinary research by 32%, (ii) preparation for upper division research by 25%, (iii) acquisition of laboratory problem solving and scientific communication skills by 28%, and (iv) scientific research literacy by 32%. The survey also reveals that the biological concepts were slightly more difficult for the students than the mechanical engineering ones (65% v/s 62.5%).
During the summer of 2019 a novel two-week Increasing Diversity in Engineering and Labor-force (IDEAL) online summer outreach workshop on Development of DNA Nano-mechanisms was offered as a part of the cross-disciplinary program. The main objectives were to increase middle and high-school female students’ self-confidence and motivation in pursuing future cross-disciplinary STEM careers. On a scale from 1 (low) to 5 (high), results from pre- and post- summer surveys showed that all five student participants enhanced their ability/skills in (i) working on a STEM research project (from average 2.8 to 4.8 score), (ii) reading/understanding STEM research papers (from an average 3 to 4.2 score) and (iii) performing literature review on a STEM research topic (from an average 2.8 to 3.8 score). In addition, an NSF Middle/High School Student Attitudes Towards STEM (S-STEM) Survey was used to assess the overall impact of the outreach program on the female students’ self-confidence and motivation. From the Math, Science, Engineering and Technology, Your Future, and 21st Century Skills sections of the S-STEM survey, the results show that the 21st Century skills (related to self-confidence) was the section with the most radical improvement. In addition, for all five middle/high school participants, the Your Future section (related to interest in STEM careers) showed increased interest in Mathematics, Medicine, Medical Science and Engineering.
Acknowledgements: The authors gratefully acknowledge the support of 2017 CSUPERB Curriculum Development Grant Program, NSF CAREER grant # 1751770, and NSF Award CHE-1508801.

Poster #: 36
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Microstructure, Mechanical stiffness, Cell migration
Project Title: Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds
Author List:
Boyd, Alexander; Graduate, Manufacturing Systems Engineering and Management, California State University, Northridge, Presenting Author
Moldthan, Matthew; Undergraduate, Manufacturing Systems Engineering and Management, Presenting Author
Li, Bingbing; Manufacturing Systems Engineering and Management, California State University, Northridge

Abstract: 3D bioprinting shows great promise not only as a potential means of creating an unlimited supply of biocompatible organs and tissues but also for studying a wide variety of cell behavior, including motility, metastatic potential, and using 3D bioprinted tissue constructs as a means of administering various regenerative treatments, including gene therapy. This study’s objective is to study the growth and development of cancer cells in tissues with different geometries, orientations, and cell concentrations relative to the amount of stromal cells in these cancerous tissues. We also evaluated the efficacy of the tissue models produced by the microextrusion 3D bioprinting technique relative to the growth and development of malignant cells. We used the microextrusion 3D bioprinter to print tissue constructs with different geometries with the goal of studying the motility of malignant and stromal cells. The microextrusion 3D bioprinter utilized two types of biomaterials: sacrificial material and a cell-laden alginate hydrogel. The sacrificial material contained a 10% v/v glycerol solution and pluronic f-127, while the cell-laden hydrogel contained hyaluronic acid (HA), glycerol, gelatin, sodium alginate, and stromal or malignant cells. Using the sacrificial material as support and the cell-laden material as a means of forming the preliminary tissue construct, the cell-laden hydrogel was chemically crosslinked using chitosan as a catalyst to convert the viscous material to a solid, cell-laden tissue construct. These tissue constructs were then observed using microscopy and various imaging/staining techniques and the motility of the malignant cells, juxtaposed against the motility of the stromal cells, was measured and recorded.

Poster #: 37
Campus: CSU Los Angeles
Poster Category: Bioengineering
Keywords: EMG, modelling, automate
Project Title: A Model for Automating Thresholds in a Muscle Activity-Based Exergaming App
Author List:
Garcia, Abigail; Undergraduate, California State University, Los Angeles, Presenting Author
Enciso, James; Graduate, California State University, Los Angeles, Presenting Author
Lopez-Juarez, Luis; Undergraduate, California State University, Los Angeles
Velasco, James; Graduate, California State University, Los Angeles
Won, Deborah; Electrical and Computer Engineering, California State University, Los Angeles

Abstract: A mobile application was designed and developed to track fitness and play games, called “exergames”, in which the players are required to perform physical activity. The app tracks muscle activity levels using wireless electromyography (EMG) sensors, which control three different exergames tailored for exercising in wheelchairs. To calibrate the games, the user must undergo a tedious process of setting EMG thresholds upon each session. These thresholds are used to detect muscle contractions during exergames. To improve the user friendliness of the app, we are attempting to automate the process of setting the EMG thresholds. In this study, we sought to determine how well the thresholds could be linearly modelled based on data that could be entered once on first use of the app.
Ten subjects performed exercises on our app while wearing EMG sensors on the bicep and anterior deltoid of the subject’s dominant arm. Three exercises were performed with increasing intensity until maximum heart rate was reached: air boxing (punching), bicep curls and military press using elastic resistance arm bands. For each muscle and each exercise, an upper and lower threshold were approximately set based on a percentage of the maximum voluntary contraction (MVC). Then, the thresholds were adjusted manually to achieve the most accurate detection possible, and this process was repeated for each game to arrive at the “true” upper and lower threshold for boxing, bicep curls, and military presses. These manually derived thresholds were regressed against the following anthropometric and physiological variables: age, height, weight, max heart rate, and MVC. Multiple linear regression was carried out using Matlab’s regress function, and the coefficient of determination (R2) values were computed as a measure of the goodness of fit. We then applied the regression models to test data and computed % accuracy of contraction detections as the percentage of actual contractions which were correctly detected.
Multiple linear regression of EMG thresholds based on user height, weight, age, maximum heart rate, and MVC yielded R2 values as high as 0.99. The R2 values across all 3 exercises and muscles averaged 0.73 and ranged from 0.38 to 0.99. The models yielded an average 80% correct detection rate. These results lend confidence to providing a way to automating threshold calibration for EMG-based exergaming to make for a more user-friendly mobile exergaming experience.

Poster #: 38
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Photonics, Optical trapping, Red Blood Cells
Project Title: Waveguides of Light in Red Blood Cell Suspensions
Author List:
Perez, Nicolas; Graduate, Physics and Astronomy, California State University, Northridge, Presenting Author, Eden Award Nominee
Chambers, Jacob; Graduate, Physics and Astronomy, California State University, Northridge
Chen, Zhigang; Physics and Astronomy, San Francisco State University
Bezryadina, Anna; Physics and Astronomy, California State University, Northridge

Abstract: Biological samples often have various absorption bands that need to be either targeted or avoided in opto-fluidic micromanipulation or biomedical imaging. With nonlinear optics, it is possible for light to self-induce a waveguide. By using light, living cells can be manipulated to form structures that are able to guide light through scattering media. However, the desired wavelengths may not be suitable to exhibit nonlinear self-guiding due to the absorption bands or the light-bioparticle interaction is not strong enough.
Here we study the formation of waveguides in red blood cell suspensions for a range of different wavelengths over a distance of several centimeters, with minimal significant photodamage of the cells. We utilize nonlinear optical response for self-trapping of a laser beam, forming light guides in RBCs suspended in a phosphate buffer solution. In particular, cells in colloidal suspensions get attracted toward the center of the laser beam due to the optical gradient force, and meanwhile move along the beam due to the scattering force. Since living cells usually have a slightly higher index of refraction than the ambient media, the optical force-induced nonlinearity leads to a self-lensing effect along the beam path, which allows for the formation of a biological “fiber”. To improve the number of usable light wavelengths over purely self-guided propagation, we use the master-slave relation, in a manner similar to the pump-probe experiment: a master beam creates a waveguide first in a scattering bio-soft-matter suspension over a few centimeters, and then a “slave” beam uses this waveguide to propagate through the medium. The slave beam, injected simultaneously, has no appreciable nonlinear self-action itself, but experiences the master waveguide akin to an optical fiber. This new approach can provide a path to guide a wide range of wavelengths, including those in the absorption bands at lower power so as not to damage the sample.
The fact that we can guide a wide range of wavelengths with low photodamage may bring about new applications in medicine and biology, for instance, in developing alternative solutions to transmit energy and information through scattering media, as needed in deep-tissue imaging, treatment and diagnostics.

Poster #: 39
Campus: Sonoma State University
Poster Category: Bioengineering
Keywords: Biosensors, x-ray, automation
Project Title: Automating X-ray footprinting mass spectrometry to effectively investigate protein structure
Author List:
Russell , Brandon ; Undergraduate, Engineering Science , Sonoma State University, Presenting Author
Rosi , Matthew; Graduate, Engineering Science, Sonoma State University, Presenting Author
Farahmand, Farid; Engineering Science, Sonoma State University

Abstract: Bound water within protein is an important factor in protein structure and interaction. X-ray footprinting mass spectrometry (XFMS) is a synchrotron-based method which is able to identify bound water in protein while in the solution state. XFMS offers benefits in drug research and understanding protein interactions. Each sample is exposed to x-rays for different lengths of time to obtain a dose response curve. The samples can then be tested again at specific exposure times to create hydroxyl radicals which can be further processed with mass spectrometry. The PROBLEM with XFMS is that there is no automated process and it requires a significant investment in time and labor. In an experiment, numerous samples are exposed to x-rays individually at each exposure time and requires researchers to move samples by hand. The GOAL of the project is to build an automated XFMS system which can run 20 samples in a single experiment, reduce overall experimentation time, and lower the volume of a sample needed for each experiment. The METHOD for accomplishing this is to use a syringe pump to create a jet of protein sample, which will be exposed to x-rays, and collect the sample in a sample tube. The exposure time for each sample is controlled by the flow rate of the pump and is provided by the researcher at the beginning of each experiment. The fluorescence of a dye within the solution, which is degraded by x-ray exposure, is measured and data is automatically logged. The individual sample tubes are automatically moved after each exposure to preserve each sample. The automation of the system is controlled by a LabVIEW virtual instrument (VI) created specifically for the project. As a RESULT of this project we have created an automated system which can: 1) automatically handle 20 samples at a time, 2) perform an experiment 25 times faster than before, and 3) reduced the per sample volume to the microliter range. The system achieves a dose response comparable to samples performed using previous manual methods. The completed system is currently implemented at beamline 3.2.1 at Lawrence Berkeley National Laboratory and is being used by XFMS researchers. The NEXT STEP of the project is to further reduce the volume of sample used, and to add additional improvements such as automatic jet alignment and detecting experiment-stopping jet clogs.

Poster #: 40
Campus: CSU Los Angeles
Poster Category: Bioengineering
Keywords: activity monitoring, EMG analysis, energy expenditure
Project Title: Monitoring and analysis of muscle activity during resistance-based exergaming on a mobile fitness app for individuals in wheelchairs
Author List:
Juarez-Lopez, Luis; Undergraduate, Electrical and Computer Engineering, California State University, Los Angeles, Presenting Author
Velasco, James; Graduate, Electrical and Computer Engineering, California State University, Los Angeles, Presenting Author
Enciso, James; Undergraduate, California State University, Los Angeles
Garcia, Abigail; Undergraduate, California State University, Los Angeles
Won, Deborah; Electrical and Computer Engineering, California State University, Los Angeles

Abstract: Popular approaches to encourage physical fitness include turning exercises into games called “exergames” and providing feedback on one’s physical activity. An exergaming mobile app was developed for individuals with lower mobility impairment to promote and facilitate exercising at home. Resistance arm band exercises have been recommended as a central component in exercise routines for people who use wheelchairs. Therefore, one of the exergames on the app implemented resistance arm band exercises. The app not only provides games which are tailored for exercises that are recommended and feasible for people in wheelchairs, but also provides the capability to monitor physical activity. For individuals with lower mobility impairment, an effective way to measure energy expenditure is by monitoring electromyography (EMG), or muscle activity. In this study, we tested the capability to monitor EMG activity from four individuals with spinal cord injury (SCI) during exergaming over an 8-week period using our app at home, and from this recorded activity, compare the energy expenditure across three different exergames: 1) boxing, 2) resistance arm bands, and 3) spinning.
Data was imported into Matlab using the MySQL Java Database Connection driver. Custom Matlab scripts were written to parse and analyze the EMG data across sessions, weeks, and games. Since caloric expenditure is directly related to muscle activity, integrated raw EMG values (iEMG) were tabulated as a measure of energy expenditure for each participant and all app sessions in a week. The efficiency of the games in terms of the rate of energy expenditure per unit time spent playing the game (iEMG/tSession, where tSession is the total duration of the sessions in seconds) was also calculated.
The average weekly iEMG ranged 2.4 to 4.5V for boxing; from 2.4 to 7.1V in resistance training; 1.7 to 8.7V in spinning. The total energy expenditure (iEMG) during resistance exergaming was 35% higher than boxing and 5.5 times higher than spinning. The efficiency of the resistance exergame was 37% higher than that of boxing and 73% higher than spinning. We have demonstrated the ability to monitor EMG through our mobile fitness app during 8 weeks of in-home exergaming, and determined that users expend more energy during the resistance-based exergames and also need to spend less time on this game to expend the same amount of energy than in the boxing and spinning exergames.

Poster #: 41
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: DNA origami, Nano-mechanisms, Drug therapy
Project Title: A DNA Origami Nano-Mechanism Functioning as a Drug Carrier
Author List:
Salazar, Arturo; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Manalad, Nico; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Herrera, Suzette; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Lopez, Kimberly; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Serrano, Allison; Undergraduate, Biology, California State University, Fullerton
Alvarez-Loya, Axel; Undergraduate, Mechanical Engineering, California State University, Fullerton
Robson, Nina; Mechanical Engineering, California State University, Fullerton
Rasche, Madeline ; Chemistry and Biochemistry, California State University, Fullerton

Abstract: DNA origami is a method of folding DNA at the nanoscale level with the ability to manipulate the geometry of the structure. Single-stranded DNA (M13mp18) is mixed with DNA fragments called staples or primers that have complementary nucleotide sequences to fold the DNA strand into a desired shape. These DNA nanostructures possess the versatility and potential to act as highly specific cancer therapeutic delivery systems. The research goal is to construct a DNA nano-mechanism by forming a DNA nanostructure with the ability to bind the chemotherapeutic drug doxorubicin and deliver it specifically to cancer cells. Gel electrophoresis was run on two samples of nanostructures constructed using two different thermocycler programs. The 75-minute program showed two discrete bands at approximately 6000 bp and 5000 bp on the agarose gel, while the 11-hour program produced a smear in the gel demonstrating the sample had a range of band sizes. Atomic force microscopy showed rectangular nanostructures formed with the 11-hour thermocycler program. The addition of fastener primers/staples to roll the rectangles into nanotubes resulted in images consistent with about 10% efficiency in folding. A fluorometer assay confirmed the binding of doxorubicin to the DNA nanostructure, based on an increase in the absorbance at 590 nm when doxorubicin was incubated with DNA. These results indicate that the rectangular DNA nanostructure has been successfully produced and bound to the drug doxorubicin. Future experiments will focus on rolling the DNA with doxorubicin bound into a nanotube with specificity for attachment to cancer cells. This research serves to further the application of DNA origami as a potential anticancer treatment by focusing on the successful formation of DNA origami nano-mechanisms that can serve as efficient drug delivery systems. [Funded by a 2017 CSUPERB Curriculum Development grant and NSF grant CHE-1508801]

Poster #: 42
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: tissue on a chip, microfluidics, in vitro modeling
Project Title: Microfluidic Device Design for Mammalian Cell Culture and Microscopy
Author List:
Henigman, Lauren; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Adettiwar, Aayushi; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Heylman, Christopher; Biomedical Engineering, California Polytechnic State University, San Luis Obispo

Abstract: Microfluidics is a growing field of study that deals with the movement of tiny amounts of liquid to analyze biological and chemical processes in a more controlled environment for applications such as medical diagnosis, drug delivery, stem cell analysis, and artificial organs. Goal: Our goal is optimizing and developing protocols for cell loading, culture, imaging, and analysis within microfluidic devices. To achieve this, we designed, simulated, and tested microfluidic devices to enable cell culture and analysis. Methods: After creating a solid model, we used the finite element analysis software COMSOL to validate that model by simulating the fluid flow velocity of cell loading and cell culture medium perfusion (cell loading: 4.33E-3 m/s, medium perfusion: 1.31E-5 m/s). After manufacturing the devices, we empirically validated our simulation data by driving fluorescent beads and solutions through the devices and used a fluorescent microscope to measure fluid velocities (cell loading average: 2.40E-3 m/s, media perfusion average: 7.94E-6 m/s). The real results were consistently higher than simulation results, telling us that the devices exhibit slightly more resistance to fluid flow than expected from the simulation. To characterize our ability to load and culture cells in the device, we began by testing an active (constant flow rate) loading of a cell solution in comparison to a passive (pressure-driven) loading of the same cell solution. After optimizing the loading technique, we fixed cells within the device and stained cell nuclei with Hoechst, a fluorescent dye that binds DNA. We imaged the devices on an upright fluorescent microscope using QCapture Pro software and quantified cell nuclei using Image J software. We have qualitatively observed that cells can be loaded into the device, grown over 24 hours, and then fixed and stained with Hoechst. This was observed by carrying out the optimized protocols and analyzing the images taken from the upright fluorescent microscope using the “Sandwich Method” (a technique to image the cells on the bottom of the device). Future plans for this project include testing functionality of the device for cell and gel matrix loading as well as developing robust, streamlined protocols for perfusion channel flow, image capture, and cell staining analysis.

Poster #: 43
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: Biomechanics of Uterine Tissue, IVF biomechanics, Elasticity
Project Title: Toward designing a biologically relavant embryo culture environment optimized for In-Vitro-Fertilization (IVF)
Author List:
Mesgun, Sara; Undergraduate, Engineering, San Francisco State University, Presenting Author
Safi, Sabrina ; Undergraduate, Engineering, San Francisco State University, Presenting Author
Rivera, Jose; Undergraduate, Engineering, San Francisco State University
Ruggeri, Elena; University of California San Franciscoq, San Francisco State University
Rinaudo, Paolo; University of California San Francisco
Azadi, Mojtaba; San Francisco State University

Abstract: In this work, we detected a key biomechanical property of the uterine tissue. Although In-Vitro-Fertilization (IVF) techniques, has led to over five million birth as of 2012, significant challenges remain. Available embryo culturing is highly inefficient with as many as 80% of all embryos transferred, failing to yield a live birth. IVF is costly, can be emotionally draining, and has a low success rate for the majority of patients over 35 years old. As a result, improving IVF including its embryo culture environment is crucial for the emotional and financial well-being of many families experiencing fertility problems. This collaborative research between SFSU and UCSF aims to develop an optimized bioinspired embryo culture substrate with biomechanical properties similar to that of the uterine tissue to increase blastocyst development and the success rate of IVF.
We used novel Atomic Force Microscopy (AFM) to determine a key time-independent biomechanical property of three murine tissue as a quantitative parameter called elasticity. The uterine tissues of three outbred mice (CF1xB6D2F1) were tested by AFM. Mice were super-ovulated and the uterine tissues were collected on the day of ovulation. The tissues were kept in a -80 C freezer and were thawed at room temperature right before the AFM experiments at SFSU. The AFM probe used to find these measurements was a BudgetSensor (AIOAL) conical probe with a nominal stiffens of 0.2 N/m. The diameter of the conical AFM tip was less than 10 nm. AFM calibration was performed in the Protease Inhibitor Cocktail solution before beginning the test on the tissues. About 30 slow (quasi-static) displacement AFM experiment tests were performed on each sample. The collected force-displacement curves were fitted using the Hertzian contact model, the elasticity of three samples was found as following” 40.24±1.50 KPa, 52.95±2.27 KPa, and 23.25±3.42 KPa. The small standard deviation indicates the repeatability and sensitivity of the experiments. The values found from these tissues are within epithelial tissue elasticity values, but, they are substantially lower than the uterine elasticity values speculated for uterine tissue in the literature (100-1000 KPa). In the next step of the study, we will detect time-dependent biomechanics (i.e. Poro-Visco-elasticity) as well as the heterogeneity of the tissue to replicate and design a biologically relevant substrate within the values measured. Funding Acknowledgement: NSF and CSUPERB.

Poster #: 44
Campus: San José State University
Poster Category: Bioengineering
Keywords: Blood Clots, Uniaxial Strain, Platelets
Project Title: The role of platelet cytoskeleton to the response of blood clots to uniaxial strain
Author List:
Alzaghari, Jinann; Undergraduate, Chemical Engineering, San José State University, Presenting Author
Aguirre Resnikowski, Luciana; Undergraduate, Chemical Engineering, San José State University, Presenting Author
Pathare, Suyog; Graduate, Biomedical Engineering, San José State University
Eng, Wilson; Graduate, Mechanical Engineering, San José State University
Lee, Sang-Joon; Mechanical Engineering, San José State University
Ramasubramanian, Anand; Chemical Engineering, San José State University

Abstract: Hemostasis is achieved when blood clots are formed at the location of vascular injury and seal the wound in order to arrest bleeding. This process is dependent on the strength of clots that form and their contractility. Therefore, understanding the biomechanical characteristics of clots is integral for the treatment of clotting disorders and cardiovascular diseases. Unlike numerous previous investigations, we have focused on tensile tests as they provide direct interpretation of clot mechanics.
In this work, we investigated the stress-strain and stress-relaxation relationships of in vitro blood clots through tensile testing. Clots were initiated by the addition of thrombin to 500 uL of recalcified blood obtained from five healthy donors. In some cases, to evaluate the effect of contractility on clot strength, platelet-cytoskeletal inhibitors blebbistatin and cytochalasin D (300 µM and 10 µM respectively), were added before clot initiation. After 1 hour, the clots were placed in in-house fabricated microextensometer apparatus, and stretched uniaxially for up to 50% strain to measure nonlinear stiffness. This was followed by a series of 1mm extensions, of the original 6 mm length, to impose prescribed strain increments and observe the corresponding stress relaxation. All the experiments were performed at least in duplicate, the results from five donors were analyzed using Students’ t-test to establish statistical significance.
We observed that the clots showed a strain-stiffening response that is best approximated by a Mooney-Rivlin model, and the relaxation is best approximated by an exponential decay. We also observed that the blebbistatin had a more significant effect on clot mechanics than cytochalasin suggesting the critical role of actomyosin contraction in determining clot strength. In summary, we have determined, for the first time, the tensile mechanical properties of the blood clots; and also established the role of actomyosin contraction in determining clot strength.

Poster #: 45
Campus: CSU Sacramento
Poster Category: Clinical
Keywords: Veterinary, Cancer, Hyperthermia
Project Title: Clinical pilot study of improved Radio Frequency (RF) heating system to shrink tumors in veterinary patients- a multi-center study
Author List:
Ramalakshmanan, Subbiah; Graduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author, Eden Award Nominee
Alhassan, Abdulaziz; Graduate, Electrical and Electronic Engineering, California State University, Sacramento
Alshehri, Meshari; Graduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Aung, Nan; Graduate, Electrical and Electronic Engineering, California State University, Sacramento
Hershey, Betsy ; CVA Integrative Veterinary Oncology, Phoenix, AZ , Electrical and Electronic Engineering, California State University, Sacramento
Mills, Brenda ; Integrative Veterinary Care,Livermore, CA
Skiwski, Sara ; The Western Dragon Integrative Veterinary Solutions, San Jose, CA
Patel, Jayesh; Graduate, Electrical and Electronic Engineering, California State University, Sacramento
Kakade, Gaurav; Graduate, Electrical and Electronic Engineering, California State University, Sacramento
Kumar, Preetham; Electrical and Electronic Engineering, California State University, Sacramento

Abstract: Hyperthermia heats body tissue to ~ 108°F using a high power RF generator e.g. the Mettler Autotherm ©) system. This RF power, in the ISM (International Scientific Medical) band of 27.12 MHz, can target cancer cells, usually with minimal injury to normal tissue. By damaging proteins and structures within cells, hyperthermia can shrink tumors, or make them susceptible to radiation or chemotherapy. Hyperthermia can also reduce chemo dosage when used in combination. Hyperthermia, though FDA approved, still faces challenges: reducing heating time from current 30 minutes and accessing deep-seated tumors. Our students have developed two tools towards this effort: a treatment planning software that uses patient scan data to adjust power and device orientation, and a 3-d printed RF focuser that complements the existing Mettler system to increase heating efficiency. Rigorous simulation with patient CT scan data and heating tests with the focuser led to a pilot clinical study with 3 volunteer vet clinics. The study typically involved our university contracts office signing an agreement with the clinic for equipment rental/training, followed by our team visit with the RF unit. The clinical results are summarized (responded well implies 50 % or higher tumor reduction): At the CVA Integrative Veterinary Oncology, Phoenix, AZ, each hyperthermia session was 30 minutes at a temperature of ~ 106°F(6 weeks, 2 sessions/week) for 5 patients. Overall response improved when chemotherapy was given in conjunction. Typical cases were a cat with an oral sarcoma that responded well to hyperthermia and low dose cyclophosphamide chemo; a Chihuahua dog with a nasal tumor; and another cat with an undifferentiated tumor that shrank well with Lomustine chemo and hyperthermia. At the Integrative Veterinary Care , Livermore, CA, hyperthermia alone was given for 30 minutes (4 weeks, 2 sessions/week) at ~ 105°F for 5 patients; typical case was a dog with chest wall sarcoma with metastases . Hyperthermia provided palliative help only to patients, since all patients had very advanced cancer. At the Western Dragon Integrative Veterinary Solutions, San Jose, CA, 5 patients on chemotherapy responded well to hyperthermia. Thus, the pilot clinical study using hyperthermia has been completed with promising results, especially when used along with chemotherapy. The next effort is aimed at expanding the pilot study to investigate hyperthermia therapy for specific veterinary cancers.

Poster #: 46
Campus: Cal Poly Pomona
Poster Category: Clinical
Keywords: Rabbit, Trazodone, Drug trial
Project Title: Trazodone hydrochloride use in rabbits: A 28-day drug treatment safety evaluation
Author List:
Rickerl, Kaitlin; Undergraduate, Biology, California State Polytechnic University, Pomona, Presenting Author
Reed, Jessica; Undergraduate, Biology, California State Polytechnic University, Pomona, Presenting Author
Brundage, Cord; Animal and Veterinary Sciences, California State Polytechnic University, Pomona

Abstract: Trazodone HCl (TZN), a serotonin modulator commonly used to treat depression and anxiety in humans, was recently found to provide sedation and improve handling in laboratory rabbits (Oryctolagus cuniculus). Clinical use of TZN however in rabbits requires an understanding of the safety and long term health consequences TZN. We evaluated the systemic effects of repeated TZN administration New Zealand white laboratory rabbits (n = 12) given 40 mg/kg TZN orally every 12 hours (±1 hour) for 28 days. A physical examination was performed by a veterinarian prior to TZN trial onset and every 7 days over the course of treatment. Animals were evaluated daily including heart and respiratory rate monitoring. Blood was collected for cell counts and serum chemistry analysis prior to and every 14 days during the TZN trial. No appreciable changes were noted in physical examination parameters over the trial period (p > 0.05). Tachycardia has been reported in humans, however fluctuation in heart and respiratory rate in rabbits did not significant change (p = 0.201 and p = 0.984 respectively). Significant elevations in liver alanine aminotransferease (ALT; p = 0.024), liver alkaline phosphatase (ALP; p = 0.003) and albumin (p=0.003) were noted at 14 days of TZN treatment, these values were not elevated at 28 days. Overall kidney function did not appear to be affected as levels of blood urea nitrogen (BUN; p = 0.626), sodium (p = 0.654), and potassium (p = 0.994) did not significantly change, not did any other measured blood parameter (p > 0.05). These results suggest that 40 mg/kg TZN can be administered every 12 hours repeatedly to rabbits for up to 28 days with a high margin of safety. This research was supported by Cal Pol Pomona Animal & Veterinary Science Department foundation research funds.

Poster #: 47
Campus: CSU Sacramento
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: metalloenzyme, density functional theory (DFT), catalytic mechanism
Project Title: Biomimetic Modeling of Non-Heme Iron Halogenase Enzymes
Author List:
Mcanally, Mason; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Lindsay, Dylan; Undergraduate, Chemistry, California State University, Sacramento
Gherman, Benjamin; Chemistry, California State University, Sacramento

Abstract: CytC3 and SyrB2 are non-heme iron halogenase metalloenzymes that catalyze halogenation reactions of natural products. Halogenation is useful in synthesizing organic compounds; however, typical organic halogenation reactions involve toxic chemicals, posing environmental danger. In order to explore the thermodynamics behind these enzymes, we focus on a biomimetic model developed by Que et al ([FeIV(O)(TQA)(Cl/Br)]+ (TQA= tris(quinolyl‐2‐methyl)amine)), which is the first synthetic model showing chemoselective halogenation reactivity (J. Am. Chem. Soc., 2016, 138, 2484-2487). This contrasts with previous biomimetic models based upon the TMG2dien=N-methyl-1,1- bis{2-[N2 -(1,1,3,3-tetramethylguanidino)]ethyl}amine and TPA=tris(pyridyl-2-methyl)amine ligands which failed to show halogenase reactivity. The halogenation reaction catalyzed by the TQA biomimetic model was studied using computational chemistry utilizing density functional theory (DFT) with cyclohexane and toluene as substrates. Both high spin (S=2) and low spin (S=1) states for the FeIV center were considered as Que et al documented that successful halogenation depended on the FeIV spin state. The reaction was modeled in two steps: 1) hydrogen atom abstraction from the substrate produces a substrate radical and iron(III)-hydroxyl complex as intermediates; 2) halogenation or hydroxylation of the substrate radical occurs from the intermediates. Beginning with high-spin FeIV, calculations show the hydrogen abstraction step had similar activation energies for the TQA ligand system with either halide ligand (Cl or Br) and with either substrate. However, the intermediates formed were lower in energy for toluene than cyclohexane due to resonance stabilization in the toluene radical. For the second step, results show that halogenation occurs spontaneously while the hydroxylation step contains a small energy barrier, evidencing that the halogenation reaction can be energetically favorable versus hydroxylation. In addition, comparing results with the TQA ligand system and low spin FeIV shows that reaction energetics are overall less favorable compared to those with the high spin state for FeIV.
Acknowledgements: Funding for this project has been provided by: (a) California State University, Sacramento College of Natural Sciences and Mathematics to M.M. through a SURE Award; and (b) by California State University, Sacramento chemistry department to D.J.L. through a Russell-Forkey Research Award.

Poster #: 48
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Mathematical modeling, Toxicology, Developmental deformities
Project Title: Mathematical and computational models analyzing the effect of common toxicants on embryonic development in the zebrafish, Danio rerio
Author List:
Schwartz, Ashley; Graduate, Mathematics and Statistics, San Diego State University, Presenting Author, Eden Award Nominee
Sully, Emma ; Undergraduate, Mathematics and Statistics, San Diego State University
George, Uduak; Mathematics and Statistics, San Diego State University
Sant, Karilyn; Environmental Health, San Diego State University

Abstract: The prevalence of increased levels of man-made chemicals including perfluorobutanesulfonic (PFBS) and tris(4-chlorophenyl)methanol (TCPMOH) in our environment is a recent area of concern for its presence in water reserves and marine mammal tissues respectively. The roles these chemicals play in embryonic development is largely unknown for their recent appearance in environmental samples. In this study, we use mathematical models, computational models and image analysis to quantify and automate discovery of various abnormalities in Danio rerio (zebrafish) embryonic organs due to toxicant exposure. Zebrafish embryos were exposed to various PFBS or TCPMOH levels each day post fertilization while microscopy images were captured. We assessed pancreatic islet morphology and yolk sac magnitude using MATLAB for image analysis and computational modeling. Additionally, a mathematical model is developed that incorporates observed effects of TCPMOH on yolk sac utilization and validated with experimental data. Embryos exposed to PFBS showed decreased islet size and abnormal morphology, resembling congenital defects in humans relating to an increase risk for diabetes. Modeling of yolk sac utilization revealed an indication of decreased embryonic nutrition for those exposed to TCPMOH, contributing to developmental progression barriers. Our results show that common environmental pollutants can disrupt pancreatic organogenesis and decrease embryonic nutrition, raising concerns for disease development.

This research was supported by the Howell-CSUPERB research scholars program.

Poster #: 49
Campus: CSU Channel Islands
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Molecular docking, Ribonucleotide reductase enzyme inhibition, Nucleoside analogues
Project Title: In silico molecular docking studies and ADMET predictions of novel sulfonamide-nucleoside analogues as potential anticancer drugs
Author List:
Salvador, Prince ; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Krajeck, Hannah ; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Chemistry, California State University Channel Islands

Abstract: Ribonucleotide Reductase (RNR) is an enzyme that catalyzes the reduction of nucleotide diphosphates to deoxynucleotide diphosphates, classifying it as a key enzyme in the DNA replication pathway. RNR specific activity has been positively correlated to tumor growth rate and thus it is a key target for chemotherapeutic agents. The objective of this study was to design a new class of RNR inhibitors as potential anticancer drugs. Therefore, in this work, the synthesis of novel sulfonamide-nucleoside derivatives is reported, and their inhibitory effect on the RNR catalytic site was evaluated. Mitsunobu reaction was utilized for the coupling of sulfonamide derivatives with the uridine nucleoside, and products were isolated in quantitative yields. Through an in silico molecular docking study using internal coordinate mechanics algorithm (ICM), the conformation, H-bonding formation and edoc scores of the sulfonamide-nucleoside analogues were assessed. Furthermore, the pharmacokinetic, bioavailability, and toxicity profiles of these compounds were determined with ADMETSAR and SWISSADME algorithms. Analysis of the data demonstrated that the derivatives which possessed electron withdrawing groups, 5′-uridyl 2-chloro-4-fluoro-5-sulfamoylbenzoate and 5′-uridyl 4-chloro-3-sulfamoylbenzoate, formed the strongest H-bonding within the catalytic pocket of RNR, scored better edoc scores of -36.3 and -35.7, respectively, and adapted a “U” conformation that all contributed to their superior affinity. Moreover, these compounds showed enhanced binding to residues that participate in the catalytic process: Tyr 737 and Tyr 738 resulting in a hypothesized disruption of electron flow, thus interrupting the enzyme mechanism to induce apoptosis. The drugs were predicted to show positive human intestinal absorption coinciding with excellent aqueous solubility scores of at least -3.0 logS, and were classified as non-AMES toxic and non-carcinogenic. Lastly, the LD50 values of 2.413 mol/kg and 2.366 mol/kg in rats make them suitable for application in human models. In conclusion, the proposed analogues showed a positive enzyme-drug interaction and met drug likeness criteria, making them excellent drug candidates and viable for further exploration via in vivo and in vitro studies.

Poster #: 50
Campus: CSU San Marcos
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Lanius ludovicianus, Population Genetics, Morphometrics
Project Title: Population Genetics, Form, and Function of Loggerhead Shrikes in California
Author List:
Wulf, Gwendalyn; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Mey, Ken; Undergraduate, Biological Sciences, California State University San Marcos
Sustaita, Diego; Biological Sciences, California State University San Marcos
Sethuraman, Arun; Biological Sciences, California State University San Marcos

Abstract: Population genetics studies of Loggerhead Shrikes (Laniidae: Lanius ludovicianus) in California have indicated considerable intraspecific genetic differentiation. Other morphological and behavioral studies have also shown geographic phenotypic variation. However, the concordance between genetic and phenotypic differentiation remains obscure. Here we explore the extent to which genetic differences among populations are correlated with phenotypic differences (beak shape and bite force) among populations of Loggerhead Shrikes throughout California. Feather samples were collected from shrikes in locations along an approximately 950 km range. Genomic DNA was then extracted and we genotyped each individual at 7 different nuclear microsatellite loci. Preliminary pairwise Fst values, along with MULTICULST analyses show evidence of population structure among some of these locations, suggesting a potential genetic basis to the observed phenotypic differences among some, but not all, populations. We expect that by increasing our genotyping with more microsatellite loci and sampling more individuals across California, we can perform more rigorous analyses of population structure that will shed light on the relative roles of local adaptation and plasticity in shaping differences in feeding morphology and function.

Poster #: 51
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Computational Chemistry, Organic Chemistry, Reactive Intermediates
Project Title: Computational Investigation of the Photooxidative Cyclization of Oxime Ethers Containing a Built-In Azole Group: A Greener Route to Heterocycles
Author List:
Torres, Danielle; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Tran, Thao; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Petit, Andrew; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Heterocycles are a common motif in organic chemistry and a prevalent component of pharmaceuticals and biomolecules. Working in collaboration with the de Lijser laboratory at CSUF, we are developing an environmentally friendly approach towards synthesizing complex heterocycles using oxime ethers containing a built-in nucleophile along with an organic photocatalyst. More specifically, the oxime ether is photooxidized, generating a radical cation reactive intermediate which subsequently cyclizes. Previous experimental work in the de Lijser group has demonstrated the feasibility of this reaction when the built-in nucleophile is an aryl, thiophene, or furan group. In collaboration with these efforts, we have employed density functional theory to understand the reaction mechanism and explain trends in experimental reactivity.
In this poster, we describe our recent efforts to use computational chemistry to investigate the overall scope of the reaction with respect to both the identity of the built-in nucleophile and the presence of substituents. We first demonstrate that benzaldehyde oxime ethers containing a built-in oxazole, thiozole, or imidazole group will cyclize under the photooxidative conditions of the de Lijser lab experiments. We then show that substituents on the central benzene ring can have a significant effect on the oxidation potential of the oxime ethers and hence the propensity of forming the radical cation through photooxidation. In contrast, the substituent effects are much smaller for the cyclization barrier. Throughout this work, we make clear predictions about the feasibility of reactions which have not yet been experimentally explored. Overall, in conjunction with the experimental efforts in the de Lijser lab, our work has the potential to lead to an environmentally friendly and effective route to synthesize the core heterocyclic scaffold of pharmaceuticals.
This work is supported by the American Chemical Society Petroleum Research Fund (59497-UNI4) and an allocation of supercomputing time through NSF XSEDE (TG-CHE180057).

Poster #: 52
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Computational Chemistry, Organic Chemistry, Reactive Intermediates
Project Title: A Joint Theory-Experiment Study of the Cyclization of Alkynylacetophenone Oxime Radical Cations: A Greener Way to Synthesize Heterocycles
Author List:
Tran, Thao; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Castro, Victor; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
de Lijser, Peter; Chemistry and Biochemistry, California State University, Fullerton
Petit, Andrew; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Heterocycles are common components of biologically relevant molecules and pharmaceuticals. One common way to synthesize heterocycles involves the cyclization of oximes or oxime ethers using either transition metal catalysts or strong acids. Recent experiments in the de Lijser lab demonstrated that benzaldehyde oxime ethers containing an aryl group will cyclize under photoinduced electron transfer (PET) conditions to form complex heterocycles. As these reactions make use of light and an organic photoredox catalyst, they are more environmentally friendly than other approaches.
We are working with the de Lijser laboratory in a joint theory-experiment collaboration aimed at understanding the scope of this approach and in particular how changing the aryl group to another built-in nucleophile affects the reaction outcome. In this poster, we describe our efforts to understand the PET-induced cyclization of 2’-alkynylacetophenone oximes. Using a combination of experimental and computational NMR analysis, we show that the cyclized product is a highly unusual isoindole N-oxide. To the best of our knowledge, this is the first example of such a compound in the literature. Our computational analysis of the reaction mechanism shows that the ioxime radical cation formed after PET undergoes rapid cyclization to generated a radical cation cyclized intermediate. Back-electron transfer from the photoredox catalyst followed by intramolecular proton transfer leads to the final product. We will show that our proposed mechanism provides a clear explanation for the regioselectivity observed in the experimental data. Overall, our collaborative efforts with the de Lijser laboratory have the potential to lead to an environmentally friendly synthesis of a key component of pharmaceuticals.
This work is supported by the American Chemical Society Petroleum Research Fund (59497-UNI4) and an allocation of supercomputing time through NSF XSEDE (TG-CHE180057).

Poster #: 53
Campus: Stanislaus State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Data-mining, Wine Chemistry, Tannins
Project Title: Data Visualization and Correlations between Tannins and Important Wine Chemicals
Author List:
Hernández, Vanessa; Undergraduate, Computer Science and Chemistry, California State University, Stanislaus, Presenting Author
Gutiérrez, Cristian; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Alemán, Elvin A.; Chemistry, California State University, Stanislaus

Abstract: Tannins are from a family of phenolic compounds. They cause wines to exhibit bitterness and astringency, making them an extremely important chemicals during the fermentation process. The goal of this project is to develop a visualization method and analyze correlations between wine chemicals and sensory data using different data-mining techniques in order to improve the overall quality of future wine making, and to better understand how chemicals in wine affect each other and the wine value. This research contains data from Pinot Noir and Cabernet Sauvignon wines from 2013 and 2017. First, we focused in finding how tannins correlated with glycosides, Brix (potential alcohol to sugar ratio), temperature and Anthocyanins. A comparison between Cabernet Sauvignon and Pinot Noir was analyzed to show the amount of chemical levels. This research used 1,729 wine samples. The software used in this project was RStudio with the programming language R and Jupiter Notebook using the programming language Python.
Using this visualization approach, we found that Cabernet Sauvignon grapes are affected more by temperature as the data shows that there is a high increase in tannins when the temperature reaches 87˚F. Anthocyanins also had a positive correlation with tannins and Cabernet Sauvignon was found to have higher concentration of anthocyanins than Pinot Noir. Brix had a negative correlation with tannins and Pinot Noir grapes were found to have a higher amount of Brix than Cabernet Sauvignon grapes which suggested that Pinot Noir grapes have higher amounts of sugar. Another finding is that Pinot Noir wine has less amount of tannins than Cabernet Sauvignon, this can explain why Cabernet Sauvignon is a thick and heavy wine. We have explored other properties of the chemical compounds found in these wines and we have used same visualization approach to investigate how anthocyanins are affected by temperature and how harvesting regions affect the chemical data.

Poster #: 54
Campus: San Francisco State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Cancer immunotherapy, T Cell receptor, Machine Learning
Project Title: A Comparative Study of Machine Learning Methods on Next-Generation Sequencing of T Cell Repertoire Data
Author List:
Kato, Chiemi ; Undergraduate, Mathematics, San Francisco State University, Presenting Author
Utterback, Nicholas; San Francisco State University, Presenting Author
Yang, Hai; University of California, San Francisco
Zhang, Li; University of California, San Francisco
He, Tao; Mathematics, San Francisco State University

Abstract: In recent decades, immunotherapy has been demonstrated as a significant clinical activity in cancer studies. T cells represent a crucial component of the adaptive immune system and are thought to mediate the anti-tumoral immunity. Antigen-specific recognition is realized via T cell receptor (TCR), which is the product of somatic V(D)J gene recombination, plus some random addition/subtraction of nucleotides at recombination junctions. In this study, we aggregated the clones based on V/J gene segments, which overcomes the limitation and thus can build machine learning models across subjects. Here we presented a comparative study of different feature selection (chi-square filter, correlated-based filter, support vector machine wrapper SVM-RFE, random forest wrapper Boruta), classification (SVM, random forest, bagging, boosting, RBF neural nets, MLP neural nets) and clustering methods (K-means, expectation-maximization) on two multiclass Next-Generation Sequencing TCR repertoire data in cancer studies. Both data are from cells samples of a group of cancer patients receiving the immunotherapy.

Poster #: 55
Campus: San Francisco State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Prosthesis, Design, Amputees
Project Title: Low-Cost 3D Printed Modular Powered Prosthesis for Shoulder Disarticulation and Transhumeral Amputees
Author List:
Stewart, Jennifer; Undergraduate, Engineering, San Francisco State University, Presenting Author
Lile, Richard; Undergraduate, Engineering, San Francisco State University
Bryant, Aaron; Undergraduate, Engineering, San Francisco State University, Presenting Author
Mesgun, Sara; Undergraduate, Engineering, San Francisco State University
Quintero, David; Engineering, San Francisco State University

Abstract: In 2005 1.6 million people in the United States were living with limb amputation, 320,000 of which were upper limb amputees. The LUKE arm from Mobius Bionics is the only commercially available upper limb powered prosthetic to include the shoulder joint and starts at $100,000, which is prohibitively expensive for the majority of the population. The design presented is for a low-cost, lightweight modular prosthesis intended to increase accessibility for amputees. The arm can be configured for transhumeral or shoulder disarticulation amputees. Recent advancements in 3D-printing have been utilized to create an arm that weighs less than 10 pounds, costs less than $2500 and can be offered as an open source design. The arm is comprised of a two high torque DC brushless motors, a two stage planetary gear transmission at the elbow and a cycloidal transmission at the shoulder. Both transmissions were designed in SolidWorks computer-aided design (CAD) to be 3D printed, eliminating the need for heavy metal gear boxes. The arm housing shell was also 3D printed further reducing weight. The elbow transmission has a gear ratio of 17.5:1, which was designed to allow the transhumeral configuration to lift a load of 5 lbs in the hand and achieve a theoretical max joint velocity of over 200 rpm at the elbow with no load. The elbow transmission weighs less than 0.6 pounds and allows a range of motion of 0 to 130 degrees, approximating 90% of able bodied range of motion. The shoulder transmission has a gear ratio of 35:1, which provides enough torque to lift a 5 lb load in the hand and a theoretical max joint velocity of 80 rpm under no load conditions. The mechanical design of the shoulder allows a range of motion of 190 degrees. The joint velocity and range of motion of the shoulder required to perform daily living tasks are 190 degrees and 55 rpm. The weight of the shoulder transmission is less than 0.7 pounds. A Exii Hackberry open source hand completed the arm as the end-effector. Both the transhumeral modular the shoulder disarticulation configuration meet or exceed the requirement to perform daily living tasks, ensuring a robust and useful design for upper limb amputees.

Poster #: 56
Campus: CSU Fullerton
Poster Category: Diagnostics/Imaging/Analytical
Keywords: SELEX, Antibiotics, Biosensors
Project Title: Development of DNA Aptamer-based Fluorescent Biosensor for Antibiotic Sulfamethoxazole Detection
Author List:
Lee, Loreece ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Nguyen, Dani; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Alonzo, Leodantez; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Pecic, Stevan; Chemistry and Biochemistry, California State University, Fullerton, Presenting Author

Abstract: Sulfamethoxazole (SMX) is an antibiotic that is used in treating a variety of bacterial infections. However, increased and inappropriate use of antibiotics in humans and animals is the main cause of antibiotic resistance. According to the World Health Organization antibiotic resistance is one of the biggest threats to global health and development today. Thus, it is necessary to continuously, sensitively, and accurately monitor the levels of antibiotics in biological samples and soil to protect the environment and public health. The goal of our lab is to develop a DNA aptamer-based fluorescent biosensor for detection of SMX using a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). We are using a protocol for the isolation of aptamers by solution-phase selection. The method is based on attaching to agarose-streptavidin matrix a biotin-strand complementary to one of the PCR primers in the single-stranded DNA library and then attaching a library through these complementary interactions. In each round, we introduced a solution of the SMX to collect all the specific DNA that binds to it. In order to isolate high binding affinity aptamers, we lowered the concentration of the SMX throughout the multiple rounds. We completed 20 SELEX rounds and the target concentrations were decreased from 100 uM to 2.5 uM. The PCR amplification yielded 30-100 ng/uL of DNA products, and the double-stranded DNA was separated into single-stranded DNA. The concentration of the ssDNA was measured and 100 pmol was used as the starting library for the next SELEX round. After we completed SELEX, cloning was performed. We incorporated the purified PCR product (789.165 ng/uL) directly into plasmid vector. The plasmids were subsequently transformed into competent cells, and positive colonies containing recombinant plasmid DNA were screened via blue/white screening. We randomly picked 24 colonies and cultured them in LB media. We isolated plasmids from the cultured cells and sent the samples to the DNA sequencing service facility. We received 18 sequences and proceeded with secondary sequence analysis. Currently, we are performing the individual clone SELEX and analyzing the elution profiles of each sequence that responds to our target at low concentration. These findings will help us to design and develop the first DNA aptamer-based biosensor for detection of antibiotic sulfamethoxazole. This project is supported by the CSUPERB NIG and the CSUF Start-up funds.

Poster #: 57
Campus: CSU Fullerton
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Brucellosis, zoonotic diseases, diagnostic testing
Project Title: The Development of a New Diagnostic Test to Detect Human Brucellosis Highlights the Severity of Neglected Urban Zoonotic Disease
Author List:
Azenon, Jonathan; Undergraduate, Biological Science , California State University, Fullerton, Presenting Author
Rey Serantes, Diego; IIB, Universidad Nacional de San Martin
Comerci, Diego; IIB, Universidad Nacional de San Martin

Abstract: Neglected zoonotic diseases such as Brucellosis are affecting communities around the world. Brucella canis, the etiological agent found in canine brucellosis, is also pathogenic to humans, causing flu like symptoms, joint pain, weight loss, and in rare cases cardiovascular complications. There is no serological test that effectively detects antibodies against B. canis in individuals, leaving its epidemiological effect on human populations unknown. Currently, blood banks use screening methods that detect the presence of antibodies against the smooth lipopolysaccharide (sLPS) found in B. abortus, B. melitensis and B. suis, which is absent in B. canis. Since B. canis has a rough lipopolysaccharide (rLPS) it is unable to cross react with the sLPS of Brucella spp used for clinical screening of the disease. Rising incidents of B. canis among domestic and free-ranging dogs is a growing concern in urban communities, increasing the potential for human infection through blood transfusion and animal contact. For this reason, the purpose of this study is to evaluate the development of a new high-performance diagnostic test for human brucellosis caused by B. canis. Using an Enzyme Linked Immunosorbent Assay called Brucella canis iELISA, this study analyzed the detection of IgG antibodies against the rLPS of B. canis in blood samples from healthy blood donors across Argentina. The evaluation of this diagnostic test showed that 2.80% of the population have reactivity against B. canis, whereas only 0.10% were reactive against sLPS of Brucella. These results highlight the need for a new diagnostic tool that can effectively detect antibodies against B. canis in order to understand its epidemiological effect on urban populations.
JA was funded by the LA Basin Minority Health and Health Disparities .Research Training Program (MHRT), 5T37MD001368, NIMHD, National Institutes of Health

Poster #: 58
Campus: San José State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: luminescent bioprobe, Europium(III) complex, Biological molecule recognition
Project Title: Luminescence Property Investigation of Europium(III) Coordinated to Oxytetracycline as a Potential Bioprobe of Biological Molecules
Author List:
Ma, Shing Cho; Undergraduate, Chemistry, San José State University, Presenting Author
Villafuerte, Cassie; Undergraduate, Chemistry, San José State University, Presenting Author
Thiri, Thant Mon; Undergraduate, Chemistry, San José State University
Muller, Gilles; Chemistry, San José State University

Abstract: Due to the lanthanide, Ln(III), ions ’ brilliant emissive properties and their line-like emission bands, they are commonly used as bioprobes in biology, bio-chemistry, medicine and related biomedical disciplines. Due to the large demand, further investigation on Ln(III) and its coordinated complex is being commanded. However, when Ln(III) ions are dissolved in water alone, they tend to have weak absorption and emission intensities. The Ln(III) luminescence is quenched by the presence of water molecules through deactivation processes and resulting in a significant amount of energy lost during the process. This energy loss can be prevented by coordinating the Ln(III) ion to a ligand, which ideally possesses an aromatic structure, to protect the Ln(III) ion from external quenching processes such as the coordination of water molecules. Also, due to the conjugation of aromatic structures, ligands that possess aromaticity can provide extra energy to the Ln(III) ion, thus increasing the intensity of luminescence observed. For this project specifically, oxytetracycline (OT) is used in this experiment as it possesses a tetracyclic structure.
The potential bioprobe was studied by first creating samples of Eu(III): OT in aqueous solution in a variety of ratios. Next, each ratio was tested at a pH of 4, 8, and 10 to explore how the environment affected the energy transfer between the ligand and metal. Then, steady-state and time-resolved excitation and emission spectra were measured for each resulting sample. Furthermore, for each ratio, the experiment was repeated with an added biological molecule, L malate or bovine serum albumin (BSA) to investigate the influence of the latter molecule on the local environment of the Eu(III) ion and its luminescence properties.
The intensity of the light emitted allowed us a better understanding of how the environment such as pH, and ratios of concentration altered the energy transfer between the Ln(III) and OT. As a result, the preferred Eu(III):OT:L-malate or BSA complex species was obtained. This study also helped us further our understanding on the recognition of the added biomolecules, since the environment of Eu(III) in the Eu(III):OT bioprobe is very sensitive to changes in the surrounding of the chiral environment. It confirmed that such a bioprobe is well suited for projects aimed at the recognition of chiral biological molecules, since the spectral data observed are influenced by the added biomolecules.

Poster #: 59
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Resistive Pulse, Surface Plasmon Resonance Microscopy, Parkinson’s Disease
Project Title: Monitoring of single particle delivery via surface plasmon resonance microscopy
Author List:
Morgan, Georgia; Undergraduate, Chemistry and biochemistry , California State University, Los Angeles, Presenting Author
Wang, Yixian ; Chemistry and biochemistry , California State University, Los Angeles

Abstract: We are working to develop a method of controlled single nanoparticle delivery using the resistive pulse technique and surface plasmon resonance microscopy (SPRM). The resistive pulse technique utilizes an ionic flow through a nanopipette to detect nanoparticles. By applying an appropriate voltage to the reference electrode inside the nanopipette, individual nanoparticles can be pushed out and delivered to desired locations. SPRM incorporates an optical microscope into SPR, a label-free sensing technique that utilizes the collective excitation of electrons in a thin metal film (e.g. Au and Ag) to probe small changes occurring at the solid/solution interface, which enables visualization of delivered nanoparticles when they reach the substrate. We have successfully performed controlled delivery of single liposomes (100-500nm in diameter) and measured their sizes through a theoretical fitting of the resistive pulses. We also have monitored the delivery of gold nanoparticles via SPRM. The current goal is to synchronize the two approaches. This method will potentially benefit our studies on the interaction between alpha-synuclein aggregates and cell membranes, which is hypothesized to be critical in the development and progression of Parkinson’s disease.
This research was supported by the CSULA Startup Fund, CSUPERB New Investigator Grant, and the National Institute of General Medical Sciences of the National Institute of Health under Award Number R25GM061331.

Poster #: 60
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: microfluidics, glucose, point-of-care (POC) devices
Project Title: 3D microfluidic paper-based analytical devices for colorimetric bioassays
Author List:
Fernandez, Alyssa; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wong, Alyssa; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Neris, Natalia; Graduate, Chemistry and Biochemistry, California State University, Los Angeles
Gomez, Frank; Department of Research, California State University, Office of the Chancellor

Abstract: Microfluidic techniques have been incorporated in many fields due to their portability, compatibility, ability to multiplex, speed to analysis, and ability to analyze samples at low expenses. Microfluidic devices (MDs) can reduce time, reagents, and the complexity of previous lab techniques. The use of paper in MDs has been demonstrated to give promising results in point-of-care (POC) diagnostics due to their small volume requirements, portability, low cost, and technique compatibility. Herein, we describe the development and use of two “mini” 3D microfluidic paper-based analytical devices (3D-µPADs), a mini wrapped (mw)-μPAD multiple inlet design and a mini tree (mt)-µPAD single inlet design. The two “mini” 3D-µPADs were fabricated to detect glucose and assess acetylcholinesterase (AChE) activity via colorimetric-based assays. For the glucose assay, a solution of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) was flowed through the device by vertical capillary flow and, upon contact with pre-spotted glucose, generated a yellow-brown color corresponding to the oxidation of iodide to iodine. For the AChE assay, a mixture of AChE and acetylthiocholine (ATC) was flowed through the devices and, upon contact with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), resulted in a yellow color change due to the thiol reacting with DTNB to produce the yellow anion TNB2-. The devices consisted of multi-wax-printed paper. Saturation curves were generated by flowing reagents through the devices via capillary action and subsequently drying, scanning, and analyzing yielding a correlation between color intensity and substrate concentration. Both mini-µPADs yielded promising results in detecting colorimetric bioassays. The mini chip demonstrates the potential of conducting colorimetric assays using these miniature devices. The use of 3D-based devices incorporating paper for POC diagnostic devices is a viable alternative to more expensive microfluidic-based patterning techniques. For future work, the ease of fabricating 3D MDs demonstrates the potential for developing other devices and for conducting multiple analytes simultaneously.

Poster #: 61
Campus: CSU Fresno
Poster Category: Diagnostics/Imaging/Analytical
Keywords: XRF, bone, lead
Project Title: Estimating lead concentrations in bone phantoms using the L-shell x-ray fluorescence and strontium Kβ/Kα ratio measurements
Author List:
Jardenil, Joshua; Undergraduate, Physics, California State University, Fresno, Presenting Author
Kroeker, Sarah; Graduate, Physics, California State University, Fresno, Presenting Author
Lawson, Alex; Undergraduate, Physics, California State University, Fresno
Gherase, Mihai; Physics, California State University, Fresno

Abstract: Lead (Pb) is a well-known toxic element which accumulates in the human bone following years or decades of exposure. Therefore, long-term Pb exposures are assessed by in vivo bone Pb concentration measurements. In vivo bone Pb concentration is measured using non-invasive x-ray fluorescence (XRF) in the tibia bone to minimize the x-ray attenuation in the overlying soft tissue (ST). In XRF, the sample is exposed to x-rays and its compounding atoms are identified by detection of the emitted characteristic energy (or fluorescence) x-rays from each atom. Intense x-rays are emitted following photoabsorption-induced vacancies in the K or the L shells of the Pb atom. Bone Pb L-shell XRF (LXRF) detects the Pb Lα (10.5 keV) and Pb Lβ (12.6 keV) x-rays and can use compact x-ray tube and detector systems which may lead to practical bone Pb surveys.
In our lab, using a table-top microbeam system, we developed an optimal grazing-incidence position (OGIP) method which enhanced Pb detectability by mitigating the x-ray scatter. Another issue is the conversion of the in vivo bone Pb LXRF data into Pb concentration using Pb-doped bone phantom data; a procedure known as calibration. The main obstacle is the unknown attenuation of the Pb x-rays in the ST. In past LXRF studies, ST attenuation was evaluated using an average ST x-ray linear attenuation coefficient and ST thickness measured by ultrasound, but the method gave inaccurate results.
In our new approach, we used the measured ratio between the Kβ (15.8 keV) and Kα (14.1 keV) peak heights of strontium (Sr) to calculate the ST x-ray attenuation of the Pb x-rays. For each x-ray spectrum, the measured Sr Kβ/Kα ratio reflected the different ST attenuation of the two Sr photon energies. The OGIP setup was used to sequentially expose, in 3 x 300 s trials, 7 cylindrical plaster-of-Paris (poP) doped with Pb in concentrations of 0, 8, 16, 29, 44, 59, and 74 µg/g which were paired with 6 cylindrical-shell-shaped polyoxymethylene (POM) soft tissue phantoms of thicknesses 0, 1, 2, 3, 4, and 5 mm. The Pb and Sr peak height data was used to compute the Pb concentration for each POM thickness.
The concordance correlation coefficients between the computed and the known Pb concentration values for the 6 POM thicknesses were: 0.998, 0.984, 0.869, 0.682, 0.727, and -0.444, respectively.
Research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award #SC2GM121187.

Poster #: 62
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: fluorescence, viruses, nucleic acids
Project Title: Fluorescent Labeling of DNA and RNA by Viral Reverse Transcriptases
Author List:
Turner, Marc; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Wyllie, Mackenzie; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Slemons, Danielle; Graduate, Department of Biology, San Diego State University
Cizmic, Julian; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Wolkowicz, Roland; Department of Biology, San Diego State University
Purse, Byron; Department of Chemistry and Biochemistry, San Diego State University

Abstract: Fluorescent tags and probes for nucleic acids have been pivotal in biochemical research by providing new capabilities in visualizing cellular structures and processes. Using tricyclic cytidine (tC) analogues, which have different photophysical properties depending on structural modifications, we aim to attain the advantage of dynamically imaging the nucleic acids of retroviruses while avoiding any conjugation chemistry. We have performed in vitro kinetics measurements with tC-derived triphosphates and viral reverse transcriptase (RT) enzymes under steady-state parameters and using Michaelis-Menten modeling. When using an RNA template, the RT from two oncogenic viruses showed 1.5-50 fold higher efficiency using the tC analogues, whereas HIV-RT exhibited an average 1.3 fold increase when using a DNA template compared to RNA. For live cell studies, we synthesized derivatives of tC that can traverse cell membranes and be enzymatically converted to active triphosphates intracellularly. The delivery of these molecules to 298T and SUPT1 human T-cells was studied using flow cytometry and the median hepatocyte fluorescence increased by 104 after 24 hours, suggesting retained intracellular fluorescence. We have begun work performing live-cell studies with prokaryotic E. coli but with different delivery methods. Bacterial cells were transformed with a plasmid encoding a nucleoside triphosphate transporter membrane protein that allows the import of extracellular nucleotides, like tC (deoxy)ribose nucleoside triphosphates, into bacterial cells. The rate of uptake and integration into host DNA or RNA will be measured by fluorescence spectroscopy and liquid chromatography mass spectrometry. The results from these studies may provide additional insight into nucleic acid metabolism in both viruses and cells using labeling technology that better preserves natural biological context.

Poster #: 63
Campus: CSU Sacramento
Poster Category: Diagnostics/Imaging/Analytical
Keywords: kinematics, aerodynamics, glider
Project Title: Development and validation of camera methods for quantifying wing shape of flying squirrels during active glides (Glaucomys oregenosis)
Author List:
Espino, Cyrrus; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Bahlman, Joseph; Biological Sciences, California State University, Sacramento

Abstract: Gliding flight is a non-flapping form of flight that allows gliders to quickly, efficiently, and safely travel distances, compared to closely related arboreal species. Originally, scientists assumed that gliders, such as the flying squirrels, glided passively with little ability to vary their aerodynamic forces in the air. However, more recent research has shown that flying squirrels can and do actively vary the aerodynamic forces produced by their wings in a continuous and repeatable behavior. Aerodynamic force is a function of wing shape, and the wing shape of flying squirrels is determined by the posture of the four limbs. Shape and posture are usually described with high-speed video analysis in a controlled laboratory setting, with trained animals wearing distinct, trackable markers. However, studying gliders requires working in the field, with untrained squirrels, without distinctive joint markers, and cameras located farther away from the animal than in a lab. Because these experimental conditions are considerably different from standard motion analysis conditions, it is prudent to verify that the video analysis method can accurately quantify the wing shape under these conditions.
The first objective of this study was to validate the method for quantifying wing shape without trackable markers. To achieve this, I used a physical model of known dimensions moving on a zipline. The model was recorded with multiple high-speed cameras, and I optimized protocol to track body features, such as the paws. I reconstructed the position of the body features into 3D coordinates, and wrote an analysis code to calculate size and shape dimensions to compare with the known dimensions of the physical model. To assess the camera method’s accuracy we compared the metrics recovered by the method to the true values measured on the model. This represents how much the method’s measurements represented the true measurement on the model. The camera method recovered the model dimensions with % accuracy (deviation/true value) ranging from 75 to 100 percent of the true measurement.
The second objective of this study was to incorporate the measurement uncertainty into a computational model that predicts measurement error as a function of camera distance. This allows the lab to predict which wing metrics cannot be resolved above a certain distance, and assists the lab in placing cameras on uneven terrain.

Poster #: 64
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: pharmacokinetics, obesity, antifungals
Project Title: Effects of Obesity and Type II Diabetes on Pharmacokinetics and Toxicity of the Antifungal Drug AmBisome
Author List:
Pomytkina, Ielyzaveta; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author, Eden Award Nominee
Won, Kyi; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona
Slarve, Matthew; Graduate, Biological Sciences, California State Polytechnic University, Pomona
Olson, Jon; Staff, Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract: The World Health Organization estimates that the number of obese and diabetic adults is 650 million and 422 million, respectively. These conditions can negatively impact immune function, increasing susceptibility to microbial infections, including fungal infections. To date, little is known about the pharmacokinetics (PK) and toxicity of the antifungal drug AmBisome (AmBi) in these diverse metabolic populations. In the present study, a murine model was used to investigate how obesity and obesity with diabetes type 2 (TD2M) effects serum PK and toxicity of AmBi. Male ICR mice were fed a high (60%) fat diet for 4 weeks to make them obese (40-45g). Some obese mice (n=45) were induced with nicotinamide (60mg/kg) and streptozotocin (100mg/kg) to become T2DM (blood glucose>200mg/dL), and other mice were not induced (n=45). Control mice (n=45) were fed a normal (10%) fat diet. Obese T2DM, obese only, and normal mice received AmBi (5mg/kg/day) for 7 days, and other mice (n=5/group) were untreated. Blood was collected at 0.5h, 1h, 2h, 6h, 12h, 24h, 48h, and 72h after the last drug treatment (n=5 mice/timepoint). Serum drug concentrations were determined via a standardized bioassay. The 12h serum samples were analyzed for liver and kidney toxicity parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. At 0.5h, 1h, and 12h, obese only mice had significantly higher serum AmBi levels than normal mice (p=0.0079) while at similar early timepoints (0.5h-6h) obese T2DM mice had significantly lower serum AmBi levels than normal mice (p< 0.0159). AmBi treated obese only mice had significantly higher AST, ALT, and BUN levels than non-drug treated mice (p=0.0079, 0.0079, 0.0238, respectively) but there was no significant difference in these toxicity parameters between treated and non-drug treated obese T2DM mice. These results show that obesity alone leads to AmBi-associated kidney and liver toxicity when the drug is given for one week, and this same toxicity is not present in obese mice with T2DM. The more rapid clearance of AmBi from the serum in the obese T2DM mice suggests that less drug is accumulating in the kidneys and liver.
Acknowledgements: Gilead Sciences Inc.

Poster #: 65
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Klebsiella oxytoca, Microbiology, Growth assay
Project Title: Nitrate promotes growth of Klebsiella oxytoca in low and no oxygen conditions that mimic the mouse colon
Author List:
Ramirez, Briana; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Olsan, Erin; Biological Sciences, California State University, Sacramento

Abstract: Klebsiella oxytoca is a common commensal of the human colon, but little is known about the niche within the gut that it occupies. Through studying the metabolic niche, we hope to understand how Klebsiella normally colonize the gut, which may lead to new methods for preventing opportunistic infections by Klebsiella spp.
Hypothesis: Klebsiella spp. are members of the Enterobacteriaceae family. Previous research shows that other family members, such as E. coli, can utilize nitrate respiration for ATP production that provides a growth advantage within the mouse gut after antibiotic treatment. We hypothesize that K. oxytoca uses nitrate respiration under hypoxic (low oxygen) and anoxic (no oxygen) conditions that mimic the oxygen levels found in the mouse colon, and that this provides a growth advantage for K. oxytoca.
Methods: To test our hypothesis, first we performed growth curve assays to measure differences in growth of K. oxytoca cultures under varying concentrations of sodium nitrate. We did this under normoxic (20% oxygen), hypoxic (1% oxygen), and anoxic (no oxygen) conditions at 37°C. Growth of K. oxytoca was measured by collecting 1 mL samples of cultures and measuring the OD600 values on a spectrophotometer at specific time points. Second, we tested for the activity of nitrate reductase enzymes to reduce nitrate to nitrite from the cultures described above. Nitrate reductase enzymes take the electron from the electron transport chain and use it to convert nitrate to nitrite during nitrate respiration. In this assay, cells were lysed to release nitrate reductase enzymes, incubated to allow time for reaction, then measured for nitrite using a spectrophotometer.
Results: Growth assay results show that in hypoxic (1% oxygen) and anoxic (no oxygen) conditions, nitrate provides a growth advantage for K. oxytoca. In normoxic conditions (20% oxygen), there was no difference in growth between 40mM Sodium Nitrate and no Sodium Nitrate cultures. In hypoxic conditions, K. oxytoca increased growth 30.1% in 40mM Sodium Nitrate compared to no Sodium Nitrate. In anoxic conditions, there was increased growth of 34.2% between 40mM Sodium Nitrate and no Sodium Nitrate cultures. Nitrate reductase activity results show 21-fold the enzyme activity in cultures with 40mM sodium nitrate compared to no sodium nitrate. These results show that in little to no oxygen environments, nitrate promotes growth of K. oxytoca and activity of nitrate reductase enzymes is increased.

Poster #: 66
Campus: CSU San Bernardino
Poster Category: Disease (Pathogens)
Keywords: Influenza virus , antivirals, protein interactions
Project Title: Influenza Nucleoprotein (NP) and Polymerase Basic 2 (PB2) Interactions as Novel Antiviral Target
Author List:
Atkins, Cody ; Graduate, Biology , California State University, San Bernardino, Presenting Author
Morales, Kimberly; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Sayers, Sarah; Undergraduate, Biology , California State University, San Bernardino
Tahmasbi, Arash ; Graduate, Biology , California State University, San Bernardino
Newcomb, Laura ; Biology , California State University, San Bernardino

Abstract: Influenza A viruses cause outbreaks and pandemics of life threatening respiratory infections. Due to many hosts, a segmented genome, and lack of proofreading; the virus undergoes changes, making vaccines ineffective and antivirals unusable. Viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is highly conserved across influenza subtypes, making it a good antiviral target. The vRNP is comprised of nucleoproteins (NP), RNA genome, and the RNA dependent RNA polymerase consisting of PA, PB1, and PB2. We use NP mutants defective in vRNP function to investigate essential NP interactions with the goal to identify novel antiviral targets. Here we report NPbd3, a 5 amino acid glycine substitutions within the body domain of NP, and nonfunctional for vRNP activity in reconstituted vRNP assays, does not interact with PB2, while WT NP does. 293T cells were transfected with wild type NP or NPbd3 mutant in combination with PB2. Cells were collected 48 hours post transfection and total protein was isolated. Protein extracts were separated on a 5-40% sucrose gradient with ultracentrifugation. Sedimentation fractions were collected and analyzed by Western blot. Our repeated trials demonstrate that while NP co-sediments with PB2, NPbd3 does not. These NP amino acids are also implicated in binding to the small molecule inhibitor nucleozin. To observe if nucleozin inhibits NP-PB2 binding, cells were again transfected, but this time nucleozin was added 24 hours post transfection. After protein isolation and ultracentrifugation, fractions were collected and are currently undergoing analysis. We also examined the influence of nucleozin on reconstituted vRNP function. Cells were transfected to express reconstituted vRNPs with GFP-MvRNA template. 48 hours post transfection GFP was observed by fluorescent microscopy and total RNA was isolated and subjected to RT-qPCR analysis. Multiple trials confirm that the presence of nucleozin results in decreased GFP-M expression from reconstituted vRNPs. This data supports our hypothesis that NPbd3 is non-functional for vRNP activities because its interaction with PB2 is disrupted, and examines the role nucleozin plays in disrupting protein-protein interactions. Future research includes examination of double mutants within NP body domain to better characterize NP-PB2 interactions and nucleozin inhibition, along with screening additional small molecules for inhibition of vRNP function.

Poster #: 67
Campus: San Diego State University
Poster Category: Disease (Pathogens)
Keywords: mouse model of melanoma, cancer immunotherapies, metastasis prevention
Project Title: VAX014 Efficacy in the B16F10 Mouse Model: A New Oncolytic Immunotherapy for Melanoma?
Author List:
Reil, Katherine; Graduate, Biology, San Diego State University, Presenting Author, Eden Award Nominee
Tsuji, Shingo; Vaxiion Therapeutics Inc.
Giacalone, Matthew J.; Vaxiion Therapeutics Inc.
McGuire, Kathleen L.; Biology, San Diego State University

Abstract: The B16F10 mouse melanoma is a unique preclinical model resembling many human tumors that are highly aggressive, poorly immunogenic, and have high metastatic potential. Therefore, this model is clinically relevant and ideal for testing potential immunotherapies. Here, a clinical stage oncolytic immunotherapy developed by Vaxiion Therapeutics, called VAX014, is being tested as a locally administered therapy for B16F10. This therapy is a novel E. coli derivative, known as recombinant bacterial minicells (rBMCs), that target and deliver therapeutic molecules directly to tumor cells. VAX014 rBMCs incorporate invasin, a rBMC surface protein that targets integrins expressed on tumor cells, and perfringolysin O, an oncolytic bacterial toxin that facilitates tumor cell membrane lysis and death.
In vitro studies demonstrate rapid VAX014-mediated oncolysis of B16F10, which set the stage for in vivo anti-tumor efficacy studies against intradermal B16F10 in immune-competent mice. All mice treated intra-tumorally 1x/week with VAX014 responded either completely (58%, n=22, p<0.0001) or partially (42%, n=16, p<0.0001) with an overall enhanced survival compared to placebo (p<0.0001). When survivors were re-challenged with B16F10 on the opposite flank, >60% (n=32, p<0.0001) had reduced tumor growth, suggesting generation of anti-tumor adaptive immunity in response to therapy. Depletion of CD8+ cells prior to treatment resulted in a total loss of VAX014 efficacy (n=11, p<0.0001), suggesting cytotoxic T cells play a critical role in VAX014-mediated tumor clearance. Flow cytometry was then utilized to identify potential differences in immune cell populations within the tumor microenvironment (TM) between complete and partial responders vs. placebo-treated controls. Consistent with depletion study results, complete responders had a significant increase in T cells, particularly CD8+ cells, compared to partial responders (p<0.05) with a concomitant decrease in CD11b+ myeloid cells (p<0.01). Immunohistochemistry is now being used to determine the location of CD8+ cells within the TM while VAX014 complete responders are being evaluated for long-term anti-tumor durability.
To date, VAX014 stimulates CD8+ T cell mediated anti-tumor immunity in this poorly immunogenic model of melanoma, inducing complete tumor regression in the majority of mice. These data encourage future studies on the investigation of VAX014 as a locally delivered immunotherapy for melanoma patients.

Poster #: 68
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Aspergillus, Poultry, Vaccine
Project Title: Effect of Delivery Route on the Efficacy of a Liposomal Aspergillus Vaccine against Pulmonary Aspergillosis in Dexamethasone treated Broiler Chickens
Author List:
Markson, Jordan; Undergraduate, Animal & Veterinary Science, California State Polytechnic University, Pomona, Presenting Author
Holznecht, Nickolas; Graduate, Biology, California State Polytechnic University, Pomona
Adler-Moore, Jill P.; Biology, California State Polytechnic University, Pomona
Brundage, Cord; Animal & Veterinary Science, California State Polytechnic University, Pomona

Abstract: Introduction: Pulmonary aspergillosis (PA) is a poultry infection with limited treatment options. Antifungal drug treatments are restricted due to cost and difficulty in delivering drugs to poultry. A better treatment option would be an effective vaccine. To determine if an Aspergillus vaccine could decrease fungal burden in PA, we set up a dexamethasone sodium phosphate (Dex SP) immunosuppressed PA model in male and female broiler chickens for testing a liposomal Aspergillus protein vaccine (LAsV) (VesiVax®, Molecular Express Inc).
Methods: SPF eggs were incubated for 21 days, hatched and moved to BSL2 rated cages. Birds were vaccinated (n = 8/group) on day 4 (d4) subcutaneously (sq) or mucosally (mu) and again d10 mu and d17 mu with LAsV using 5ug Aspf3 protein/dose and 5ug Aspf9 protein/dose with 5ug/dose of the adjuvant lipidated tucaresol. Control birds received phosphate buffered saline. Animals were immunosuppressed intramuscularly with 8mg/kg Dex SP twice daily on d19, d20, d21, d22, and d23 and challenged mucosally d20 with 5.4X10ex8 Aspergillus fumigatus conidia (ATCC 13.073). Birds were monitored for disease signs until d24, when lungs and tracheas were collected for fungal burden (CFU/g tissue). Blood was also collected on d24 for an anti-spore agglutinating antibody assay.
Results: The Mann-Whitney test was used for statistical analysis. Chickens vaccinated with LAsV were protected against infection, with lower CFU/g lungs versus non-vaccinated, fungal challenged birds (327 vs 546 CFU/g lungs, P=0.005 with the sq, mu ,mu regimen; 357 vs 546 CFU/g lungs, P=0.07 with the mu, mu, mu regimen). In comparison, the fungal burden in the tracheas of chickens vaccinated with LAsV showed better efficacy for birds given the mu, mu, mu regimen versus birds given the sq, mu, mu regimen (53 vs 347 CFU/g tracheas, P = 0.04). Based on disease scores, only the LAsV mu, mu, mu regimen yielded significantly lower scores versus non-vaccinated birds (P = 0.05). In addition, the anti-spore antibody titers were higher for the mu, mu, mu regimen versus the non-vaccinated chickens (70 vs 10, P = 0.015) and this was not the case for birds given LAsV sq, mu, mu.
Conclusion: LAsV given mu, mu, mu provided better protection against PA in chickens than the LAsV given sq, mu, mu, based on higher levels of anti-A. fumigatus spore antibodies and marked reduction of fungal burden in both lungs and tracheas of infected birds.
Acknowledgement: CSU ARI grant 18-04-250

Poster #: 69
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Tuberculosis, Antimicrobial Peptides, Innate Immunity
Project Title: Determining the mode of action of Human Beta-Defensin 2, an epithelial antimicrobial peptide, against Mycobacterium smegmatis
Author List:
Powell, Tyler; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author, Eden Award Nominee
Porter, Edith; Biological Sciences, California State University, Los Angeles

Abstract: Tuberculosis, most often caused by Mycobacterium tuberculosis (Mtb), remains a global healthcare burden. Mtb infects nearly 10 million people each year, causing 1.3 million deaths annually. The lack of an effective vaccine and the rise of multi-drug and extreme-drug resistant strains have limited progress in eliminating this disease. Considering that only 10 % of the infected individuals will develop tuberculosis, our lab is interested in understanding the normal functioning of the mucosal immune system as a means to design novel approaches to control tuberculosis. Epithelial cells line the inner body surfaces and are an integral part of the mucosal immune system through the production of antimicrobial compounds, including human beta-defensin 2 (HBD2), and other factors. Preliminary data from our lab suggested that HBD2 has strong activity against Mycobacterium smegmatis (Ms), a model bacterium for Mtb, as demonstrated with metabolic assays. However, it is unclear how HBD2 interacts with Ms. We hypothesized that HBD2, like other antimicrobial peptides, has a membrane-targeted action leading to pore formation and membrane depolarization. In this study, we first confirmed the metabolic inhibition and extended the observed inhibitory activity to include biofilm reduction at concentrations as low as 0.313 µM (72.03% ± 5.23% metabolic inhibition and 94.31% ± 4.03% biofilm reduction, p < 0.001 for control versus HBD2). Subsequently, we adapted a protocol to measure membrane permeability with the DNA sensitive fluoroprobe propidium iodide (PI) and membrane depolarization with the fluoroprobe Dibac(4)3. Since PI also stained extracellular DNA that accumulated in biofilm during long-term incubation, we employed this fluorescence-based assay to short term exposure (1.5 h) within the time frame of known antimicrobial peptide action. There was no evidence for membrane permeabilization and, in contrast to the depolarizing agent gramicidin, HBD2 did not affect membrane polarization. These data suggest that the observed metabolic and biofilm inhibition by HBD2 does not reflect killing by pore formation and experiments to test dormancy induction are currently under way. This research may lead to novel prophylactic or therapeutic measures for tuberculosis.
Acknowledgements: Dr. Wuyuan Lu for providing us with HBD2, Louis Stokes Alliance for Minority Participation in Research NSF Grant#HRD-1807387.

Poster #: 70
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Trichomonas vaginalis, Neutrophils, CRISPR/Cas9
Project Title: The Role of Complement Receptor 4 in Trogocytic Killing of the Parasite Trichomonas vaginalis by Neutrophil-like Cells
Author List:
Moran, Jose; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Mercer, Frances; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Trichomoniasis is a sexually transmitted infection (STI) caused by a parasite called Trichomonas vaginalis (Tv) that infects around 3.7 million people in the United States. Roughly 70% of people affected by trichomoniasis are asymptomatic and thus do not seek treatment. Trichomoniasis can increase the risk of infertility, spontaneous abortion, cervical cancers, and secondary STIs, such as HIV. Metronidazole, a non-specific antiparasitic, is the current treatment for Tv infection. However, Metronidazole does not prevent reinfection, and up to 10% of Tv strains are resistant. In order to develop more effective preventative strategies, the cellular and molecular immune responses against Tv need to be better understood. We recently showed that immune cells called neutrophils attack Tv through a recently discovered process called trogocytosis, in which the neutrophil “nibbles” the Tv until its membrane is breached. Trogocytosis is contact-dependent and is thought to be initiated by proteins in human serum priming the parasite for trogocytosis. An abundant protein in sera is iC3b, which coats the surface of Tv. This indicates the presence of a receptor on host neutrophils that bind iC3b. A receptor of interest is complement receptor (CR) 4, which is known to bind iC3b. CR4 is composed of two subunits, CD11c and CD18. We have shown that both subunits are present in neutrophil-like cells (NLCs). We found NLCs to be a suitable model for this study as primary neutrophils are very short lived. To generate NLCs, we will differentiate the pro-myelocytic cell line PLB-985. We hypothesize that deletion of CR4 in PLB-985 cells will decrease trogocytic activity of the resultant NLCs, and their ability to kill Tv. We will knockdown CR4 using CRISPR/Cas9, which will allow for functional deletions of CR4. We designed a CD11c-specific oligonucleotide to serve as the guide RNA and ligated it into a plasmid containing the Cas9 gene. We will repeat this for CD18. With these location-specific edits, we predict that CR4 protein will be rendered non-functional. Future experiments will determine whether CR4-lacking NLCs trogocytose and kill Tv parasites in a cytotoxicity assay, and whether the reintroduction of CR4 utilizing lentiviral stable transfection can rescue the knockout phenotype. Determining the surface receptors involved in the initiation of trogocytic killing of Tv will better characterize this novel antimicrobial process and will better inform novel Tv therapies.

Poster #: 71
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Candida glabrata, adaptive laboratory evolution, environmental stressors
Project Title: CgSTE11 mediates cross tolerance to multiple environmental stressors in Candida glabrata
Author List:
Huang, Mian
Khan, Jibran
Kaur, Manpreet ; Graduate, Chemical and Materials Engineering, San José State University
Ramasubramanian, Anand; Chemical and Materials Engineering, San José State University
Kao, Katy; Chemical and Materials Engineering, San José State University, Presenting Author

Abstract: Candida glabrata is a human commensal and an opportunistic human fungal pathogen. It is more closely related to the model yeast Saccharomyces cerevisiae than other Candida spp. Compared with S. cerevisiae, C. glabrata exhibits higher innate tolerance to various environmental stressors, including hyperthermal stress. Here we investigate the molecular mechanisms of C. glabrata adaptation to heat stress via adaptive laboratory evolution. We show that all parallel evolved populations readily adapt to hyperthermal challenge and exhibit extensive cross-tolerance to various other environmental stressors such as oxidants, acids, and alcohols. Genome resequencing identified mutations in CgSTE11 in all parallel evolved populations. The CgSTE11 homolog in S. cerevisiae plays crucial roles in various mitogen-activated protein kinase (MAPK) signaling pathways, but its role is less understood in C. glabrata. Subsequent verification confirmed that CgSTE11 is important in hyperthermal tolerance and the observed extensive cross-tolerance to other environmental stressors. These results support the hypothesis that CgSTE11 mediates cross-talks between MAPK signaling pathways in C. glabrata in response to environmental challenges. In addition, we show that Ste11 in C. glabrata also plays a role in biofilm formation, which is an important aspect of pathogenesis.

Poster #: 72
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Metastasis, Breast Cancer, Molecular Heterogeneity
Project Title: eIF5A1/2 mediates TGFb/FN1-induced metastasis and associates with prognostically unfavorable SOX2/TP53 genomic alterations in breast cancer patients
Author List:
Geller, Cameron; Graduate, Biology, California State University, Northridge, Presenting Author, Eden Award Nominee
Güth, Robert; Postdoc, Biology, California State University, Northridge
Adamian, Yvess; Graduate, Biology, California State University, Northridge
Maddela, Joanna; Undergraduate, Biology, California State University, Northridge, Presenting Author
Bahkta, Kishan; Undergraduate, Biology, California State University, Northridge
Kutscher, Lindsay; Undergraduate, Biology, California State University, Northridge
Agajanian, Megan; Graduate, Biology, California State University, Northridge
Kim, Sa La; Graduate, Biology, California State University, Northridge
Kelber, Jonathan; Biology, California State University, Northridge

Abstract: Metastatic breast cancers carry a 5-year survival prognosis of less than 20%. Thus, it is highly desirable to identify therapeutic strategies that specifically target both primary and metastatic tumors. It is generally accepted that epithelial-mesenchymal transition (EMT) is an important regulator of therapy responsiveness, contributing to intratumoral heterogeneity and systemic dissemination in solid tumor types such as breast cancer. Since we have previously reported that posttranslational hypusination of eukaryotic Initiation Factor 5A (eIF5A1/2) is required for Pseudopodium-Enriched Atypical Kinase One (PEAK1) translation and PEAK1 mediates malignant phenotypes of Transforming Growth Factor β (TGFβ) signaling, we hypothesized that TGFβ may directly regulate eIF5A1/2 activity during EMT and that targeting this pathway may effectively impair metastatic breast cancer cells. We show evidence of an active eIF5A/PEAK1 pathway in undifferentiated, mesenchymal breast cancer tissue. Notably, inhibition of eIF5A hypusination blocks PEAK1 translation, cell viability, and TGFβ/fibronectin-induced EMT and metastasis of breast cancer cells. Further, assessment of subcellular eIF5A1/2 in response to inhibitors of the hypusination pathway revealed cell line-dependent patterns that suggest eIF5A1/2 functions to drive intercellular heterogeneity. Using a gene set defined by PEAK1-induced EMT and eIF5A1/2 pathway markers, we generated an instructive interactome containing 12 nodes represented in the initial gene set and an additional 74 interacting nodes. Bioinformatic analyses identified SOX2 and PT53 as DHPS interactors within this PEAK1-eIF5A1/2 interactome that significantly contribute to diminished patient survival when genomically altered in combination with DHPS. Taken together, these results suggest that combinatorial therapies targeting DHPS and PEAK1-SOX2/TP53 axes may reduce intratumoral heterogeneity and improve breast cancer outcomes.

Poster #: 73
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microbiome, antibiotic resistance, DNA sequencing
Project Title: Establishment of Coastal Microbe Culturing and Analytical Techniques with the Purpose of Developing a Coastal Microbial Mock Community Standard
Author List:
Zanesco, Dennis; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Baker, Kellen; Undergraduate, Biological Sciences, California State University San Marcos
Rivera, Uriel; Undergraduate, Biological Sciences, California State University San Marcos
O’Brien, Erin; Undergraduate, Biological Sciences, California State University San Marcos
Becket, Elinne; Biological Sciences, California State University San Marcos

Abstract: Development of mock microbial community standard (MCS) is crucial for standardizing microbiome analyses to detect workflow biases, which can cause inaccurate results in studying microbial populations. Such mock MCSs have been produced for a variety of microbial niches, but not for marine environments. As microbes have unique characteristics and therefore unique analytical biases in different niches, we sought to create a marine-specific MCS. In order to accurately create an MCS containing marine microbes, the development of culturing, strain collection, and analytical techniques on marine microbial strains was required. Culturing was done using modified marine media recipes to culture a larger number of strains and used to build a strain collection. Cultured isolates and total coastal microbiomes were analyzed using 16S rRNA sequencing (Sanger and Next-Generation Sequencing) in order to type isolates and establish community profiles, respectively. Although the microbial standard is still in development, multiple strains of bacteria have been isolated and characterized, such as Vibrio spp. and Rhodopseudomonas palustris, and a first-pass of a community profile has been created, revealing a roadmap for what strains should be included in the MCS. Fluorescence-based quantification methods have also been established for the accurate counting of each culture to be included in the final standard. The developed marine MCS is to be used to standardize internal lab analytical pipelines, as well as made available to all CSU labs studying marine microbial populations.

Poster #: 74
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Tandem gene duplication, Arabidopsis thaliana, CRISPR/Cas9
Project Title: CRISPR/Cas9-mediated mutagenesis of tandem gene arrays in Arabidopsis thaliana
Author List:
Ford, Cristina; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Fernandez, Francisco; Undergraduate, Biological Sciences, California State University San Marcos
Sanchez, Kassandra; Undergraduate, Biological Sciences, California State University San Marcos

Abstract: Tandem gene duplications are very prevalent in plant genomes. For example, ~10% of the genes in the Arabidopsis thaliana genome are present in arrays containing two or more consecutive gene copies. Determining the function of tandemly duplicated genes can be challenging because the individual homologs often have a high degree of sequence similarity and functional redundancy. Thus, inactivating one gene copy in a tandem array may have no functional effect, because the remaining homologous gene copies can fulfill a similar or identical function. Our research objective was to use CRISPR/Cas9 as a tool to target and mutagenize entire tandem gene arrays in A. thaliana, thus allowing their functional characterization. We compared two CRISPR/Cas9 mutagenesis strategies: 1. Designing guide RNAs to target opposing ends of a tandem array in the hope of creating a large chromosomal deletion, and 2. Using sequence conservation within tandem gene coding sequences to design guide RNAs that mutagenize all gene copies simultaneously. For these studies, we targeted a tandem array of five highly conserved glutaredoxin genes on A. thaliana chromosome 4. Previous research suggested that these glutaredoxins are involved in root system development and the uptake of nitrate from the soil. We generated a set of transgenic A. thaliana plants expressing “large deletion” CRISPR/Cas9 vectors (Group 1) and a separate set of transgenic plants expressing “multi-gene target” CRISPR/Cas9 vectors (Group 2). Based on PCR screening, less than 5% of transgenic plants in Group 1 showed evidence of large chromosomal deletions (~12 kb) that removed the entire glutaredoxin gene array. Further, these rare deletion events were probably occurring in small numbers of somatic cells, because they were not consistently inherited in subsequent generations. In contrast, plants displaying small insertions and deletions in the coding sequences of multiple target glutaredoxin genes were relatively common in Group 2. One transgenic line was fully characterized by sequencing, and was shown to have homozygous knockout mutations in all five target glutaredoxin genes. Our results suggest that using CRISPR/Cas9 to target conserved regions of gene coding sequences is an effective strategy to mutagenize tandem gene arrays. Now that a quintuple glutaredoxin mutant has been generated, we are in the process of functionally characterizing root development and nutrient uptake in the mutant line.

Poster #: 75
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: pyomelanine, Vibrio, ochronosis
Project Title: Characterization of a pigmented Vibrio anguillarum isolate
Author List:
Batallones, Veronica; Undergraduate, Biological Science , California State University, Fullerton
Fernandez, Jennifer; Graduate, Biological Science , California State University, Fullerton
Farthing , Brett; Undergraduate, Biological Science, California State University, Fullerton
Shoemaker, Jordan; Undergraduate, Biological Science, California State University, Fullerton
Qian, Keizen Li ; Undergraduate, Biological Science, California State University, Fullerton
Phan, Kimberly; Graduate, Biological Science, California State University, Fullerton
Fung, Eric; Undergraduate, Biological Science, California State University, Fullerton
Rivera, Ashley; Undergraduate, Biological Science, California State University, Fullerton
Van, Kevin; Undergraduate, Biological Science, California State University, Fullerton
de la Cruz, Francesca; Undergraduate, Biological Science, California State University, Fullerton
Ferreri, Alexandra J.; Undergraduate, Biological Science , California State University, Fullerton
Burinski, Krystle; Undergraduate, Biological Science, California State University, Fullerton
Zhang, Jackie; Undergraduate, Biological Science, California State University, Fullerton
Lizarraga, Vicente; Undergraduate, Biological Science, California State University, Fullerton
Doan, Kevin; Undergraduate, Biological Science, California State University, Fullerton
Roca, Kenneth; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Traglia, German; Universidad de Buenos Aires, Buenos Aires, Argentina
Ramirez, Maria S.; Biological Science, California State University, Fullerton
Tolmasky, Marcelo E.; Biological Science, California State University, Fullerton

Abstract: Background: The Gram-negative bacterium Vibrio anguillarum causes vibriosis, a haemorrhagic septicaemic disease that affects marine and fresh/brackish water fish and is responsible for significant loses to aquaculture industries. A V. anguillarum strain isolated in the East coast produces large quantities of a brown pigment. We identified the molecular nature of this unique phenotype and propose that it is an ochronotic pigment. Methods: Cloning vehicles used for molecular cloning of chromosomal DNA were pCR-BluntII-TOPO (expression vector) or pACYC177. DNA fragments for cloning were generated by PCR or restriction endonuclease digestion. Analysis of the phenotypes produced by different recombinant clones was carried out by visual inspection after transformation of E. coli TOP10 or BL21(DE3) strains. Results: Inspection of the colonies formed after plating the original isolate showed that about 99% were dark brown and 1% were clear, the usual color of V. anguillarum colonies. Dark and clear colonies were isolated for further analysis. Cloning of DNA fragments corresponding to the location of the hpdA and hgdA genes resulted in dark or clear E. coli TOP10 colonies. DNA sequencing showed that the dark variant had a 410-bp duplication within the hgdA gene that resulted in disruption of the gene. Then, this strain could not catalyze the conversion of homogentisate to 4-maleylacetoacetate. As a consequence, homogentisate (an intermediate in tyrosine metabolism) is accumulated and converted to ochronotic pigment through non-enzymatic oxidation and polymerization. Complementation with a clone carrying hgdA restored the clear phenotype. Comparison of the dark and clear colonies by whole genome sequencing and restriction endonuclease mapping of their signature pJM1-like plasmids showed that they were practically identical with the exception of the deletion that causes the dark phenotype. Fitness experiments did not show differences in laboratory growth conditions. Conclusions: A mutation that disabled the hgdA gene is responsible for the high production of pigment. This is a novel variant of V. anguillarum, A search of all V. anguillarum sequenced genomes available showed that this is a novel, unique variant. Future infection experiments may reveal if the dark phenotype confers any advantage with respect to virulence.
This work was funded by California State University Fullerton and Public Health Service Grant 2R15AI047115-05A1, NIAID, NIH

Poster #: 76
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Apoptosis, Autophagy, Glucocorticoids
Project Title: Interactions Between Apoptosis and Autophagy in Glucocorticoid–evoked Death of Human Leukemic CEM C7-14 Cells
Author List:
Fierro, Manuel; Undergraduate, Biology, California State University, Northridge, Presenting Author
Zhou, Shou; Undergraduate, Biology, California State University, Northridge
Medh, Rheem; Biology, California State University, Northridge

Abstract: Apoptosis and autophagy are two overlapping pathways for programmed cell death (PCD). While cells may undergo autophagic cell death (PCD II) in addition to apoptotic cell death (PCD I), autophagy has been proposed to facilitate cell survival by protecting them from apoptosis. Suppression of autophagy causes cells to switch to apoptosis. Anti-leukemic agents such as glucocorticoids (GCs) target PCD pathways to eliminate cancer cells and autophagy may promote or inhibit drug-induced cell death. We hypothesize that autophagy plays a protective role in GC-evoked apoptosis of human leukemic CEM C7-14 cells in culture exposed to the synthetic GC, Dexamethasone (Dex). To uncouple apoptosis and autophagy, we are using an apoptosis inhibitor, NS3694 (NS), and an autophagy inhibitor, Cpd18 (Cpd). Cell viability in the presence of ethanol vehicle, Dex, NS, Cpd, or combinations of these agents was tested via MTT cell proliferation assays. Dex induced cell death was partially blocked by NS, while Cpd promoted basal and Dex-induced cell death, supporting out hypothesis that inhibition of autophagy promotes apoptosis. Dex-mediated cell death is associated with upregulation of a panel of genes including BCL2L11, BIRC3, NFIL3, and RCAN1. BCL2L11 (pro-apoptotic, anti-autophagy) and BIRC3 (pro- or anti-apoptotic, pro-autophagy) play important roles in both apoptosis and autophagy, while NFIL3 and RCAN1 are associated with GC-induced apoptosis of CEM cells. Our goal is to test the hypothesis that inhibition of autophagy will promote apoptosis by enhancing expression of BCL2L11, BIRC3, NFIL3 and RCAN1. We are currently evaluating the ability of NS and Cpd to alter Dex-mediated upregulation of transcript levels of these genes. Preliminary results suggest that Cpd mediated inhibition of autophagy potentiates Dex-mediated upregulation of BRIC3 and BCL2L11, supporting our hypothesis that blocking autophagy may facilitate GC-mediated upregulation of pro-apoptotic genes in human leukemia cells. The dual role of BIRC3 in promoting apoptosis and autophagy is being further investigated.

Poster #: 77
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sensory Physiology, Muscle Spindle Afferent, Proprioception
Project Title: The Essential Role of Glutamate in Maintaining Muscle Spindle Afferent Sensitivity in Adult Mice
Author List:
Kim, Enoch; Graduate, Biological Sciences, San José State University, Presenting Author
Salazar, Alexandra; Undergraduate, Biological Sciences, San José State University, Presenting Author
Than, Kimberly; Graduate, Biological Sciences, San José State University
Bustos, Cebrina; Graduate, Biological Sciences, San José State University
Klier, Nikola; Undergraduate, nklier38@gmail.com , San José State University
Nguyen, Tyler; Undergraduate, Biological Sciences, San José State University
Occiano, Alyssa; Undergraduate, Biological Sciences, San José State University
Ortiz, Serena; Undergraduate, Biological Sciences, San José State University
Villegas, Natanya; Undergraduate, Biological Sciences, San José State University
Wilkinson, Katherine; Biological Sciences

Abstract: Muscle spindles are the sensory receptors in muscles responsible for proprioception or the ability to sense one’s body position in space. Innervating these structures are group 1a and II muscle spindle afferents (MSA), sensory neurons critical to detecting muscle movement and transmitting the sensory information to the central nervous system. Though it is well documented that during muscle stretch, firing rates of MSAs increase and glutamate is released by synaptic-like vesicles (SLV) at MSA nerve endings, the precise role of this glutamate is still uncertain. Previous studies have observed exogenous glutamate to increase the MSA sensitivity to stretch, but the activity of individual MSAs was not measured, suggesting that the observed change in firing rates may have been from other neurons like nociceptors. To address this shortcoming and test the hypothesis that glutamate released by SLVs affects MSAs by enhancing their sensitivity, we isolated the extensor digitorum longus (EDL) muscle and its sciatic nerve from mice and recorded the firing activity of individual MSAs during exogenous glutamate treatment. The muscle-nerve preparation was perfused in an oxygenated synthetic interstitial fluid tissue bath, an extracellular recording electrode was placed on the nerve to measure the activity of individual MSAs, and a battery of 4s ramp-and-hold stretches were performed on the muscle. Glutamate addition led to a significant increase in MSA firing rate during stretch (1mM; n=12, 18.8%). We also decreased glutamate release by blocking the vesicular glutamate transporter 1 (VGLUT1) with the inhibitor xanthurenic acid (XA), and found a significant decrease in firing rates (3mM; n=17, -39%), with 5 MSAs exhibiting a complete absence of firing. However, we noticed heterogeneity in MSA responses to glutamate and XA treatment, potentially due to drug degradation. To confirm our pharmacological findings, we obtained a transgenic mouse line lacking a single copy of VGLUT1 since total loss is lethal. We observed two MSAs responding normally to stretch, while another two failed to sustain firing at the end of stretch. We are currently increasing our sample size, but our preliminary results support our hypothesis that glutamate is critical to maintaining MSA firing during maintained stretch. A better understanding of how muscle spindle stretch sensitivity is regulated can inform treatment targets for patients with compromised muscle spindle function.

Poster #: 78
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Motor Control, Motor Neurons, Muscle Spindle
Project Title: Development of an optogenetic method to stimulate gamma motor neurons in vitro
Author List:
Karekal, Apoorva; Graduate, Biological Sciences, San José State University, Presenting Author, Eden Award Nominee
Byri, Sai Kiran; Undergraduate, Biological Sciences, San José State University, Presenting Author
Masri, Sameer; Undergraduate, Biological Sciences, San José State University
Villegas, Natanya ; Undergraduate, Biological Sciences, San José State University
Hochman, Shawn; Emory University School of Medicine
Wilkinson, Katherine; Biological Sciences, San José State University

Abstract: The muscle spindle is a sensory organ located in skeletal muscle that is critical for motor control and proprioception, or the sense of body position in space. It is innervated by the muscle spindle afferents which respond to changes in the length of the muscle. The length of the muscle spindle is controlled by gamma motor neurons innervating the intrafusal muscle fibers of the spindle. However, it has been challenging to study the gamma motor neurons since it is hard to specifically stimulate the small gamma but not the alpha motor neurons that control the force generating extrafusal fibers. A previous study shows that alpha motor neurons were recruited from small to large diameter with increasing optical intensities in mice expressing the blue light gated Channelrhodopsin2 (ChR2) in motor neurons. We hypothesized that gamma motor neurons, which are smaller than even the smallest alpha motor neurons, will be recruited first using low optical stimuli (≤1mW) and that this will allow us to characterize their effects on muscle spindle afferent sensitivity.
Methods: The extensor digitorum longus muscle and peroneal branch of the sciatic nerve from adult mice expressing ChR2 in choline acetyltransferase (ChAT) positive motor neurons were dissected and placed in an in vitro tissue bath with oxygenated synthetic interstitial fluid. The sciatic nerve was attached to an extracellular suction electrode that recorded muscle spindle afferent activity. Blue LED light (470nm; 0.5mW-5 mW) was delivered to the end of the nerve.
Results: We found that, as expected, the lowest optical intensities of 0.5-1mW recruited the more slowly conducting gamma motor neurons. Higher optical intensities of 2-5mW recruited the shorter latency alpha motor neurons, evidenced by the presence of twitch contraction. At the lowest optical intensities we observed an increase in muscle spindle afferent firing rate in the absence of twitch contraction, confirming that gamma motor neurons alone had been recruited. We are currently using this technique to characterize gamma motor neuron control of muscle spindle afferent tone. In summary, we have developed an optogenetic technique to recruit gamma motor neurons in vitro that can be used to study fusimotor control of the muscle spindle. Future studies will develop this approach for use in in vivo to better study gamma motor neurons and their contribution to motor control under normal and disease conditions.

Poster #: 79
Campus: Cal Poly San Luis Obispo
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: recombinant protien, coronavirus vaccine, protein expression level
Project Title: PLANT PRODUCTION OF A RECOMBINANT SUBUNIT VACCINE AGAINST PORCINE EPIDEMIC DIARRHEA VIRUS
Author List:
Dizon, Mark; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Clark, Makenna; Applied Biotechnology Institute, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Contreras, Brittney; Applied Biotechnology Institute, Biological Sciences, California Polytechnic State University, San Luis Obispo
Hayden, Celine; Applied Biotechnology Institute
Manjarín, Rodrigo; Animal Science, California Polytechnic State University, San Luis Obispo
Egelkrout, Erin; Applied Biotechnology Institute
Keener, Todd; Applied Biotechnology Institute
Fake, Gina; Applied Biotechnology Institute
Howard, John; Applied Biotechnology Institute
Maj, Magdalena; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Protein synthesis using recombinant gene technologies in plants is currently the primary strategy for developing novel vaccines in a safe and economical way. The overall goal of our study is to create a broadly applicable, plant-produced, orally delivered subunit vaccine against porcine epidemic diarrhea virus (PEDV), a highly contagious disease in pigs caused by coronavirus, leading to substantial losses in the swine industry. The spike glycoprotein (S1) located on an envelope of coronaviruses has been identified as the most critical antigen to obtain protection and develop neutralization antibodies.
Here, we focused on determining the optimal extraction conditions of S1 antigen and selection of maize lines with highest expression of PEDV-related proteins.
We separately inserted five different S1 antigen DNA constructs in expression vectors under the control of seed preferred promoters. The differences in the DNA constructs were intended to increase the accumulation and immunogenicity in the animals. Each construct has been transformed into maize seed by electroporation. After growing the first generation of seed for 4 months in a greenhouse, we collected the seed to identify high expressing lines from each of the constructs.
At first, we tested several different extraction procedures by changing buffer, its pH, and adding detergents. Based on Western blotting analysis, we concluded that the optimal extraction condition for S1 recombinant antigen from maize seed is the phosphate buffered saline at pH 7 with 1% of sodium dodecyl sulfate at room temperature. Next, we used a house-made ELISA assay to measure antigen concentration for every DNA construct. This method however made it difficult to distinguish between background optical density and positive signal optical density, providing inconclusive data. Alternatively, we used Western blotting and determined that the 3 maize lines called PDC, PDK, and PDM, each encoding S1 antigen and targeting either endoplasmic reticulum (PDC), cell wall (PDK and PDM), or being fused with dendritic cell peptide to increase immunogenicity (PDM), have the highest concentration of S1 antigen. Using immunochromatography columns, we are currently working on S1 antigen purification.
In conclusion, using a maize system we produced a recombinant subunit vaccine candidate against PEDV coronavirus; the effectiveness of the antigens as vaccines will now be evaluated in the animal trial. ARI #58913, Mazen #18-536

Poster #: 80
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Course-based Research, Gibson assembly, antibodies
Project Title: Modular Cloning and Expression of Antibodies in Chinese Hamster Ovary Cells: A Course Based Research Experience for Senior Biochemistry Students
Author List:
Brooks, Teresa; Chemistry, California State University, Fresno, Presenting Author
Brooks, Cory; Chemistry, California State University, Fresno

Abstract: Integration of course based research as part of the undergraduate curriculum is a high impact approach to providing genuine research experiences to a large and diverse student population. We have implemented a research module involving the cloning and expression of therapeutic antibodies as the culmination of a senior level Biochemistry lab course (CHEM 156) at Fresno State. We have designed a cloning system that takes advantage of the modular domain structure of antibodies in order to facilitate changes in an antibody’s affinity, isotype, or allotype. A system was developed for readily swapping the antigen-binding N-terminal sequences of different antibodies into a set of vectors encoding the conserved regions of the heavy and light chains, all in the background of a strong mammalian expression vector. Gene fragments corresponding to the constant regions of each immunoglobulin chain, as well as the antigen-binding regions of four unique anti-MUC1 antibodies were designed, and produced by gene synthesis. Students from a senior biochemistry lab course (CHEM156) designed oligonucleotides to create sequence overlaps between the variable and constant gene fragments and the destination vector pcDNA3.1. Using a high fidelity Gibson Assembly reaction, seven of the eight intended constructs were prepared by the class in just a few weeks. Of these constructs, three of the four plasmid pairs were transfected into Chinese Hamster Ovary cells for recombinant production of IgG. All three IgG were found to be expressed, though to varying degrees. These constructs are now being using our research lab as part of the investigation of the nature of antibody recognition of the cancer-specific antigen MUC1. This project allowed our senior undergraduates to contribute to an active research program, while earning credit toward a required course. It exposed them to modern cloning methodologies, bioinformatics tools, cell culture techniques and permitted them to participate in a local research conference. It is also a readily scalable project as only the variable regions of new antibodies of interest need be synthesized. Funding for this project was provided by a charitable donation to fund undergraduate research to the Fresno State College of Science and Math.

Poster #: 81
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Induced Pluripotent Stem Cells, Regenerative Medicine, Flow Cytometry
Project Title: Induced Pluripotent Stem Cells Express Major Histocompatibility Complex Class I Genes in its Pluripotent Stage
Author List:
Nguyen, Kayla; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Briggs, Amy; Undergraduate, Biological Sciences, California State University San Marcos
Northington, Kyle; Undergraduate, Biological Sciences, California State University San Marcos
Mothé, Bianca; Biological Sciences, California State University San Marcos

Abstract: Induced pluripotent stem cells (IPSCs) have created a plethora of opportunities in the field of regenerative medicine. Any type of adult cell on the human body can be reprogrammed back to its pluripotent stem cell state, thus calling them induced pluripotent stem cells. These IPSCs can then be differentiated into almost any cell type desired, which can ultimately be used clinically for cell-based therapy. Although this method is promising, it requires a lot of time, effort, and money. Research has recently moved towards the idea of a universal stem cell line that can be mass-produced and readily available to any patient in need. To do this, universal stem cell lines would need to have a lack of Major Histocompatibility Complex (MHC) Class I genes, otherwise, the human immune system would reject these cells. Previous studies show low levels of MHC expression on ES and IPS cells, therefore similarly we hypothesized that at the pluripotent stem cell stage, there would be little or no protein expression of MHC on the surface of the cells. There is no current evidence of a timeline of MHC expression in IPSCs. We tested a commonly used, commercially available, IPSC line for MHC expression. Human B-cell line, 721.221, was used as our MHC-negative control line. Whereas, the 721.221 B-cell line expressing HLA-B*57 and the 721.221 B-cell line expressing HLA-A*02 were both used as MHC-positive control lines. These cells were tested for MHC expression using flow cytometry with the W6/32 antibody. Pluripotency was also tested in the cells using the TRA-1-60 antibody. We now have evidence that there is MHC expression in an IPSC line, even in the pluripotency stage. An ANOVA test was used to determine the statistical significance of our data from our repeated flow cytometry experiments. We found that there is a statistical significance between our MHC-negative control line with our IPSC line (p<0.001) and there is no significant difference between our MHC-positive control line with our IPSC line (p=ns). Thus, our hypothesis was not supported by our data and these IPSCs express MHC at the pluripotent stage. We are currently testing these IPSCs further to better understand the nature of MHC class I genes and to help guide research in the production of universal stem cell lines.

Poster #: 82
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila melanogaster, autism, microbiome
Project Title: The autism-associated chromatin modifier, Chromodomain Helicase DNA Binding Protein 8, affects gastrointestinal phenotypes in Drosophila melanogaster
Author List:
Niosi, Angelo; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Vo, Nguyen; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Amin-Rahbar, Takhmina; Undergraduate, Biological Sciences, California State University, Sacramento
Ardon-Castro, Any; Undergraduate, Biological Sciences, California State University, Sacramento

Abstract: Autism spectrum disorder (ASD) refers to a group of heterogeneous neurodevelopmental disorders that afflict 1 in 59 children in the United States. Mutations in Chromodomain Helicase DNA Binding Protein 8 (CHD8) are among the most common de novo mutations associated with ASD. CHD8 is a chromatin modifier that influences the transcription of many other ASD-risk genes; thus, it is regarded as a master regulator that defines a common ASD subtype, characterized by macrocephaly and gastrointestinal (GI) problems. Yet, the cellular phenotypes caused by CHD8 mutations have not been completely explored. Our research is aimed at determining both neural and GI phenotypes in Drosophila to elucidate how mutations in CHD8 impair development to cause ASD. We have previously shown that loss of function mutations in the Drosophila ortholog of CHD8, kismet (kis), impairs courtship behavior and causes axon outgrowth defects. Here, we aimed to characterize GI phenotypes caused by loss of CHD8/kis expression—specifically, rate of digestion and gut microbiota composition. To determine the rate of digestion, we fed wild-type and CHD8/kis mutant Drosophila food containing a dye tracer. The time from feeding to excretion was tracked. We found that kis mutant flies exhibited a significantly longer rate of digestion compared to wild-type flies, consistent with studies of CHD8 in vertebrate model organisms. Metagenomic sequencing was used to compare the microbial composition of the midguts of CHD8/kis and wild-type Drosophila. Our results show the gut microbiomes differ across these two genotypes at the species level. Further, when we compared the bacterial abundance and composition of anterior and posterior midguts, we found the greatest variation occurred within the posterior region of the midgut. This is the first research to our knowledge in which the gut microbiome of Drosophila carrying an autism risk gene has been characterized.
FUTURE DIRECTIONS: We plan to characterize the cellular composition of CHD8/kis GI tracts in an effort to understand how this gene impacts the rate of digestion and gut microbiome to cause GI problems associated with the CHD8 subtype of ASD. In addition, now that we have characterized the gut microbiome of CHD8/kis flies relative to wild-type, we will alter the composition of the microbiomes in each genotype in order to measure potential changes in behavioral phenotypes. FUNDING: CSUPERB New Investigator Award.

Poster #: 83
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Methanogen, Greenhouse gas inhibition, Enzyme kinetics
Project Title: Using Liquid Chromatography-Mass Spectrometry to study new substrates catalyzed by 4-(β-D-Ribofuranosyl)aminobenzene 5′-phosphate synthase
Author List:
Aguilar, Edwin; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Nominee
Campos, Reianna; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Lin, William; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Hudson, Paula; Chemistry and Biochemistry, California State University, Fullerton
Rasche, Madeline; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Methanogens are microorganisms that produce methane, which is released into the environment as a greenhouse gas. In the human gut, methanogens have also been correlated with obesity. These microorganisms contain an important one-carbon carrier known as tetrahydromethanopterin (H4MPT) needed for biological methane production. The first step of H4MPT side-chain biosynthesis is catalyzed by 4-(β-D-ribofuranosyl)aminobenzene 5′-phosphate synthase (RFAP synthase). This enzyme from the methanogenic archaeon Methanocaldoccus jannaschii (MJ1427) catalyzes the reaction between phosphoribosylpyrophosphate (PRPP) and p-aminobenzoic acid (pABA) to form RFAP, CO2, and inorganic pyrophosphate. Inhibition of this enzyme is a proposed strategy for decreasing methane production. Interestingly, the RFAP synthase inhibitor p-hydroxybenzoic acid has also been shown to act as a substrate for RFAP synthase, yet the kinetic values have not been determined and the prospect that other inhibitors may also serve as substrates has not been explored. In this work, a Liquid Chromatography-Mass Spectrometry (LC-MS) assay protocol was developed to identify additional substrates and analyze their kinetic parameters (Km and Vmax). Following the 2-hour RFAP synthase assay at 70°C, centrifugation and hydrophobic column chromatography, RFAP was detected using its exact mass of 306.06642. Next, the pABA analogs p-hydroxybenzoic acid (pHBA) and p-methylaminobenzoic acid (pMBA) were used to test for enzyme promiscuity. The LC-MS results indicated that 4-(β-D-ribofuranosyl) hydroxybenzene 5’-phosphate (RFHP) and 4-(β-D-ribofuranosyl) methylaminobenzene 5’-phosphate (RFMP) were products of catalysis. The MJ1427 enzyme concentration profile for RFMP and RFHP generated a linear range from 0 to 10 ug of enzyme added. Using LC-MS, the Km for pHBA and pMBA was found to be 1.2 mM and 2.4 mM, respectively after fitting the equations to a hyperbola. The Km values for these substrates are higher than their previously reported Ki values 590 and 150 µM, respectively, and higher than the Km for pABA (150 µM). This implies, as substrates, pHBA and pMBA bind less tightly to RFAP synthase compared to the biological substrate pABA. Discovering new substrates for RFAP synthase will contribute to our understanding of the mechanism of RFAP synthase and may be useful for designing pharmaceutical treatments for decreasing human obesity in the future. [Supported by NSF grant CHE-1508801]

Poster #: 84
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetobacter, gene amplification, replica-printing
Project Title: Replica-print experiments demonstrate gene amplification mutations arise prior to selective stress in Acinetobacter baylyi
Author List:
Davis, Daniel; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Dominguez, Constancia; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Herrmann, Jennifer; Graduate, Biological Sciences, California State University, Sacramento
Reis, Elizabeth; Graduate, Biological Sciences, California State University, Sacramento

Abstract: Gene amplification mutations are key contributors to cancer, antibiotic resistant pathogens, and evolution. Recent studies suggest these mutations are induced by stress imposed during growth-limiting environments. An alternative hypothesis predicts these mutations are present prior to the selective environment and provide a fitness advantage after selection is imposed. In this study, we test both hypotheses using a model system in the bacteria Acinetobacter baylyi where exclusively gene amplification mutants continually accumulate under prolonged growth-limiting selective conditions in benzoate minimum media. We tested two parent strains, both requiring gene amplification mutations to form colonies on benzoate agar plates. One parent strain must amplify a 10kb region encompassing two clustered transcriptional units, encoding catA and catBCIJDF, while the second parent strain must amplify an overlapping 15kb region including an additional third operon, benABCDE. For each parent strain, approximately 30,000 micro-colonies were cultivated on non-selective rich media agar plates, where gene amplification is not required for growth. These plates were replica printed onto one non-selective master plate and three selective benzoate agar plates. Our results demonstrate gene amplification mutant colonies arose continually in the same positions on all three benzoate replica-plates, strongly suggesting the mutants existed before selection was imposed. Nine sets of same position sibling colonies were collected from one parent strain and two additional sets were collected from the other parent strain. For each set, the corresponding position of these three sibling mutant colonies were traced back to the location on the non-selective master plate to collect the original ancestral cells. These enriched cells were resuspended, serial-diluted, and plated onto non-selective agar plates. After incubation, the plates were replica-printed onto one non-selective and three benzoate plates. Our results show the majority of these purified colonies on nonselective plates grew in the same positions on the benzoate replica-plates. Pulse-Field Gel Electrophoresis and qPCR analyses showed that while selected sibling colonies contain high gene amplification (between 8-45 copies), the enriched non-selective cells contain the precursors to gene amplification, such as duplications or triplications. This research was supported by the CSUPERB New Investigator Award.

Poster #: 85
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Blood, Cancer, human
Project Title: Proliferation of Chronic Myeloid Leukemia Cells is Prevented by Antagonizing Growth Receptor Bound Protein-2
Author List:
Griffin, Kallie; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Engbretson, Isabella; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Arpin, Carolynn; Chemistry and Biochemistry, California State University, Chico
Stachura, David ; Biological Sciences, California State University, Chico

Abstract: Chronic myeloid leukemia (CML) is a disease that affects the normal growth of myeloid progenitor cells, which give rise to red blood cells, platelets, and granulocytes. Most CML cases are caused by a chromosomal translocation that links the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1) gene, which is translated into the mutant protein BCR-ABL1. BCR-ABL1 binds to an SH2 domain on growth receptor bound protein-2 (GRB2), an intracellular adaptor protein involved in cell growth and differentiation. This binding initiates progenitor cell proliferation, causing leukemia. Imatinib, a current CML therapy, is a tyrosine kinase inhibitor that targets BCR-ABL1 and prevents its activation. However, most patients become resistant to imatinib, so an alternative or complementary treatment for CML is needed. To prevent and treat oncogenesis in imatinib-resistant patients, we developed a novel GRB2 SH2 antagonist, NHD2-15. With surface plasmon resonance assays, we determined that NHD2-15 binds to GRB2 with a KD of 119uM. Additional experiments indicated that it specifically bound the SH2 domain, rendering BCR-ABL1 inactive when measured by western blotting. We found that NHD2-15 decreased metabolic activity in human CML cells overexpressing BCR-ABL1 (called K562 cells). Due to these findings, we performed proliferation assays on K562 cells to determine the effect of NHD2-15 on CML cell proliferation. Significant growth reduction was observed after the addition of uM amounts of NHD2-15. Furthermore, adding NHD2-15 and imatinib combinatorially showed decreased growth compared to imatinib alone. Since most patients develop imatinib resistance, we created an imatinib-resistant K562 cell line and found that NHD2-15 also significantly reduced their proliferation, indicating that NHD2-15 may be effective at treating this resistant type of leukemia. NHD2-15 appears to be specific for reducing proliferation of leukemic cells, while having little effect on non-cancerous cell lines we have examined. Future research will offer insight into the molecular mechanisms involved in BCR-ABL1+ leukemias. Importantly, since many cancers overexpress GRB2, this novel compound also potentially provides effective treatment for a wide range of different cancers.

Poster #: 86
Campus: CSU Bakersfield
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: plant pathogen, motility, transposon mutant
Project Title: A TetR regulator is involved in the newly discovered motility of the plant pathogen Rhodococcus fascians
Author List:
Ramirez, Favian; Graduate, Biology, California State University, Bakersfield, Presenting Author
Vereecke, Danny; Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces
Francis, Isolde; Biology, California State University, Bakersfield

Abstract: The plant pathogenic bacterium Rhodococcus fascians is known to infect a large variety of plants through the production of a mix of cytokinins. These plant hormones will trigger abnormal growth on above-ground plant parts leading to the formation of a leafy gall at the infection site. Members of the genus Rhodococcus have consistently been described as non-motile. Hence, our understanding of how R. fascians reaches its host remains unknown. Recently, we have demonstrated that R. fascians is motile as bacterial populations were able to significantly spread on softer medium (0.25% agar concentration) typically used to test twitching or gliding motility in comparison to the standard 1.5% agar medium on which bacteria can only move through swimming with flagella. Seven mutants exhibiting reduced motility on 0.25% agar were retained upon screening of a transposon (jumping gene) mutant library. The goal of this project is to identify the interrupted gene in the mutant with the most severe phenotype and confirm its involvement in the bacterium’s ability to move. Genomic DNA was extracted and digested with an enzyme that does not cut in the transposon. The generated fragments were circularized through self-ligation and transformed into Escherichia coli. E. coli bacteria that took up the plasmid containing the transposon were selected through their ability to grow on the antibiotic kanamycin. Their plasmid DNA was extracted and sequenced from the transposon out into the unknown neighboring region hereby identifying that the disrupted gene coded for a TetR transcriptional regulator. In order to show that this regulator is indeed involved in motility, a complementation plasmid containing the tetR gene with its promoter was introduced into the R. fascians mutant. A motility assay on 0.25% agar successfully showed that the complemented mutant exhibited motility similar to that of the wild type bacterium. Hence, we have identified the first component, a TetR transcriptional regulator, that actively contributes to the motility of R. fascians. Future work aims to uncover the target genes controlled by this regulator and identify the disrupted genes in the remaining transposon mutants in order to create a working model on how the pathogenic bacterium reaches its host.

Poster #: 87
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: copper, enterocytes, uptake, ,
Project Title: New findings on how copper is absorbed by cells of the intestinal mucosa, determined with the Caco2 cell model grown in bicameral chambers
Author List:
Azenon, Jonathan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Corona, Luis; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Ricarte, Adrian; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Jaime, Alexis; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Kidane, Theodros; Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Much is still unknown about how dietary Cu is absorbed. The current view is that Cu(II) is taken up by Cu transporter 1, the only as yet identified transporter, after being reduced to Cu(I) by brush border reductases. However, inhibition of CTR1 by excess silver ions does not eliminate Cu uptake, indicating it is not the only uptake system. The objectives here were to document the contributions of CTR1 and a potential chloride-dependent process in Cu absorption, using the Caco2 cell intestinal model, and to explore whether cellular iron status or the ability to reduce Cu(II) impacts these processes.
Caco2 cells monolayers were grown on collagen-coated filters in Transwell plates to form monolayers with tight junctions (trans-epithelial electrical resistance =>250 Ohms). Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu-labeled Cu-nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Ascorbate (1 mM) was added to reduce Cu(II). Control uptake medium contained all chloride salts. The contribution of CTR1 to uptake was measured by the fall in uptake rate due to 50-100 uM Ag(I). Cu(II) reduction was inhibited by an excess of Fe(III). Chloride dependent uptake was determined by the fall in uptake rate when sulfate was substituted for chloride. Effects of substituting other halide ions for chloride were also recorded. Cellular Fe status was varied by preincubating monolayers for 48 h with 5 uM ferric ammonium citrate, or depletion with desferrioxamine. Levels of mRNA for CTR1, DMT1, and reductases (STEAPs 2 and 3) were determined by qPCR, relative to 18S rRNA.
The presence of excess Ag(I) decreased rates of Cu(I) and Cu(II) uptake about 30%. The same was true when sulfate was substituted for chloride. Silver and sulfate effects were additive. Substituting bromide for chloride made no difference, while iodide enhanced uptake rates. Fe pretreatment reduced expression of CTR1 and DMT1 mRNAs; extreme Fe deficiency greatly enhanced it but with no marked changes in Cu uptake. Inhibition of Cu/Fe reductases with excess Fe(III) did not reduce rates of Cu(II) uptake.
We conclude that CTR1 and a halide-dependent uptake process each account for about one third of Cu uptake carried out by this enterocyte model, and that uptake of Cu(II) per se is also occurring.

Poster #: 88
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: ceruloplasmin, estrogen, mRNA expression, ,
Project Title: How beta-estradiol and estrogen mimics increase levels of the multifunctional copper protein, ceruloplasmin, in the blood plasma
Author List:
Savoy, Aleeyah; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Hanamoto, Nicole; Undergraduate, Chemistry and Biochemistry , California State University, Fullerton
Goodrum, Mark; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Kidane, Theodros; Chemistry and Biochemistry, California State University, Fullerton
Linder, Maria; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Ceruloplasmin (Cp) is the main Cu binding protein in the blood plasma, with functions that include facilitation of iron release from cells (as a ferroxidase) and oxidation/inactivation of serotonin and other amine hormones. Cp is also a major transporter of copper to cells throughout the organism, and particularly to the placenta during gestation, which explains the increased levels of this protein that occur during pregnancy and in women taking estrogen-containing oral contraceptives. It has generally been accepted that the Cp in plasma is produced by hepatocytes in the liver, where Cu is incorporated in the trans Golgi network delivered by the Cu “pump” ATP7B and then exocytosed into the plasma. The kidney also expresses Cp mRNA, but its potential role in Cp production has not previously been explored. Our goals were thus to determine whether estrogen increases levels of blood plasma Cp by enhancing expression of Cp and ATP7B mRNAs in kidney as well as liver epithelial cells.
Cultured rat hepatoma or kidney epithelial cells, were treated for 7 days with 17-beta estradiol or thalidomide (5 or 10 uM) or vehicle (ethanol or DMSO). Adult, female Fisher or Sprague Dawley rats were injected subcutaneously twice daily for two weeks, with estradiol (2 mg) or vehicle before euthanization and collection of kidney, liver and blood plasma. Relative levels of plasma Cp were determined by rocket immunoelectrophoresis. Tissue and cell culture expression of mRNA for Cp, ATP7B, and homolog ATP7A, were determined by qPCR after extraction of RNA and reverse transcription into cDNA, using primers and probes from Dharmacon, and comparing levels to 18S rRNA.
Estrogen treatment doubled the levels of Cp protein in the plasma. In liver, estrogen slightly increased expression of Cp and ATP7B mRNAs, but also increased that of ATP7A – not previously thought to provide Cu to Cp. Changes in kidney levels of all three mRNAs were more pronounced. In cultured kidney and hepatic cells, estrogen and thalidomide consistently caused small increases in Cp and ATP7A mRNA expression, and larger increases in ATP7B expression.
These results suggest that estrogen is mainly regulating Cp formation through ATP7B, and that kidney production of Cp may rival that of the liver and be a major source of the increased Cp circulating in pregnancy and in women taking estrogenic oral contraceptives.

Poster #: 89
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Dengue virus protease, Cellular assay, Screen
Project Title: Development of a novel secondary screen that enhances the power of a cellular platform aimed at revealing antivirals against Dengue virus protease
Author List:
Slemons, Danielle ; Graduate, Biology, San Diego State University, Presenting Author
Fealy, Alex; Undergraduate, Biology, San Diego State University, Presenting Author
Smurthwaite, Cameron; Staff, Biology, San Diego State University
O’Hanlon, Ryan; Graduate, Biology, San Diego State University
Wolkowicz, Roland; Biology, San Diego State University

Abstract: Proteolytic cleavage, either by viral or host proteases, is essential for maturation and full activity of many viral proteins. Consequently, antiviral development often targets viral encoded proteases. Our research group developed a cell-based assay to monitor viral protease activity, using HIV-1 protease (PR) as proof of principle. The assay relies on the inducible expression of PR fused between the DNA-binding and transactivation domains of the yeast transcription factor, Gal4. In the presence of an effective PR inhibitor, the fusion protein binds the Gal4 responsive element, and activates the green fluorescent protein (GFP) reporter. Dengue virus (DenV) is the causative agent of DenV Shock Syndrome and Hemorrhagic Fever. There are no antivirals against DenV on the market, leaving bedside care as the only treatment option available against a virus that is spreading together with global warming and mosquitos. Previously, we adapted the HIV-1 PR assay (referred to as cytosolic assay) to the DenV PR non-structural protein 3 (NS3). DenV NS3 is a serine PR that requires the NS2B co-factor for enhanced proteolytic activity and proper folding. NS2B has four transmembrane domains (TM), two on either side of a hydrophilic cytoplasmic-exposed region. In order to monitor the activity of DenV PR in the context of the assay, we had to remove the NS2B TMs to allow the fusion protein to travel to the nucleus.
We have now developed a secondary assay to be used as a counter screen. This assay utilizes Gal4 and GFP in a novel manner, where rather than deleting the NS2B TMs, we exploit them to anchor our constructs in the Endoplasmic Reticulum membrane. It is worth noting that the virus naturally embeds its proteome in the Endoplasmic Reticulum membrane exploiting its many TMS, including those of NS2B. For this assay, referred to as anchored assay, we engineered an intact Gal4 downstream NS2B/NS3 and a well-known NS3 cleavage site. Gal4 is released upon cleavage, thus activating GFP only when PR is active. This novel assay, in conjunction with the cytosolic assay, which has an opposite readout, will increase the potential of finding inhibitors when we embark in drug screening. The opposite readouts of the cytosolic and anchored assays have been corroborated through flow cytometry, western blotting, and confocal microscopy.

Poster #: 90
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microbial genetics, bioremediation, transposon mutagenesis
Project Title: Assembling a model for sheath production in Leptothrix cholodnii SP-6
Author List:
Steiner, Kirstie; Graduate, Biological Sciences, California State University, Chico
Martell, Sandra; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Tamietti, Betsey; Staff, Biological Sciences, California State University, Chico
Fleming, Emily; Biological Sciences, California State University, Chico

Abstract: Leptothrix spp. are aquatic metal-oxidizing bacteria ubiquitous in freshwater environments. These bacteria generate an extracellular tube-like sheath structure to avoid predation, acquire nutrients, and evade toxic metabolic byproducts. Sheaths aggregate into floating mats that clog water distribution systems and enhance the corrosion of steel. Despite their reputation as nuisance bacteria, Leptothrix and other sheathed bacteria have been considered for bioremediation due to the sheath’s ability to attract and bind aqueous inorganic materials. While the structure and chemical composition of the sheath are characterized, the genes associated with sheath formation have not been determined due to a lack of a tractable genetic system in sheath-forming Leptothrix spp. To establish a genetic system in Leptothrix cholodnii SP-6, we first tested the antibiotic sensitivity of a wild type strain and determined cells were sensitive to kanamycin. We next determined L. cholodnii is able to conjugate with E. coli DH5α with the pRL27 plasmid. Transposon mutagenesis with a Tn5 transposon generated mutants at a frequency comparable to the literature, 2.9 x 10-4 transconjugants per recipient cell. We then established a protocol to screen transconjugants for sheath loss based on colony morphology. Sheathless mutants had a smooth-edged colony morphology, while wild type colonies had filamentous, rough-looking edges. Of the 1,167 transconjugants produced, 20-40% presented a sheathless phenotype. Using sequence similarity searches, we assigned putative protein functions to each disrupted gene. Mutants were classified into categories based on the developmental stage affected: monomer synthesis, polymerization, secretion, assembly, and mineral deposition. We hope to use these data to build a model for sheath production. Preliminary analysis suggests that sheath production is multi-step process where components are transported across the membrane and assembled outside the cytoplasmic membrane.

Poster #: 91
Campus: CSU Monterey Bay
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: cell cycle , G1 , Temperature sensitive mutants
Project Title: The Generation of Temperature Sensitive Mutants of Genes Involved in the Early G1 Checkpoint in the Yeast Saccharomyces cerevisiae
Author List:
Wilson , Donny; Undergraduate, Biology and Chemistry , California State University, Monterey Bay, Presenting Author
Rosas, Carlos ; Undergraduate, Biology and Chemistry , California State University, Monterey Bay, Presenting Author
DiSilva, Dominic ; Undergraduate; UC Santa Cruz Department of Molecular, Cell and Developmental Biology , Biology and Chemistry , California State University, Monterey Bay
Kellogg, Douglas R.; UC Santa Cruz Department of Molecular, Cell, and Developmental Biology , Biology and Chemistry , California State University, Monterey Bay
Sreenivasan, Aparna ; Biology and Chemistry , California State University, Monterey Bay

Abstract: The model organism Saccharomyces cerevisiae, also known as budding yeast, has a number of ceramide synthetase genes involved in the synthesis of sphingolipids used to build and expand the cell wall. Three specific genes, LAC1, LAG1, and LIP1 have been identified as major players in the ceramide synthase pathway (Vallée and Riezman 2005), and subsequently are implicated in the early G1 cell size checkpoint. Studying these genes and other early G1 events can have great implications, because gaining further understanding of how cells normally progress through the cell cycle can shed light on how cells become cancerous.
Our lab designed a series of PCR primers to amplify the LIP1 and LAC1 genes from yeast genomic DNA as well as tag them with selectable markers. We skewed our PCR parameters to attempt to induce mutations via Taq Polymerase. We have now successfully amplified our genes with potential point mutations. The next step will be to take advantage of yeast’s inherent ability to undergo homologous recombination and transform the potentially mutant PCR products into wildtype S. cerevisiae to generate a series of LAC 1 and LIP1 temperature sensitive mutants. After the mutants are checked via PCR and sequencing, we will use methods like western blotting to further analyze the effects of the mutant proteins against other known proteins involved in early G1 events. We expect our cells, upon switch to the non permissive temperature, to have an abnormally small cell size and potentially shape, as the ceramide synthase proteins will not function properly, and cause the cell to arrest at the G1/S checkpoint.
1. Bähler, Jürg, Jian‐Qiu Wu, Mark S. Longtine, et. al. “Heterologous Modules for Efficient and Versatile PCR‐based Gene Targeting in Schizosaccharomyces Pombe.” Yeast 14, no. 10 (1998): 943-51.
2. Vallée B, Riezman H. Lip1p: a novel subunit of acyl-CoA ceramide synthase. 2005;24(4):730–741.

Poster #: 92
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microgravity, human stem cells, differentiation
Project Title: Simulated Microgravity Affects Numb Localization in Human Adipose-Derived Stem Cells
Author List:
Javier, Areli Jannes; Undergraduate, Biology, California State University San Marcos, Presenting Author
Roufaeil, Daniel; Undergraduate, Biology, California State University San Marcos, Presenting Author
Solis, Emmanuel; Undergraduate, Biology, California State University San Marcos
Burch, Shalise; Graduate, Biology, California State University San Marcos
Kumar, Rosaline; Undergraduate, Biology, California State University San Marcos
Day, Holly; Undergraduate, Biology, California State University San Marcos
Luna Lopez, Carlos; Biology, California State University San Marcos

Abstract: One of the ultimate goals of human space exploration aims is to travel to Mars and beyond in search of scientific knowledge. However, little is known about the long-term effects of microgravity on the human body. A current problem in space exploration is that astronauts tend to develop osteoporosis or loss of bone mass. To recover bone mass, some tissue engineering applications include the use of adult stem cells. Adult stem cells are known for their unique characteristic of being able to self-renew and asymmetrically divide into a specialized cell like bone, muscles, cartilage, etc. However, our own results presented that stem-cells exposed to simulated microgravity did not differentiate into specialized cells. It is not known why stem cells demonstrated inhibited differentiation on microgravity. We decided to study Numb, a cell fate determinant protein due to its potential role in osteogenesis. Numb promotes differentiation by inhibiting the Notch signaling pathway through localization changes in asymmetric stem cell division. Thus, we hypothesized that Numb localization is affected under microgravity.
We approached this by plating stem cells under standard gravity and simulated microgravity to observe how Numb is localized inside the cells. We used a ground-based simulated microgravity device based on lab-on-a-chip and clinorotation technology, as previously published. Cells were stained using immunofluorescence for intracellular Numb and imaged in a fluorescence microscope. Our results showed that there was highly localized Numb in elongated cells, whereas in stem cells simulated in microgravity did not. Numb localization is essential for asymmetric cell division which leads to stem cell differentiation.
Our next step was to explore how to control Numb localization to increase differentiation. We used a micropattern method that allowed us to force the cells to take on specific shapes like squares, triangles, and circles. By controlling cell-substrate adhesion, we found that we were able to increase Numb localization. The potential use of adult stem cells to treat osteopenia in microgravity maybe inhibited due to the lack of numb localization and osteogenic differentiation. A potential approach could be to use the micropatterning method to understand and promote Numb localization. Next, we will use this method to observe long-term Numb localization and osteogenic differentiation in simulated microgravity.

Poster #: 93
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Chlamydomonas reinhardtii, Cryptochromes, Pull-Down
Project Title: The use of cryptochrome CPH1 as bait in a pull-down assay may allow for the identification of a potential phytochrome in Chlamydomonas reinhardtii
Author List:
Anguiano, Pedro; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Arce, Laura; Staff, Biological Science, California State University, Fullerton
Cohen, Amybeth; Biological Science, California State University, Fullerton

Abstract: Chlamydomonas reinhardtii is a unicellular green alga often used as a model organism to study light-based processes. C. reinhardtii possesses several genes induced by red light and Ca2+/calmodulin. While these signaling molecules are associated with the red-light activated/far red-light deactivated phytochrome photoreceptor, no phytochrome-like photoreceptor has been identified in C. reinhardtii, despite it being present in cyanobacteria, fungi, and vascular plants. In Arabidopsis thaliana, the plant-type cryptochromes CRY1 and CRY2 directly interact with phytochromes when either photoreceptor is in its inactive conformation. If C. reinhardtii possesses a phytochrome-like photoreceptor, then phytochrome-cryptochrome interactions may have been conserved. In the current study, a pull-down assay will be performed using the C. reinhardtii plant-type cryptochrome CPH1 as bait to explore potential cryptochrome-binding partners. The full-length CPH1 cDNA and the truncated CPH1 cDNA, encoding only the Photolyase Homology Region, were subcloned into the expression vectors pGS21a and pET28a, respectively, and then used to transform BL21 (DE3) Escherichia coli cells. The polyhistidine-tagged recombinant CPH1 proteins were induced in the dark and total soluble protein was extracted after treatment with lysozyme and sonication. The recombinant proteins were then purified using Immobilized Metal Affinity Chromatography and subsequently desalted. Next, the purified full-length and truncated CPH1 recombinant proteins will be used in a pull-down assay under red, blue, or far red light along with soluble protein obtained from dark-grown C. reinhardtii cells. If phytochrome-cryptochrome interactions are conserved, then we expect to pull down a phytochrome-like photoreceptor under far red light, but not red or blue light. Any proteins isolated via the pull-down assay will then be sent for sequencing and bioinformatic analyses will be performed to characterize the proteins. Together, these results will help elucidate if red-light signaling pathways in C. reinhardtii are mediated by a yet to be identified red-light photoreceptor.

Poster #: 94
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Nitric Oxide, Mechanically-gated calcium ion channels, Myogenesis
Project Title: Stretch Activates Mechanically-Gated Calcium Channels for Nitric Oxide Formation in the Ectoderm of Chicken Embryos
Author List:
Martin, Adrian; Graduate, Biology, San Francisco State University, Presenting Author
Denetclaw, Wilfred; Biology, San Francisco State University

Abstract: Embryology is the study of fundamental mechanisms that address the origins of tissues and organs and their interactions following fertilization and early development. However, the mechanistic changes that control gene expression and tissue differentiation are only now being understood. For example, calcium-dependent activation of NOS (nitric oxide synthase) in elevation of NO (nitric oxide) was recently identified to signal in myogenesis, but how this rise of NO occurs is poorly understood. Therefore, we hypothesize the ectoderm layer under stretch forces activate mechanically-gated calcium channels to raise calcium (Ca2+) and signal to NOS for NO. To investigate, controlling tension stretch activity was done using HH10 embryos that were egg extracted and pinned to a silicon dish on one side and pulled on the other by a custom made “rake” device. Live-imaging through spinning disc confocal microscopy was used on embryos labeled for Ca2+ using calbryte520 and NO using DAF-2. Stretch-forces induced in the embryo cranial midline of the ectoderm show an immediate two-fold increase of Ca2+ within 3 seconds that lasts for 1 minute before returning to normal levels. However, this strategy, while invoking a Ca2+ rise, did not increase NO. To show the functionality DAF-2 probe and the overall health of the embryo, 1mM ATP added to the ectoderm layer resulted in a substantial increase of NO 30 minutes after treatment. Despite these results, stretch-forces produced a slight rise in Ca2+ and a more sharp increase in NO through isolated sheets of ectoderm cultured on a stretchable PDMS membrane which demonstrate the ability of the ectoderm under stretch to generate NO. The embryo stretch may also result in NO elevation after additional modifications of embryo loading with the DAF-2DA probe. In conclusion, these results suggest that the ectoderm possesses the ability to raise NO levels that signal in myogenesis through mechanically-gated calcium channels.
NSF STC CCC: 1548297

Poster #: 95
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Metastasis, Cell based assay, Matrix Metalloproteinase MMP-14
Project Title: A Cellular Based Assay to Monitor Cleavage of the Extracellular Matrix by Matrix Metalloproteinases as a Tool for Drug Discovery Against Metastasis
Author List:
Doyle, Ryan; Undergraduate, Biology, San Diego State University, Presenting Author
Canter, Ben; Graduate, Biology, San Diego State University, Presenting Author
Nader, Joy; Graduate, Biology, San Diego State University
Tandon, Ruby; Staff, Biology, San Diego State University
Smurthwaite, Cameron; Staff, Biology, San Diego State University
Wolkowicz, Roland; Biology, San Diego State University

Abstract: One of the most important genes up-regulated in cancer is known as Matrix Metalloproteinases (MMPs). MMPs are enzymes critical for the remodeling of the protein matrix which surrounds and supports cells throughout the body known as the extracellular matrix (ECM). While this protein can be either secreted or membrane bound, it functions to remodel the extracellular environments in healthy individuals. However, in cancerous cells, its overexpression leads to the breakthrough of cancer cells from their original niche and the spread in metastasis.

In order to investigate novel ways to block metastasis,, our lab has engineered a new cellular Two-Tag Assay as a tool for drug discovery, enabling the search for inhibitors. The assay, based on a system we developed for HIV and Dengue Virus, was adapted to MMP-14, a critical membrane-bound MMP responsible for ECM remodeling. The Two-Tag system relies on a scaffold composed of two antibody epitopes, HA and FLAG, flanking a protein substrate. As the FLAG epitope is encoded upstream of the substrate and HA, FLAG is lost when cleavage occurs while HA remains attached to the cell membrane. We then use flow cytometry to monitor the presence of the HA and FLAG tags. Based on the presence of one or two tags, we can determine the robustness of cleavage. Previously, we have inserted viral proteases between the tags and showed autocatalytic cleavage or lack of while the protein travels through the classical secretory pathway. For the MMP-14 assay, we have replaced the viral protease with a consensus substrate of MMP-14 and then obtained a cell line through retroviral technology. In addition, we have expressed the MMP-14 enzyme, in the cell expressing the substrate or in a naive cell. Comparing substrate-expressing cells with substrate-and-enzyme expressing cells we have shown that MMP-14 cleaves the substrate. In order to corroborate that cleavage occurred specifically at the cell surface, we have performed mixing experiments with substrate-expressing cells and enzyme-expressing cells. Results were corroborated with confocal microscopy and western blotting. Decrease in FLAG expression in the presence of MMP-14 proved the robustness of the assay and its utility for the discovery of new inhibitors of MMP-14 in an attempt to block metastasis.

Poster #: 96
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer Metastasis, Premetastatic Niche, Signaling Mechanisms
Project Title: Protumorigenic mechanisms of LCN2 action within the premetastatic lung
Author List:
Sanchez, Francesca; Graduate, Biology, California State University, Northridge, Presenting Author, Eden Award Nominee
Nadales, Nathalie; Undergraduate, Biology, California State University, Northridge, Presenting Author, Nagel Award Nominee
Aguayo, Analine; Undergraduate, Biology, California State University, Northridge
Geller, Cameron; Graduate, Biology, California State University, Northridge
Kelber, Jonathan; Biology, California State University, Northridge

Abstract: Mortality for patients with metastatic breast cancer is over 60% at five years post-diagnosis. Thus, there is a critical and urgent need to identify targetable mechanisms that support breast cancer cell dissemination to and expansion within the metastatic niche. In this regard, previous work has established mechanisms by which cancer cells prime these premetastatic tissues, and we recently established a tumor cell-free syngeneic in vivo breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues. Using this model, we reported that secretomes from metastatic breast cancer cells, enriched for a prognostically unfavorable lipocalin 2 (LCN2) axis, induced anti-inflammatory MSC actions and a tumor-supportive premetastatic lung. Interestingly, previous work has demonstrated that LCN2 expression in hypoxic murine kidneys requires deoxyhypusine synthase (DHPS)-dependent hypusination/activation of the eukaryotic translation initiation factor 5A (eIF5A1/2) – an established tumorigenic node. Thus, we asked whether eIF5A1/2 activity is necessary for LCN2 expression in metastatic breast cancer cells. Notably, LCN2 expression in the metastatic triple-negative Py230 breast cancer cells was abrogated following treatment with a DHPS inhibitor. To further characterize potential LCN2 regulatory and/or effector pathways, we analyzed the prognostic significance for a focused set of common eIF5A1/2 and tumor heterogeneity markers. Of these proteins, only genomic alterations in LCN2 or HSPA5 (GRP78 or BiP) genes significantly correlated with decreased median survival of breast cancer patients. In combination with our ongoing studies aimed at identifying Py230 secretome-induced transcriptome changes within the premetastatic lung, these data promise to identify novel targetable mechanisms by which primary breast tumors communicate with the premetastatic niche to promote breast cancer progression and patient mortality.

Poster #: 97
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: antibiotic resistance, microbiome, DNA sequencing
Project Title: Development of a Next-Generation Sequencing Bioinformatics Pipeline to Analyze Antibiotic Resistomes in Coastal Microbial Communities
Author List:
Stretch, Ebony; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
O’Brien, Erin; Undergraduate, Biological Sciences, California State University San Marcos
Apodaca, Leanna; Undergraduate, Biological Sciences, California State University San Marcos
DeNamur, Nicole; UC Berkeley
Zanesco, Dennis; Undergraduate, Biological Sciences, California State University San Marcos
Becket, Elinne; Biological Sciences, California State University San Marcos

Abstract: The ocean is an endpoint for anthropogenic runoff containing antibiotics and other wastes, and because of this, coastal microbiomes are acquiring more antibiotic resistance genes (ARGs). The environment, notably marine niches, is an under-studied wellspring of antibiotic resistance elements. Through horizontal gene transfer, ARGs are spread between bacteria, increasing the number of antibiotic-resistant microbes in these environments, which poses ecological and clinical health impacts. A wealth of publicly available metagenomic (whole-community DNA sequencing) data exists, though specific analyses of ARGs in these datasets have yet to be performed. In order to analyze this vast array of data, an open-source effective bioinformatics pipeline with low computational demand needed to be developed. Only one maintained open-source pipeline currently exists, though preliminary testing resulted in high-computational demand and under-calling of ARGs. Therefore, we combined an established multiple sequence alignment pipeline, Bowtie2, with the data visualization outputs from another pipeline, GROOT, to produce a rapid pipeline that can identify ARGs from next-generation sequencing (NGS) data on any standard laptop. In this study, we adapted this pipeline to our internal sample data, as well as to some publicly available NGS metagenomic ocean data, to analyze ocean resistomes. From this, we uncovered a number of antibiotic resistance traits stemming from both acquired genes or gene mutations in these ocean populations. This pipeline will be utilized for future metadata studies in hundreds of publicly available ocean metagenome datasets to uncover patterns of ARG spread.

Poster #: 98
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, small RNAs, Northern hybridizations
Project Title: Using Northern Blots to Study Transcriptional Regulation of sRNA Genes by the ExoS/ChvI Signaling Pathway in Sinorhizobium meliloti
Author List:
Mack, Laurena; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Chen, Esther; Biological Science, California State University, Fullerton

Abstract: Sinorhizobium meliloti is an alpha-proteobacterium that forms a symbiosis with the plant host Medicago sativa (alfalfa). S. meliloti infects the roots of the plant host, induces nodule formation, and performs nitrogen fixation in return for carbon compounds. This symbiosis allows robust plant growth and is an alternative to manufactured nitrogen fertilizers which contribute to greenhouse gas emissions. The ExoS/ChvI two-component signaling pathway in S. meliloti regulates genes required for the symbiosis. Small RNAs (sRNAs) are short transcripts that play an important role in gene regulation in bacteria. The aim of this research is to use Northern blots to determine whether ExoS/ChvI transcriptionally regulates sRNA genes in S. meliloti. Previously, our lab had identified several sRNA genes that may be regulated by ExoS/ChvI. The expression of these sRNA genes was studied by purifying total RNA from three S. meliloti strains (wild-type, chvI gain-of-function mutant, and chvI partial loss-of-function mutant), enriching for the sRNA fraction, and Northern blotting. So far, we observed that the SmelC337 sRNA gene has higher expression in the chvI partial loss-of-function mutant and lower expression in the chvI gain-of-function mutant compared to wild-type. This result indicates that ExoS/ChvI negatively regulates the expression of SmelC337. In future work, the expression of additional sRNA genes will be analyzed to determine if they are ExoS/ChvI-regulated. Studying sRNA genes regulated by ExoS/ChvI can help us better understand the symbiosis, which can lead to new ways to decrease agricultural usage of manufactured nitrogen fertilizers.

Poster #: 99
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: diabetes, microRNA, insulin
Project Title: miR- 375’s Effect on Pancreatic β-cell Development, Function and Insulin Secretion
Author List:
Frias, Stephanye; Graduate, Biology, California State University, Chico, Presenting Author
Garcia, Jesse; Undergraduate, Biology , California State University, Chico, Presenting Author
Keller, David ; Biology, California State University, Chico

Abstract: Over 30 million Americans have been diagnosed with diabetes, with roughly 90% of the diagnoses being Type II diabetes (T2D). This pancreatic disease is caused by the inability to produce or efficiently use the hormone, insulin. The pancreas regulates metabolism via specialized endocrine islet cells. Islets contain β-cells that regulate blood sugar by means of insulin secretion. Studies show that microRNA-375 (miR-375) is overexpressed in T2D, leading us to hypothesize that β-cells with elevated and reduced levels of miR-375 will result in decreased and increased insulin secretion rates, respectively. To investigate the role of miR-375 in β-cells an insulin enzyme-linked immunosorbent assay (ELISA) was performed on MIN-6 cells. We overexpressed miR-375 or a control microRNA (miRNA) in different glucose concentrations, to see if insulin secretion rates varied. Results showed that when miR-375 was overexpressed, insulin secretion rates decreased 154% when compared to a control in 25 mM glucose. A second insulin ELISA was performed on MIN-6 cells under-expressing miR-375 or a control miRNA in different glucose concentrations. Results showed that when miR-375 was under expressed, insulin secretion rates increased 27% when compared to a control in 25 mM glucose. Studies have shown that overexpression of miR-375 in T2D decreases β-cell quantity, thus we hypothesize that the overexpression of miR-375 in T2D will impair β-cell proliferation or increase apoptosis. To investigate this, a cell counting assay was performed on MIN-6 cells. Cells were transfected with a miR-375 inhibitor or a control and were counted over 4 days to examine the rate of proliferation. Results showed that under-expression of miR-375 does not affect β-cell proliferation. Another consideration is that miR-375 induces apoptosis. To test this, we have developed a positive control using ultraviolet light. Irradiated MIN-6 cells were stained with FITC Annexin V and Cytotox Red to monitor apoptotic stages. Flow cytometry results showed a 10% increase in early stage apoptosis and an 11% increase in late stage apoptosis, when comparing 10 and 40 second time exposures. Our data supports the hypothesis that overexpression of miR-375 contributes to reduced insulin secretion in T2D. However, our data does not yet support the hypothesis that overexpressed miR-375 in T2D impairs β-cell proliferation or apoptosis.

Poster #: 100
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: sirtuin, mammary gland, hormones
Project Title: Estrogen and Progesterone Rescues Defective Mammary Development in Mice Lacking Sirtuin 4
Author List:
Nguyen, Albert; Undergraduate, Department of Biological Sciences, San José State University, Presenting Author
Ouyang, Michael; Undergraduate, Department of Biological Sciences, San José State University, Presenting Author
Lin, Zhihong; Duke Molecular Physiology Institue
Hirschey, Matthew; Duke Molecular Physiology Institute
Huynh, Frank; Department of Biological Sciences, San José State University

Abstract: Sirtuin 4 (SIRT4) is a mitochondrial enzyme that can remove several different post-translational modifications (PTMs) on proteins in mitochondria. By removing PTMs in mitochondria, SIRT4 is able to regulate the metabolism of a variety of nutrients, including glucose, lipids, and amino acids. Unexpectedly, we have found a novel role for SIRT4 in mammary gland biology. Our preliminary data show that female mice lacking the SIRT4 gene (SIRT4KO mice) can have normal pregnancies and give birth to normal pups. However, nearly 100% of the pups born to SIRT4KO females, regardless of pup or sire genotype, do not survive past postnatal day 2. We suspected that the pups were not receiving enough maternal breast milk and indeed, we found that SIRT4KO females had impaired mammary ductal development during puberty and therefore do not develop enough milk-secreting alveoli during pregnancy. We now seek to determine how a genetic loss of SIRT4 leads to impaired mammary gland development. Since the ovarian hormones 17-beta estradiol and progesterone are vital to the development of mammary glands during puberty and pregnancy, we hypothesized that insufficient production of 17-beta estradiol and progesterone in SIRT4KO mice may be responsible for their defective mammary gland development. In order to test this hypothesis, our goal was to treat SIRT4KO mice with high levels of 17-beta estradiol and progesterone to see if we could restore normal mammary gland development. Methods: We treated SIRT4KO mice for 3 weeks with 17-beta estradiol and progesterone and then dissected the mammary glands. Next, we performed histological stains to observe changes in mammary ductal development. Results: Treatment of SIRT4KO mice with 17-beta estradiol and progesterone was able to restore mammary gland development such that it was similar to wildtype mice. These results indicate that SIRT4 plays an important role in the secretion or signaling pathways of female ovarian hormones. Ultimately, the fact that SIRT4KO females are unable to develop normal mammary glands and support offspring suggests that SIRT4 could have evolved as an indispensable gene for mammalian survival. Further, since many mammary developmental pathways are also perturbed in breast cancer, our work could have broad implications for the treatment and prevention of breast cancer in the future.

Poster #: 101
Campus: CSU Long Beach
Poster Category: Other
Keywords: taste, alcohol, rat
Project Title: The role of taste status and opioid signaling in ethanol intake in male and female rats
Author List:
Shakeshaft, Morgan; Graduate, Psychology, California State University, Long Beach, Presenting Author
Gonzalez, Clarissa; Graduate, Psychology, California State University, Long Beach, Presenting Author
Treesukosol, Yada; Psychology, California State University, Long Beach

Abstract: Individual variability in taste responsivity influences food and fluid selection. Like humans, when presented a “sweet”-tasting solution, rats will increase intake in a concentration-dependent manner. A similar response is observed in many non-nutritive sweeteners which have become popular sugar alternatives. Sucralose, an artificial sweetener marketed as “Splenda”, is reported by humans to taste primarily “sweet” but with a concentration-independent bitter taste. Similarly, rats display individual variability in sucralose acceptance. In a two-bottle test in which rats are simultaneously presented water and sucralose, as sucralose concentration increases some rats drink more sucralose than water (sucralose preferers; SP) while some drink less sucralose than water (sucralose avoiders; SA). Individual differences in processing oral cues and reward may contribute to variability in alcohol intake. Ethanol is described by humans as being primarily bitter and is innately avoided, yet when paired with postoral cues, ethanol can be learned to be preferred. We tested the hypothesis that sucralose acceptance predicts how rats respond to ethanol. Here, male and female rats were categorized as SA/SP in a two-bottle test by presenting water and a sucralose concentration (0.0001, 0.001, 0.01, 0.25, 0.5, 1.0 and 2.0 g/L). Next, rats were given access to 1, 2, 4, 6, 8, and 10% ethanol in ascending order in daily 1-h sessions across 6 days. As ethanol concentration increased, all rats decreased in ethanol intake and no significant SA/SP group differences were observed. Next, to assess differences in opioid signaling, 0.1 and 1.0 mg/kg naltrexone, an opioid receptor antagonist, were administered before presentations of 4 and 6% ethanol. At 0.1 mg/kg naltrexone, SA females drank significantly less 4% ethanol than SP females (p = .005), but this difference was no longer apparent at 1.0 mg/kg naltrexone suggesting that SA females are more sensitive to naltrexone than SP females. For males, intake of 4% ethanol decreased similarly following administration of 0.1 and 1.0 mg/kg naltrexone for both SA/SP. Taken together, these findings suggest SA/SP phenotype does not predict ethanol responses in tests involving oral and postoral cues. Additionally, SA/SP group differences were observed for female, but not male rats for reward signaling. Thus, the association of SA/SP status with ethanol responsivity may be partially attributed by differences in endogenous opioid signaling.

Poster #: 102
Campus: CSU San Bernardino
Poster Category: Other
Keywords: fentanyl, developmental, addiction
Project Title: One trial fentanyl-induced sensitization in adolescent and adult rats
Author List:
Lopez, David; Graduate, Psychology, California State University, San Bernardino, Presenting Author
Diaz, Henry; Undergraduate; California State University, San Bernardino Alumni, psychology, California State University, San Bernardino, Presenting Author
Orozco, Oscar; Undergraduate, Psychology, California State University, San Bernardino

Abstract: Opioid misuse has reached epidemic proportions in the United States, primarily as a result of the rise in availability of synthetic prescription
opioids like fentanyl. Despite the growing problems of fentanyl abuse, few preclinical investigations have assessed the addictive properties of this
compound. Thus, the goal of the current study was to assess the abuse liability of fentanyl using a one-trial behavioral sensitization paradigm. In
this experiment, adult and adolescent male and female Sprague-Dawley rats (n=7-8) were injected once with fentanyl (200 or 400 µg/kg, sc) or
saline and placed immediately in locomotor activity chambers for 60 min. After a 48-h abstinence period, all rats were injected with fentanyl (200
µg/kg, sc) and placed in the locomotor activity chambers for 120 min. On the first injection day (i.e., the pretreatment day), fentanyl reduced the
locomotor activity of all groups as rats injected with fentanyl (200 and 400 µg/kg) were less active than rats treated with saline. On the second
injection day (i.e., the test day) rats pretreated with fentanyl exhibited more locomotor activity than rats pretreated with saline; however, like on
the pretreatment day, fentanyl had a suppressant effect on locomotor activity during the first h. Interestingly, while all rats exhibited a sensitized
response, fentanyl-treated adult male rats were only significantly more active than the controls on the first time block (i.e., the first 10 min). In
contrast, adolescent rats and adult female rats treated with fentanyl exhibited increased locomotor activity when compared to saline-treated rats
on time block 1 and the last 12 time blocks (i.e., the final 60 min of testing). Regardless of pretreatment condition, adult female rats exhibited
more activity than male rats on the test day. Our results suggest that adult female and adolescent rats show an enhanced behavioral response to
repeated fentanyl exposure and may be more susceptible to fentanyl use and abuse.

Poster #: 103
Campus: CSU Sacramento
Poster Category: Other
Keywords: Drosophila melanogaster, Neurodevelopment, Toxicology
Project Title: Bisphenol-A Exposure Differentially Impairs Neurodevelopmental Phenotypes in Wild Type Drosophila and a Drosophila model of Fragile X Syndrome
Author List:
Tinsley, Brendan; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author, Eden Award Nominee
Palacios, Yomira; Undergraduate, Biological Sciences, California State University, Sacramento
Ceballos, Alex; Undergraduate, Biological Sciences, California State University, Sacramento
Stein, Jacqueline; Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Undergraduate, Biological Sciences, California State University, Sacramento
Chu, Daniel; Undergraduate, Biological Sciences, California State University, Sacramento

Abstract: BACKGROUND & RATIONALE: Neurodevelopmental disorders (NDDs) have complex multifactorial etiologies, including genes and environmental factors. While hundreds of genes have been linked to NDDs, much less is known about environmental factors that may impact NDD pathophysiology. The gene x environment hypothesis suggests that many environmental factors may only confer risk of NDDs when combined with NDD risk genes. Thus, identification of environmental factors that confer risk of NDDs and examination of how they interact with genetic risk factors is critical to improve our understanding of NDD etiology and to enact preventative measures. The goal of this project was to examine the environmental chemical bisphenol-A (BPA) in combination with fragile X mental retardation 1 (fmr1). Loss of FMR1 expression in humans causes fragile X syndrome, and is the most common monogenic cause of intellectual disability and autism spectrum disorder. The endocrine-disrupting chemical BPA is a high-production chemical commonly used in the production of plastics and epoxy resins; it is found in a vast number of products ranging from baby bottles to thermal receipt paper. The structure of BPA allows it to easily cross the placental and fetal blood brain barriers. METHODS & RESULTS: Here, we exposed wild type and fmr1 Drosophila melanogaster to BPA during embryonic and larval development. We used immunofluorescence and confocal microscopy to examine axon outgrowth in the adult brain. In wild type flies, we found that millimolar concentrations of BPA caused midline crossing defects in fasciculating axons of the mushroom body, an adult neural structure required for higher sensory integration. Counterintuitively, BPA exposure rescued the midline crossing defect in fmr1 flies, suggesting a neuroprotective effect of BPA in this specific mutant background. We used immunofluorescence to quantify the number of mitotically active cells in the larval central brain. We found that BPA exposure caused a significant reduction in the proliferative capacity of neural stem and progenitor cells in the central brains of wild type, but not fmr1 larvae. Finally, we conducted a lethality assay and found that BPA exposure significantly increased lethality in wild type, but not fmr1 mutant flies. FUTURE DIRECTIONS: We are currently investigating how BPA impacts the transcriptome and translatome using RNA-Seq and ribosomal profiling. FUNDING: NIH SCORE SC2 Award (1SC2GM132005-01)

Poster #: 104
Campus: CSU East Bay
Poster Category: Other
Keywords: Biosorption, Spirulina maximus, Bisphenol A
Project Title: Biosorption of Bisphenol A by the Cyanobacteria Spirulina maximus
Author List:
Bouligny, Jonathan; Graduate, Biological Sciences, California State University, East Bay, Presenting Author
Tamulac, Eriko; Undergraduate, Biological sciences, California State University, East Bay
Yousefvand, Azadeh; , FDA Alameda CA 94502
Curr, Kenneth; Biological Sciences, California State University, East Bay

Abstract: Bisphenol-A (BPA) is an estrogen mimetic that is commonly used in epoxy resins used to coat the inside of metal products, such as food cans. Additionally, BPA is found in one use plastic bottles, bottle tops, water supply lines and used in the treatment of many paper products, such as receipt paper. Exposure to BPA is a concern because of possible health effects of BPA on the brain and prostate gland of fetuses, infants and children, contrary to the FDA’s assessment that low concentrations of BPA is quite safe. The widespread consumption of BPA causes significant concentration of BPA to be directly discharged into aquatic environment causing severe toxicity to aquatic microorganisms. Arthrospira maximus, commonly known as Spirulina, is a cyanobacteria that is used as a popular dietary supplement containing high concentrations of proteins, vitamins, omega-3 and 6 fatty acids and works as a strong anti-oxidant.
Our research has measured the biosorption properties of Spirulina maximus to bind BPA in controlled condition. A range of BPA concentrations (0-100ppm) was added to Spirulina cultures in growth curves ranging from 0-5 days. Our growth curves indicated that Spirulina is able to tolerate BPA in solution, at physiologically high concentrations over five days at the largest concentration tested (100 ppm). Using FTIR analysis, several functional groups were identified in the binding of BPA to cell surfaces, while early HPLC analysis indicates Spirulina can uptake BPA at the cell surface or through absorption into the cell. Currently, our preliminary investigation, as to whether Spirulina can remove BPA from BPA contaminated solutions, is giving us positive results and may indicate, yet, another health benefit attributed to Spirulina.

Poster #: 105
Campus: CSU Sacramento
Poster Category: Other
Keywords: marine invertebrate, climate change, growth rate
Project Title: Assessing the Phenotypic Effects of Heat Stress and Starvation in the Economically Important Red Abalone Haliotis rufescens
Author List:
Romero, Carissa ; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Gleason, Lani; Biological Sciences, California State University, Sacramento

Abstract: El Niño events, which will be more frequent in the coming years, increase water temperatures and negatively impact coastal organisms. In particular, bull kelp, a primary food source for the economically important red abalone Haliotis rufescens, has drastically declined due to these warming conditions. Traditionally, red abalone supported a recreational fishery in California. However, due to starvation and declining populations of red abalone, the fishery closed in 2018, resulting in an economic loss of $44 million per year. Red abalone also support an aquaculture industry in California, but juveniles in these farms experience high mortality during El Niño events. Research on how H. rufescens responds to El Niño conditions is therefore crucial to prevent eradication of the species. The goal of this project is to investigate how 1) warm water temperatures and 2) the combined factors of warm water and starvation affect the growth of juvenile red abalone. Juveniles were obtained from three abalone farms: Monterey Abalone Company, The Cultured Abalone Farm, and American Abalone Farm. Abalone (~ 19 individuals per treatment) were then separated into three different food treatment conditions (no food, medium food, and high food), and all three treatments were exposed to a 24-day heat stress simulating warm temperatures experienced during a very strong El Niño event. The shell length of each individual was measured before and after exposure to treatment conditions and daily growth rate was calculated to assess how heat stress and starvation conditions impact growth. Our results indicate that heat stress alone (high food treatment) significantly reduces growth by 40% compared to pre-experimental control conditions (p < 0.001). Exposure to starvation in addition to heat stress (no food treatment) causes an additional 50% reduction in growth compared to heat stress alone (p < 0.001). Individual variation in growth rate among individuals from the same farm was also observed. We conclude that starvation exacerbates the negative effects of heat stress on juvenile red abalone growth. In addition, growth variation within farms suggests that some individuals are more tolerant of heat stress than others. Ultimately, this research can provide crucial information to maintain H. rufescens’ survival and economic value in the face of climate change. CSU Sacramento and the CSU Council on Ocean Affairs, Science & Technology (COAST) provided financial support for this project.

Poster #: 106
Campus: San Francisco State University
Poster Category: Other
Keywords: marine natural products, actinomycetes, cytotoxic compounds
Project Title: Chemical Investigation of Cytotoxic Secondary Metabolites Produced by the Marine-Derived Streptomyces sp. CP55-76
Author List:
Pham, Jessica; Graduate, Chemistry and Biochemistry , San Francisco State University, Presenting Author
Luu, Gordon ; Undergraduate, Chemistry and Biochemistry , San Francisco State University
Campit, Scott; Undergraduate, Chemistry and Biochemistry , San Francisco State University
Bray, Walter; UC Santa Cruz
Lokey , R. ; UC Santa Cruz
Valeriote, Frederick; Jesephine Ford Cancer Center
Amagata, Taro; Chemistry and Biochemistry , San Francisco State University

Abstract: Our group has developed a unique in vitro cancer screening method designated as CP-DDA that rapidly identifies cytotoxic compounds with excellent solid tumor selectivity as well as their mode of actions. We have tested a number of organic extracts prepared from marine sediment-derived actinomycetes. One of the hit strains, Streptomyces sp. CP55-76, has been selected for further study. The organic extract from a large-scale culture (90L) was applied to a reversed-phase flash column chromatography to separate secondary metabolites based on their polarity. This chromatography generated five fractions (20%, 40%, 60%, 80% and 100% MeOH in H2O). Each fraction was applied to molecular networking to analyze all the secondary metabolites produced by this strain. Detailed analysis of the result of molecular networking led to identification of two potentially new compounds in 20% and 40% MeOH in H2O. In addition, these two compounds showed significant selective cytotoxicity against the prostate cancer cell line, LNCaP. These two compounds were isolated using reversed-phase HPLC. Compound 1 isolated from 20% MeOH in H2O fraction turned out to be a novel heptaketide derivative whereas compound 2 isolated from 40% MeOH in H2O was a monoterpene alkaloid. The molecular formulas of compounds 1 and 2 were established as C18H32O3 and C26H30N2O5, respectively, based on their HRESIMS data. The structures of compounds 1 and 2 were characterized by comprehensive 1D and 2D NMR experiments. Details of analysis of molecular networking, isolation of the new compounds and structure elucidation will be presented.

Poster #: 107
Campus: Cal Poly Pomona
Poster Category: Product-focused Innovation
Keywords: Edible insects, Extruded rice , Consumer acceptance
Project Title: Effects of Edible Insect Ingredients on Eating Quality and Sensory Properties of Extruded Rice Products
Author List:
Tao, Jaynie ; Graduate, Nutrition & Food Science, California State Polytechnic University, Pomona
Li, Yao Olive; Nutrition & Food Science, California State Polytechnic University, Pomona, Presenting Author

Abstract: Entomophagy, or the practice of consuming insects, can provide a nutritious relief to many malnourished people. Currently, two billion people from over 113 countries consume some 2,000 recorded edible insects as part of their traditional diets. Many of these insects contain adequate amounts of protein, fat, vitamins, and minerals that are comparable to commonly eaten livestock. Accordingly, the goal of this study was to investigate the feasibility of incorporating edible insect flours in an extruded rice product. Four formulations were developed, including cricket or locust flours at 10 or 15% addition levels, respectively. The extrusion was easier to proceed when making the two cricket formulations than making the two locust formulations. This could be attributed to higher fat content and lower dietary fiber in cricket flour, which may cause more lubricating effect. Physico-chemical properties of the novel insect rice products were assessed against a reference – the market brown rice, over a 3-month storage under the ambient conditions. Overall, the insect rice was found to have darker color than the reference and to be softer and stickier in texture after cooking. All physico-chemical properties remained shelf stable, esp. with a low water activity (insignificant increase from 0.16 to 0.27 after 3 months) indicating a long shelf life (still much below 0.6). Nutritional analysis suggested these insect rice formulations are good sources of protein (ranging from 12.6% to 15.7% in four formulations vs. 7.5% in the reference), fat (7.4% to 9.7% vs. 3.2%), dietary fiber (12.2% to 21.8% vs. 3.6%), and iron (2.8% to 6.7% vs. 1.3%). Sensory evaluations involving 120 untrained panelists were conducted to predict consumer acceptability, including a preference test on 7-point Hedonic scale and a ranking test. Results suggested cricket formulations at 10% and 15% levels were better accepted based on color, aroma, flavor and mouthfeel than the two locust formulations (overall liking scores > 4.5 vs. < 3.5 out of 7). Both cricket formulations warrant continuing optimization based on a penalty analysis. As a staple food providing 20% of the world’s dietary energy and consumed by more than 1 billion people, rice is an ideal vehicle to deliver nutrients carried by edible insects. The incorporation of insect flours in processed foods such as extruded rice products can greatly promote the consumer acceptance by disguising the ‘yuck’ factor associated with intact insects.

Poster #: 108
Campus: Cal Poly San Luis Obispo
Poster Category: Product-focused Innovation
Keywords: protein functionality, hot-boning, low fat
Project Title: Reduction of fat content in processed meat using hot-boning and cold-batter mincing technology
Author List:
Wonderly, Morgan; Graduate, Animal Science, California Polytechnic State University, San Luis Obispo, Presenting Author
Lee, Hongchul; Postdoc, Animal Science , California Polytechnic State University, San Luis Obispo
Pokharel , Siroj; Animal Science , California Polytechnic State University, San Luis Obispo
Kang, Ike ; Animal Science, California Polytechnic State University, San Luis Obispo

Abstract: Processed meats have received negative publicity due to high fat contents that have been linked to adverse effects on human health. The purpose of this study was to generate low-fat meat products using the combination of hot-boning (HB)/crust-freeze-air-chilling (CFAC) (HB-CFAC) and cold-batter mincing technologies. Ham muscles of eviscerated pork carcasses were subjected to either HB-CFAC or chill-boning (CB). The muscles were then pre-chopped (3 min) and chopped (6 min) for control batters using 2% salt, 2% starch, 20% back-fat, and 16% ice, whereas low-fat batters were similarly pre-chopped and chopped for 6, 24, and 36 min using the same ingredients except 0% back-fat and 36% ice. Data in four replications were evaluated by one-way ANOVA, using PASW 18 statistic program and a completely randomized design. A post-hoc analysis was performed using Duncan`s multiple range test to evaluate difference among treatments at P < 0.05. After chilling, the pH of HB muscle was 6.27 at 80 min post-mortem that was significantly higher than the CB muscle (5.63) at 24 h post-mortem (P < 0.05). Similarly, the pH (6.0) of cooked HB gels was higher than the pH (5.7) of cooked CB gels, regardless of fat content (P < 0.05). The fat and moisture contents of control gels were 11 – 12% and 65%, respectively, while those of low-fat gels were 1.6 – 3.0% and 76 – 78%, regardless of HB or CB. The hardness of pork gels was higher in HB gels (53 – 57 N) over CB gels (24 – 30 N), indicating the gel-forming ability of HB batter is better than the CB batter, regardless of fat content (P < 0.05). The value of cohesiveness was the highest in HB low-fat gel, followed by HB or CB control gels, and CB low-fat gel. There was no significant difference in springiness, regardless of boning time or fat content. The values of gumminess and chewiness were significantly higher in HB low-fat gels than CB low-fat gels, with the intermediate values observed for control gels. Based on these results, the crust freezing of HB muscles demonstrated superior raw meat quality to CB muscles, and the combination of HB-CFAC and cold-batter mincing technologies showed improved protein functionality and gel-forming ability compared to the cold-batter mincing of CB muscle (P < 0.05).

Poster #: 109
Campus: Sonoma State University
Poster Category: Product-focused Innovation
Keywords: precision apiculture, internet of things, bee colony monitoring
Project Title: Remote Monitoring of Bee Hive Health Factors For Rural Apiaries
Author List:
Aboumrad, Anthony; Undergraduate, Engineering Science, Sonoma State University
Chesbrough, Sarah; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Lawrence, Logan; Undergraduate, Engineering Science, Sonoma State University, Presenting Author

Abstract: Humans have been keeping and cultivating honey bees since prehistory, and value of these colonies as pollinators and producers of honey (and other hive products) makes their continued health one of the most crucial ecological challenges today. Since the start of the 21st century, significant die-offs of European honey bee colonies have been occurring in North America (in some regions, up to 70% of hives). Contemporary research into the alarming spread of the phenomenon called “colony collapse disorder” (CCD) suggests that a variety of environmental factors may be responsible for the decline. Thus, there is an urgent need for the development of research tools that both scientists and civilian beekeepers alike can use to study the health of their hives in the pursuit of meaningful corrections for repairing the damage done by CCD. One such tool we are developing is an internet-of-things (IoT)-connected, sensor-packed bee hive module that can be used to track real-time environmental metrics within and without the hive. Whereas most existing technology-enhanced bee hive monitoring systems are designed for boutique beekeepers with a stable WiFi connection in close proximity to their backyard farms or gardens, this solution implements a rugged, modular, solar-powered digital sensor array system that can be deployed at minimal cost on new or existing apiaries in remote locations. The modules are designed with a particular focus on providing insight into the health of bee hives in rural areas, and as such they are completely solar-powered and make use of LoRa, a long range, low power wireless communication technology that is ideal for regions with poor internet infrastructure. Many LoRa applications are also open-source, which makes this project readily adaptable for academic research. We are working closely with bee keepers and biologists in our community to identify the most crucial aspects of a colony’s health that ought to be monitored. This project has the potential to make a significant impact on the study and cultivation of bee colonies in remote locations, as its successful completion will provide rural beekeepers with a novel tool for better understanding their hives and many variables on which the health of their hives depend.

Poster #: 110
Campus: Cal Poly San Luis Obispo
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: drug development, in vitro modeling, colon cancer
Project Title: Optimization of Media Mix, Cell Ratios, and Antibody Concentrations for Co-Culture of Human Colorectal Cancer and Vasculature
Author List:
Cole, Gabrielle; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Heylman, Christopher; Biomedical Engineering, California Polytechnic State University, San Luis Obispo

Abstract: A key contributor to the massive cost of drug development lies in the inadequacy of current pre-clinical models that do not recapitulate the complexity of the human body. This has driven the production of revamped in vitro disease models to better predict in vivo cellular and molecular responses to novel therapeutics. Presently, the co-culture of human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts (HDFs), and a metastatic human colon cancer line (SW620) was initiated in 2D and 3D cultures to create a vascularized colon cancer model for drug screening. The effects of different ratios of cell media were analyzed with a luminescence assay, which indicated that the greatest cell survival occurred when the cells were grown in 80% EGM and 20% RPMI. This media ratio was applied in follow-up studies for determining the ratio of SW620 to 5:1 HUVECs/HDFs within fibrin gels, where ideal conditions were vessel formation and controllable tumor growth. Vessel formation was not observed at any of the ratios due to insufficient seeding densities, and above 0.1:5:1 SW620/HUVECs/HDFs tumor growth overtook the culture. Immunostaining of the HUVECs within the 100-150 μm thick gels proved unsuccessful, with diffuse antibody staining within the gel and poor staining of the cell junctions observed regardless of antibody concentrations. The duration of the staining and washing steps likely needed to be extended to observe cell staining as was seen in 2D and to eliminate the excess antibodies. Overall, a media formulation was identified that allowed for survival of all three cell types and a cell ratio of tumor cells to vasculature cells was selected that kept tumor growth in check. Future work to develop the vascularized colon cancer model will include optimizing both the seeding density for vessel formation and staining protocols for visualization of the 3D culture. This work was supported by the CIRM Bridges to Stem Cell Research 2.0, RFA 15-05.

Poster #: 111
Campus: CSU Northridge
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: BUILD-PODER, C. elegans, Sleep
Project Title: Finding the elusive sleepless animal: the C. elegans male?
Author List:
Orselli, Kostantina; Undergraduate, Biology, California State University, Northridge, Presenting Author
Van Buskirk, Cheryl; Biology, California State University, Northridge

Abstract: The nematode Caenorhabditis elegans (C. elegans) is a model organism ideal for the study of conserved behaviors, including sleep. Human sleep is regulated by circadian cues (sleep timing) and by homeostatic control (need-based regulation); however, sleep in C. elegans is not under circadian control, and thus ideal for the study of sleep need. Interestingly, these worms sleep following exposure to damaging conditions, a phenomenon known as stress-induced sleep (SIS). Populations of these animals consist mainly of hermaphrodites, with males arising rarely that are capable of mating with hermaphrodites. As a result, previous studies have overlooked male sleep behavior. To determine if sleep is sexually dimorphic in C. elegans we used a special male-enriched population to examine SIS. We found that males remained awake under conditions that put hermaphrodites to sleep. To determine if this sleeplessness was due to a competing drive to mate, we examined sleep in males that had been isolated from hermaphrodites, and we found that males were still sleepless. Our genetic analyses have narrowed down the possible mechanisms by which this difference arises. Our results have broader implications for the emerging understanding of behavioral plasticity in sleep regulation across species.

Poster #: 112
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: clustering, dendritic cells, myeloid cells
Project Title: Survival of Mature Dendritic Cells Dependent on Clusterin Expression
Author List:
Mendoza, Roy; Undergraduate, Biological Science , California State University, Fullerton, Presenting Author
Lopez Malizia, Alvaro; INBIRS, University of Buenos Aires
Ailin Garcia, Guadalupe; INBIRS, University of Buenos Aires
Sabatte, Juan; INBIRS, University of Buenos Aires

Abstract: Clusterin is a multifunctional glycoprotein found in various tissues and body fluids. It is involved in a number of physiological and pathological processes such as apoptosis, protein homeostasis, cancer and Alzheimer’s disease. Although clusterin expression in myeloid cells has been reported, its influence in dendritic cell (DC) function has yet to be analyzed. Therefore, the purpose of our experiment was to determine the role of clusterin in DC survival. First, we analyzed whether activation of human monocyte-derived DCs resulted in clusterin production. This was done by culturing cells for 48 hours with or without LPS (10 ng/ml). Clusterin was analyzed by western blot and quantified by ELISA. A basal expression level of clusterin was observed in unstimulated DCs, while an increase in production was noted upon activation of these cells by LPS: clusterin concentration in cell supernatant was 0.04 ± 0.1 vs 7.47 ± 2.14 ng/ml (control vs LPS, mean ± ES, n=5, p<0.01 vs unstimulated cells). In order to understand the dynamics of production after activation, we constructed a production curve measuring the level of clusterin post stimulation over a period of four days. A peak in the production of intracellular and secreted clusterin was noted 48 – 72 hours post stimulus. Finally, a knockdown (KD) strategy using a lentiviral gene silencing protocol that delivered a clusterin and scramble (SCR) shRNA was used to determine the role of clusterin in DC function. Two lentiviral combinations were used due to their ability to knockdown >85% of clusterin expression, as evaluated by western blot (in HeLa cells) and rtPCR (DCs). Puromycin was used as a selection drug and a Ficoll gradient was used to separate live and dead cells. We found that silencing clusterin expression (CLU-KD) resulted in a massive death of DCs upon stimulation with LPS, as evaluated by anexin-V/propidium straining: 63.7 ± 11.8, 19.7 ± 4.9, and 25.2 ± 6.5% of dead cells, for LPS-stimulated CLU-KD DCs, LPS-stimulated scramble DCs, and unstimulated DCs, respectively (mean ± ES, n=4, p<0.01 CLU-KD vs scramble or unstimulated DCs). These observations uncover a novel function of clusterin as a key player in the control of the adaptive immune response.
RM was funded by the LA Basin Minority Health and Health Disparities Research Training Program (MHRT), 5T37MD001368, NIMHD, National Institutes of Health

Poster #: 113
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: Computational Biochemistry, Molecular Biophysics,
Project Title: Development of New Methods for Enhanced Conformational Sampling of GPCRs
Author List:
Serrano, Erik; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Khleif, Rafeed; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Chemistry and Biochemistry, California State University, Northridge

Abstract: G protein coupled receptors (GPCRs) are integral membrane proteins that enable a cell to respond to extracellular stimuli like light, small molecules, peptides, and proteins. These receptors have evolved as highly flexible proteins that possess multiple functionally important conformational states, which enable these receptors to activate pleiotropic signaling events inside the cells. Recent progress in membrane protein structural biology techniques and cryo-electron microscopy is providing structural evidence of two or more distinct receptor conformations for these receptors. Computational structural modeling approaches can complement and supplement these studies by predicting all functionally important states of a receptor, however, they run into the classical protein conformational sampling problem. We are developing a computational biophysical method called Enhanced Conformational Markov-state Sampling in Membrane BiLayer Environment (EnCoMSeMBLE) to solve this sampling problem for GPCRs. We are also developing general higher-order GPCR topology parameters (TMHTOP2) that can capture the conformational diversity seen during GPCR activation to find functional-state specific structural signatures. We are utilizing Markov-State-Models to combine the Boltzmann sampling of receptor conformations from molecular dynamics (MD) with the brute-force sampling of receptor conformations from our previously developed ActiveGEnSeMBLE method. As a proof-of-principle, we wanted to see if EnCoMSeMBLE can identify the pre-active (intermediate) state of the adenosine A2A receptor starting from the inactive or the active state conformations. The method was able to predict the pre-active state of the A2A receptor starting from the active state using only MD simulations but required the combination of both MD and ActiveGEnSeMBLE methods to predict the pre-active state from the inactive state. This is not surprising because the pre-active state is present downhill in energy from the active state but is present uphill from the inactive state, which classical MD methods cannot sample. The newly developed TMHTOP2 topology parameters were able to identify unique conformational signatures of the pre-active state of the A2A receptor compared to its inactive and active states, that can be used to identify the pre-active states of other receptors as well.

Poster #: 114
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: membrane proteins, transmembrane protein folding, structural bioinformatics
Project Title: A three-state model of helical membrane protein folding: The role of membrane-water interface as a vestibule for helices before membrane insertion
Author List:
Kawamala, Bridget; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Chemistry and Biochemistry, California State University, Northridge

Abstract: Integral membrane proteins are dominated by transmembrane alpha-helical (TMH) proteins, which play critical roles in cellular signaling processes. These proteins display a wide range of sizes from one TMH domain to 24 TMH domains and a diversity of structural folds. A common feature of most of these folds is the transmembrane orientation of the helical domains and the approximately parallel packing of these domains into helical bundles of varying stability and flexibility. Even though this packing (being the result of the lipid bilayer’s constraints) reduces enormously the folding landscape of TMH proteins, it has been challenging to study the folding of these proteins experimentally. Originally, a two-state model of helical membrane protein folding was proposed, in which, first the helices fold within the membrane or the translocon complex during cotranslational folding and then they assemble within the membrane to create the final folded topology of the membrane proteins. In this study, we have computed the contribution of helix insertion energies to the TMH protein folding landscape, to understand the role of the membrane-water interface in this energy landscape, for the full range of TMH protein sizes from 1-TMH proteins to 24-TMH proteins with available structures. The TM helix insertion stabilization energies from the Water phase to the membrane-water Interface (W→I energies) are smaller in general than those insertion stabilization energies from the Water phase to the transMembrane orientation (W→M energies). However, the W→I energies correlate well the W→M energies for all TMH proteins considered in this study, suggesting a potential role of the membrane-water interface as a holding vestibule for the TM helices during the folding of those proteins. This necessitates the modification of the original two-state model of helical membrane protein folding into a three-state model, where the new (intermediate) state resides in the membrane-water interface. This three-state model helps explain the diversity of folding mechanisms seen for this protein family, where the consecutive TM helices can insert in the membrane either sequentially or in a random order from the membrane-water interface vestibule, dictated by the kinetics of helix insertion into the membranes, helix stabilization by lipids, and interhelical interaction energies.

Poster #: 115
Campus: San Francisco State University
Poster Category: Synthetic Chemistry
Keywords: spermidine, fluorescent label, synthesis
Project Title: Regiospecific Synthesis of Acyl and Fluorescence-labeled Derivatives of Spermidine and Analogues
Author List:
Ochoa, Biviana; Undergraduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Gonzalez, Joseph; Graduate, Chemistry and Biochemistry, San Francisco State University
Lin, Sin Tung Constance; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Wu, Weiming; Chemistry and Biochemistry, San Francisco State University

Abstract: Polyamines are extremely important due to the roles that they play in cell functions including cell growth, survival, protein synthesis, and other activities having to do with regulating the cell. Spermidine and spermine in the cell fall within the polyamine family. The enzyme, spermidine/spermine-N (1)-acetyltransferase or (SSAT) is responsible for the homeostatic condition of polyamines. In order to elucidate the biochemical and molecular mechanism of SSAT, it is imperative to investigate the interactions of SSAT with acylated and fluorescence-labeled derivatives of spermidine and analogues such as norspermidine. These derivatives may function as alternative substrates or inhibitors of SSAT. These results will give insights on the mechanism and substrate specificity of SSAT. The aim of this project involves the synthesis of the regiospecifically acylated and fluorescence-labeled derivatives of spermidine and analogues. Spermidine is an asymmetric molecule and it is thus important that the acyl group is attached to a specific amino group for the biochemical results to be meaningfully interpreted. The synthesis is achieved through the reactions of diamines with amino-containing alkyl halides. The amino groups in the two starting materials are differentially protected so they can be removed under different conditions. Upon the coupling of the protected diamines and the amino-containing alkyl halides, one of the amine protecting groups is selectively removed to allow the attachment of acyl groups or fluorescence labels at a specific site. The subsequent removal of the second protecting group yields the regiospecifically labeled spermidine and analogues. The presentation includes the synthesis and characterization by nuclear magnetic resonance spectroscopy (NMR) of eight derivatives of spermidine and analogues labeled with acyl or fluorescence groups. The synthesis of these derivatives will allow the investigation of the molecular mechanism and substrate specificity of SSAT in the near future.

Poster #: 116
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: gold catalysis, benzotriazole, antifungal
Project Title: Design, Synthesis, and Evaluation of Novel Diarylmethane 1,2,3-Benzotriazole Derivatives using a Gold Catalysis Protocol
Author List:
Shoemaker, Kaylee; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Aguilar, Emily; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Iafe, Robert; Chemistry & Biochemistry, California State University San Marcos

Abstract: Candida species cause a majority of superficial and disseminated fungal infections in humans. The most commonly prescribed antifungal used for a majority of C. albicans infections is fluconazole, a member of the triazole class of antifungals. Unfortunately, there is extensive documentation in clinical settings of developed resistance to triazole antifungals of several Candida species. This situation has stimulated an urgent need to develop novel compounds with intriguing activities, broad biological spectra, and fewer adverse effects. A bioisostere of triazole is 1,2,3-benzotriazole (BT). A bioisostere is a compound with similar physical, chemical or biological properties. BT derivatives possess three sites available for hydrogen bonding and a larger conjugated system which allow pi-pi stacking interactions. It has been shown this variety of intermolecular interactions allows BT derivatives to bind with a diverse group of receptors of biological systems and could possess bioactivity to overcome triazole drug resistance. Because of this, many BT derivatives are currently being used to treat a wide range of infectious diseases and cancers.
Our first aim was to prepare a library of novel compounds containing the BT motif that can potentially show activity against Candida species. A series of 12 diarylmethane BT derivatives were designed, synthesized, and characterized by proton nuclear magnetic resonance, carbon-13 magnetic resonance, infrared, and mass spectroscopy. The BT derivatives were synthesized in two steps starting from commercially inexpensive aldehydes. First, a Grignard reaction was performed using phenymagnesium bromide and a substituted aldehyde to afford a diarylmethanol. Second, using a microwave-assisted gold-catalysis methodology developed in our lab, the benzylic alcohol was displaced with BT. Yields range from 60-98%, and all reactions showed excellent regioselectivity (>95%) at the N1-position of 1,2,3-benzotriazole, which is superior to other methods involving acid catalysis. Our second aim includes disk diffusion assays to establish the in-vitro minimum inhibitory concentration (MIC) against standard species of Candida available from the ATCC repository. The long-term goal of the project is to test the BT derivatives against clinical isolates known to be resistant to fluconazole.
This project is supported by CSUPERB Presidents’ Commission Scholarship and the Dreyfus Foundation.

Poster #: 117
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: anti-cancer, medicinal chemistry, organic chemistry
Project Title: Total Synthesis of a novel marine Anti-Cancer Natural Product Palmyramide A isolated from the Pacific Ocean Palmyra Atoll
Author List:
Bender, Ana; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Moser, Austin; Undergraduate, Chemistry and Biochemistry, San Diego State University
Walsworth, Kevin; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Bergdahl, Mike; Chemistry and Biochemistry, San Diego State University

Abstract: Background and Significance: Palmyramide A is a cyclic depsipeptide isolated from the cyanobacteria Lyngbya majuscule found near the Palmyra Atoll in the Pacific Ocean. Its molecular structure is composed of three amino acids and three hydroxy acids. It’s activity against colon cancer is spectacular (IC50 8.9 nM against HCT-116 colon cancer cells); however, the supply of Palmyramide A harvested from natural sources is too low to sustain further research. We will report a highly efficient and economical synthesis of the target molecule.
Methods: Our approach first involves the synthesis and modification of the necessary natural and unnatural amino acids and specific hydroxy acids as building blocks. Preparation of various N-methylated amino acids as well as synthesis of rare hydroxy acids will be presented using novel aldol type methodologies applied in organic chemistry. Phenyllactic acid can be synthesized using a simple conversion from L-phenylalanine using diazo chemistry. With these uncommon pieces in hand, we will use modern approaches to connect these molecular fragments to form the crucial linear intermediate in the final step(s). This crucial fragment will then set the synthesis of the final cyclic shape of the molecule to form the Palmyramide A structure. With an ample amount of compound made, we will be able to better characterize the biological activity of Palmyramide A. Our reported route also allows for future modification and protein binding (biotinylation), which will allow for identification of the molecular target as well as the design of structural analogs through SAR experiments.
Results: Excellent progress to the completion of the target molecule has been achieved. Currently five of the six monomers have been made and progress is currently being made towards the remaining fragment prior to assembly of the main molecular chain.

Poster #: 118
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: hepatitis C, virus-RNA, Medicinal chemistry
Project Title: Synthesis of Small Novel Methylsulfoximine Derivatives Active Against the Hepatitis C Virus
Author List:
Hall, Tyler; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Molina, Josiah; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Walsworth, Kevin; Graduate, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; Chemistry and Biochemistry, San Diego State University
Hecht, David; Chemistry and Biochemistry, San Diego State University

Abstract: Synthesis of Small Novel Methylsulfoximine Derivatives Active Against the Hepatitis C Virus
Background and Significance: Approximately 3.2 million people in the United States are currently living with hepatitis C virus (HCV). As a positive-sense RNA virus, HCV is prone to mutations, which makes it difficult to design drugs that can target either the viral proteins or the genome itself. It has been discovered that a small section of the 5’ non-coding RNA, called the internal ribosome entry site (IRES) subdomain IIa, acts as a molecular switch. The IRES recruits human ribosomes to bind, which causes a conformational change in the RNA, and allows for the ribosome to translate the RNA into viral proteins. Notably, this small section of RNA is highly conserved; as previously discovered, the virus showed only two point mutations in the subdomain IIa. Due to this high degree of conservation, it is an invaluable target for drug design that mimics the natural ligand and forces the IRES into a conformation that will not recruit ribosomes, rendering the virus unable to translate its RNA into proteins.
Methods: We have devised an improved, efficient synthesis of diverse methylsulfoximine compounds using novel techniques developed in our laboratory. We have subsequently initiated new analog syntheses designed to improve affinity of the targets for the HCV RNA. We plan to conduct rapid assays of the new compounds against the IRES-IIa subdomain, allowing us to iteratively refine SAR and leverage our new synthetic route toward obtaining compounds with increased potency.
Results: We will report the syntheses of heterocyclic methylsulfoximine derivatives and discuss the advantage of space-filling interactions in the virus RNA. Multiple scaffolds have been tested against the IRES construct, and we are currently designing and synthesizing new derivatives based on these results, and these discoveries will be presented. We will show that the precise shape of our novel molecules is complementary based solely on the hydrophobic interactions that can significantly improve ligand binding even in hydrophilic target sites such as the virus RNA.

Conclusions: The efficient new synthetic route we have developed has made it practical to obtain enough material to optimize for inhibition, replication, favorable pharmacokinetics, and bioavailability, thus advancing the prospects of this class of compounds as potential medications against HCV.

Poster #: 119
Campus: Sonoma State University
Poster Category: Synthetic Chemistry
Keywords: antibiotic, protein synthesis, bacteriocin
Project Title: Synthesis and purification of a fragment of the antibacterial protein piscicocin V1b
Author List:
Uribe, Alejandro; Undergraduate, Chemistry, Sonoma State University, Presenting Author
Ng, Ethan; Undergraduate, Chemistry, Sonoma State University, Presenting Author
Lillig, Jennifer; Chemistry, Sonoma State University

Abstract: Bacteriocins are proteins used by bacteria as a countermeasure against other bacteria. We study the bacteriocins piscicocin V1a and V1b that kill Listeria monocytogenes (L. mono), a food-born pathogen. Our previous work has shown that the full amino acid (AA) sequence is unnecessary for killing L. mono. Originally, we synthesized the first 18 AA (N-term) of V1a and discovered it can inhibit L. mono individually. Our goal in this work was to synthesize and purify the V1b N-term. Method: We used solid-phase protein synthesis to prepare the V1b N-term. In order to add AA to the solid-phase resin, we first used methylpiperdine to chemically remove a protecting group (FMOC) which is bound to all individual AA at the reaction site to prevent unwanted side reactions. After FMOC removal, the resin was rinsed with dimethylformamide (DMF) and the next AA was added. This process was repeated until all AA were added to complete the N-term. Removal of the FMOC was monitored by UV spectroscopy at 280 nm. Reaction of each AA was monitored using ninhydrin which would turn yellow at attachment. Our final N-term was detached from the resin using trifluoroacetic acid. This N-term solution was added to ether and the resulting solid was filtered and freeze-dried. The crude N-term was then purified using a nonpolar HPLC column with a mixture of polar and nonpolar solvents. Results: FMOC removal was found to be successful after six DMF rinses evidenced by a decrease in light absorbance from > 2 absorbance units (AU) in the first rinse to <0.01 AU in the last. Initial ninhydrin tests were purple which suggested incomplete reaction of the first AA. Additional experiments showed that DMF reacted with the ninhydrin to yield false negatives. Therefore, we allowed the attachment reaction to proceed two hours and then drained the resin of DMF prior to the ninhydrin test. This new method yielded a yellow ninhydrin test indicating successful attachment of each AA. The HPLC chromatogram of the crude showed a single large peak that released from the purification column at 21 minutes with a 68.9% peak area. We isolated this fraction and freeze-dried it to leave a fluffy white protein. The presence of the large HPLC peak coupled with the appearance of the resulting protein suggests we achieved our goal of successfully synthesizing and purifying the V1b N-term. Future work includes confirming the V1b N-term mass and measuring its ability to kill L. mono for comparison to V1a.

Poster #: 120
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: anti-cancer, angiogenesis, medicinal chemistry
Project Title: Our Adventurous Journey to Azaspirene; a Powerful Angiogenesis Agent against Cancer
Author List:
Acuna, Anthony; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Bender, Ana; Undergraduate, Chemistry and Biochemistry, San Diego State University
Hall, Tyler; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Bergdahl, Mike; Chemistry and Biochemistry, San Diego State University
Hecht, David; Chemistry and Biochemistry, San Diego State University

Abstract: Background: Azaspirene, a natural product isolated from the soil fungus Neosartorya sp., is a new powerful and promising anti-cancer agent that inhibits the creation of new blood vessels for growth of cancer cells via a process called angiogenesis. In a normal cell growth angiogenesis is also the pinnacle of creation of new blood vessels for growth and wound repair. Tumors reroute these natural functions to support their uncontrolled growth and Azaspirene has been shown to disrupt the inordinate signaling pathway associated with tumor angiogenesis, thus stopping tumor growth without harming normal body functions. Due to its mechanism of action, Azaspirene, unlike most modern cancer drugs, has also extremely low level of cytotoxicity and has therefore a high potential for use as a synergistic form of chemotherapy. Currently, the supply of Azaspirene from either natural sources or synthetic methods is too small for further biological research or drug use. As such, we developed an efficient synthetic route is to alleviate these challenges.
Results: This work demonstrates two efficient asymmetric syntheses of Azaspirene. Moreover, this reported route is designed to allow the access of pseurotin analogs, a group of highly bio-active natural products related to Azaspirene. Utilizing scalable reactions and dirt-cheap starting materials, this work may allow for easy access to these elusive molecules, allowing for in depth biological testing and potential large scale drug synthesis. A sufficient quantity of azaspirene will allow for the characterization of azaspirene’s biological binding site and further evaluation of azaspirene as an anti-angiogenic and anti-tumor agent.
We present two unique and novel routes towards Azaspirene. The first synthesis, which starts with L-phenylalanine, has been completed in a 6% overall yield in 11 steps, compared to over 30 steps previously reported in the literature. Efforts are currently being made to circumvent the use of mercury without a reduction in the overall yield of Azaspirene. The second synthesis is initiated with D-malic acid and establishes an even shorter route, increasing the accessibility to Azaspirene further.

Poster #: 121
Campus: CSU Sacramento
Poster Category: Synthetic Chemistry
Keywords: HIV, Glycodendrimer, Synthesis
Project Title: DEVELOPMENT AND OPTIMIZATION OF THE FIVE STEP SYNTHESIS OF A TETRA-AMINE DENDRIMER CORE FOR GLYCODENDRIMER SYNTHESIS
Author List:
Raskovalov, Aleksey; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Cerney, James; Graduate, Chemistry, California State University, Sacramento
Silva, Madeline; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
McReynolds, Katherine; Chemistry, California State University, Sacramento

Abstract: Background and Significance:
In 2017, over 36.9 million people were found living with Acquired Immunodeficiency Syndrome (AIDS), with 940,000 people dying each year. Cures for this viral infection are in high demand, however, historically, the best cure has always been prevention. Acquiring the disease requires an individual to contract the Human Immunodeficiency Virus (HIV), and involves contact with specific body fluids, such as the blood, semen, and vaginal fluids of an HIV carrier. A recent study has shown that sugar-based polymers, known as glycodendrimers, have a considerable potential in inhibiting the binding of HIV to its host cell. Therefore, the ability to produce these glycodendrimers at high yields can be of great interest to the general public to curtail the spread of HIV.
Results and Future Work:
In this study, a five-step synthetic method was developed to produce a tetravalent amine terminated dendrimer molecule, an intermediate used towards the synthesis of an octavalent glycodendrimer. These reactions have proven to be successful, producing each compound in relatively high yields up to the tetravalent nitrile dendrimer. In the first reaction, ethylene glycol was reacted with acrylonitrile producing Compound 1 at an 88.9% yield. Compound 1 was then reacted with tosyl chloride to produce the mono-tosylated linker, Compound 2 at an 81.3% yield. Compound 2 was then reacted with the central core molecule, 2,2’-oxybis(ethylamine), producing the tetra-nitrile core, Compound 3 at a yield of 63.4%. Finally, compound 3 was reacted with acetyl chloride and methanol, producing the tetra-methyl ester, compound 4, which was then immediately reacted with 1,3-diaminopropane in a fifth step, producing the tetra-amine terminated dendrimer compound, Compound 5, in a two-step yield of 9.8%. Future work will aim towards optimizing the reaction conditions of the combined fourth and fifth reaction steps to hopefully increase the 9.8% yield, by extending the reaction duration time from overnight to five days.

Poster #: 122
Campus: Cal Poly Pomona
Poster Category: Synthetic Chemistry
Keywords: bioinorganic, X-ray absorption spectroscopy, hyponitrite
Project Title: Characterization of Copper Hyponitrite Complexes using X-ray Absorption Spectroscopy
Author List:
Henriquez, Brenda; Graduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Ghosh, Pokhraj; Georgetown University, Chemistry
Kundu, Subrata; Indian Institute of Science Education and Research, Thiruvananthapuram
Warren, Timothy H.; Georgetown University
Stieber, S. Chantal E.; Chemistry & Biochemistry, California State Polytechnic University, Pomona

Abstract: Nitrous oxide (N2O) is the main contributor to the ozone layer depletion, has almost 300 times the warming potential of carbon dioxide, and lingers in the atmosphere for an average of 114 years. Biological systems can both produce N2O via NO reductive coupling, and reduce N2O through reactions at copper and iron centers, but not at sufficient rates to mitigate pollution. The mechanisms are not fully understood, and a more complete understanding would benefit pollution remediation efforts. Hyponitrite (O2N22-) intermediates are proposed, but there are few isolated examples. In the current work, discrete -diketiminate (iPrNNF6) copper complexes formed in solution from NO coupling were analyzed by X-ray absorption spectroscopy (XAS) to determine the metal oxidation state, coordination environment, and assess hyponitrite formation. The X-ray absorption near edge structure (XANES) analysis determined the oxidation state of the complexes [(iPrNNF6)CuO2N2][CoCp2] and (iPrNNF6)CuO2N2 to be +1, while (iPrNNF6)CuO2N2Ph has an oxidation state of +2. The [(iPrNNF6)CuO2N2][CoCp2] complex had a lower rising edge feature making the oxidation state assignment more nuanced. The extended X-ray absorption fine structure (EXAFS) allowed for the copper coordination environment to be determined, using (iPrNNF6)CuO2N2Ph as a comparison for which the 3-D atomic structure is reported. The EXAFS for [(iPrNNF6)CuO2N2][CoCp2] suggest dimer formation. The EXAFS for (iPrNNF6)CuO2N2 are consistent with O2N2 bound to copper, and suggest that the hyponitrite complex was synthesized and isolated. This has implications for understanding reactivity and reduction of N2O and NO and is one of the first examples of of an isolated copper hyponitrite complex. Future work includes understanding the mechanism by studying other intermediates formed in NO coupling reactions and has implications for understanding pollutant reduction. This work was supported by a CSUPERB New Investigator Grant and NSF CAREER Award (1847926).

Poster #: 123
Campus: CSU Northridge
Poster Category: Synthetic Chemistry
Keywords: chemotherapy, transport, synthesis
Project Title: Preparation and toxicity of 9,10-phenanthrenediimine based anti-tumor agents
Author List:
Terzian, Armenuhe; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Rios, Erick; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge

Abstract: 2,2’:6’,2”-Terpyridines (Tpys) have been reported as promising anti-tumor agents that are believed to interfere with DNA replication and transcription by intercalation (inserting themselves into the base-pair stack). Interestingly, Tpys exhibit selectivity for tumor cells over normal ones suggesting the additional exploitation of an active means of cellular uptake elevated in tumor cells. Our prior work with poly-Tpy complexes such as (Tpy)Ru(TpyTpy)(2+) (1) [where TpyTpy=4’,4””-bis(2,2’:6’,2”-terpyridine) demonstrated toxicity toward several breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7) with EC50s from 35 to 6 microM. The toxicity of Complex (1) in particular was attenuated by spermine (a competitive inhibitor for active polyamine transporters) suggesting that it had an affinity for these transporters and likely gained entry into cells through them. This behavior is consistent with the nitrogen spacing of Complex (1)’s pendant Tpy group chemically resembling a polyamine. This principle has been applied to a minimalist design of new anti-tumor candidates that feature polyamine-like structures directly connected to organic DNA intercalating groups. The first three of these compounds, 2,3-bis[(E)-(2-pyridyl)methylideneamino]phenazine (2), 9,10-bis{[(2-pyridyl)methyl]imino}-4,5-diazaphenanthrene (3), and 9,10-bis{[(2-pyridyl)methyl]imino}phenanthrene (4) were prepared by Schiff-base condensation of commercial reagents and surveyed for toxicity against breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7). Surprisingly, compound (3) exhibited excellent toxicities (EC50s ~ 250 nM) while the others were practically non-toxic (EC50s > 100 µM). Numerous variations of Compound (3) were prepared including 9,10-bis(benzylimino)phenanthrene (5) and 9,10-bis(butylimino)phenanthrene (6) that exhibited similar toxicities even when the polyamine-like nitrogen spacing within the appendage groups was removed. This could be a consequence of the nitrogen atom arrangement within the 9,10-phenanthrenediimine core resembling a conformationally restricted polyamine. This common core may allow all of these compounds to interact with polyamine transporters. The preparation of these compounds and their structure-to-toxicity correlations will be presented. We hope that new anti-tumor agents that target cancer cells through elevated transport processes will lead to more effective chemotherapies with fewer side-effects.

Poster #: 124
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: natural product, anticancer agent, synthesis
Project Title: Two Generations of Syntheses toward a Macrocyclic Ketone Mimic of Zampanolide
Author List:
Jiang, Ziran; Graduate, Chemistry, California State University, Fresno, Presenting Author, Eden Award Nominee
Sekhon, Arman; Undergraduate, Chemistry, California State University, Fresno
Oka, Yogeshwari; Undergraduate, Chemistry, California State University, Fresno
Chen, Guanglin; Staff, Chemistry, California State University, Fresno
Kaur, Jasleen; Undergraduate, Chemistry, California State University, Fresno
Chen, Qiao-Hong; Chemistry, California State University, Fresno

Abstract: (-)-Zampanolide is a structurally and medicinally intriguing macrolide isolated from marine sponge. Its medicinal properties have been demonstrated by its low nanomolar cytotoxicity against various cancer cell lines, especially against multidrug resistant cancer cell lines. Mechanistically, zampanolide has been established as a microtubule stabilizing agent with a unique covalent bond to residue His229 on β-tubulin. Nonetheless, the lactone moiety in zampanolide will contribute to metabolically instability in vivo, which has been exemplified by epothilones and E7389. This study thus aims to synthesize a macrocyclic ketone mimic of zampanolide in hope to enhance its metabolic stability and simplify the synthesis process. The mimic was designed by replacing the lactone moiety with a ketone, as well as removing the tetrahydropyran ring. Two generations of syntheses toward this mimic have so far been performed in our laboratory. In the first generation of synthesis, an advanced intermediate with Fragments C1-C8 connected to C13-C18 through a critical Grignard reaction has been successfully synthesized through a 13-step transformation. However, attempt to attach Fragment C9-C11 to this advanced intermediate via a Williamson ether synthesis failed. We then turned to the second generation of synthesis. At this point, Fragment C9-C11 was successfully incorporated into Fragment C13-C18 via a Williamson ether synthesis, giving us Fragment C9-C18. To this end, a highly advanced intermediate with Fragment C1-C8 connected to C9-C18 through the critical Grignard reaction has been achieved. Only two more steps are needed to reach our desired macrocyclic ketone core structure of zampanolide. The chemical structures for all synthesized intermediates have been characterized by interpreting the 1H and 13C NMR spectra. This project is supported by NIH/NIGMS under Award Number SC2GM121185.

Poster #: 125
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: Grapevine Rootstock, Stomatal Conductance, Water Potential
Project Title: Characterization of Commercial Grapevine Rootstock Hydraulic Response to Water Stress
Author List:
Palmer, Nathaniel; Undergraduate, Wine and Viticulture , California Polytechnic State University, San Luis Obispo, Presenting Author
Stauch, Samantha ; Graduate, Crop Science Graduate Group, Wine and Viticulture Department , California Polytechnic State University, San Luis Obispo
Ling, Carter ; Undergraduate, Wine and Viticulture , California Polytechnic State University, San Luis Obispo
Wootten, Jennifer ; Undergraduate, Wine and Viticulture , California Polytechnic State University, San Luis Obispo
Dodson Peterson, Jean ; Wine and Viticulture , California Polytechnic State University, San Luis Obispo, Presenting Author

Abstract: Vascular plants respond to water stress through a variety of physiological and biochemical mechanisms. The primary physiological response to drought is the closure of stomata to prevent decreases in plant water potential. This response limits water losses through gas exchange and transpiration. Isohydric plants maintain constant water potential when water is abundant as well as under drought conditions through rapid stomatal closure. Conversely, anisohydric plants tolerate greater decreases in water potential before closing stomata. This hydraulic behavior is potentially damaging to plant health and productivity due to water losses allowed by the delayed stomatal closure. Grapevines (Vitis vinifera L.) are known to exhibit both spectrums of hydraulic behaviors and are thought to respond to grapevine rootstock (Vitis spp.) selection. Categorically mapping grapevine rootstock hydraulic responses to water stress may allow for informed selection of grafted grapevines away from potentially damaging anisohydric behaviors. This study investigated the hydraulic response of five commercial grapevine rootstocks (101-14, 1103P, 110R, 420A, 5C), of varying parentage crosses, to water stress. Hydraulic responses were evaluated using gas exchange parameters, and vine water potential. Gas exchange parameters, stomatal conductance (gs) and photosynthetic rate (net CO2 assimilation, An), of rootstock leaves were measured with an infrared gas analyzer. Leaf stem water potential (Ψstem) was measured with a pressure chamber to determine the degree of vine water stress. The relationship between gs and Ψstem were compared by rootstock. While rootstocks were not significantly different (p>0.05), rootstocks were correlated with varying degrees of gs under water stress. 101-14 was correlated with limited gs (p=0.13) whereas 1103P was correlated with comparatively much higher gs (p=0.20) as Ψstem decreased, suggesting isohydric and anisohydric behaviors respectively. Photosynthetic rates (An) were directly related to gs (p<0.0001). Rootstock specific hydraulic behaviors can be correlated. Future work will focus on establishing a spectrum of rootstock hydraulic behavior and evaluating the influence of rootstock selection on vine grafted to Sauvignon blanc. Funding was generously provided by the Baker Koob Endowment and the CSU Agriculture Research Institute. The researchers also thank CSUPERB for the conference travel support and Wonderful Nurseries for the plant material.

Poster #: 126
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: foodborne microbes, active packaging, microbial enumeration
Project Title: The antifungal effects of chitosan and natamycin
Author List:
Rivera Penafiel, Julia; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Padilla, Bryan; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Shin, Joongmin; Industrial Technology and Packaging, California Polytechnic State University, San Luis Obispo
Yeung, Marie; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Aspergillus spp. and Penicillium spp. are common food spoilage microbes that can be responsible for massive agricultural product loss. Some mycotoxin-producing fungi are also human and animal pathogens. Therefore, it is imperative for the food and dairy industry to protect the food supply from fungal growth. The best food safety approach is “hurdle technology” in which a combination of food protection methods is employed. Our group plans to design an active packaging film that can be used to wrap finished food products to inhibit spoilage and pathogenic microbes during storage. The focus on this study was to determine the effective concentrations of two promising antimicrobial agents – chitosan and natamycin – that will be used to graft onto the surface of a cling-wrap. Aspergillus spp and Penicillium spp were grown on potato dextrose agar at 25 degree C for 3 days. Spores were collected after mycelia were filtered. Fungal spores were suspended in tryptic soy broth, enumerated using a counting chamber, and to dilute to yield a spore solution of ~1e5 spores/mL. Different concentrations of chitosan and natamycin were prepared in tryptic soy broth. They were added to the fungal spores to reach a concentration of (4%, 2%, 1%) chitosan, or (1%, 0.5%, 0.25%) natamycin. Viable fungal cell counts were determined by using the plate count and the most probable number methods after the treatment. Even at the highest concentration, chitosan was found to be ineffective on both species as >9x1e4 CFU/mL were recovered. On the other hand, all natamycin concentrations reduced the fungal count to an undetectable level (<100 MPN/mL). Our results show that natamycin is a more effective antifungal agent. Low concentration (0.25%) of natamycin on a packaging film is potentially sufficient to inhibit fungal growth on the food surface.

Poster #: 127
Campus: CSU Fullerton
Poster Category: Agriculture/Biofuels/Environment
Keywords: manganese, photosynthesis, cyanobacteria
Project Title: Trading manganese for water in photosynthesis
Author List:
Kim, William; Undergraduate, Biology, California State University, Fullerton, Presenting Author
Johnson, Hope; Biology, California State University, Fullerton

Abstract: The development of oxygen producing photosynthesis is arguably one of the most important events in Earth’s life history. Not much is known about how photosynthesis evolved to produce oxygen; however, manganese (Mn) oxidation is proposed to be an evolutionary step that transitioned anoxygenic photosynthesis to produce oxygen. The research here focuses on detecting Mn oxidation in the modern cyanobacterial strain Synechocystis sp. PCC 6803. We hypothesize that Mn(II) is oxidized by photosystem II (PSII) and electrons are transferred into the electron transport chain, the same way a water molecule’s electrons are used in oxygenic photosynthesis. Therefore, we expected oxygen production would decrease if cells are also oxidizing Mn by PSII. Cells were incubated in the light and oxygen production was continuously measured using an oxygen electrode and oxidized Mn(II) (as Mn(III)) was measured at discrete time points by a spectrophotometric assay. Although cells oxidized Mn, oxygen production did not decrease. Future experiments include utilizing non-oxygen producing PSII mutants to detect electron flow through PSII during Mn oxidation.

Poster #: 128
Campus: Stanislaus State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: Biomass, Biofuels, Catalyst
Project Title: Mesoporous Silica Catalysts for Biomass Conversion
Author List:
Brown, Lucas ; Undergraduate, Chemistry, California State University, Stanislaus
Gandhi, Shivam; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Manchanda, Amanpreet; California State University, Stanislaus, Presenting Author

Abstract: Acid catalysts are used extensively for biomass conversion. These catalysts can be divided into two categories namely homogenous and heterogeneous catalysts. Homogenous catalysts such as sulfuric acid are in the same phase as the reactants and have toxicity, filtration and recovery problems. Heterogeneous acid catalysts are considered environmentally friendly and are desired for the generation of biofuels, biomass conversion. There has been a lot of research going on to develop heterogeneous catalysts with good catalytic activity and regenerability. In our research, we synthesized sulfonic-acid functionalized mesoporous silica heterogeneous catalysts for the esterification reaction between ethanol and acetic acid. The synthesized catalyst showed good catalytic activity and had BET surface area of 550 m2/g, and pore diameter of ~20 nm. The acid capacity of the catalyst is 0.98 mmol/g. We plan to further develop these catalysts and use for other esterification and transesterification reactions.

Poster #: 129
Campus: CSU Los Angeles
Poster Category: Agriculture/Biofuels/Environment
Keywords: Metal-organic frameworks, hydrogels, hybrid materials
Project Title: Synthesis and characterization of metal-organic framework hydrogel composites
Author List:
Klein, Shirell; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Liu, Yangyang; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: Metal-organic frameworks (MOFs) are an emergent class of porous crystalline materials composed of metal ions or nodes and organic linkers. Their high surface area and tunable structures make them candidates in applications ranging from drug delivery and catalysis to gas sensing and storage. However, lack of stimuli responsiveness and macroscopic ordering of MOFs pose issues in integrating them into functional devices or sensors. To mediate these issues, MOF hybrid materials are of special interest. One such class of hybrids are MOF hydrogel composites which utilize the metal nodes of a MOF to crosslink hydrophilic polymer chains and form a gel. Previous work suggests a wide variety of functionalities are possible for these gels, but no work has previously been done on stimuli responsiveness or incorporation of Zr-based MOFs into the gel systems. In this study, we synthesize a Zr-trimesic acid (BTC)-alginate (Alg) hydrogel composite and confirm the incorporation of both the zirconia node and linker into the gel. The Zr-BTC-Alg gel shows increased loading capacity compared to both the corresponding hydrogel and MOF alone. Additionally, stimuli response testing of methylene blue loaded Zr-BTC-Alg gels indicate selective release or extended encapsulation in stimuli that cause gel swelling or shrinking, respectively. Methylene blue encapsulation trials suggest enhanced stability in acid for the composite gels and display greater distinctions in material response to environments of varying pH. Further testing of stimuli response and gel state characterization are needed to evaluate the complete functionality of the hybrid materials for environmental sensors or devices.

Poster #: 130
Campus: Stanislaus State University
Poster Category: Biochemistry
Keywords: monooxygenase, chlorobenzene, green chemistry
Project Title: Oxidation of chlorobenzene to 4-chlororesorcinol, chlorohydroquinone, 3-chlorocatechol and 4-chlorocatechol by toluene o-xylene monooxygenase
Author List:
Phul, Onkar K; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Roth, Owen S; Undergraduate, Chemistry, California State University, Stanislaus, Nagel Award Nominee
Soria-P, Gerardo; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Vardar-Schara, Gönül; Chemistry, California State University, Stanislaus

Abstract: Toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 belongs to a remarkable family of bacterial multicomponent monooxygenases and has been shown to have a great potential for biotechnological and environmental applications. Chlorobenzene (CB) is a commonly used industrial solvent listed as a priority environmental pollutant by the US Environmental Protection Agency (EPA). Long-term exposure of humans to CB affects the central nervous system and may damage lungs, liver, and kidneys. Therefore, EPA regulations require companies and governmental agencies to detect its presence in effluents and remove them from contaminated sites. Direct enzymatic oxidation of CB for the synthesis of potentially useful dihydroxy chloroaromatics such as 4-chlororesorcinol (4-CR, precursor for hair dye formulations) and/or chlorohydroquinone (CHQ, photographic developer, stabilizer, and antioxidant) is attractive compared to organic-based clean-up efforts. In this study, the goal was to characterize the enzyme ToMO for its ability to oxidize CB and convert this harmful chemical into valuable dihydroxy chloroaromatics for the very first time. Oxidation activity levels and regiospecificities were determined using reverse-phase high-performance liquid chromatography. Compounds were identified by comparison of retention times to those of authentic standards and by co-elution with the standards. Escherichia coli strain TG1 was used as the host to express ToMO. The negative controls used in these experiments contained TG1 cells expressing ToMO with no substrate as well as empty TG1 cells with substrates (no-ToMO control). Experiments were performed with several independent ToMO cultures for each substrate, and six injections were made for each substrate. We found that ToMO oxidized CB to form 2-CP (4%), 3-CP (12%), and 4-CP (84%) with a total product formation rate of 1.2 ± 0.2 nmol/min/mg of protein. We also found that ToMO forms 4-CC from 3-CP and 4-CP with a formation rate of 0.5 ± 0.1 and 0.4 ± 0.1 nmol/min/mg of protein, respectively, and 4-CR (3%), CHQ (13%), and 3-CC (84%) from 2-CP with a total product formation rate of 1.1 ± 0.3 nmol/min/mg of protein. The significance of this work lies in the ability to degrade environmental pollutants while, at the same time, produce valuable chemicals using environmentally-benign biological methods rather than expensive, traditional chemical processes. This project was supported by the CSUPERB New Investigator Grant.

Poster #: 131
Campus: Stanislaus State University
Poster Category: Biochemistry
Keywords: toluene monooxygenases, chlorobenzene, protein engineering
Project Title: Regiospecific oxidation of chlorobenzene by toluene monooxygenases and engineered toluene o-xylene monooxygenases of Pseudomonas sp. OX1
Author List:
Roth, Owen S; Undergraduate, Chemistry, California State University, Stanislaus, Nagel Award Nominee
Phul, Onkar K; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Tlatelpa, Areli; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Vardar-Schara, Gönül; Chemistry, California State University, Stanislaus

Abstract: Bacterial toluene monooxygenases (TMOs) are known for their ability to oxidize a wide range of aromatics, degrade a variety of organic pollutants, and produce industrially important compounds. Chlorobenzene (CB) is a commonly used aromatic solvent listed as a priority environmental pollutant by the US Environmental Protection Agency. Because of its toxicity, it is also regulated under the Safe Drinking Water Act. Direct microbial oxidation of CB is attractive because it should reduce waste relative to organic-based methods. Furthermore, control of regiospecific oxidation of aromatics is of vital importance for industrial applications. In this study, regiospecific oxidation of CB was examined with Escherichia coli strains expressing wild-type TMOs including toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4, toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, and toluene 4-monooxygenase (T4MO·BTAi1) of Bradyrhizobium sp. BTAi1 for the very first time. Oxidation activity levels and regiospecificities were determined using reverse-phase high-performance liquid chromatography (HPLC) on a C18 column using a water/acetonitrile solvent system. We found that ToMO oxidized CB faster than the other wild-type enzymes (1.3- to 30-fold) and produced 4% of 2-chlorophenol (2-CP), 12% of 3-CP, and 84% of 4-CP. TOM oxidized CB primarily at the ortho position and formed 93% of 2-CP, 3% of 3-CP, and 4% 4-CP. TpMO and T4MO·BTAi1 oxidized CB primarily at the para position and formed 91 and 100% 4-CP, respectively. After these new reactions were discovered, four engineered enzymes containing mutations in ToMO alpha hydroxylase (TouA) were examined in order to generate further improvements in ToMO regiospecificity. We found that the selectivity of 2-CP was enhanced from 4% to 68% and the selectivity of 4-CP was enhanced from 84% to 98% by TouA variants; hence, ToMO was converted into an ortho- and a better para-hydroxylating enzyme through protein engineering. The results of the present study clearly show the capacity of TMOs and ToMO variants for regiospecific oxidation of CB using environmentally-benign biological methods rather than expensive, traditional chemical processes. This study also confirms the advantages of protein engineering approaches and adds to the list of research on probing the remarkable family of bacterial TMOs.This project was supported by the CSUPERB New Investigator Grant.

Poster #: 132
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: CDK5, Prostate Cancer, Akt pathway
Project Title: Effect of Cyclin-Dependent Kinase 5 inhibition on Akt Signaling in DU145 Prostate Cancer Cells
Author List:
Khatib, Jude; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Cyclin-dependent kinase 5 (CDK5), a serine/threonine protein kinase, is ubiquitously expressed and plays a crucial role in embryonic development. Dysregulation of CDK5 has been implicated in several diseases including cancer, neurodegeneration, and type II diabetes. In prostate cancer, CDK5 promotes cell migration and invasion through both androgen receptor (AR)-dependent and -independent mechanisms. Previous studies have proposed that the AR-independent effects of CDK5 are mediated via the pro-survival Akt pathway in a prostate cancer cell line, PC3. The goal of our study was to determine if CDK5 mediates activation of the Akt pathway in another AR-deficient prostate cancer cell line, DU145. To achieve this goal, we treated DU145 cells with roscovitine, an ATP competitive inhibitor of CDK5, prior to lysing the cells and analyzing the lysates using immunoblotting. Our results showed that under basal conditions roscovitine treated cells have marginally higher level of activated Akt than the control treated cells. To further study the effect of CDK5 inhibition on the Akt pathway in DU145 cells, we serum starved the cells to minimize the basal level of activated Akt and then determined Akt activation downstream of stimulated epidermal growth factor receptor. Serum-starved cells were treated with epidermal growth factor for 0, 5, and 15 minutes followed by cell lysis and immunoblotting. Our results showed that roscovitine treated cells have significantly higher levels of Akt upon epidermal growth factor receptor stimulation. As a complementary approach, we used RNA interference to determine that CDK5 depletion also enhanced Akt activation in DU145 cells. Together, our results conclude that as opposed to the reported CDK5-dependent Akt activation in PC3 cells, DU145 cells show an opposite trend. Our current and future experiments are aimed at studying the effect of CDK5 depletion and inhibition on cell migration in DU145 cells.
This project is supported by the NIGMS-NIH grant #SC2GM121246. The content of this abstract is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Poster #: 133
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: ER stress, Unfolded protein response, Akt
Project Title: Inhibition of Akt Kinase Differentially Regulates the Unfolded Protein Response
Author List:
Nguyen, Anh; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Bhandari, Deepali; Chemistry and Biochemistry, California State University, Long Beach

Abstract: The endoplasmic reticulum (ER) is an organelle in eukaryotic cells that plays a vital role in many important cellular functions including proper folding of secretory and membrane proteins. Any disruption of ER function triggers ER stress leading to activation of an evolutionarily conserved signaling program called the Unfolded Protein Response (UPR). UPR signaling initially aids in re-establishing normal ER functions and promotes cell viability. However, if the homeostasis is not restored in a timely manner, UPR eventually commits the cells to death. Cancer cells are known to be able to evade ER stress-induced cell death, which also helps them acquire resistance to therapy. Our laboratory has previously shown that Akt kinase is activated in ER stressed cancer cells leading to enhanced cell survival. The goal of this project was to test if Akt activation modulates the outcome of UPR signaling. To achieve our goal, we treated cells with a commercially available small molecule inhibitor of Akt to reduce its activation. Changes in the expression or post-translational modification of known UPR signaling proteins were then analyzed by immunoblotting. We tested the effect of Akt inhibition on two cell lines, HeLa – a cervical cancer cell line, and HEK293 – human embryonic kidney cells. ER stress was triggered using chemical inducers tunicamycin or thapsigargin. Densitometric analysis of our immunoblotting data confirmed that in both cell lines, Akt inactivation leads to differential effects on UPR signaling dampening one ER stress sensor pathway whereas constitutively upregulating another. Our results also showed significant reduction in the ER stress-induced expression of a key chaperone in cells treated with the Akt inhibitor. Together, our results confirmed that Akt regulates the outcome of UPR signaling. Our current experiments are focused on studying the effect of Akt inhibition on cell viability of multiple cancer cell lines exposed to ER stress.
This project is supported by the NIGMS-NIH grant #SC2GM121246. The content of this abstract is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Poster #: 134
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Reactive Oxygen Species, Particulate Matter, Quinones
Project Title: Particulate Matter Composition as a Determinant in Subcellular ROS Production
Author List:
Lee, Devon; Undergraduate, Environmental Sciences, California State University, Fresno, Presenting Author
Olivas, Micah; Undergraduate, Chemistry, California State University, Fresno
Flores, David; Graduate, Biology, California State University, Fresno
Hasson, Alam; Chemistry, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno

Abstract: Identifiable links have been established between ambient atmospheric particulate matter (PM) exposure and adverse health effects in humans. PM smaller than 2.5 micrometers in diameter pose significant risk to the respiratory system of humans, triggering inflammatory stress responses at the distal ends of the tract upon interaction with alveolar macrophage cells (AM). This issue has necessitated further examination of the intracellular stress dynamics of PM2.5 in humans in order to provide a more thorough explanation of the health risks implicated by these highly concentrated aerosols. Understanding the contribution of organelle-specific ROS production is imperative to developing treatments tailored to combat cellular oxidative stress.
Past iterations of the study were successful in quantifying the oxidative stress potential of particulate matter samples originating from filter samples collected from the areas of Fresno and Claremont during summer and winter months. We first showed that the amount of PM2.5 collected in these samples is sufficient to induce significant ROS responses in NR8383 AM. Interestingly, the specific cellular ROS response was dependent on the sample origin; this strongly suggests that the chemical composition of PM2.5 is a major determinant of AM ROS metabolism. We elected to study more specifically three commonly PM2.5 -occurring cyclic diones (9,10 phenanthrenequinone, 1,2 naphthoquinone, and 1,4 naphthoquinone) and one commonly PM2.5 -occurring transition metals (Cu2+) for their propensity to generate oxidative stress within a cellular context. Regression analysis of the experimental stress response in AM suggested that cell populations treated with prepared solutions of quinones on average reached a peak intracellular ROS level at comparatively lower treatment concentrations than cells treated with prepared solutions of the two transition metals. As represented in initial filter sample assessments, quinones were identified as large components of ambient particulate matter. Given the oxidative role of the quinone-derivative Coenzyme-Q in mitochondrial metabolism, we suggest that quinones contained in PM2.5 may interfere with the activity of the Electron Transport Chain, leading eventually to an increase in ROS production.

Poster #: 135
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Lipoprotein lipase, Fatty acid metabolism, skeletal muscle cells
Project Title: Regulation of Fatty Acid Metabolism in Lipoprotein Lipase Knock-Down Skeletal Muscle Cells
Author List:
Pashanyan, Davit; Undergraduate, Biology, California State University, Northridge, Presenting Author
Ohana, David; Undergraduate, Biology, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge

Abstract: Background: The enzyme lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides to free fatty acids and glycerol. The free fatty acids are taken up by the mitochondria of the cell and oxidized to be used as energy. In earlier reports, we demonstrated that reducing the expression of LPL in skeletal muscle cells increased insulin-stimulated glucose oxidation but reduced oxidation of fatty acids to carbon dioxide. The rate-limiting enzymes of fatty acid biosynthesis and oxidation are, respectively, carnitine palmitoyl transferase-1 (CPT1) and acetyl CoA carboxylase (ACC). ACC catalyzing the synthesis of malonyl-CoA, the precursor of all fatty acids. Malonyl CoA allosterically inhibits CPT-1 which is required for transport of FFA into the mitochondria prior to beta-oxidation.
Objective: To compare the transcript and protein levels of Carnitine palmitoyl transferase-1 and Acetyl-CoA Carboxylase in the wild type (WT) and LPL-knockdown (LPL-KD) L6 rat skeletal muscle cells.
Methods: WT and LPL-KD L6 cells were propagated in DMEM supplemented with 10% fetal bovine serum (FBS). Myoblasts were differentiated into myotubes by depriving them of FBS. Total RNA was isolated to make cDNA libraries. Specific primer pairs were used for end-point and real-time quantitative PCR amplification of LPL, CPT-1, ACC-2. beta-actin was used as a house keeping gene. The comparison of CPT-1 and ACC-2 protein levels by Western blot analysis is in progress.
Results: Quantitative PCR confirmed that LPL expression in the LPL-KD cells was <1% of that of WT L6 cells due to shRNA silencing. Silencing the LPL gene also altered the transcription of CPT1 and ACC. The threshold cycle (CT) values were used to calculate the fold change of the target gene relative to beta-actin. We found that ACC-2 expression in LPL-KD cells was 36% of that in WT cells. CPT-1 transcript levels in LPL-KD cells was 1326% higher than that in WT counterparts.
Conclusion: The knockdown of LPL in L6 cells increases the expression of the CPT-1 while decreasing the expression of ACC-2. This is contrary to our observation that beta-oxidation of FFA is reduced in LPL-KD cells. We hypothesize that the level of transcription of these enzymes may be countering any post-translational regulation of enzyme activities in LPL-KD cells. Future studies will include comparing ACC-2 and CPT-1 enzyme activities in WT and LPL-KD cells.

Poster #: 136
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: enzyme kinetics, cancer, IDH
Project Title: Kinetic and Cellular Consequences of pH on Metabolic Enzyme Activity
Author List:
Luna, Lucas; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Lesecq, Zach; Undergraduate, Chemistry and Biochemistry, San Diego State University
Hoang, An; Staff, Chemistry and Biochemistry, San Diego State University
Sohl, Christal; Chemistry and Biochemistry, San Diego State University

Abstract: Changes in the intracellular environment, such as changes in pH, oxygen levels, and oxidative stress can reroute metabolism by altering the activities of metabolic enzymes like isocitrate dehydrogenase 1 (IDH1). IDH1 is responsible for catalyzing the reversible NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to -ketoglutarate (KG) and NADPH. The conversion of KG to ICT is a key step in the reductive metabolism of glutamine that drives the growth of many cancers. As a cell makes the transition from normal to cancerous, the intracellular pH deviates from normal, which can have drastic effects on enzyme activity and metabolites present. Here, we sought to determine how pH regulates IDH1 catalytic activity using steady-state kinetic assays with recombinant protein, and by assessing the cellular consequences of IDH1 upon encountering lowered intracellular pH. We have kinetically assessed the effect of pH on recombinant WT IDH1 by monitoring the increase or decrease in absorbance of NADPH at 340 nm using a spectrophotometer. We found that the rate of the forward reaction was pH-dependent and decreases as the pH becomes more acidic, with kcat values ranging from 39.1 s-1 at a pH of 8.0 to 20.2 s-1 at a pH of 6.2. However, the rate of the reverse reaction does not show pH dependence and peaks at a pH of 6.8 with a kcat of 5.0 s-1. In cellular assays, we treated HT1080 cells with proton pump inhibitors to lower intracellular pH. Then the HT1080 cells were then incubated with the pH-sensitive dye BCECF, and fluorescence was measured as a ratio of 490 nm to 440 nm excitations. Thus far, esomeprazole has the best effect, lowering the intracellular pH from 7.57 to 7.28. We expect that metabolic quantitation analysis will show that reductive metabolism is increased at more acidic pHs. Overall, the data elucidates the effect of pH on IDH1 catalytic activity, the cellular consequences that pH has metabolites present, and hints at how they aid in cancer growth and progression.

Poster #: 137
Campus: San José State University
Poster Category: Biochemistry
Keywords: protein-protein interactions, binding affinity, fluorescein labelling
Project Title: Elucidating Intramolecular Interactions within SIRT1 by Quantifying Binding Affinity
Author List:
Peralta, Carla; Graduate, Chemistry, San José State University, Presenting Author
Rizvi, Fatima; Undergraduate, Biology, San José State University, Presenting Author
Wang, Ningkun; Chemistry, San José State University

Abstract: SIRT1 is an NAD+-dependent deacetylase that regulates many cellular pathways. This protein is often studied for its regulatory effects on transcription and has been identified as a potential therapeutic target for many age-related degenerative diseases such as Alzheimer’s and Parkinson’s. Though SIRT1’s catalytic core has been well documented, less is known about the flanking disordered regions in both the N- and C-terminus. Motif A, an intrinsically disordered region located in the N-terminus has been seen to trans-activate SIRT1-dependent activities. However, it has only been studied in vivo, and not in vitro as it has never been expressed and purified separately. Understanding Motif A’s role within SIRT1 is imperative in order to develop drugs that can activate SIRT1’s activity. We aim to illuminate Motif A’s allosteric interactions within SIRT1 by recombinantly expressing Motif A and analyzing the binding interactions between the catalytic core and Motif A. We have successfully expressed a construct of Motif A within E.coli and utilized a Ni-NTA column to extract the peptide at 95% purity, with a concentration of 84.67mg/L cell culture. This construct of Motif A includes a cysteine at the end so that we may attach the thiol-reactive dye fluorescein-5-maleimide as a label. Presently, we are exploring the optimal conditions for labeling Motif A, utilizing a fluorimeter to quantify labeling efficiency. Once labeled, we will be able to quantify Motif A’s binding affinity to the catalytic core of SIRT1 using microscale thermophoresis. These studies will provide an increased understanding of SIRT1’s overall mechanism of action that can be utilized for the development of therapeutics against neurodegeneration.

Poster #: 138
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, self-association, lipid binding
Project Title: Probing the Lipid Binding and Self-Association Properties of N-terminal and C-terminal Helices of Apolipoprotein A-I using Chimera Proteins
Author List:
Patel, Nairuti; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Alcala, Diego; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Human apolipoprotein A-I (apoA-I) is a major component in high-density lipoprotein and plays a critical role in reverse cholesterol transport. The 28 kDa protein (243 residues) is composed of an N-terminal (NT) helix bundle domain and a less structured C-terminal (CT) domain. The CT domain contains helices responsible for high-affinity binding to phospholipids which initiates apoA-I lipidation and also mediates self-association. Previous studies have suggested that the NT domain may also initiate lipid binding. To better understand the role of NT and CT helices of apoA-I in lipid binding and self-association, four chimera proteins were designed. A chimera system has been developed in which monomeric insect apolipophorin III (apoLp-III) is used as a scaffold to present segments of apoA-I. Lipid binding of apoLp-III has been disabled by introducing two cysteine residues locking the protein in a closed inactive conformation. The apoLp-III/ apoA-I chimera can thus adopt affinity for lipids and acquire self-association properties. Thus, the chimera protein approach allows for determination of the functional properties of NT and CT helices of apoA-I. In the present study, residues 1-43 or 44-65 of apoA-I were attached to the NT of apoLp-III to assess the role of apoA-I NT helices. In addition, CT domain residues 209-243 or residues 220-243 of apoA-I were attached to the CT of apoLp-III. Circular dichroism showed that the chimeras displayed an α-helical content of ~60%, which is similar to apoLp-III, indicating that the structural integrity of apoLp-III was not affected by the addition of apoA-I segments. Lipid binding was analyzed by phospholipid vesicle solubilization. Phospholipid solubilization rates for apoA-I(1-43)/apoLp-III (0.0168/s), apoA-I(44-65)/apoLp-III (0.0086/s), apoLp-III/apoA-I(209-243) (0.0091/s), apoLp-III/(G4S)2/apoA-I(220-243) (0.0064/s) presented a 6 to 17 fold increase in comparison to apoLp-III (0.0010/s). To analyze the self-association state of the chimeras, proteins were crosslinked with dimethylsuberimidate. This showed that only apoLp-III/apoA-I(209-243) formed oligomers, while the NT segments of apoA-I and CT segment 220-243 were not able to induce self-association. Thus, the NT and CT helices of apoA-I have high-affinity for phospholipids, indicating the potential to initiate lipid binding. However, only specific CT residues of apoA-I can mediate self-association.

Poster #: 139
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: nanobody, humna serum albumin, protein structure
Project Title: Crystal structure of a cross-species reactive nanobody in complex with human serum albumin
Author List:
Jian, Mike; Graduate, Chemistry, California State University, Fresno, Presenting Author
Hussack, Gregory ; Human Health Therapeutics Portfolio, National Research Council of Canada
Brooks, Cory; Chemistry, California State University, Fresno

Abstract: Nanobodies are single-domain antibodies derived from the unique heavy-chain antibodies found in camelid species. Nanobodies are attractive therapeutics as they are easy to produce, are highly stable, and can potentially bind targets inaccessible to traditional antibody formats. However, due to their small molecular weight, one disadvantage associated with nanobodies is rapid renal clearance resulting in a short serum half-life. One innovative solution to increasing the serum half-life of nanobodies is to make use of bispecific nanobodies. In this format, a therapeutic nanobody is fused to a second nanobody specific for the highly abundant plasma protein, human serum albumin. Such bispecific nanobody constructs targeting serum albumins have been shown to increase the half-life of therapeutic nanobodies. The use of an albumin specific nanobody that cross reacts with albumin found in commonly used research animals is particularly desirable as the bispecific nanobody could be used pre-clinical animal trials. Previously, we have generated a panel nanobodies which cross-react with the serum albumins of humans, macaques, mouse, and rat, through immunization of a llama with human serum albumin. To determine the basis of the cross-species albumin reactivity of the nanobodies we have determined the X-ray structure of the nanobody M79 in complex with human serum albumin. M79 is cross reactive with serum albumins from macaques, mouse, and rat. The structure revealed of the three nanobody binding loops, only two of the loops (CDR2 and CDR3) interacted with human serum albumin. The M79 nanobody bound to the apex of subdomain IIB on human serum albumin, specifically forming polar contacts with residues D308, D314, N318, and E321. Comparison of the serum albumin sequences from human, macaques, mouse, and rat show that the residues in serum albumin that bound M79 are highly conserved amongst the species explaining the nanobodies’ cross-reactivity. These results give insights on the cross-species specificity of M79 towards serum albumin, and can bolster the use of M79 in animal model experiments.

Poster #: 140
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: transient state kinetics, flavin monooxygenase, enzymology
Project Title: Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin-dependent N-oxygenase
Author List:
Huynh, Katherine; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Baluch, Muneeba; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge

Abstract: Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a derivatized form of doxorubicin with an additional seven-carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N-oxygenase that oxidizes an sugar-linked exocyclic amine, triggering a nonenzymatic retro oxime-aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ-catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.
Our transient-state experiments confirm that DnmZ catalyzes the formation of the C4a-(hydro)peroxyflavin intermediate (C4a-OOH). We have identified the appropriate wavelengths to monitor to observe C4a-OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 μM, which compares well with other Class D monooxygenases. We are also investigating the effect of pH (pH 5.5 to 10.5) on C4a-OOH formation and elimination. Our preliminary results suggest that as pH increases, the rate of C4a-OOH elimination also increases, but no effect is observed on the intermediate’s formation.

Poster #: 141
Campus: CSU Northridge
Poster Category:
Keywords: chaperone protein, NMR spectroscopy, disulfide bond
Project Title: The role of a disulfide bond in the structural plasticity and function of an acid-stress chaperone
Author List:
Aguirre Cardenas, Imex; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author, Nagel Award Nominee
Geddes-Buehre, Dane; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge

Abstract: HdeA is a chaperone protein found within the periplasm of many infectious bacteria. It is responsible for protecting periplasmic proteins as bacteria pass through the acidic environment of the stomach, en route to colonize the intestines. Unlike other proteins, HdeA is inactive at physiological pH and unable to bind other proteins. However, in response to acidic conditions (pH 1-3), HdeA unfolds into a disordered state thereby allowing the binding of client proteins and aiding bacterial survival. Our lab seeks to understand the mechanism by which HdeA unfolds into its active monomeric state in an attempt to target HdeA and render infectious bacteria more vulnerable to antibiotics.
The disulfide bond between residues 18 and 66 is believed to play an essential role in HdeA’s structural plasticity. My research project investigates the effects of mutating the cysteines in HdeA’s disulfide bond to serines. The HdeA-C18S-C66S mutant was purified via a protocol modified from wild-type, using first a SP Sepharose cation exchange column and then a HiLoad Superdex size exclusion column. The pure sample was subjected to NMR (Nuclear Magnetic Resonance) spectroscopic analysis. Preliminary inspection of the 1H-15N correlation (HSQC) spectrum indicated that the mutant HdeA was at least partially unfolded at pH 6. Given this, additional 2D and 3D multinuclear NMR experiments were recorded to permit assignment of chemical shifts in the HSQC spectrum. These assignments were used to calculate the Secondary Structure Propensity (SSP) of HdeA-C18S-C66S. SSP results show that the mutant holds very little secondary structure (even at near-physiological pH), as the protein almost entirely adopts a random coil configuration. Regions that are partially folded are prominent near the N-terminus and show a slight propensity to adopt a β strand configuration in this normally all-helical protein.
Given that our mutant is unfolded at pH 6, it is clear that the disulfide bond plays a vital role in HdeA’s structural dynamics. Moving forward, it would be interesting to see if this unfolded mutant has any function as a chaperone. It is also possible that the mutation of cysteine to serine interferes with the network of intramolecular interactions normally found in wild type HdeA; a mutation of cysteine to alanine may prove to yield different results.
We acknowledge support from the NIH for research funds (SC3-GM116745), and the NSF for funding our NMR spectrometer (CHE-1040134).

Poster #: 142
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: flavin monooxygenase, enzymology, transient-state kinetics
Project Title: Preliminary biochemical characterization of Flavin Dependent Monooxygenase DszA
Author List:
Sanchez, Misael; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge

Abstract: Sulfur dioxide production through fossil fuel combustion is a major contributor to atmospheric pollution. This molecule has been known to cause respiratory illness in humans and land deterioration through acid rain formation. Fortunately, the biodesulfurization pathway (BDS) in R. erythropolis and other bacterial species have shown promise in becoming a useful tool for the removal of sulfur from crude oil. However, improvements are still necessary for this pathway to become competitive and industrially applicable. Our current objectives are to rationally design the BDS pathway in R. erythropolis, starting with enzymes DszC and DszA. The project goal is to decrease substrate specificity in the pathway, allowing it to work on a larger selection of organosulfur compounds found in fossil fuels. Here we exhibit preliminary biochemical characterization of the second enzyme in the BDS pathway, DszA, a flavin dependent monooxygenase. Stopped-flow spectrophotometric studies are being used to monitor the reduced flavin dissociation constant(Kd) as well as its rate of oxidation in the presence or absence of DszA and its native substrate, DBTO2. A 20.3 ± 0.451 M Kd value was determined for the reduced form of flavin. As expected, flavin oxidation increases with oxygen concentration in the presence of DszA. The rate of flavin oxidation also increases in the presence of both DszA and DBTO2 compared to DszA alone, suggesting that the oxidation we observe is enzymatically mediated. Contrary to previous studies, which suggested the production of a flavin-N5-oxide intermediate during catalysis, we have not observed the formation of flavin-N5-oxide. This intermediate should be detectable through stopped-flow experiments. Our work towards confirming our enzyme’s activity and ability to generate either the N5-oxide or the better-understood C4a-(hydro)peroxyflavin intermediates is ongoing.

Poster #: 143
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: alternative splicing, RNA binding proteins, recombinant DNA technology
Project Title: Identification and Characterization of a Minimal Functional Splicing Regulatory Protein
Author List:
Hernandez, Luis; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Gonzalez, Stephen ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Nguyen, Haylena; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton

Abstract: In higher eukaryotes, alternative splicing of a single gene transcript into more than 1 final spliced mRNA contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that bind to regulated exons and influence the assembly of a functional spliceosome at adjacent splice sites. Splice variants have been identified in many neurodegenerative diseases and cancer, underscoring the importance of alternative splicing. Detailed mechanistic and atomic understanding of how splice variants are generated will provide new targets for therapeutic intervention. The Polypyrimidine Tract Binding Protein 1 (PTBP1) is a well characterized RNA binding protein with roles in alternative splicing regulation, mRNA localization, and IRES-mediated translation initiation. Studies highlight that PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript (Amir-Ahmady et al., 2005). PTBP1 contains four RNA recognition motifs (RRMs) joined via three linker regions which bind to pyrimidine rich sequences with varying affinity and structural preferences (Oberstrass et al., 2005). How PTBP1 binds to its target RNA and the role of each RRM during exon inclusion/exclusion is unclear and hinges on obtaining atomic structures of PTBP1 bound to a target RNA molecule.
Attempts to crystallize RNA-bound PTBP1 are hindered by a flexible N-terminal (57 aa’s) and two linker regions between RRM1 and 2 (linker 1, 42 aa’s) and RRM 2 and 3 (linker 2, 81 aa’s). Previously, we characterized a minimal functional PTBP1 protein with deletions in Linker 1 (Δ40) and Linker 2 (Δ71) regions. In this study, we cloned an N-terminal deletion mutant (Δ50) into a bacterial expression plasmid and assayed the mutant for protein expression in vitro. Our data highlight the mutant is soluble and well-expressed. We assayed the mutant for RNA binding via electrophoretic mobility shift assays. Our results indicate the minimal PTBP1 binds with high affinity to target RNA sequences. We are currently carrying out large-scale expression and purification to set up crystallization trials.

Poster #: 144
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Drug delivery, Nanodiscs, Lipoproteins
Project Title: Incorporation of Antioxidant Luteolin into Apolipoprotein-containing Nanodiscs
Author List:
Anderson, Kyla; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Benedicto, Vernon; Graduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Ly, Brendan; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach
Seo, Kevin; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach

Abstract: High-density lipoprotein (HDL) is a large spherical protein-lipid complex that is composed of proteins such as apolipoprotein (apo) AI and apoE3, several molecules of lipids, such as phospholipids and cholesterol. Amongst the apolipoproteins, only apoE3 has the ability to bind the low density lipoprotein receptor (LDLr) family of proteins that facilitate cellular uptake of lipoproteins via receptor-mediated endocytosis. Other ways of cellular entry for lipoproteins involves LDLr-related protein, and scavenger receptor B1 (SR-B1); the latter recognizes apoAI, apoE3 and other apolipoproteins. We propose to capitalize these features of apolipoproteins to deliver luteolin (3′,4′,5,7-tetrahydroxyflavone), a non-polar flavonoid with antioxidant properties, to breast cancer cells (MDA-MB-231) with an immediate goal to incorporate luteolin into reconstituted high-density lipoproteins (rHDL). The objectives of the study are to: (i) overexpress and purify recombinant human apoE3 encompassing residues 1-191 of the N-terminal domain (apoE3NT) and apoAI, both bearing a His-tag; (ii) reconstitute HDL in the presence of synthetic phospholipids, apoE3NT or apoAI, and luteolin; (iii) perform biophysical and biochemical characterization of the rHDL. Overexpression of apoE3NT and apoAI was accomplished using E. coli BL21-Gold (DE3) pLysS cells and the proteins purified by affinity chromatography. SDS-PAGE analysis revealed that the proteins were ~ 95% pure. The proteins were mixed with one of the following phospholipids: POPC, DMPC, DMPE, or DMPG in the absence or presence of luteolin and sodium deoxy cholate, and dialyzed to obtain rHDL. The samples were subjected to density gradient ultracentrifugation, fractionated and protein-containing fractions (with visible yellow coloration due to luteolin) pooled. Non-denaturing polyacrylamide gel electrophoresis confirmed formation of high molecular mass complexes (~ 600 kDa). The next step is to determine the efficiency of incorporation of luteolin into rHDL by HPLC, and assess the effect of phospholipid head group variation on the incorporation. Subsequently, rHDL/luteolin will be examined by electron microscopy and the effect of the presence of luteolin on cellular uptake of rHDL, and the antioxidant activity against cellular oxidative stress assessed in MDA-MB-231 cells. The findings from this study will aid in developing nanodiscs as a viable drug delivery vehicle of other hydrophobic drugs such as chemotherapeutic agents.

Poster #: 145
Campus: San Francisco State University
Poster Category: Biochemistry
Keywords: styrene monooxygease, flavin monooxygenase, electrochemistry
Project Title: Development of an Electrode-Based Supply-Line of Reduced Flavin Adenine Dinucleotide for Flavin Monooxygenase Biocatalysts
Author List:
Amongre, Robert; Graduate, Chemistry and Bichemistry, San Francisco State University, Presenting Author
Gassner, George; Chemistry and Biochemistry, San Francisco State University

Abstract: Two-component flavin monooxygenases (TCFMOs) represent a diverse group of enzymes that catalyze the reductive activation of molecular oxygen and its addition to substrates. These enzymes offer unique potential as biocatalysts with applications ranging from bioremediation, green chemistry to biosensor development. Despite their utility, TCFMOs derive their electrons for catalysis from pyridine nucleotides by using a discreet flavin reductase, which is too costly and complex for industrial applications. As an alternate means of flavin reduction, it has been demonstrated that the monooxygenase component can efficiently bind reduced flavin adenine dinucleotide (FAD) directly from solution in a reaction that is rapid compared with the reaction of unbound reduced-flavin with dissolved oxygen. An electrode capable of efficient transfer of electrons to free flavins in the bulk solution can thus replace the pyridine nucleotide dependent reductase in these systems and greatly contribute to the practical implementation of FMOs in biocatalytic applications. In the work to be presented, we focus on the epoxidase component of styrene monooxygenase as a model system and have constructed a low-volume spectroelectrochemical cell, which allows us to test electrode materials under different conditions. We find graphene electrodes doped with electron carriers communicate with FAD under aerobic and anaerobic conditions in a reaction that is reversible as demonstrated by cyclic voltammetry. We have determined that the rate limiting steps in bulk FAD reduction is desorption of reduced FAD. The rate of bulk FAD reduction under anaerobic conditions is 1-2µM/min indicating that electron-transfer from flavin adsorbed at the electrode surface to bulk FAD in solution is rate limiting. We further established that under aerobic conditions, molecular oxygen diffuses rapidly and reacts with reduced FAD at the electrode surface resulting in the production of hydrogen peroxide at 5µM/min. In addition, FAD adsorbed on the electrode surface is inaccessible to the epoxidase. We are presently evaluating graphene electrodes doped with polyethylene glycol that readily communicate with FAD, but which have superior FAD-desorption characteristics. We are also investigating and will report the use of organocopper complexes such as copper-EDTA, copper-citrate, and copper-glutamate as mediators to efficiently transmit electrons from the electrode surface to bulk FAD solution.

Poster #: 146
Campus: CSU Monterey Bay
Poster Category: Biochemistry
Keywords: Neural Crest Cells, Tubulin Beta-III, Neurons
Project Title: The Role of Tubulin Beta-III in Cranial Neural Crest Cell Determination
Author List:
Chacon, Jose; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal; Biology, California State University, Northridge

Abstract: Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of amniote embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone and cartilage and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has recently discovered the expression of a marker of differentiated neurons, Tubulin Beta-III (TUBB3), in newly specified premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during embryonic development. Using the model organism, Gallus gallus, we performed immunohistochemistry experiments and have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. In our early gain and loss of function assays, we have determined that loss of TUBB3 results in a reduction of NC cells and an expansion of the neural progenitor cells. After TUBB3 knockdown, Sox2-positive cells invade the area normally occupied by neural crest cells. We hypothesize that TUBB3 can function in neural crest domain maintenance, migration, and neuronal specification. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.

Poster #: 147
Campus: San José State University
Poster Category: Biochemistry
Keywords: light-driven biocatalysis, directed evolution, medium throughput screening
Project Title: Expanding the Synthetic Scope of Light-Driven Hybrid P450 Enzymes using Directed Evolution
Author List:
Vu, Julia; Undergraduate, Chemistry, San José State University, Presenting Author
He, Eric; Undergraduate, Chemistry, San José State University, Presenting Author
Foley, Bridget; Undergraduate, chemistry, San José State University
Kato, Mallory; Staff, Chemistry, San José State University
Cheruzel, Lionel; Chemistry, San José State University

Abstract: As biotechnology attempts to address the rising demand for green and sustainable synthesis, biocatalysts like Cytochrome P450 enzymes have been of great interest due to their ability to functionalize C-H bonds with high regio- and stereoselectivity. These heme thiolate enzymes use molecular dioxygen and two reducing equivalents to catalyze a number of selective oxidation reactions. Our laboratory has developed hybrid P450 enzymes featuring a Ru(II)-polypyridyl complex covalently attached to non-native single cysteine residues of P450 BM3 heme domains. This approach has enabled the rapid injection of electrons into the heme active site leading to high photocatalytic activity towards a range of substrates. In an effort to expand the substrate scope of the hybrid enzymes and optimized the photocatalytic activity, we have implemented a directed evolution approach to evolve the light-driven hybrid enzymes. Directed evolution, recently recognized in the 2018 Nobel Prize, is a proven methodology to engineer unique properties in enzymes, especially cytochrome P450, and expand their substrate scope.
Using methodology established in our laboratory and a valuable colorimetric assay with 96 well plate reader, we have implemented a medium throughput screening. We thus recently identified several randomly mutants with enhanced photocatalytic activity after several rounds of screening. The randomly generated mutants were then sequenced, expressed and purified on a larger scale to confirm their enhanced photocatalytic activity. In addition, we recently showed using HPLC that the enhanced photocatalytic in the hybrid enzyme mutants results from improved coupling efficiency in the electron delivery to the heme active site and substrate binding. We will present our latest results on some interesting randomly evolved hybrid P450 BM3 mutants. This research is currently supported by a grant from the National Science Foundation.

Poster #: 148
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: cytoskeleton, actin, neuronal
Project Title: Mapping molecular interaction interface between neuron-specific drebrin A and diaphanous formin-2
Author List:
Srapyan, Sargis; Undergraduate, Chemistry & Biochemistry, California State University, Long Beach, Presenting Author
Ramirez, Yajaera; Staff; CSU Long Beach -, Chemistry & Biochemistry, California State University, Long Beach
Grintsevich, Elena; Chemistry & Biochemistry, California State University, Long Beach

Abstract: In order to assess the root causes behind neuronal degradation, we need a molecular level understanding of regulatory interactions among neuronal proteins. Actin cytoskeleton is critical for neuronal shape and function. Neuron-specific drebrin A and ubiquitous diaphanous formin-2 (mDia2), are active regulators of the actin cytoskeleton. Drebrin A is highly enriched in postsynaptic terminals of mature neurons (dendritic spines, DS) and mDia2 formin is upregulated during dendritic spine initiation. Recently, inhibitory interaction between drebrin A and mDia2 was reported. It led us to the hypothesis that this interaction can trigger DS maturation during brain development and also contribute to de-evolution of DS in disease. The goal of this project is to map the molecular interaction interface between drebrin A and mDia2 formin. First, we used limited digestion of drebrin combined with the mass spectrometry (MS) analysis to identify drebrin fragments that can bind to formin immobilized on the beads via affinity tag. Our next step was to clone such fragments with glutathione transferase (GST-) affinity tag and test their interaction with formin in pull-down assays. Following our top MS leads, we started by cloning drebrin fragment corresponding to amino acid sequence 446-551. Originally, this recombinant construct was found in insoluble fraction after cell lysis. We optimized its expression in ArcticExpress cells to produce soluble protein. We purified GST-tagged drebrin 446-551 construct and tested its interaction with formin in pull-down experiments. In these experiments, GST tag alone and GST-tagged full length drebrin were used as negative and positive controls, respectively. Analysis of elution fractions (GST-drebrin bound) and flow through (unbound) showed very weak (if any) binding of formin to drebrin fragment 446-551 as opposed to very strong binding to full length neuronal drebrin. This indicates that formin binding site on drebrin A locates outside its 446-551 region. We now use this experimental design to test other drebrin fragments suggested by MS analysis. In conclusion, our pull-down experiments with drebrin fragment 446-551 narrowed down drebrin-mDia2 formin interaction interface.

Poster #: 149
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Drug Delivery, Metal-Organic Frameworks, Surface Chemistry
Project Title: Biodegradable Metal-Organic Frameworks as Drug Delivery Systems
Author List:
Bui, Angela; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Tian, Fangyuan; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Coronary artery disease is the number cause of death nationally. One of the efficient treatments of this disease is balloon angioplasty along with stent implantation damaged artery to restore blood flow. We have prepared one type of iron-containing metal-organic frameworks (Fe-MOF) thin films that have superior properties for drug delivery. In this work, we identified the chemical and structural features that have the greatest impact on drug loading and release properties. We modified several suitable surface platforms (with specific chemical features) that can be used to construct selective Fe-MOF films. This biodegradable porous film material is composed of Fe (III) and various organic ligands that serve as binding sites to the metal surface as well as the connection nodes within the framework itself. The drug loading/releasing with Fe-MOF materials were studied in terms of drug encapsulation amount and mass transfer efficiency. Two types of Fe-MOFs were studied: MIL-53 and MIL-88B. Both consist of iron (III) ligands connected by benzene dicarboxylate linkers. MIL-53 is formed through homogeneous nucleation while MIL-88B through heterogeneous nucleation. The different nucleation pathways result in MIL-53 with one-dimensional channels and MIL-88B with a three-dimensional cage. By controlling the crystal structure, we were able to tune drug loading and releasing patterns with ibuprofen as a model drug. We found that MIL-53 can take up to 372.2 +/- 10.2 mg of ibuprofen per gram while only 194.6 +/- 50.2 mg of ibuprofen with the same amount of MIL-88B. However, the drug elution process is much slower observed for the MIL-88B system compared to its analog material. This indicates complex 3D cages are beneficial for regulating drug release in a controlled manner. Additionally, MIL-88B film drug delivery systems were studied at a COOH-terminated gold and OH-functionalized stainless-steel substrates. A similar drug-releasing pattern (second-order) was observed in both cases. The knowledge acquired from these initial studies will be beneficial for the future in vivo studies of applying a Fe-MOF thin film as a drug-eluting stent coating. In addition to the creation of new thin-film materials with potential applications in medical devices, this work aims to enhance the scientific understanding of surface and interface interactions between general three-dimensional materials and assorted therapeutic agents.

Poster #: 150
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Drosophila melanogaster, neurodegeneration, neurotoxin
Project Title: A Locomotor Activity Study of the Effects of L-serine to Neurodegeneration Impacts of Beta-Methylamino-L-alanine in Drosophila melanogaster
Author List:
Nguyen, Quang T.; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Rivera, Lemuel Vince S.; UCSF Medical School
Goto, Joy J.; Chemistry, California State University, Fresno

Abstract: Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS-PDC) is a neurodegenerative disease that described by loss of motor function, tremors, and dementia due to neurodegeneration. The disease is thought to be caused by Beta-Methylamino-L-Alanine (BMAA), a neurotoxin from cyanobacteria. Since it has a structure that is similar to glutamate, BMAA act as an agonist to the glutamate receptor N-methyl-D-aspartate which lead to excitotoxicity and degenerate neural cells. L-serine is an essential amino acid that has shown to interfere with the effect of BMAA in vivo studies. The purpose of this research is to further test the novel impact of L-serine to BMAA-induced fly in long term study. The interaction of BMAA and L-serine was analyzed through circadian rhythm activity and viability of wild-type Drosophila melanogaster in ALS-PDC model. We used two Drosophila Activity Monitor (DAM) systems to measure activity of 64 fruit flies (Gender and Age-Match). The experimental groups were fed with L-serine, BMAA, BMAA+L-serine and the control group was fed with standard fly agar. Fly activity was recorded by number of time fly broke the IR beam in the monitor for 10-day (12L/12D at 22°C). After 10 days, circadian rhythm data showed BMAA flies increased activity over the first few days, and decreased gradually to the end of the period. BMAA-induced flies (n = 12) does not show pattern of regular activity in L/D as compared to Control (n = 15) and L-serine (n = 16). The co-fed BMAA+L-serine (n = 7) showed a similar pattern as the Control. For daytime vs nighttime activity (mean ± s.e.m), Control (n = 15) shown regular activity (day: 137 ± 13%, night: 21 ± 5%) as compared to BMAA (n = 12) that shown high activity in dark cycle (day: 162 ± 10%, night: 155 ± 21%). Moreover, the cofed L-serine+BMAA (n = 7) exhibited normal daytime activity but not nighttime activity (day: 138 ± 14%, night: 167 ± 31%).The data from DAM shows the abnormal activity of flies in long term when inducing with BMAA such as abrupt sleep due to high activity during dark phase. L-serine shows to reduce down the high activity when cofed with BMAA. This supports our hypothesis of L-serine ameliorates the effects of BMAA in fly. This preliminary data is a starting point for further research in drug discovery for ALS-PDC. Acknowledgements: This work was funded by the Fresno State Undergraduate Research Grant Award, and Howard P. Kubo, M.D. Award for Outstanding Achievement in Biomedical Science.

Poster #: 151
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: transfer-RNA, cryo-EM, kinase
Project Title: Structural and functional characterization of threonylcarbamoyl adenosine biosynthesis in bacteria
Author List:
Paranagama, Naduni; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Luthra, Amit; Postdoc, Chemistry and Biochemistry, San Diego State University
Bayooz, Susan; Undergraduate, Chemistry and Biochemistry, San Diego State University
Jeon, Youngmin; Salk Institute
Lyumkis, Dmitry; Salk Institute
Swairjo, Manal; Chemistry and Biochemistry, San Diego State University

Abstract: tRNA, the central molecule of translation, is heavily modified post transcriptionally with noncanonical nucleosides that constitute ~83% of all RNA modifications. Modifications of the anticodon stem-loop (ASL) of tRNA are required for translational fidelity and codon diversification. The ASL modification N6-threonylcarbamoyl adenosine (t6A) at position 37 in ANN-decoding tRNAs (N is any nucleobase) is universally conserved and its deficiency in humans causes renal-neurological disease. Due to its essentiality in bacteria, the t6A pathway has emerged as a potential antibacterial target. Bacterial t6A biosynthesis begins with synthesis of the intermediate threonylcarbamoyl adenylate (TC-AMP) from threonine, ATP and bicarbonate by the TsaC enzyme, followed by transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNA by the theonylcarbamoyl transfer (TCT) complex, comprised of the three proteins TsaB, TsaD and TsaE. We recently showed in T. maritima that while the TsaBD complex is the substrate binding platform responsible for TC transfer, the complex becomes inactivated after each t6A synthesis cycle, and TsaE-dependent ATP hydrolysis “resets” the platform for multi-turnover catalysis. However, the nature of the inactivation and reset steps remain unclear. Recent reports of a kinase activity for TsaE prompted us to investigate the role of this activity in the t6A cycle. Using a radiochemical kinase assay and mutagenesis experiments we show that T. maritima TsaE switches between a highly stable multimeric form possessing a tyrosine autokinase activity and a monomeric form lacking kinase activity and functioning as an ATPAse in t6A synthesis. Further, to elucidate the structural basis of the specificity of t6A biosynthesis to ANN-decoding tRNA substrates, we initiated cryo-EM studies of the tRNA-bound TCT complex. The preliminary maps reveal tRNA bound on the back surface of the TsaD subunit with its anticodon lodged in the TC-transfer site and acceptor stem stabilized by the C-terminal tail of TsaB, consistent with previous biochemical data. Collectively, the results provide insight into the mechanism of TC transfer, and suggest a regulatory role of TsaE that links t6A synthesis to other cellular processes.

Poster #: 152
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: breast cancer, metabolomics, NMR
Project Title: Investigating the Biochemical Impact of HER2 on Breast Cancer Cell Metabolism
Author List:
Leon, Nicole; Undergraduate, Biology, California State University, Fresno, Presenting Author
Abuelreich, Sarah; Graduate, Biology, California State University, Fresno, Presenting Author
Krishnan, Krish; Chemistry, California State University, Fresno
Bush, Jason; Biology, California State University, Fresno

Abstract: HER2-positive breast cancer represents about 20% of breast cancer cases and although treatments are available, some patients develop resistance to multiple exposures of anti-HER2 therapies. New strategies need to be developed for a more accurate selection of patients who can benefit from these therapies. While much is known about HER2 biology, the biochemical impact of HER2 on breast cancer cell metabolism is not well understood. The goal of this project is to better understand HER2 metabolomics and associated molecular mechanisms using Nuclear Magnetic Resonance (NMR) spectroscopy. A series of eight different breast cancer cell lines ranging from low to high HER2 expression were adapted to a common media with negligible morphological change or alteration in HER2 expression levels. Metabolite profiles for replicates of each cell line were determined using high-resolution 1H-NMR, principal components, and pathway analyses. We identified an upregulation of the pentose phosphate pathway (PPP) which is vital in supporting cancer cell survival and growth by generating pentose phosphate for nucleic acid synthesis and providing nicotinamide-adenine dinucleotide phosphate (NADPH), which is needed for fatty acid synthesis and cell survival under stress conditions. We believe that this upregulation is due to the presence of glucose and glycine in the common media. Ultimately, we hope to exploit this discovery for metabolic targeting as a therapeutic strategy against HER2-positive breast cancer cells.

Poster #: 153
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: DNA Repair, yeast, fluorescence microscopy
Project Title: Investigating Key Proteins, Saw1 and Rad1-Rad10, in Single-Strand Annealing via Fluorescence Microscopy
Author List:
Odango, Jane; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Alon, Alex; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author, Nagel Award Nominee
Fregoso, Fred; Graduate, Chemistry and Biochemistry, California State University, Northridge
Camberos, Juan; Graduate, Chemistry and Biochemistry, California State University, Northridge
Rakibova, Yulduz; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge

Abstract: Unrepaired DNA double-strand breaks (DSBs) can result in cancer. Cells possess repair pathways such as single-strand annealing (SSA) to repair DSBs. In SSA, DNA repeats flank the DSB site and the ends are resected in a 5ʹ to 3ʹ manner. The exposed repeats are annealed by Rad52, thus generating overhanging 3ʹ “flaps”. Prior studies revealed that Saw1 recruits the Rad1-Rad10 endonuclease to cleave these flaps in a flap length-dependent manner. Saw1 recruitment timing is not precisely known but presumably follows annealing. This project had two goals: first, to elucidate the flap-length dependence of protein recruitment at a higher resolution, and second, to create a yeast strain enabling us to peg Saw1 recruitment to the annealing step of SSA.
For the first goal, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB-RFP), allowing us to monitor the recruitment of Rad10-YFP via fluorescence microscopy. The percentages of cells containing colocalized YFP/RFP foci in strains either wild-type or mutant in Saw1 were quantified, revealing that Saw1-mediated recruitment of the Rad1-Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to DSBs across all flap length substrates via experiments with a Saw1-CFP strain. Our findings indicate that Saw1 is necessary for Rad1-Rad10 recruitment in flaps that are ∼20-30 nt or longer and therefore that management of medium/longer flaps is a critical step, and likely the key function Saw1.
For the second goal, we modified a positive control yeast strain possessing Rad52 with two inducible DSB sites with distinct fluorescent markers, by incorporating incomplete HIS3 repeats situated next to inducible DSB sites on different chromosomes. Simultaneous induction of both DSB sites is expected to result in repair by chromosomal translocation, triggering convergence of the fluorescent signals (YFP and RFP) during annealing. We are thus able to peg the arrival of Rad52-CFP in the positive control strain, which serves as a proxy for Saw1-CFP recruitment. Preliminary experiments have shown Rad52-CFP arriving at the annealed repair site (YFP/RFP colocalized foci), but YFP label persistence was lower than expected. We are currently generating a modified strain to improve YFP persistence, and subsequently will generate Saw1-CFP labeled strains to test the timing of Saw1 recruitment to the repair site.

Poster #: 154
Campus: San José State University
Poster Category: Biochemistry
Keywords: intracellular pH, Drosophila, cell death
Project Title: A new paradigm for regulation of cell death by intracellular pH
Author List:
Khomych, Snizhana ; Undergraduate, Biological Sciences, San José State University, Presenting Author
Mancilla, Tania; Undergraduate, Biological Sciences, San José State University, Presenting Author
Peralta, Jobelle; Graduate, Biological Sciences, San José State University
Gates, Hillary; Graduate, Biological Sciences, San José State University
DuPriest, Blake; Graduate, Biological Sciences, San José State University
Grillo-Hill, Bree; Biological Sciences, San José State University

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 showed that the apoptotic 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 in these studies is to directly test this prediction in vivo.
Methods: We used transgenic Drosophila lines that overexpress the Na-H exchanger DNhe2 specifically in the eye (DNhe2-OE), which increases pHi and resulted in a smaller, mispatterned adult eye. We performed cell counts in pupal eyes midway through metamorphosis. We also used a published chemical method to increase pHi in Madin-Darby Canine Kidney (MDCK) cells, and performed cell counts.
Results: In the developing fly eye we found a significant decrease in the number of interommatidial lattice cells at the end of pattern formation from an average of 15 cells in control to 11.4 cells with DNhe2-OE. We performed temperature shift experiments to temporally regulate transgene expression, and determined that the critical phase for DNhe2-OE is pupal eye development, which coincides with two well-studied waves of apoptotic cell death. We next tested for genetic interactions between DNhe2-OE and apoptotic genes. We found that the pH-dependent cell death is p53-dependent but caspase-independent, which is inconsistent with apoptosis, but suggests autophagy. We also confirmed that higher pHi causes a decrease in cell number in MDCK cells, suggesting that this is a conserved process.
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. Funding from CSUPERB and NIH.

Poster #: 155
Campus: CSU Bakersfield
Poster Category: Biochemistry
Keywords: Hops, Soxhlet, Cancer
Project Title: The Contribution of Hops Oil Extracts on Colon Cancer Cell Growth
Author List:
Gonzales, Alexander; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Rotella, Donna; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Webster, Garrett; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Martinez-Iniguez, Samantha; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Chen, Meishan; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Song, Thomas; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Kaur, Navdeep; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Singh, Henna; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Pineda, Nicholas; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Forester, Sarah; Chemistry and Biochemistry, California State University, Bakersfield

Abstract: According to the American Cancer Society, colon cancer is the third most common type of cancer in the United States for both men and women. Consumption of certain phytochemicals in foods and beverages may help prevent this disease. Terpenes, phenolics, alpha-acids, and beta-acids derived from hops (Humulus lupulus) contribute to the flavour, and preservative characteristics of craft beer. This study investigates the contribution of these intrinsic bioactive components from hops in the potential anticancer activity of beer. Two beers were chosen as precedence for hops selection, El Cerrito a Mexican lager with a 22 IBU index, and Hollow Body an Indian Pale Ale with a 44 IBU index. From Hollow body, the hops used were Citra & El Dorado hops; and from El Cerrito, the hops used were Saaz hops. To extract the essential oils an ethanol soxhlet extraction was carried out to extract the alpha- and beta-acids, winterized to precipitate the lipids, and distilled to get rid of any remaining ethanol. The two oil extracts, Citra/El Dorado and Saaz, were dissolved in dimethyl sulfoxide (DMSO) before being resuspended in cell culture media at varying diluted concentrations. HT-29 and Caco-2 human colon cancer cells were incubated with various concentrations (0 – 1000 ppm) of the oil extract treatments for 24 hours. DMSO was added to all treated and control cells at a concentration of 0.1% (v/v). Following incubation, cell viability was quantified using the thiazolyl blue tetrazolium bromide (MTT) assay. Both the Saaz and Citra/El Dorado oil extracts inhibited growth of the HT-29 cells in a dose-dependent manner. At a concentration of 500 ppm, the Saaz and Citra/El Dorado oil extracts inhibited cell viability by 88.6% and 83.1%, respectively. At the same concentration, the Saaz and Citra/El Dorado oil extracts inhibited cell viability of Caco-2 cells by 91.8% and 90.0%, respectively. This level of inhibition was comparable to a previous experiment in our lab where HT-29 cells were treated with aqueous hops extracts. We conclude that hops may contribute to the potential anticancer effects of beer, whether it is made using ethanol-based oil extracts or water-based freeze-dried extracts. The anti-proliferative effects of the hops treatments potentially resulted from bioactive molecules such as alpha/beta-acids and polyphenols. In future experiments, alpha- and beta-acids will be isolated and used to treat colon cancer cells.

Poster #: 156
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Combination Chemotherapy, Drug Efficacy, Breast Cancer
Project Title: Development of Robust Protocols in Efficacy Determination for Combination Chemotherapy Using Hybrid Peptides in MCF-7 Cancer Cells
Author List:
Alvarez, Andrew; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Slowinska, Katarzyna; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Combination chemotherapy studies in metastatic breast cancers have shown increased efficacy compared to single-agent chemotherapy. Unfortunately, most multi-agent treatments need to be administered sequentially rather than in one combination due to the difficulties of delivering agents with different chemical properties; therefore, limiting the possible synergistic effects of combination chemotherapy. We have designed a hybrid collagen/cell-penetrating peptide drug carrier and hypothesize that this carrier will be able to deliver multiple agents simultaneously and in a controlled manner. The drugs to be tested will include Paclitaxel (PTX), Doxorubicin (DOX), 5-Fluorouracil (5FU), and a combination containing the three drugs. To test their effectiveness, the hybrid peptide drug carrier will be evaluated on MCF-7 cells, a model breast cancer cell line, with drug efficacies expressed as half-maximal inhibitory concentration (IC50). The IC50 of PTX, DOX, and 5FU will be determined individually and in combination using luminescence cell viability assay (CellTiter-Glo 2.0). A challenge in determining IC50 of drugs on MCF-7 cells is their strong tendency to form clumps, preventing accurate measurements in cell counting. We try to overcome this challenge by separately treating the cells with a commercially available anti-clumping agent (Gibco) and a concentrated cell dissociation reagent (TrypLE Select 10x Enzyme). Preliminary data show that clumps were still observed in treatment groups and standard deviations of countable cells in treatment groups were higher compared to countable cells in the control group, which signifies that both reagents (anti-clumping and TrypLE) were not effective in obtaining accurate MCF-7 cell counts. We plan to further optimize and change parameters for our anti-clumping protocols to improve cell viability assays and advance drug delivery methods.

Poster #: 157
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Organelle, Metabolism, Vesicular transport
Project Title: Rab GTPase accelerating protein Gyp8 opposes signaling by Rab1/Ypt1 and optimizes peroxisome-dependent carbon utilization
Author List:
Vuong, Chau; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author, Nagel Award Nominee
Mora, Adrian; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Connolly, Sean; Undergraduate, Biology, California State University, San Bernardino
Nickerson, Daniel; Biology, California State University, San Bernardino

Abstract: Rab GTPases (Rabs) are lipid-anchored proteins that regulate membrane docking and fusion of vesicles and membrane-bound organelles. Rabs change activity based on their association with GTP (active) or GDP (inactive), but the intrinsic rate at which GTP is hydrolyzed is slow. Rabs require Rab GAPs (GTPase accelerating proteins) to trigger GTP hydrolysis and inactivate Rab signal. Using the yeast S. cerevisiae, we have identified a Rab GAP, Gyp8, that localizes to peroxisomes via a transmembrane domain. Peroxisomes are membrane-bound organelles that serve several functions, including breakdown of toxic waste products and fatty acids. This is the first report in any experimental system of a Rab GAP localized to peroxisomes, demanding investigation of possible physiological roles of Gyp8 in regulating peroxisome function and dynamics.
It was previously reported (DeAntoni et al, 2002) that an allele of Rab1/Ypt1, ypt1Q67L, with severely impaired intrinsic ability to hydrolyze GTP could still be stimulated to hydrolyze GTP in the presence of a Rab GAP, so the mutation ypt1Q67L renders cells more dependent on Rab GAPs for inactivation and provides an effective background for exploring defects resulting from loss of a single GAP. While cells expressing ypt1Q67L exhibit slow growth at 15°C, overexpression of GFP-GYP8 rescues the growth defect, indicating that the GFP tag does not disrupt Gyp8 function and that Ypt1 is an in vivo substrate for Gyp8.
We used a quantitative colorimetric assay to explore peroxisomal cargo import defects. Cells lacking Gyp8 showed no overt deficiencies when compared to wild type cells, indicating that Gyp8 on its own is not essential for peroxisomal cargo import.
Microbial growth assays revealed that cells lacking Gyp8 suffer a disadvantage when grown in media that requires peroxisome function to utilize a carbon source (oleic acid). Cells lacking Gyp8 saturated their growth at a consistently lower density than wild type, indicating that Gyp8 cells struggle to fully utilize energy from oleic acid. The growth defect is so far restricted to peroxisome-dependent carbon sources, since growth assay using either glycerol (mitochondrion-dependent) or glucose (no stress) reveal no defects in Gyp8-deficient cells.
Phenotypic analyses continue, exploring which domains of the Gyp8 protein are essential for function and screening for synthetic genetic defects arising from loss of both Gyp8 and known peroxisomal proteins or other Rab GAPs.

Poster #: 158
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: Biomechanics of tissues , Frequency domain biomechanics, dynamic elastic modulus
Project Title: Developing an engineering tool to detect time and frequency dependent biomechanics of gels and tissues
Author List:
Maningding, Alvin; Undergraduate, Engineering, San José State University
Border, Neal; Undergraduate, Engineering, San Francisco State University, Presenting Author
Selimovic, Hasan; Undergraduate, Engineering, San Francisco State University, Presenting Author
Azadi, Mojtaba; Engineering, San Francisco State University

Abstract: This work addresses part of a research project funded by CSUPERB in early 2019 to develop biomechanical technologies to study embryo development and to optimize the process of culturing embryos on gel substrates. The optimized gel substrate should have biomechanical properties similar to that of uterine tissue and will be eventually used for In-Vitro-Fertilization (IVF) process. The technique developed here will enable us to quantitatively capture time and frequency dependent properties of gel and soft tissues using Atomic force microscopy (Funded by NSF) indentation.
The response of poroelastic materials found by indentation, such as stress relaxation and creep experiments, are known to be time- and space-dependent (i.e., a function of indenter shape and size). Despite the complexity of the poroelastic response and in contrast to viscoelastic mechanics, poroelastic mechanics can be captured in terms of several intrinsic mechanical properties, such as elasticity, permeability and Poisson ratio. These intrinsic properties can be found from time-domain or frequency-domain master curves. This work (1) proposes using the instantaneous elastic modulus, depicted over normalized time, as a unifying and understandable set of master curves for time-domain tests on poroelastic gels for cylindrical, conical, and spherical shapes; (2) shows how to map time-domain data to the frequency domain with an easy-to-implement numerical method for thick and thin gels as well as poro-visco-elastic gels; (3) shows an easy, but useful method to express poroelastic characteristics in the frequency domain by describing a gel as a linear, time-invariant system with a dedicated transfer function; and (4) suggests the dynamic elastic modulus, depicted over normalized frequency, as a set of master curves for presenting poroelastic mechanics of gels in the frequency domain.
Due to the complexities of tissues (i.e. heterogeneity) as well as experimental challenges, the linearized dynamic elastic modulus is usually found experimentally instead of the dynamic elastic modulus discussed in this work. In this work, we have shown that the linearized and non-linearized dynamic elastic moduli are identical if (1) the indentation used for the linearized dynamic elastic modulus is much smaller than the initial indentation, and (2) The linear-system assumptions, including homogeneity and overall-small indentations, are also valid.

Poster #: 159
Campus: CSU San Marcos
Poster Category: Bioengineering
Keywords: Microfluidics, Stem Cells , Back Pain
Project Title: Fabrication of a Microfluidic Device to Study Interactions Between Human Chordoma UCH-1 Cells and Human Adipose Derived Stem Cells
Author List:
Day, Holly; Undergraduate, Biotechnology, California State University San Marcos, Presenting Author
Kumar, Rosaline ; Undergraduate, Biotechnology, California State University San Marcos, Presenting Author
Carter, Kristian; Undergraduate, Biological Sciences, California State University San Marcos
Javier, Areli; Undergraduate, Biological Sciences, California State University San Marcos
Jelinek , Steven; Undergraduate, Biological Sciences, California State University San Marcos

Abstract: According to the National Institute of Health, 80 percent of adults suffer low back pain at some point in their lifetimes. Degenerative disc disease is correlated with patients suffering from low back pain. The loss of intervertebral disc elements, such as the nucleus pulposus, leads to abnormal spine mechanics, inflammatory signaling and compression of neural structures that lead to pain. Recently, undifferentiated stem cells have been used to rescue specialized cells via transfer of mitochondria and growth factors. However, it is not known how undifferentiated stem cells can aid in the regeneration of human nucleus pulposus cells. Partly this is due to the fact that human nucleus pulposus cells are hard to obtain. In this work, we used a microfluidic device to co-culture human adipose-derived stem cells (ADSCs) next to human chordoma UCH-1 cells. We hypothesized that UCH-1 cells will grow more when cultured next to ADSC cells. Human chordoma derived UCH-1 cells have the same genetical and morphological properties as nucleus pulposus cells and thus, serve as a model for in vitro studies. We made a microfluidic device that consisted of two wells separated by a channel, each well allows for the culture of a specific cell type. When the channel is opened, it mixes the media from each cell line. The microfluidic device was fabricated using SLA resin 3D printing to create a mold. Polydimethylsiloxane (PDMS) was used to create the device which was then bonded using deep UV to a glass slide. In average we saw about 50% increase in cell number when UCH-1 cells were cultured with ADSC cells than just UCH-1 cells alone. These results indicate that even at a distance stem cells (ADSC) are able to influence the growth of UCH-1 cells. In the future, we want to check for the growth factors that affect cell proliferation, the maximum distance between cell groups at which an effect can be seen as well as the potential bidirectional effect that UCH-1 cells can have on stem cells.

Poster #: 160
Campus: CSU Sacramento
Poster Category: Bioengineering
Keywords: Neonatal Jaundice, Hyperbilirubinemia, Bilirubin Monitor
Project Title: A Flexible Testbed for Developing a Noninvasive Continuous Blood Bilirubin Monitor for Neonates
Author List:
Dupart, Emmanuel; Graduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Smith, Warren; Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Thomas, Douglas; Electrical and Electronic Engineering, California State University, Sacramento

Abstract: Jaundice, caused by hyperbilirubinemia (elevated blood bilirubin), is common in neonates, especially when premature. Excessively high bilirubin levels, which can cause permanent brain damage, often are treated with light therapy. Existing noninvasive bilirubin monitors cannot provide accurate readings during light therapy, so blood samples are obtained from the neonate by “heel stick” (cutting the neonate’s heel) and analyzed in the laboratory to monitor the therapy. Heel sticks cause blood loss in the neonate and are difficult for the parents to bear. We are developing a novel non-invasive, continuous bilirubin monitor that can track the progress of light therapy, thus preventing the need for heel sticks. The monitor is based on the analysis of the transmittance of blue and green light through the neonate’s foot to track blood bilirubin. To develop this monitor, we need a flexible testbed that will allow us to determine the best means of obtaining the light transmittance values in the presence of ambient light and processing these values to provide meaningful bilirubin information. Our plan is to rapidly cycle through the application of blue, then green, then no light and measure photodetector output for each of these three conditions, taking care to ensure that the conditions do not interfere with one another. We need to cycle fast enough to faithfully capture the cardiac cycle waveform shapes of transmittance for the fastest neonatal heartrate (as high as 200 beats per minute). We then need to develop suitable digital signal processing to extract the bilirubin information. We are investigating whether National Instruments LabVIEW software and associated hardware can provide us with a suitable testbed to support our bilirubin monitor development. We chose LabVIEW because it provides rich resources for rapid prototyping and development. Our experimental results are promising. We have demonstrated that LabVIEW can successfully control the cyclic generation of the three light conditions, without mutual interference, and perform the acquisition and display versus time of the corresponding photodetector samples to provide at least 30 light cycles per heart cycle for the highest expected neonate heart rate. We believe this performance is sufficient to let us proceed with our next step of using LabVIEW to develop suitable signal processing to extract the desired bilirubin information.

Poster #: 161
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Biomedical Device, Cooling Techniques, Heat Exchanger
Project Title: Thermal Analysis of Foam Filled Heat Exchangers with Application in Cooling of Biomedical Devices
Author List:
Kashkuli, Sina; Graduate, Mechanical Engineering, California State University, Northridge, Presenting Author
Mahjoob, Shadi; Mechanical Engineering, California State University, Northridge, Presenting Author

Abstract: Increasing the demand for more power and advanced technologies in biomedical devices have resulted in high usage of microprocessors and electronics. That have caused dramatic increase in generated heat flux and temperature values in these devices which can threaten safety, functionality, and accuracy of the devices. As such, development of advanced cooling techniques are critical in designing advanced biomedical devices such as diagnostics imaging equipment, polymerase chain reaction (PCR) machines, surgical instruments, and ultrasound equipment. Some of the main advanced cooling techniques to keep the temperature of devices below maximum operating temperature include heat sinks, fans, direct and indirect cooling, heat pipes, and spray cooling. It is shown that jet impingement and microchannel cooling can provide a high efficiency cooling. On the other hand, employment of conductive metal foams in cooling systems improves temperature control and heat transfer. In this work, an advanced conductive foam-filled heat exchanger employing jet impingement technology and non-uniform channels is investigated with application in cooling of biomedical devices. The coolant enters the heat exchanger through a vertical inlet channel, passes through the foam filled non-uniform channel, and exits through two lateral horizontal outlet channels. The base of the heat exchanger is subjected to a high heat flux leaving from the device to be cooled. For numerical modeling, governing equations of continuity, momentum and energy are solved using advanced computational fluid dynamics techniques. Two equation energy model is employed for thermal transport in foam filled region utilizing local thermal non-equilibrium model. In this model, volume averaging are performed over each of the fluid and solid phases in porous media resulting in two energy equations, one for solid phase and one for fluid phase. Grid independency study and code verification are performed against experimental data and data in literature. Multiple design parameters are investigated to develop the designed heat exchanger, such as the shape of the jet inlet cross section (circular cross section and square ones with rounded corners versus square one) for different coolant flow rates, inlet velocity, heat flux values, and coolant types including nanofluids. Flow field characteristics, temperature distribution, temperature uniformity and thermal effectiveness are investigated and analyzed in all studied cases.

Poster #: 162
Campus: San José State University
Poster Category: Bioengineering
Keywords: Isotachophoresis, microfluidics, bacteria detection
Project Title: Development of a microfluidic approach for rapid and continuous detection of pathogens in food and water samples
Author List:
Klar, Gurpreet; Undergraduate, Chemical Engineering, San José State University, Presenting Author
Rosales, Niko; Undergraduate, Chemical Engineering, San José State University
Rosenfeld, Liat; Chemical Engineering, San José State University
Han, Crystal; Mechanical Engineering, San José State University

Abstract: Continuous monitoring and detection of waterborne pathogens is crucial for public health. It can be achieved with specific binding reaction between bacteria’s surface protein and fluorescently labeled peptide-based probes. However, the detection sensitivity and sample processing volume has been limited in particular for low concentration samples. In our research, we aim to develop a microfluidic device for rapid and high sensitivity detection of pathogens from a larger sample volumes. We leverage strong preconcentration of isotachophoresis (ITP) and a controlled sample flow in microfluidic channels to create a stationary and locally concentrated zone of fluorescently labeled bacterial probes within a microchannel, in which the reaction kinetics between the probe and target proteins on the bacteria surface is enhanced. The cell-counts of labeled bacteria is to be performed downstream of the channel through automatic fluorescence image processing. We have accomplished the first phase of the project where we developed a fluidic interface that enabled control of the pressure driven flow using a syringe pump to achieve stationary ITP for up to 2 minutes at 200 µl/min flow rate. This is already 200-fold larger processing volume flow rate compared to the previously reported value of 1 µl/min. In an independent experiment, we developed a custom MATLAB code for image processing, which enabled counting of the microparticles flowing in a microchannel. In addition, the uniformity of the ITP focusing across the channel width is required for efficient labeling of bacteria regardless of their location within the flow. After a set of testing with variations in channel designs, we found that two parallel channels of 190-µm width separated by a 20-µm-width wall resulted in the most uniform shape of focused zone. The ITP-focusing control method developed and tested with fluorescent dye will be applied to the reaction between fluorescently labeled antimicrobial peptide (AMP) and bacteria mixed in the sample solution. We are currently optimizing ITP buffers to focus AMP and further increasing the volume flow rate with higher electric field. Future work will focus on integrating the individual components of flow control, image processing, and ITP-based reaction together with bacteria containing sample solution. We acknowledge the funding from College of Engineering at San Jose State University (SJSU) is greatly acknowledged.

Poster #: 163
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: Bionenginered Muscle, Vascularization, 3D Mdels
Project Title: Engineering Vascularized Muscle Tissue in 3D Hydrogels
Author List:
Al-Lali, Aymen; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Kane, Madison; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Bomar, Briana; Undergraduate, Biomedical Engineering, California State University, Long Beach
Nguyen, Jeffrey; Undergraduate, Biomedical Engineering, California State University, Long Beach
Vu, Bryan; Undergraduate, Biomedical Engineering, California State University, Long Beach
Ayala, Perla ; Biomedical Engineering, California State University, Long Beach

Abstract: Patients who sustain traumatic muscle loss or injury often have limited treatment or rehabilitation options due to a lack of vascularized bioengineered substitutes. Artificial tissues grown in vitro are usually developed without blood vessels. To overcome this challenge, an engineered vascularized muscle tissue developed in vitro could be used as a potential artificial transplant option. The purpose of this work is to develop a fully functional bioartificial tissue. Cells were cultured on a collagen type 1 hydrogel on a 3D mold with parallel micro-channels. Co-culture experiments of HUVECs and myoblasts were conducted to study the development of endothelial tubules and lumen in the presence of myoblasts. Ten-day culture experiments were performed; immunostaining and fluorescent imaging showed that collagen biomaterial supported the proliferation of each cell type. The micro-channels helped to direct the formation of myofibers with similar orientations, however robust vessel formation was not observed. Simulated 3D models in vitro using a hydrogel as scaffold seeded with (HUVEC) and co-cultured with human Mesenchymal Stem Cells (hMSC) were developed to further investigate formation of robust vascular networks. Membrane staining of each cell was done to track the proliferation and differentiation of each cell type for a 5-day culture period. Preliminary results indicate that the hMSCs and HUVECs start to form small vessel-like structures around day 3. Current work is focused on investigating tri-cultures in the collagen-based hydrogel to determine the optimal conditions to generate vascularized muscle tissue that mimics human physiology. Results from this work can be used for future development of artificial engineered organs or tissue grafts.

Poster #: 164
Campus: San José State University
Poster Category: Bioengineering
Keywords: Rare earth elements, methylotrophs, evolution
Project Title: Evolution of Electronic Waste Resistant Methylotrophic Bacteria for Rare Earth Bio-recycling
Author List:
Lepe, Jennifer; Undergraduate, Biological Sciences, San José State University, Presenting Author
Huang, Alice; Undergraduate, Biological Sciences, San José State University, Presenting Author
Grace, Joseph Michael; Graduate, Biological Sciences, San José State University
Vu, Huong; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Biological Scienes, San José State University

Abstract: Methylobacterium extorquens AM1 is a model organism for the study of methylotrophy and is of interest for production of value-added chemicals from single-carbon feedstocks. It was recently discovered that XoxF methanol dehydrogenases use rare earth elements (REE) for methanol oxidation. REE are essential components of our industrial science; they are found in smartphones, computers, hybrid cars, medical devices, and many other technologies. However, mining for REE is costly and environmentally destructive. We demonstrate that M. extorquens can obtain REE from mining ores, fly ash, and electronic waste suggesting it could be engineered as a platform for REE recovery from a variety of sources. However, REE sources like electronic waste can contain toxic levels of heavy metals which present an obstacle to the development of a biological recycling platform. To obtain a toxicity baseline, a strain requiring REE was challenged with blended smartphone; 0.5% facilitated growth while 5% caused cell death. Additional tests showed that toxicity varies based on model and time after blending. To obtain electronic waste resistant mutants, strains were mutagenized with either ethylmethane sulfonate or UV-C, then exposed to 0% -14% blended smartphone. Survival was assessed by plating for colony forming units at 0, 24, 48, and 72 hours post challenge and the process was repeated for three iterations. Three resistant populations were isolated and streaked onto methanol plates containing smartphone. Colonies arose only from one population suggesting either the resistant cells did not survive or smartphone in solid medium is more toxic. A clonal strain was isolated and showed a significantly increased growth rate and the ability to grow without REE suggesting it may be a contaminant. 16srRNA sequencing determined that the isolate is M. extorquens, however, identity at the strain level was not possible. Sequencing of mxaF and xoxF alcohol dehydrogenase genes will being carried out to identify the isolate at the strain level. If the strain is AM1, stress tests suggest that smartphone resistance is not due to an increased general stress response. If the isolated strain is not AM1, it will be explored for its commercial and industrial uses as it grows quickly in methanol media and is more resistant to electronic waste. This work has the potential to advance the development of a biological REE recycling platform to decrease U.S. reliance on foreign REE suppliers.

Poster #: 165
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: tissue engineering, in vitro assays, medical devices
Project Title: Creation of Blood Vessel Mimics using Endothelial Cells on Nanofiber Scaffolds
Author List:
Dozois, Cami; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Youngblood, Abigail; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Yakligian, Evan; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Cardinal, Kristen; Biomedical Engineering, California Polytechnic State University, San Luis Obispo

Abstract: Tissue engineering continues to gain popularity as a method for developing early stage in vitro preclinical models. The advantages of such models include decreased cost, increased reproducibility, and increased control over variables. Kristen Cardinal’s tissue engineering lab at Cal Poly works to create and optimize in vitro tissue-engineered blood vessels, known as “Blood Vessel Mimics” (BVMs), with the goal of testing vascular devices, such as coronary stents and cerebral flow diverters, before using them in typical in vivo settings. With cardiovascular disease being the number one cause of death in both men and women, improvements to these devices could impact a significant part of the population. The current BVM model consists of a polymer-based, electrospun scaffold with a layer of Human Umbilical Artery Smooth Muscle Cells (HUASMCs) followed by a layer of Human Umbilical Vein Endothelial Cells (HUVECs). During testing of stents and flow diverters in this model, both HUASMCs and HUVECs can contact the tested devices. Although this dual-cell type model can be useful for certain applications, a HUVEC-only model would allow for less complicated vessel preparation and analysis, making it desirable for simpler applications. Thus, the goal of this project was to establish parameters for creating HUVEC-only BVMs. Accomplishing this goal included two main aims: 1) optimizing the required sodding density of HUVECs on nanofiber scaffolds and 2) establishing an optimal timepoint for vessel cultivation. The methods for the project included: culturing and expanding endothelial cells, obtaining electrospun nanofiber scaffolds made of Poly Lactic-co-Glycolic Acid (PLGA) and salt, aseptically connecting scaffolds to bioreactors, conditioning scaffolds with proteins, sodding each scaffold with cell densities ranging from 250,000-2,500,000 cells/cm2, cultivating vessels under dynamic conditions for 1-7 days, and performing analysis with scanning electron microscope (SEM) and fluorescent imaging. After experimentation and analysis, we concluded that 1,000,000 cells/cm2 at a 36-48 hour timepoint produced optimal cell coverage. Having determined these parameters, next steps for research are to deploy devices into vessels engineered with such parameters to better understand endothelialization of the devices.
Acknowledgements: We would like to acknowledge the CSU Presidents’ Commission Scholars Program for funding CD and this project

Poster #: 166
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: Cell-free protein synthesis, In-vitro transcription and translation, Biotechnology
Project Title: Redesigned upstream processing enables a 24-hour workflow from E. coli cells to cell-free protein synthesis
Author List:
So, Byungcheol; Undergraduate, Department of Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Levine, Max; Graduate, Department of Biological Sciences, California Polytechnic State University, San Luis Obispo
Mullin, Alissa; Undergraduate, Department of Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Watts, Katharine; Department of Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Oza, Javin; Department of Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo

Abstract: Cell-free protein synthesis (CFPS) provides a method to produce proteins in vitro by isolating all the necessary translational and transcriptional machinery from cells, enabling a variety of applications such as therapeutics, biomanufacturing, point-of-care detection, and education. Since the introduction of Escherichia coli-based CFPS, numerous innovations have made the process of generating cell extract a streamlined process. However, producing robust crude cell lysate remains a tedious and time-consuming operation that is also technically nuanced. Fundamentally, existing media types restrict E. coli cells from growing to higher densities. This necessitates researchers to grow cells to a lower density and harvest at the mid-log phase of the growth. In order to bypass the lengthy and tedious workflow, we have developed a new upstream workflow based on cell-free specific autoinduction media (CFAI) that sustains long-term cell growth and obviates manual intervention during growth. The creation of the CFAI-based workflow sustains long-term cell growth to be harvested at the stationary phase of the growth curve by correcting potential metabolic and pH limitations that previous medias posed. This development is an advance over existing methods which aimed to maximize ribosome density within the cell extract by harvesting at mid-log phase of growth. Following this workflow, the total mass of cells is significantly increased, directly resulting in a 400% increase in cell extract volume compared to existing workflows. Ultimately, these advances enabled the creation of a streamlined workflow that allows researchers to go from cells on a streak plate to completing CFPS reactions within 24 hours while maintaining the quality of cell extracts. Presenting these innovations, we hope to reduce the barrier to entry for new researchers to implement E. coli-based CFPS in their laboratories and improve the time and cost-efficiency for existing CFPS researchers.

Poster #: 167
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: Cardiac Fibrosis, 3D Models, Co-Cultures
Project Title: Bioengineered 3D Fibrosis Model
Author List:
Suarez, Jessica; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Young, Kiera; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Hoang, Anderson; Undergraduate, Biomedical Engineering, California State University, Long Beach
Ayala, Perla; Biomedical Engineering, California State University, Long Beach

Abstract: Cardiac fibrosis is one of the leading causes of heart failure post myocardial infarction. Fibroblasts respond by rushing to the injured site and differentiating into excess proliferation of myofibroblast cells resulting in cardiac fibrosis. The purpose of this research is to investigate methods to modulate fibroblast proliferation and myofibroblast activation in an in-vitro 3D model. Hydrogels from extracted porcine collagen type I was prepared within 3D PDMS molds with different micro-topographical environments at 1 and 5 days. HL-1 cardiac myocytes were co-cultured with fibroblast cells to better simulate the environment of the myocardium. Co-cultured samples were treated using fluorescent probes of red and green cell tracker to distinguish the cell lines. Cells were stained using immunostaining of alpha-actin and F-actin. MTT Assay was used to correlate cell density. Quantitative polymerase chain reaction (qPCR) was used to detect alpha-smooth muscle actin, collagen 1 and cyclin D1. Preliminary qPCR results suggest that micro-channeled environments decrease proliferation of fibroblasts. Microscope imaging showed that cells within the micro-channels proliferate with a defined structure compared to the cells in the flat environment. This suggests that fibroblast activation and proliferation is influenced by the micro-topographical environment, which can potentially be applied to minimize cardiac fibrosis.

Poster #: 168
Campus: Sonoma State University
Poster Category: Bioengineering
Keywords: wastewater treatment, earthworms, microbiology
Project Title: A microbial fuel cell / vermifiltration system for treatment of winery wastewater
Author List:
Quintero Plancarte, Gabriel; Undergraduate, Biology, Sonoma State University, Presenting Author
Kainuma, Mami; Okinawa Institute of Science and Technology
Goryanin, Igor; Okinawa Institute of Science an Technology
Cohen, Michael; Biology, Sonoma State University

Abstract: Means to recycle wastewater are necessitated by the increasing demands on and major costs of freshwater resources. On average, wineries generate six liters of wastewater for every one liter of wine produced. Due to space limitations, various small-scale wineries are faced with the expense of trucking their wastewaters to off-site treatment facilities. Since 2015 we have partnered with a small local winery in Santa Rosa, California to test a system for onsite treatment of winery wastewater (WW) by a sequential microbial fuel cell (MFC) / vermfiltration system.
The most energy-intensive portion of the typical WW treatment is aerating the wastewater to facilitate microbial oxidation of organic compounds. MFCs are an energy-saving substitute for WW aeration since they rely on oxidation of organics by anaerobic bacteria with the ability to pass electrons to conductive material, generating electricity, as well as to neighboring methanogens that form biogas. Vermifilters also obviate the need for mechanical aeration since the earthworms (Esenia fetida) burrow through their bed medium and specifically enhance the density of bacteria having the ability to degrade recalcitrant polyphenolic compounds of the kind found in WW.
Concentrations of organics in the WW were measured as soluble chemical oxygen demand (sCOD). During the summer of 2019, while operated under a 5.7 d hydraulic retention time, our 45 L MFC lowered the sCOD of the WW feed by 71%, from 700.8 +/- 117.8 mg O2 /L to 205.6 +/- 9.8 mg O2/L (mean +/- SE, n = 5), a level well below the World Health Organization standard of 500 mg O2 /L for agricultural reuse of treated WW. The vermifiltration system receiving MFC effluent did not further reduce sCOD levels but did significantly lower levels of phenolics from 31.5 +/- 1.3 mg/L to 13.6 +/- 0.2 mg/L.

Poster #: 169
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: Addiction, psychostimulants, development
Project Title: Ontogeny of context-dependent methamphetamine sensitization using a one-trial behavioral sensitization paradigm
Author List:
Omerjee, Faiyaz; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Coyne, Brendan; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Sortman, Bo; Undergraduate, Psychology, California State University, Long Beach
Nunez, Eric; Undergraduate, Psychology, California State University, Long Beach
Iñiguez, Sergio; University of Texas, EL Paso
Zavala, Arturo; Psychology, California State University, Long Beach

Abstract: Repeated administration of methamphetamine produces an augmented behavioral response known as behavioral sensitization. Indeed, only two injections of methamphetamine are needed to produce a sensitized response, such as enhanced locomotion. The development of methamphetamine-induced behavioral sensitization in juvenile rats is characterized as context-independent sensitization, during which rats will exhibit sensitization regardless of the environmental context that is associated with methamphetamine. In contrast, adult rats will only display a sensitized response when the drug is administered in the same context. We investigated the ontogeny of METH context-dependent sensitization in adolescent rats to ascertain transition from context-independent to context-dependent sensitization. We have previously shown that postnatal day (PD) 49 rats exhibit context dependent-sensitization. In this study, we examined one-trail behavioral sensitization to methamphetamine in PD 29 and 39 rats. We hypothesized that male and female rats would exhibit adult-typical one-trial sensitization at PD 39, but not at PD 29. Male and female adolescent rats were injected either with saline or METH (3.0 mg/kg, i.p.) paired in their home cage (home-paired) or a novel activity chamber (activity-paired) on PD 28 or 29. The next day, rats in each group were given an injection of METH (0, 0.1, or 0.3 mg/kg, i.p.) immediately before being placed in the activity chamber and their locomotor activity was assessed for 90 min. Regardless of the age group, rats displayed context-dependent sensitization at the highest dose of methamphetamine. Moreover, the sensitized response was more robust in females compared to males. These findings indicate that adolescent rats exhibit adult-typical methamphetamine-induced sensitization (i.e., context-dependent sensitization) at the onset of adolescence (i.e., PD 28). Thus, the development of context-dependent sensitization begins prior to the onset of adolescence. Future studies will need to examine earlier periods of development (less than PD 28). Sensitization to drug effects is thought to play a key role in the development of addiction, and understanding the ontogeny of context-dependent sensitization is important to understand the underlying neural mechanisms that develop during adolescence.

Poster #: 170
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: cardiovascular disease, heart rate variability, 30-year Framingham risk
Project Title: Heart rate variability is associated with blood pressure but not long-term cardiovascular disease risk in young adults
Author List:
Isaacs, Justin; Graduate, Physical Therapy, California State University, Long Beach
Weng, Albert; Graduate, Physical Therapy, California State University, Long Beach, Presenting Author
Thomson, Rachel; Graduate, Physical Therapy, California State University, Long Beach
Lee, Michael; Graduate, Physical Therapy, California State University, Long Beach
Mendoza, Christopher; Graduate, Physical Therapy, California State University, Long Beach
Dawson, Jacqueline; Physical Therapy, California State University, Long Beach

Abstract: Elevated risk of cardiovascular disease (CVD) can begin as early as young adulthood. With the prevalence of CVD in the United States expected to increase as the population ages, strategies for the early identification of CVD risk are needed to improve interventions and reduce the future burden of CVD. Cardiac autonomic dysfunction, measured non-invasively through heart rate variability (HRV), has been suggested as an early marker of CVD. Reduced HRV is associated with increased CVD risk among older adults or those with existing metabolic disease, but data are limited in young adults. Thus, this study examined the association between HRV and other commonly used CVD risk prediction variables, including systolic blood pressure as a measure of hypertension and 30-year Framingham risk score as a measure of long-term CVD risk, in a cohort of young adults. A total of 18 females (24.9 ± 2.0 yr) and 14 males (26.5 ± 2.2 yr) performed study measurements in one visit. Assessments included 10-minute seated HRV collection using a wearable sensor (BioHarness-3, Zephyr Technologies), blood pressure, waist circumference and body mass index. HRV data were filtered and visually inspected for artifacts. The root mean square of successive differences in the time domain (RMSSD) was used as the variable of interest. The 30-year Framingham risk score was calculated from sex, age, systolic blood pressure, body mass index and antihypertensive treatment, smoking, or diabetes mellitus status. Multiple linear regression was used to investigate the association between RMSSD and systolic blood pressure or 30-year risk score, adjusted for sex. Elevated or hypertensive systolic blood pressure was observed in 39% of females and 79% of males. RMSSD was inversely associated with systolic blood pressure (p=0.004, 𝛃=-0.113), but not 30-year risk score (p=0.217). Sex significantly influenced RMSSD in both models (p<0.001). In this limited sample of healthy adults, our data suggest that lower HRV is associated with higher systolic blood pressure. However, 30-year risk score, designed to better discriminate long-term CVD risk from multiple factors, was not associated with HRV. Further research is needed to determine whether additional factors, such as disparities in race, physical activity level or metabolic variables, may improve the predictive capacity of HRV. If demonstrated to be effective, HRV would offer a low-cost, easily obtained measurement for the early prediction of CVD risk.

Poster #: 171
Campus: CSU Long Beach
Poster Category:
Keywords: behavior, developmental, dopamine
Project Title: Neonatal caffeine administration on the behavioral sensitization to methylphenidate (Ritalin): Implications for ADHD
Author List:
Gonzalez, Tiffany; Graduate, Psychology, California State University, Long Beach, Presenting Author
Nelson, Tyler; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Nunez, Eric; Undergraduate, Psychology, California State University, Long Beach
Coyne, Brendan; Undergraduate, Psychology, California State University, Long Beach
Mehta, Rhea; Undergraduate, Psychology, California State University, Long Beach
Iñiguez, Sergio; University of Texas, El Paso
Zavala, Arturo; Psychology, California State University, Long Beach

Abstract: About 6 million children and adolescents are diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). Interestingly, administration of methylphenidate (Ritalin©), produces a paradoxical calming effect in children and adolescents with ADHD but increases activity in those without the disorder. Children and adolescents with ADHD have significantly more sleeping issues compared to that of their healthy controls, even when controlling for exposure to stimulant medication. We hypothesize that early-life sleep disruption may underlie the behavioral symptoms in children and adolescents with ADHD. Early-life sleep disruption is difficult to achieve in neonatal rats because of stress effects. In the present study, we examined whether administration of caffeine, a psychostimulant that is known to reduce sleep, could be used to disrupt sleep in neonatal rats, as well as determine if the neonatal exposure to caffeine disrupts methylphenidate-induced behaviors later in life. Repeated administration of methylphenidate results in an enhanced behavioral response that is known as behavioral sensitization. Thus, we expected that early-life caffeine administration would disrupt methylphenidate-induced behavioral sensitization in adolescent rats, whereas saline control animals would exhibit a sensitized response. Rats were pretreated with either saline or caffeine (20 mg/kg, IP) on postnatal day (PD) 7 for seven consecutive days (i.e., PD 7-13). Beginning on PD 26, rats from each group were pretreated with saline or methylphenidate (5 mg/kg, IP) for 10 consecutive days (i.e., PD 26-35). A methylphenidate challenge was then conducted on PD 37, during which all rat rats received saline or methylphenidate (5 mg/kg, IP). Results show that the development of methylphenidate sensitization was attenuated in rats that received neonatal caffeine. Specifically, neonatal caffeine pretreated rats did not increase their locomotor response between the first and final day of pretreatment with methylphenidate (5.0 mg/kg) compared to controls. These findings suggest that neonatal caffeine exposure during PD 7-13 may have a long-lasting disruption of dopaminergic systems that persist into adolescence. Moreover, this suggest that neonatal caffeine exposure may be a novel approach to examining ADHD-like behavioral symptoms.

Poster #: 172
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: clinical decision support, knowledge discovery, tinnitus
Project Title: A Clinical Decision Support System for Tinnitus Diagnosis and Therapy
Author List:
Dispoto, Brett; Undergraduate, Computer Science, San José State University, Presenting Author, Nagel Award Nominee
Tarnowska, Katarzyna; Computer Science, San José State University, Presenting Author

Abstract: Tinnitus is a very common hearing disorder, affecting around 15% of the general population. Commonly referred to as “ringing in the ears,” it has a devastating impact on the quality of life of those affected chronically. At the moment, there are no approved drugs available to treat tinnitus, and current strategies focus on tinnitus management. Among them is tinnitus retraining therapy (TRT), which has been practiced clinically with a success rate of about 80%. This method is based on the neurophysiological model of tinnitus and the accommodation to symptoms is achieved through a combination of counseling and sound therapy. Unfortunately, the method is not widely offered, due to limited expertise in its delivery. Clinical decision support (CDS) systems are innovative tools that combine computer science and medical technology. Within this research, we propose a data-driven CDS tool that will assist audiologists in delivering TRT. To attain our overall objective, we pursue the following specific aims: (1) Automatically extract knowledge base on TRT by applying machine learning algorithms on clinical data. The extracted predictive and descriptive models are then used to generate recommendations for diagnosis and treatment. (2) Evaluate the system, by developing a human-computer interaction (HCI) component and testing the system with the users for its overall usability and predictive accuracy. Our preliminary results include discovering about 680 associations between the category of tinnitus and variables describing a patient (55 variables representing interview data and 57 – audiological measurements). The extracted rules with confidence 80% or higher have been preliminarily encoded into the system’s knowledge base and used to infer a recommendation for a hypothetical new patient. When deployed in clinical practice, the system is expected to bring advantages such as speed, accuracy, and long-term storage of information. Medical professionals will be supported with real-time advice to achieve efficient and effective treatment of tinnitus. This contribution is expected to be significant because tinnitus management is a complicated task that requires a high level of expertise and experience to operate accurately and efficiently. The proposed research is creative and original because no one has implemented a CDSS to advise medical professionals on TRT. Furthermore, this unique approach to developing a data-driven CDS system can be applied to other medical domains.

Poster #: 173
Campus: CSU Monterey Bay
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Assembly, Polishing,
Project Title: Evaluating A Deep Network Polishing Pipeline PEPPER (Program for Evaluating Patterns in the Pileups of Erroneous Reads) Based On Long Read Assembly
Author List:
Sanchez, Joaquin; Undergraduate, Mathematics and Statistics, California State University, Monterey Bay, Presenting Author

Abstract: Long read sequencing technologies like Oxford Nanopore can produce de novo assemblies that can discover large structural variations. Although long read sequencing can generate highly contiguous assemblies, the high error-rate coming from the sequencing data requires polishing pipelines that can improve the base-level accuracy of the assembly. Recent advances in polishing pipeline show that deep neural network based approaches can significantly improve the assembly accuracy. For our project, we propose to evaluate the Program for Evaluating Patterns in the Pileups of Erroneous Reads (PEPPER), a recurrent neural network based assembly polisher pipeline. We assess PEPPER and we compare it to the current state-of-the-art polisher pipeline, MarginPolish, and Homopolymer Encoded Long-read Error-corrector for Nanopore (HELEN). MarginPolish and HELEN both work in haplotype compression mode where MarginPolish generates image-like tensors, and HELEN produces polished assemblies using the images. PEPPER, on the other hand, does not have an external dependency on MarginPolish and can be extended to use with other pipelines easily. We compare PEPPER to MarginPolish and HELEN using genome HG002 (Chromosome 20) and HG00733 whole genome. PEPPER creates images like tensors for training and testing which are then used to produce models that give test error and test accuracy. Next, we choose the best model based on the test accuracy and error. In the process of validation on the best model, PEPPER creates a consequence sequence and finally its ready to assess the assembly. Using Oxford Nanopore technologies Pomoxis, we then assembly for PEPPER by looking at the percent error and the quality score. Our results demonstrate that PEPPER percentage error and quality score does a lot better more often than the state-of-the-art MarginPolish and HELEN for both the genome HG002 (Chromosome 20) and whole genome HG00733 for error. From this experiment, we validate the methods employed in PEPPER which will help us create a portable assembly pipeline.
Acknowledgements
Funding for the BD2K Biomedical Data Science program at CSUMB is supported by the Office Of The Director (OD) of the National Institutes of Health under grant number R25MD010391 and at UCSC by NIH/NHGRI 1U54HG007990. Thank you CSUPERB.

Poster #: 174
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: TAR-Tat, RNA binding, molecular dynamics
Project Title: Alternative RNA-Protein Interactions for BIV Tat Peptide Binding to TAR Determined by Complementary Computational Approaches: DOCK and GROMACS
Author List:
Fozo, Alex; Undergraduate, Chemical Engineering, San José State University, Presenting Author
Suwandi, Ethan; Materials Engineering, UC Davis, Chemical Engineering, San José State University, Presenting Author
Le, Truc T.; Neuroscience, UC Davis
Mir, Vaseem; Undergraduate, Biology, San José State University
Nguyen, An; Undergraduate, Chemistry, San José State University
Le, Thanh; Chemistry, SUNY Stony Brook
Lustig, Brooke; Chemistry, San José State University

Abstract: Viral-encoded regulatory proteins interacting with RNA target sequences control the gene expression of lentiviruses, notably the human immunodeficiency virus (HIV) and bovine immunodeficiency virus (BIV). The latter provides a simpler interaction model between the viral trans-activator protein (Tat) and trans-activation response RNA element (TAR), including Tat peptides binding to TAR RNA fragments. This may offer insights into possible treatment approaches through theoretical consideration of the role of peptide flexibility in binding as evidenced from literature-based binding assays and NMR. More recently DNA-protein binding is apparently enhanced by increases in conformational entropy. Here, our previously flexibility-identified hinge region of the BIV TAR-Tat complex has indicated key residues K75 and R78 are available for substitution by a more local-flexible glycine; these substitutions allow for alternative RNA-peptide interactions. Initially we generated 277 possible TAR RNA structures that bind Tat peptides. Then, molecular modeling by UCSF DOCK in flexible ligand mode (some three-million structures) indicated, for single and double substituted K75G and R78G 14-mer peptides, conformations partially excluded from the major groove of the RNA. The binding energies show the mutants are more stable than the wild-type peptide.
The order of binding for the peptide ligands from 100ns GROMACS (MBAR) calculations are consistent with the DOCK results, however the magnitude of the mutants’ binding energies scale as a group higher than realistically would be expected. Still, identifying hydrogen bonds indicates a set of alternative RNA-protein interactions for mutants with respect to wild-type for both GROMACS and DOCK. Moreover, the lowest thousand structures in energy for each ligand indicate a handful of dominant RNA secondary structures. Real differences in such dominant structures are noted for wild-type and the double mutant. Finally, an initial set of free-energy landscape (FEL) calculations were performed with respect to the GROMACS set of results, indicating multiple local minima for the wild-type and at least the K75G and R78G mutants. We look forward to comparing the RNA secondary structures and the peptide topology as well as the network of hydrogen bonds and van der Waals contacts from these local minima with respect to the low energy structures characterized in the DOCK results.
Acknowledgements: Funding provided by SJSU and NSF MRI 1626645.

Poster #: 175
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: protein switches, secondary structure, prediction from sequence
Project Title: Switch-Like Features in Proteins Using Logistic Regression with Sequence-Based Descriptors Compared to Regions Identified in X-ray Crystal Structures
Author List:
Strauss, Benjamin; Graduate, Computer Science, San José State University, Presenting Author
Qi, Charles; Chemistry, Emory University, Presenting Author
Oribello, Jonathan; Graduate, Computer Science, San José State University
Huynh, Angela; Graduate, Chemistry, San José State University
Millan, Edgardo; Undergraduate, Chemistry, San José State University
Cao, Khai; Undergraduate, Chemistry, San José State University
Wang, Ningkun; Chemistry, San José State University
Lustig, Brooke; Chemistry, San José State University

Abstract: Ligands can bind at specific protein locations, inducing changes in conformation such as those involving secondary structure. Identifying possible associated switches from primary amino acid sequence, including homology, is an important ongoing area of research. We attempt to predict possible switch-like regions from protein sequences using a logistic regression approach with 48 acetyltransferases as our learning set and 5 sirtuins as our test set. Validated residue binary assignments of 0 (no secondary structure change) and 1 (secondary structure change) were determined (DSSP) from 3D X-ray structures for sets of virtually identical chains crystallized under different conditions. Some 1796 residue positions were identified as structurally variable in the 13,797-residue learning set as opposed to 436 such residues in the 2754-residue test set. Our sequence descriptors include amino acid type, 6 and 20-term sequence entropy, Lobanov-Galzitskaya residue disorder propensity, Vkabat (variability with respect to predictions from sequence of helix, sheet and other), and all possible combinations. We find the optimal AUC values approaching 70% for two separate models involving either residue disorder propensity or Vkabat.
An additional descriptor involving charge did not significantly improve prediction for the test set. Calculations did suggest a significant fraction of possible switch-like regions as being adjacent to the allosteric region for Human Sirt-1. Analysis on the much larger learning set indicated the best models included both the residue disorder and Vkabat descriptors, as well as 20-term sequence entropy and charge. Overall the addition of residue disorder propensity had the most significant positive impact on model viability. Note some 2984 aligned-residue positions, determined from sets of 3D-structures for homologues under different conditions, included at least one aligned-residue that did not have an assigned secondary structure. Predictions of switch-like residues from our sequence-derived descriptors for such unassigned residues showed improved ROC AUC values exceeding 90% when applying the residue disorder descriptor. Further characterization of these unassigned residues is suggested to better classify residues as specifically disordered.
Acknowledgements: This work was supported in part by the SJSU Chemistry Research Scholars Award. And would like to thank Phuong Tran and Mai Nguyen for exploring disordered residue assignment.

Poster #: 176
Campus: CSU Channel Islands
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Antibacterial agents, Dihydrofolate synthase inhibition, Computational study
Project Title: Computational analysis of dihydrofolate synthase inhibition using sulfamoylbenzoic acid derivatives
Author List:
Uslan, Travis; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Marshall, Maddison; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Chemistry, California State University Channel Islands

Abstract: Antibacterial resistance is rapidly becoming a major concern in medicine; this is especially an issue with pre-existing treatments targeting the biosynthesis of folate in many gram-negative strains of bacteria. Shortcomings with treatments have generally exploited dihydropteroate synthase (DHPS) in the folic acid pathway. This has prompted an altered approach for new, innovative treatments targeting other enzymes within this pathway. Due to its highly conserved substrate-binding pocket, dihydrofolate synthase (DHFS) poses as an ideal target for development of novel antibacterial agents. Herein, the synthesis and antibacterial activity of sulfamoylbenzoic acid derivatives were reported, and their binding to the target DHFS was investigated. Experimental data showed that bacterial growth is halted by administration of such derivatives: 4-carboxybenzenesulfonamide (1), 4-chloro-3-sulfamoylbenzoic acid (2), 2-chloro-4-fluoro-5-sulfamoylbenzoic acid (3), 2,3-dimethoxy-5-sulfamoylbenzoic acid (4), N-benzyl-4-chloro-5-sulfamoylanthranilic acid (5), and furosemide (6). Bacterial assays were conducted on three gram-negative and three gram-positive strains, then analyzed by Kirby-Bauer disk diffusion. Four of the six compounds showed successful inhibition of bacterial growth (1-4). Substrate 1 inhibited an average of 13% bacterial growth across two of the three gram-negative strains, while substrates 2, 3, and 4 showed inhibition across all three gram-negative strains with averages of 12%, 14.67%, and 12.67% respectively. To better explain the mechanism for substrate-binding across all enzymes within the folic acid pathway, computational analysis was performed by utilizing the internal coordinate mechanism (ICM) algorithm, in tandem with pharmacokinetics properties. Enzyme docking confirmed the likelihood for DHFS inhibition, by displaying a lower average energy score in three of these four substrates; -41.73 (kcal/mol), -43.91 (kcal/mol), -23.70 (kcal/mol) for substrates 1, 3 and 4, respectively. Further laboratory testing displayed the importance of the carboxyl moiety in each substrate in order for binding to occur, reinforced by examining the binding orientation of each substrate to DHFS. Toxicity reports revealed these compounds to be non-toxic in human systems. This data shows potential for further refinement in sulfamoylbenzoic acids as viable antibacterial agents for DHFS inhibition.

Poster #: 177
Campus: CSU Monterey Bay
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: genomics, biological pathways, cancer
Project Title: Generating Interpretable Reports for a Genomics Machine Learning Algorithm
Author List:
Chavez, Gabriel; Undergraduate, Mathematics and Statistics Department, California State University, Monterey Bay, Presenting Author
Fridel, Verena; University of California, Santa Cruz
Wong, Chris; University of California, Santa Cruz
Stuart, Joshua; University of California, Santa Cruz

Abstract: Quantifying the importance of both individual biological pathways and individual genes has been a difficult task. AKLIMATE, an algorithm developed by Dr. Vlado Uzunangelov, was developed to combine the works of past experiments in order to seamlessly integrate genomic feature sets and multi-omics data. We ran AKLIMATE in order to predict cancer subtypes on a set of patient data from the TCGA (the cancer genome atlas). AKLIMATE, however, outputted non-user friendly results in the form of multiple R files. We developed a workflow to generate interpretable and informative reports for AKLIMATE results. In practice, this will help clinicians diagnose and give treatment to patients who have a certain type of cancer, but are unsure which subtype they have or how to combat that subtype. We also compared AKLIMATE’s results to another high performing algorithm Cloud Forest, and found we predict cancer subtypes with higher accuracy using AKLIMATE. After running the TCGA data through AKLIMATE and our pipeline, we discovered new features that can aid in the prediction of cancer subtypes in five separate cohorts. For our future research, we plan on running the workflow pipeline on all 33 cohorts in the TCGA to develop user-friendly reports for clinicians.
Keywords: biological Features and pathways

Poster #: 178
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Candidatus Saccharibacteria, uncultured bacteria, human oral diseases
Project Title: Lactase from Uncultured Candidatus Saccharibacteria Phylum associated with Oral Diseases show close evolutionary history among diverse sample sources
Author List:
Rapuru, Manasa; Undergraduate, Biological Sciences, San José State University, Presenting Author

Abstract: Uncultured bacteria comprise nearly 70% of the human microbiome, but yet their role in human health is vastly unexplored. Candidatus Saccharibacteria (aka TM7) is a bacterial phylum made entirely of uncultured species. TM7 has been detected in various natural environments using DNA sequence analysis as well as in association with the human skin, the oral cavity, gastrointestinal tract, and the urogenital tract. Some TM7 phylotypes have been associated with oral diseases including periodontitis, which affects 18% of the adult population in industrialized countries. The goal of this project was to use in silico genomic analysis to determine the evolutionary history of a functional gene within the TM7 phylum to better understand the diversity and potential role in sugar degradation. We generated and sequenced a genomic library from a natural mixed microbial community and identified a putative gene (533 amino acids) belonging to TM7 that can hydrolyze lactose sugar (lactase) into glucose and galactose (tested empirically). Using BLAST search and Uniprot we retrieved 341 homologous amino acid sequences with > 40% sequence similarity to our gene. Further analysis identified 22 out of 341 as TM7 proteins involved in sugar metabolism (range: 466-574 amino acids) whereas the remaining 319/341 were homologous metabolic proteins originating from other phyla (range: 466-759 amino acids). A total of 342 (341 retrieved plus our own TM7 gene) sequences plus an outgroup were aligned in a Multiple Sequence Alignment (MSA) and used to build a rooted Neighbor-Joining phylogenetic tree with a random number generator seed of 111 and 1000 bootstrap trials. The tree topology consisted of 9 distinct clades with all 23 TM7 genes (6.7% of collected data) clustering together along with a single sequence from the TM7 phylum. It is yet to be determined if the Candidatus Saccharimonas sequence was misclassified in GenBank. As for the 22 TM7 homologs, the tight clustering was surprising, considering the distinct sample sources: aquifer system (1), dolphin oral cavity (1), mining wastewater (2), human oral subgingival plaque (9), and other unknown sources (9). In conclusion, the TM7 lactase gene shows evolutionarily conserved and distinct from closely related groups. 3D images reveal novel features of TM7 lactase as compared to E coli. Future work will evaluate potential regions to design new PCR primers to retrieve more novel sequences of TM7 from human oral microbiomes.

Poster #: 179
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: density function theory, computational modelling, photosensitizer
Project Title: Development of a small cluster computational model for photosensitized degradation of diuron on titania surfaces
Author List:
Houts, Justin; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Closser, Kristina; Chemistry, California State University, Fresno

Abstract: Contamination of waterways by synthetic organic compounds, such as herbicides and insecticides, is an ongoing issue in the Central Valley. These contaminants can build up and ultimately affect tap water if not effectively removed. According to the state of California, Fresno has the greatest annual pesticide use of any county in the state, measuring at over 35 million pounds of output a year. Photosensitization reactions using ultraviolet (UV) light, can be used to purify runoff containing such contaminants by electronically exciting the photosensitizer which in turn transfers energy to the organic molecule and results in it breaking down into simpler, and ideally less harmful compounds. The goal of this project is to determine a small cluster model for the photosensitizer for use in computational simulations, in order to better understand the degradation process that takes place. We used diuron, one of the most common herbicides in use worldwide as a prototypical contaminant. This molecule in the presence of anatase titania (TiO2), which is a well-studied photosensitizer, formed the preliminary system we studied using quantum chemical methods. The chosen system was modeled using density functional theory with basis set of Gaussian functions via the Q-chem software package and results were visualized using IQmol. The first step in this process is to create a small cluster model that will interact appropriately with diuron. We successfully formed a small cluster consisting of two titanium atoms and eight oxygen atoms passivated with hydrogen, which reasonably reflects an aqueous titania surface. We screened a large number of starting configurations for this system of diuron and titania. In reality the surface is highly extended, but as the association is a local process, we use the small model in order to make these calculations more practical. We found 7 low energy configurations of our model titania structure with ground state of diuron. These configurations typically consist of hydrogen-oxygen surface bonding and others involve proton transfers from diuron to the surface. Using these as initial configurations, the next step in this project will be to investigate later steps in the photosensitization process.

Poster #: 180
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: bioinformatics, microbiology, probiotics
Project Title: Monitoring Antibiotic Resistance in Multi-strain Probiotic Supplements
Author List:
Flores, Emmanuel; Graduate, Biology, California State University, Fresno, Presenting Author
Sistrom, Mark; UC Merced
Van Laar, Tricia; Biology, California State University, Fresno

Abstract: Probiotics are live microorganisms that can competitively exclude enteropathogens and beneficially modulate bodily systems when consumed by their host. Probiotic products often appear as dietary supplements containing various bacterial strains. Although their benefits are well studied, there is a lack of reporting on their ability to resist antibiotics. In this study, we obtained and sequenced five multi-strain probiotic supplements for downstream metagenomic analysis. We assembled genomes and plasmids from our paired-end reads with SPAdes and plasmidSPAdes assemblers. We then used Kaiju metagenomics to identify bacterial species within the supplements. In some of the supplements, we found bacterial pathogens such as Bacillus thuringiensis and Streptococcus pneumoniae. However, in all the supplements we found other bacterial species not labelled on the nutritional information. We then used the Comprehensive Antibiotic Resistance Database (CARD) and integrated it with the Basic Local Alignment Search Tool (BLAST) to construct a database of antibiotic resistance (AR) nucleotide sequences provided by CARD to align with our probiotic genomic and plasmid sequences. We found various genes conferring resistance to several classes of antibiotics. Lastly, we used the Bioinformatics Application for Navigating De novo Assembly Graphs Easily (BANDAGE) to construct De Bruijin graphs of our genomic and plasmid assemblies to identify where AR nucleotide sequences are located in the assembly and which bacterial species carries that AR nucleotide sequence. Bifidobacterium spp. contained most of the AR genes we found through CARD. Alongside we found genomes of pathogens, Streptococcus pneumoniae and Bacillus thuringiensis harboring genes conferring resistance to erythromycin and fosmomycin. Results from this study will help identify overlooked risks in the use of multi-strain probiotics and can be used for future monitoring of antibiotic resistance.

Poster #: 181
Campus: San Francisco State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Exoskeleton, Stroke, Design
Project Title: Design of a Powered Upper Limb Elbow Exoskeleton for In Home Stroke Rehabilitation
Author List:
Mineart, Brian; Undergraduate, Engineering, San Francisco State University, Presenting Author
Long, Ezrael; Undergraduate, Engineering, San Francisco State University, Presenting Author
Lile, Richard; Undergraduate, Engineering, San Francisco State University
Garcia, Martin; Undergraduate, Engineering, San Francisco State University
Achor, Nikki; Undergraduate, Engineering
Quintero, David; Engineering, San Francisco State University

Abstract: In the United States, someone has a stroke every 40 seconds. Two thirds of these individuals survive but require rehabilitation to return to a normal life. Rehabilitation requires encouraging an individual with limited to no mobility to move their limb in order to restore neural connections and regain control. Robots that currently preform this task are only located at rehabilitation centers because they are big and bulky. This requires the patient to go somewhere for rehabilitation rather than having the rehabilitation come to the patient. This is an ideal case for a wearable robotic device, one that is light weight and can be used at home to do everyday tasks. Our project is a wearable powered upper limb exoskeleton that can improve the rehabilitation of stoke patients. A powered exoskeleton is a mechanical device that conforms to part or all the human body to manipulate musculoskeletal joints to assist in a task or encourage movement. This system was designed in 3D computer-aided design (CAD) comprised of Bowden cables that will drive a pulley on an arm brace to get motion in the elbow. A pulley of only 1.5 inches in radius is all that is required to achieve the about 3-pound feet of torque for a person to lift 5 pounds. All components were manufactured from 3D printed nylon material. Our design has 125 degrees of motion which is 86% of the full range of the elbow. There are limiters to protect the wearer against elbow movement that would cause injury. Overall weight of the orthotic device is less than one pound which minimize the stress on the motors and the person itself.

Poster #: 182
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Scanning Ion Conductance Microscopy, alpha-Synuclein, Parkinson’s
Project Title: Real-Time Monitoring of alpha-Synuclein-Induced Cell Membrane Disruption in Parkinson’s Disease
Author List:
Parres-Gold, Jacob; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author, Nagel Award Nominee
Chieng, Andy; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Wong Su, Stephanie; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles
Chang, Megan; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles
Wang, Yixian ; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: Parkinson’s disease (PD) affects more than one million people in the United States alone, making it the second most common neurodegenerative disorder in the US. In PD, aggregates of the natively unstructured protein alpha-synuclein (alpha-Syn) are known to induce the death of dopamine-producing cells in the substantia nigra, ultimately hindering cognitive function. Although monomeric alpha-Syn likely regulates vesicle trafficking, mutations, protein overproduction, or oxidative stress can promote its aggregation into oligomers and fibrils. These aggregates have been observed to increase membrane permeability and promote membrane damage. However, the nature and mechanism of this disruption have yet to be characterized. This work used scanning ion conductance microscopy (SICM) to image in real-time the topographies of live SH-SY5Y neuroblastoma cell membranes after the addition of alpha-Syn aggregates. The alpha-Syn aggregates were produced in-vitro and were characterized by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy. Significant damage to the cell membrane was observed, with both large defects and small pore-like structures appearing across the membrane over time. In addition, analysis of SICM current-distance curves allowed for an assessment of changes to the cell’s local ionic environment. These results suggest that the alpha-Syn aggregates may themselves be binding to and disrupting the neuronal membrane. This work shows that scanning ion conductance microscopy can be used as a novel method for monitoring real-time damage to neuroblastoma cell membranes and suggests membrane disruption as a mechanism of alpha-Syn-induced cell death.
This work was funded by the CSULA new faculty startup fund, the CSUPERB New Investigator Grant, the MBRS RISE program at CSULA (NIH Grant #R25GM061331) and the LSAMP program at CSULA.

Poster #: 183
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: breast cancer biomarker, laser wave-mixing, microfluidics
Project Title: Ultrasensitive Detection of Breast Cancer Biomarker HER2 Using Laser Wave-Mixing Detector Interfaced to Microfluidics
Author List:
Liang, Jie; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Suprapto, James; Graduate, Chemistry and Biochemistry, San Diego State University
Maness, Jessica; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Tong, William; Chemistry and Biochemistry, San Diego State University, Presenting Author

Abstract: Novel nonlinear laser wave-mixing detector interfaced to microfluidics is demonstrated as a sensitive detector for potential early diagnosis of breast cancer. Although there are no FDA-approved biomarkers for early diagnosis of breast cancer, human epidermal growth factor receptor 2 (HER2) is a candidate biomarker. Patients with lower HER2 overexpression are generally in a better condition and there are FDA-approved agents targeting HER2. Our wave-mixing technique has some inherent advantages including small (nanoliter to picoliter) probe volumes and micrometer-thin samples, and hence, high spatial resolution for single-cell analyses, and convenient interfacing to microfluidics, microarrays and capillary-based separation systems. Capillary electrophoresis allows both high chemical selectivity and sensitivity levels. Microarray-based wave-mixing detection systems allow high throughput analyses. The wave-mixing signal has a quadratic dependence on the analyte concentration, and hence, it yields big changes in signal for small changes in analyte concentration. Therefore, it is an ideal sensor for monitoring small changes or progress of a disease. In addition, the wave-mixing signal is a coherent laser-like signal and it can be collected with high signal-to-noise ratios to achieve zeptomole-level detection sensitivity that is comparable or better than those of current detection methods including immunohistochemistry (IHC) and fluorescence-in-situ hybridization (FISH). Wave mixing is also more cost-effective and does not require time-consuming sample preparation steps. In addition, wave mixing is an optical absorption-based method, and hence, it can detect both fluorescing and non-fluorescing analytes. One could use visible lasers to detect analytes labeled by fluorophores or chromophores or use a compact UV laser to detect label-free analytes in their native form. Since both UV and visible lasers are compact, our wave-mixing detectors are portable for field use.

Poster #: 184
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Electrochemistry, Microfluidics, Glucose
Project Title: Capillary Tube and Thread-Based Electrodes for the Detection of Glucose and Acetylthiocholine
Author List:
Duenas, Lauren; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Uchida, Kathryn; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Gomez, Frank; Department of Research, California State University, Office of the Chancellor

Abstract: Rapid technology growth has greatly enhanced the scope of real-time health monitoring, facilitating the incorporation of medical diagnostics into patients’ home healthcare regimens. Reusable and simple to fabricate electrochemical sensors for the detection of glucose and acetylthiocholine (ATC) using thread-based electrodes is described. Production costs of the sensors are low since they are reusable, composed of inexpensive materials, and require minimal reagents, making them well suited for resource-limited regions throughout the world. The devices contain three thread-based electrodes (reference, working, and counter) fabricated by painting pieces of nylon thread with either layered silver ink and carbon ink or silver/silver chloride ink. The thread-based electrodes were inserted into glass capillary tubes, leaving the ends of the electrodes exposed to minimize the surface area of the exposed electrode and minimize solution volumes used for glucose detection. For the first system, glucose and ATC concentrations were measured by pipetting 12 µL of solution onto wax-printed circles on chromatography paper. The capillary tube electrodes were placed within the circles to run cyclic voltammetry (CV) for a range of glucose concentrations (0-20 mM) and ATC concentrations (0-9.84 mg/mL). Glucose concentration was plotted against the oxidation peak heights at 0.45 V of each CV (R2 = 0.966), and ATC concentration was plotted against the current output at 0.6 V (R2 = 0.984). For the second system, the electrodes were placed inside bubble wrap in which 30 µL solutions of glucose or ATC solution were spotted. After CVs were obtained, glucose (0-20 mM) concentration was plotted against current output at 0.45 V (R2 = 0.991). ATC (0-9.84 mg/mL) concentration was plotted against current output at the oxidation peak at 0.6 V (R2 = 0.982). For the third system, two nylon threads (5 inches in length) were knotted, twisted together, and pressed into 5 layers of parafilm with one thread dipped into glucose (0-15 mM) and the other dipped into a solution of GOx, PBS, and potassium ferricyanide. Through capillary action, the solutions traveled and mixed within the thread matrix, and the electrodes were placed on top of the thread to obtain CVs. Glucose concentration was plotted against current output at 0.45 V (R2 = 0.9584).

Poster #: 185
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Skeletal Muscle MRI, Fat and fibrosis imaging , Aging
Project Title: Quantifying muscle, fat, and connective tissue fraction in Calf Muscle from Magnetic Resonance Images to Monitor Age Related Structural Changes.
Author List:
Herrera, Kristopher; Graduate, Physics, San Diego State University
Malis, Vadim; Physics, University of California at San Diego
Sinha, Shantanu; University of California at San Diego
Sinha, Usha; Physics, San Diego State University

Abstract: As we age, the size and composition of our muscles change which leads to a loss of strength. In addition to loss of muscle fibers as well as muscle atrophy, there is increased infiltration of fat and in connective tissue that also contributes to a loss of muscle strength. Recent developments in MRI such as Iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL)enable quantification of fat. Connective tissue has a high percentage of collagen and no appreciable signal intensity on routine MR images. Ultralow TE sequences (UTE, TE= 80 µs) can image low T2 species offering the potential to identify voxel level connective tissue in muscle.
We combine IDEAL and UTE techniques to quantify muscle, fat and connective tissue voxel level quantification to identify differences in younger and older cohorts. The specific focus is to develop a processing pipeline to identify inter, intramuscular and subcutaneous fat, muscle and connective tissue fractions. MR images including multi echo IDEAL and multiecho UTEs images were acquired in two cohorts (young: 20-30 years, old: 70 years and greater) in axial orientation covering the entire lower leg contiguously in 5 mm slice thickness. The Maximum Voluntary Contraction (MVC) under isometric contraction was measured for all subjects. Using the water only image from the IDEAL sequence, four muscles of interest (medial and lateral gastrocnemius, soleus and dorsiflexors as one region), the outer whole muscle, and subcutaneous fat are manually contoured. The four muscle regions of interested are then processed with a morphological operator, erosion, to constrain the muscle contours to lie a few voxels inside each muscle enabling the automated identification of intermuscular regions. Fat fraction for each region is computed from the fat fraction maps while recognizing that fat fractions can be erroneously high in regions of total low signal (e.g., in voxels dominated by connective tissue which has a low signal in all the IDEAL images). The identification of tissues with significant connective tissue is implemented by voxel level analysis of T2* maps computed from IDEAL as well as UTEs images and a locally adaptive threshold derived based on the total signal intensity on water + fat images. This image analysis pipeline will be used to analyze the calf muscle data from young and old subjects and to correlate muscle, connective tissue and fat fractions with the measured MVC.

Poster #: 186
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: compressed sensing, quantitative Magnetic Resonance Imaging, brain and skeletal muscle
Project Title: Compressed Sensing Simulations: Application to Quantitative Brain and Skeletal Muscle Magnetic Resonance Imaging
Author List:
Fleischman, Laura; Graduate, Physics, San Diego State University, Presenting Author
Romero, Ignacio; University of California at Merced
Sinha, Usha; Physics, San Diego State University

Abstract: Quantitative Magnetic Resonance Imaging (qMRI) extends the utility of MRI by providing reproducible indices across subjects that reflect underlying physiology as well as pathophysiology. However, qMRI is limited by the longer acquisition times of the scans. MR Compressed Sensing (CS) is a fairly recent approach to reducing acquisition times and is based on random undersampling of the k-space coupled with a non-linear reconstruction that exploits image sparsity in some transform domain. Combined with multi-coil imaging, CS provides a robust way to reduce scan times for both MRI and qMRI. Here, we report simulation studies to explore compressed sensing for two quantitative MRI techniques: Magnetization transfer saturation (MTsat) and spin-lattice relaxation (T1) mapping applied to brain and skeletal muscle.
The lower leg was imaged using a combination of spine coil and an anteriorly placed flex coil (total of 8 channels) at 3T. Three fully sampled k-space data were acquired on the calf muscle using 3DFLASH sequences with identical geometry at two different flip angles (40 and 100 with and without the MT pulse) to enable MTsat calculations. Simulation was performed by undersampling along the phase and slice encode directions using two schemes: 2 1D variable density and 2D variable density poisson disk undersampling. To evaluate CS strategies, we performed simulations on the full k-space acquisitions to determine the best undersampling pattern as well as the acceleration factors possible without degradation of image quality that also maintained the accuracy of the MTsat parameters. CS reconstruction was performed using the ESPIRiT algorithm publicly available from (https://mrirecon.github.io/bart/). ESPIRiT performs joint reconstruction of mutiple coil undersampled data. Reconstruction was performed in 2D by taking the inverse Fourier Transform along the readout direction (kx, fully sampled) and then undersampling the ky-kz space using the two undersampling schemes outlined above. A global normalization was performed to maintain the signal intensity scale across all the slices in the volume. We found anatomical images reconstructed using the elliptical variable density at acceleration factor 8 were reproduced with no visible artifacts and MTsat values were not significantly different from the full k-space acquisition. Future work will extend simulations to T1 mapping of muscle and to T1 and MTsat mapping of the whole brain.

Poster #: 187
Campus: Sonoma State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Diabetes, Wearable Sensor, Breath VOC
Project Title: Wristband for Monitoring Blood Glucose using Volatile Organic Compounds in Breath
Author List:
Hayden Town, Gaven ; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Buenrstro, Alexis; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Lawrence, Logan; Undergraduate, Engineering Science, Sonoma State University
Shrestha, Sudhir; Engineering Science, Sonoma State University

Abstract: In 2017, 9.4% of the US population or 30.3 million people had diabetes. Failure to manage diabetes can lead to acute and long-term health complications including premature death, vision loss, heart disease, kidney failure, and amputation of toes, feet, or legs. This poster will present a smart wristband that analyzes a user’s breath for diabetes signature and provides an instant feedback on blood glucose status. The device will enable patients to test blood glucose status as often as they desire without having to prick a finger, and track historical results through an user-friendly smartphone app. This will assist patients to better manage their diabetes, and to adhere to their diabetes treatment plans. By using small low-power hardware, embedded machine learning, and transmitting data via a Bluetooth-smartphone connection, the device is able to be worn on the user’s wrist. The device uses three metal oxide semiconductor based sensors to detect small amount of known diabetic volatile organic compounds (VOCs) such as acetone and ethanol. Given the number of published works with acetone or acetone and ethanol, and the controlled nature of our experiment, we were confident that the two VOCs would be adequate to produce classification results with good accuracy. We divided the blood glucose levels in two groups; high: representing reported blood glucose levels of 125 mg/dL and higher, and normal: representing blood glucose levels of 100 mg/dL or lower. The associated concentrations of the VOCs are as follows; low BG level: acetone 1-3 parts-per-million (ppm) and ethanol 0-20 parts-per-billion (ppb), and high BG level: acetone: 5-7 ppm and ethanol: 35-50 ppb. These VOC signatures were introduced into ultra pure air percolated through water to create simulated breath representing high and low blood glucose levels. The simulated breath was used for subsequent tests. Test data from the sensors with the simulated breath of high and low blood glucose levels were used to train a support vector machine. The trained algorithm classified test samples with 97% accuracy. The algorithm will be implemented on the wristband for classifying users breath as “Normal” or “High”. The results will be instantly communicated to the user. Patients will be able to track historical reading on the smartphone. The data will be saved in the cloud for further analysis. Future research plans include clinical test with diabetic patients.

Poster #: 188
Campus: Sonoma State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Vital Signs Detection, Remote Monitoring, FMCW Radar
Project Title: Remote Vital Signs Detection using Frequency Modulated Continuous Wave Radar
Author List:
Arjona, Anthony; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Shrestha, Sudhir; Engineering Science, Sonoma State University, Presenting Author

Abstract: This poster will present a remote detection of heart and breathing rates using Frequency Modulated Continuous Wave (FMCW) radar. This technology works by sending a frequency chirp and detecting reflected signals from an object. In adults, breathing and heartbeat generate chest displacements of 1-12 mm and 0.1-0.5 mm, respectively. By measuring a change in phase of the FMCW signal at millimeter wave, we can measure a small displacement at the target and correlate that with the heartbeat and breathing rates. We used Texas Instrument’s 77 GHz module and vital-signs lab. The system sends and receives signal, and feeds the data to a MATLAB program. The signal is processed to decode displacement information and correlate it to heart and breathing rates. The results are subsequently displayed on a monitor via a graphical user interface. Through testing, we were able to accurately read both heart and breathing rates, and determined that an angle of about 30 degrees normal to the radar antenna array and a distance of 1-1.5 m were optimal. Further research is aimed at improving the range of the system and study performance when there are multiple targets. The presented system has application in remotely monitoring vital signs of a patient without probes in hospital settings. We aim to achieve an ability to continuously monitor vital signs in a home setting, which would assist elderly population to live an independent life. This is increasingly becoming more important as the number of Americans aged 65 and older is projected to reach 78 million by 2030, and outnumber children. Similar demographic shifts are also projected to occur in other parts of the world. In addition, we are studying a possible application of the system in detecting breathing and human movements through debris in order to assist in locating survivors in natural disasters. In preliminary tests with obstructions, we found that the current system is unable to read the vital signs when a concrete or wood obstruction of 5 cm was placed. Thus, further research is needed to improve the system and signal analysis to detect people through debris. The methods and results from the vital sign detection research and findings from further study on detecting victims through debris will be presented and discussed at the symposium.

Poster #: 189
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: HSV-1, vaccine, liposomes
Project Title: Comparative efficacy of adjuvants MPL, MTP-PE, LT1, and CDN in gD3pep liposomes against intravaginal Herpes Simplex Virus Type-1 Infections
Author List:
Shafiq, Khadija; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Gonzalez, Edgar; Graduate, Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Herpes Simplex Virus Type 1 (HSV-1) infects 48% of individuals ages 14 to 49 in the United States. It usually causes oral or eye infections, but is now responsible for nearly half of genital herpes infections. Antiviral treatments reduce the symptoms of infection but there are no vaccines to prevent infection. To test the efficacy of a liposomal HSV-1 vaccine containing three epitopes of the HSV-1 gD protein (L-gD3pep), we first developed an intravaginal (Ivag) HSV-1 (Strain F) infection in BALB/c mice, by challenging the mice with varying concentrations of virus. Mice were observed for morbidity for 11 days and 20 uL of 1.9 X 10ex6 PFU/mL (plaque forming units) was selected for further testing as it produced morbidity in all mice by day 6. Other groups of mice (n=21/gp) were then subcutaneously vaccinated d0, d14, and d28 with L-gD3pep containing different adjuvants including MPL (monophosphoryl lipid A), MTP-PE (muramyl tripeptide phosphatidylethanolamine), LT1 (lipidated tucaresol), or CDN (cyclic dinucleotide) with phosphate buffered saline (PBS) as the control. Serum was harvested d31 (n=7 mice/gp) to assess HSV-1 neutralizing antibody titers (NAb). On d35, 14 mice/gp were challenged Ivag with HSV-1 and monitored for morbidity (mucosal and neurological symptoms, weight loss) to d57. Viral burden was determined in spinal cords collected from viral challenged mice on d42 (n=7/gp), using a PFU assay. To determine the sensitivity of the HSV-1 PFU assay, we collected spinal cords from non-vaccinated mice (n=5/gp), homogenized the samples with a known amount of HSV-1 virus (2560 PFU/mL) and performed a PFU assay on the homogenates. We found that 99% of the added virus was detectable indicating that the assay was a reliable method for analyzing viral burden in HSV-1 infected tissues. In the vaccine study with the different adjuvants, survival was 86% with CDN, 71% with MPL, 57% with MTP-PE, and 14% with both LT1 and PBS. Weight loss and symptom scores paralleled survival. CDN and MPL gps had significantly lower viral burden in their spinal cords vs the other gps (p≤0.05) while all vaccine gps had significantly higher NAb titers vs. PBS (p≤0.05). In conclusion, our results showed that protection against HSV-1 genital infection correlated with the type of adjuvant in the L-gD3pep. CDN and MPL liposomes provided the most protection with CDN targeting to TLR9 in immune cells and MPL targeting to TLR4 on immune cells.
Acknowledgements: CSUPERB Howell Award

Poster #: 190
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Candida albicans, Antifungal Drug Development, Pyrvinium pamoate
Project Title: Pyrvinium pamoate inhibits biofilm formation and endocytosis of Candida albicans
Author List:
Nguyen, Jennifer; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Zuluaga, Cipriano; Graduate, Biological Sciences, California State University, Los Angeles
Liboro, Karl; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Eivers, Edward; Biological Sciences, California State University, Los Angeles
Park, Hyunsook; Biological Sciences, California State University, Los Angeles

Abstract: Candida species are the fourth most common cause of hospital-acquired bloodstream infections in the US, with a mortality rate of 22-35%. Unfortunately, there are limited numbers of effective antifungal drugs, and many fungal species have emerged resistance to existing drugs. Thus, there are unmet demands for developing novel antifungal therapies targeting new cellular processes to overcome the options of antifungal resistance. We tested Pyrvinium pamoate (pyrvinium), an FDA approved anthelmintic drug, in the attempt of repursing pyrvinium as a novel antifungal agent. Our previous study found that pyrvinium inhibited the growth and morphogenesis of C. albicans and reduced cell wall integrity under planktonic conditions. In this study we further evaluate the antifungal effect of pyrvinium to biofilm formation and the cell viability of C. albicans. Pyrvinium decreased the extent of biofilm by 75% at 0.5μg/ml, 91 % at 1μg/ml, and 92% at 5 μg/ml, suggesting that pyrvinium effectively inhibiting biofilm formation as a dose dependent manner. However pyrvinium was less effective when it was added to preformed biofilm. Only 5 μg/ml of pyrvinium decreased the preformed biofilm by 28% as compared to the no drug control. The vacuole staining suggested that pyrinium affected endocytosis and vacuolar function. The cells treated with 0.5 μg/ml and 1.0 μg/ml pyrvinium were unable to uptake MDY-64 (yeast vacuole membrane marker), which suggests either a possible defect in endocytosis or lack of cell viability in the pyrvinium-treated cells. Pour plating was completed to test for cell viability, which showed that cells treated with 5 and 10 ug/mL remained viable at 24 hours. Pyrvinium demonstrated a fungistatic, rather than fungicidal, effect against C. albicans, with a greater effect seen in higher concentrations. Thus it is likely that the lack of MDY-64 uptake is due to pyrvinium blocking endocytosis of the live cells. In conclusion, pyrvinium shows promise as a potential antifungal agent that could potentially be repurposed as an antifungal agent to treat C. albicans infections.

Poster #: 191
Campus: Cal Poly San Luis Obispo
Poster Category: Disease (Pathogens)
Keywords: Escherichia coli, glycogen, survival
Project Title: The effect of glycogen accumulation on survival of Escherichia coli
Author List:
Andrews, Kaylee; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
D’Souza, Tamica; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Yep, Alejandra; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Glycogen is a glucose polymer that Escherichia coli cells can accumulate when entering stationary phase or under nutrient depletion as long as a carbon supply abounds. While the exact role of glycogen storage in bacteria is not fully understood, it may serve as a source of carbon and energy in poor nutritional conditions. We hypothesized that E. coli strains that are capable of accumulating glycogen have a survival advantage when transferred to nutrient deficient environments specifically filtered tap water and dry glass cover slips. To test this, we grew strains of E. coli we identified as high glycogen accumulators in earlier experiments, in Kornberg media (KM) and Luria-Bertani broth (LB). KM is a rich media supplemented with glucose that promotes bacterial glycogen accumulation while LB is also a rich media but does not promote glycogen accumulation. For survival in filtered tap water, cells were then washed in the filtered tap water and kept in tubes at room temperature. The appropriate dilutions were completed on a sample taken each day and plated on Tryptone Soy Agar (TSA) plates. Plates were incubated for 24 hours and colonies were then counted and a survival curve was constructed. For survival on glass coverslips, cells were washed and aliquots of cells were placed on multiple dry coverslips. Each day cells on one coverslip were re-suspended and the appropriate dilutions were completed to plate on TSA. Plates were then incubated for 24 hours and colonies were counted in order to construct a survival curve. Our results show that glycogen accumulation could potentially provide a survival advantage for some strains in filtered tap water and a disadvantage on dry surfaces. The graphs show that the survival of E. coli behavior varies between strains. Additionally, we show that some strains of E. coli can survive in water for longer than 30 days and on glass surfaces for longer than 2 weeks. Currently, a deletion mutant that cannot accumulate glycogen and cannot catabolize glycogen is being generated in order to test in these experiments to further confirm the role of glycogen accumulation on survival of E. coli.

Poster #: 192
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: biofilm, resistance, klebsiella pneumoniae
Project Title: Evolution of antibiotic resistance in Klebsiella pneumoniae
Author List:
Hough, Alexandra; Undergraduate, Biology, California State University, Fresno, Presenting Author
Brar, Navleen; Undergraduate, Biology, California State University, Fresno, Presenting Author
Van Laar, Tricia; Biology, California State University, Fresno

Abstract: Klebsiella pneumoniae is a pathogenic bacterium responsible for causing significant infection in clinical settings. K. pneumoniae causes a wide range of infections, varying from urinary tract infections, pyogenic liver abscesses, pneumonia, septicemia, and meningitis. K. pneumoniae virulence results from its ability to form strong biofilms, in which typically adhere to surgical sites and open wounds. These biofilms can be very difficult for the immune system to clear without the aid of antibiotics.
In recent years, K. pneumoniae infections have evolved significant antibiotic resistance, contributing to the bacterium’s increasing virulence and resistance to healing. K. pneumoniae is commonly found in hospitals and these multidrug resistant strains have been life threatening for immunocompromised communities, resulting in roughly 50% mortality rates for afflicted patients.
The goal of our study was to determine if K. pneumoniae in a biofilm could evolve resistance to an antibiotic to which the bacterium was previously sensitive.
In order to characterize the ability of K. pneumoniae to acquire antibiotic resistance when in a biofilm, we first formed biofilms on glass discs. We then treated each biofilm with either 5 or 100 times the minimum inhibitory concentration (MIC) of kanamycin for 24 hours. After 24 hours of treatment, we allowed the biofilms to recover in media without antibiotics for 24 hours. We repeated this cycle for 14 days. Every 24 hours, the biofilm on one disc in each treatment condition was disrupted via sonication and plated on LB supplemented with kanamycin to determine if the cells within the biofilm were able to acquire resistance during their exposure to antibiotics.
In biofilms treated with 5x MIC, the cells were able to develop resistance to this concentration of antibiotics following a single 24 hour period of antibiotic therapy. In biofilms treated with 100x MIC, the cells developed resistance as early as day 4 in the protocol.
We next plan to extract whole genomic DNA for sequencing in order to determine which mutations the cells have acquired allowing them to be resistant to kanamycin. By identifying genes likely to mutate during antibiotic therapy, we may be able to develop better treatments to avoid acquisition of resistance and the subsequent impairment in healing.

Poster #: 193
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Microbiome, Probiotics,
Project Title: Optimization and Standardization of Sampling and DNA Extraction Methods for Microbiome Analysis of Inflammatory Skin Dysbiosis
Author List:
Truong, Catalina; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Kumar, Sheelta; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Haas, Kelly; Postdoc, Biological Sciences, California State University, Sacramento
Sivamani, Raja; Pacific Skin Institute
Crawford, Rob; Biological Sciences, California State University, Sacramento

Abstract: Flare-ups of chronic inflammatory skin disorders are routinely characterized by an overabundance of Staphylococcus aureus (SA) within the microbiome and patients are often prescribed antibiotics as a means of course correcting. However, this strategy risks collateral damage to benign microbe populations associated with immune education and colonization resistance to infection. As such, there is great interest in developing novel probiotic and prebiotic approaches toward alleviating clinical reliance on antibiotics, a quest that requires robust data sets elucidating microbiome shifts from health to dysbiosis. A barrier remains the lack of standardization by which clinicians and microbiologists collect samples and extract DNA from complex communities prior to analysis by next generation sequencing methodologies. Industry standard kits yield low concentrations, do not include reagents to lyse cell wall components for all microbe types, and therefore suffer from an underrepresentation of total species composition. This calls to question much of the published literature characterizing microbial shifts and urges caution about probiotic and prebiotic postulations. Here we use lab cultures of SA and S. epidermidis (SE) to optimize DNA extraction methods for highly abundant skin species at all phases of bacterial growth cycles. Overall yields as determined by fluorometric quantification using were affected by lysozyme concentration during exposures at different times and temperatures. The same was true for inflammation-associated Cutibacterium acnes (CA). Importantly, pre-lysis kit conditions including choice of extraction buffer and freezing steps at -80º C dramatically altered DNA representation post-elution on standard columns. We repeated these procedures to identify microbiome shifts in follicular casts obtained from human subjects presenting with rosacea at the UC Davis Medical Center. Our results show differences in representation depending on extraction methodologies, including for keystone species CA, SA, and SE that often guide clinical treatment regimens. We therefore establish optimal collection and extraction methods and propose using them as a means of standardizing the burgeoning field of microbiome science. These studies represent collaborative efforts between UC Davis and CSUS and were supported by generous gift funding from Burt’s Bees.

Poster #: 194
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: thyroid cancer, angiogenesis, qPCR
Project Title: Examining Angiogenic Biomarkers in Aggressive Tumor Types of Thyroid Cancer
Author List:
Chavez, Nancy ; Undergraduate, Biology, California State University, Fresno, Presenting Author
Holm, David; Undergraduate, Biology, California State University, Fresno, Presenting Author
Cheatham, Jazmin; Graduate, Biology, California State University, Fresno
Slater, David; UCSF-Fresno
Maser, Christina; UCSF-Fresno
Bush, Jason; Biology, California State University, Fresno

Abstract: Thyroid cancer is the most common endocrine malignancy in the United States with an estimated 52,070 new cases diagnosed in 2019 (American Cancer Society, 2019). Roughly 2,170 Americans die from the disease each year and its incidence is steadily increasing. Incidence of thyroid cancer are higher in the Central Valley of California than other parts of the state. Recent evidence demonstrates that the second most common type of well-differentiated thyroid cancer is the follicular variant of papillary thyroid cancer (FVPTC). A critical issue in the pathology field involves refining the criteria for which lesions of FVPTC are benign and which have the potential to metastasize. The purpose of this study is to evaluate whether thyroid tumors with elevated expression of angiogenic (blood vessel-forming) biomarkers correlate with clinical features of aggressiveness. We recruited a test cohort of 20 archival FVPTC tumor tissue specimens and optimized tissue recovery for laser microdissection (LCM) by deparaffinization and staining. LCM was performed with multiple cuts between 1500-2000 micrometers to separate tumor tissue from adjacent normal control tissue. From this micro-dissected FVPTC material, RNA was extracted and quantified for downstream semi quantitative RT-PCR analyses of common angiogenic factor expression. Preliminary results demonstrate the difficulty in maximizing reliable and quantitative differences from archival tissue when comparing tumor versus control via gel electrophoresis. Therefore, we have moved to a SYBR green qPCR-based strategy that shows enhanced quantitative analysis of genetic material and avoids utilizing gel electrophoresis. Our hope is to integrate pathologic findings with molecular validation for predictive clinical utility.

Poster #: 195
Campus: CSU Monterey Bay
Poster Category: Disease (Pathogens)
Keywords: Metagenomics, Parasite, Fish
Project Title: Metagenomic Analysis of Myxozoan Parasites in Amazonian Fishes
Author List:
Miller, Samantha; Undergraduate, Biology and Chemistry, California State University, Monterey Bay, Presenting Author
Williams, Kalani; American Museum of Natural History
Jue, Nathaniel; Biology and Chemistry, California State University, Monterey Bay
Siddall, Mark; American Museum of Natural History

Abstract: Myxozoans are highly diverse (>2000 described species), global, microscopic, obligate parasites that cause infections that can yield detrimental symptoms such as whirling disease, proliferative kidney disease, and myoliquefaction in many fish and some amphibian species. Analysis of the frequency and diversity of myxozoan species present in tissue samples taken from a particular location can be an indicator of the water quality and ecological state of the locality. To determine if myxozoan DNA can be used as bioindicator metric for the parasite’s environment, we isolated, amplified and sequenced myxozoan and ciliate small subunit ribosomal genes (SSU rDNA) directly from 184 fish gill samples, without knowledge of their infection status, to identify patterns of myxozoan infection associated with different sites. The fish were collected from areas near the confluence of the Rios Amazona, Tapajós, and Arapiuns, all of which have distinct water types. Amplified DNA products were combined into 44 different pooled groups based on sample species and location. Across all of the samples from the 3 rivers, ciliate sequences present were predominantly of the genus Trichodina and 22 of the 44 pools revealed gill infections by 12 total species of myxozoans (esp. species of Ellipsomyxa, Henneguya, and Myxobolus). 36.4% of the pools had infections from multiple species of myxozoa. There were 35 myxozoan infections from 17 different species of myxozoa present in samples from the Rio Tapajós, 12 infections from 6 different species in samples from the Rio Arapiuns, and 7 infections from 8 different species of myxozoa found in the Rio Amazona samples. At least one species of infected fish from the Rio Tapajós were uninfected in the less acidic Rios Arapiuns and Amazona, while only 2 species of fish positively amplified for myxozoans in the Rio Amazona. Myxozoan infections were detected in 52.6% of fish species sampled from the Rio Tapajós, 37.5% of fish species from the Rio Arapiuns, and 30.8% of fish species samples from the Rio Amazonas had myxozoan infections. Because the 3 rivers have different environments, the rates of myxozoan infections in fishes demonstrate potential in their use as an indicator of environmental status, specifically suggesting a higher frequency of infection being correlated with high stress environments.

Poster #: 196
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: blood, CRISPR/Cas9, leukemia
Project Title: CRISPR/Cas9 Technology Indicates that ccl44 is Essential for Normal Embryonic Hematopoiesis
Author List:
Rueb, Kristen; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author, Nagel Award Nominee
Stachura, David; Biological Sciences, California State University, Chico

Abstract: Hematopoiesis (blood formation) is the process by which progenitor cells differentiate into the many different cells that constitute mature blood. By studying the molecular pathways responsible for this cellular differentiation we can better understand and treat blood diseases like anemia and leukemia, which arise from defects in these processes. The zebrafish (Danio rerio) is an ideal model organism to use for the study of hematopoiesis because their blood development is similar to humans and they have high fecundity which allows us to look at a larger sample pool in a shorter time frame. In addition, the embryos develop externally and are transparent making manipulation and observation easier. We performed an RNA sequencing screen on hematopoietic supportive zebrafish cell lines, and identified the top 100 highly expressed genes. One gene codes for a secreted chemokine previously only implicated in guiding neurons. When we reduced the levels of the chemokine ligand 44, ccl44, with morpholinos, we observed a reduction in multiple blood cell types such as red blood cells, myeloid cells, and thrombocytes. In order to validate these findings, we performed knockout experiments using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. CRISPR/Cas9 cuts double stranded DNA, allowing us to remove the whole ccl44 gene, causing a permanent genomic mutation. Zebrafish with fluorescently labeled myeloid and erythroid cells were injected with ccl44 guide RNA (gRNA) that we produced along with Cas9 protein. The removal of ccl44 was confirmed with T7 endonuclease assays and gel electrophoresis. In mutant animals, a decrease in myeloid and erythroid cells was observed with fluorescent microscopy. We confirmed the reduction in red blood cells with benzidine staining, a specific stain for hemoglobin. Many phenotypic defects were also seen, including shortened tail length and spinal curvature, consistent with a defect in mesoderm patterning. Additional defects in brain formation and spine were observed, validating the role that ccl44 plays in axon guidance. Currently we are screening animals to see if the genomic modification has gone germline, allowing further studies and the generation of a stable mutant line. Elucidating the role of ccl44 should help inform us about the evolution of the vertebrate hematopoietic system and could have clinical importance for the treatment of human blood diseases.

Poster #: 197
Campus: Stanislaus State University
Poster Category:
Keywords: CRISPR, Mutagenesis, Plasmids
Project Title: Deletion and Substitution Mutations in E. coli Plasmids using CRISPR-Cas9
Author List:
Hensley, Jordan; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Olivas, Cassandra; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Giang, Christy; Undergraduate, Biology, California State University, Stanislaus
Green, James ; Undergraduate, Biology, California State University, Stanislaus
Youngblom, Jim; Biology, California State University, Stanislaus

Abstract: The purpose of this study is to determine the types and sizes of mutations that can be produced in E. coli plasmids using CRISPR-Cas9 in conjunction with recombineering (recombination-mediated genetic engineering). We (and others) have shown that CRISPR can reliably mutate the E. coli chromosome but CRISPR effects on plasmids have not been well-documented. To initiate the project, we constructed a plasmid (pCRISPR) that produces a guide RNA directed toward another plasmid (containing chloramphenicol resistance, CmR). Following electroporation of pCRISPR into an E. coli recombineering strain harboring the CmR plasmid we demonstrated that the Cas9 nuclease is efficiently cutting and removing the CmR plasmid (i.e. when selecting on bacterial growth media the number of CmR colonies plummets). Once established that Cas9 is targeting DNA in the plasmid of your choosing, mutating that DNA fragment can be accomplished with minimal effort and for minimal cost. When the electroporation is repeated with the addition of a single-stranded oligonucleotide (59 nucleotides in length) with similarity that overlaps the nuclease target site, severed plasmids are rescued and repaired by recombineering (i.e. the number of CmR colonies significantly increases). Integration of the oligonucleotide saves the plasmid and simultaneously introduces mutations. Integration of the oligonucleotide does not require 100% similarity to the plasmid. While our initial experiment introduced a 1 nucleotide substitution mutation, recent experiments utilized oligonucleotides that were progressively more divergent. We replaced plasmid segments of 1, 5, 11, 20, and 30 nucleotides (all confirmed by DNA sequencing). Our one attempt at producing a large-scale plasmid deletion utilized a 90 nt oligonucleotide that was costly (>$200) but successfully generated a 400 bp plasmid deletion with nucleotide precision. Our current experiments are utilizing replacement oligonucleotides with randomized (mixed) bases which allows creation of hundreds of plasmid mutants in a single experiment. We are analyzing colonies by isolating and sequencing the plasmids to determine if all of the expected mutants are produced with similar efficiencies. Our long-term goal is to develop a CRISPR-based plasmid mutagenesis system that can outcompete current plasmid mutagenesis protocols in terms of cost and speed. This is particularly feasible when a large number of mutations in a small DNA fragment is the research objective.

Poster #: 198
Campus: CSU Channel Islands
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sweetener, Cancer, Growth
Project Title: Are artificial sweeteners capable of affecting cellular growth and signaling pathways?
Author List:
KHEDMATGOZAR, CHASE; Undergraduate, BIOLOGY, California State University Channel Islands, Presenting Author
RAMIREZ, MELINA; Undergraduate, BIOLOGY, California State University Channel Islands, Presenting Author
RUANO, PATRICIO; Undergraduate, BIOLOGY, California State University Channel Islands, Presenting Author
CUXIL, LILY APEN; Undergraduate, BIOLOGY, California State University Channel Islands, Presenting Author

Abstract: Artificial sweeteners such as saccharin, acesulfame, aspartame, neotame, and sucralose are found in a wide variety of foods today, particularly in zero-calorie sugar substitutes (Equal, Sweet’ N Low, Splenda). Research conducted on animals and human epidemiological studies involving use of these compounds have provided contradictory results, in some cases indicating the compounds as safe and in others, as causing cancer. In our project, we sought to analyze the impact of six different synthetic sugar substitutes (found in Whole Earth, Equal, Splenda, Sweet’ N Low, Nutrasweet and Nutame) on bacterial and mammalian cell growth. In this two-phase project, E. coli cells were grown on minimal media lacking glucose but supplemented with the substitutes to determine the impact on growth. Bacterial cells were exposed to 0.4% of the compounds (0.4% is the standard glucose concentration found in rich LB media) and grown on media plates as well as in liquid cultures. Positive controls included minimal media plates containing glucose as well as rich media LB plates; negative controls included minimal media plates lacking any sugars. Our results indicate that bacteria are able to metabolize sucralose (found in Splenda) but are unable to grow in the other sugar substitutes. Growth profiles indicate severe reduction in growth. Currently we are conducting a dose-dependent profile to monitor whether bacteria will be able to utilize these compounds at higher doses (>0.4%) or will be able to survive if exposed to much lower doses (<0.1%) in the event these are toxic to the cells. The second phase of our study will be done on normal and cancer mammalian cell lines whereby the insulin-mTOR pathway will be specifically analyzed to monitor its up- or down-regulation in response to cells grown in the presence of these substitutes at different concentrations. The mTOR pathway has been linked to a wide variety of cancers and some sugar substitutes have been linked to cancers in animals (e.g. sucralose to bladder cancer in rats). Considering the enormous use of these sweeteners today, this study will provide useful findings that could have important implications for their continued usage.

Poster #: 199
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Transcription factor, microRNA, Gene regulation
Project Title: miR-375 and ICER: A cAMP Mediated Double Negative Feedback Loop in Pancreatic Beta Cells
Author List:
Nielsen, Ryan; Graduate, Biology, California State University, Chico, Presenting Author
Perez, Isis; Graduate, Biology, California State University, Chico, Presenting Author
Keller, David ; Biology, California State University, Chico

Abstract: The area of study for this project is a microRNA called miR-375 which is overexpressed in people with type 2 diabetes (T2D) and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both were found to be regulated by cAMP pathway. Additionally, because of a sequence alignment showing possible binding of miR-375 to the ICER transcript we hypothesize that the two are in a negative feedback loop and can regulate each other’s expression. To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. INS-1 cells that were transfected with a luciferase reporter plasmid containing a cAMP recognition element (CRE) and a plasmid driving the overexpression of ICER had a 72% decrease when compared to our control (preliminary data). INS-1 cells that were transfected with a luciferase reporter plasmid the miR-375 promoter and a plasmid driving the overexpression of ICER had no significant fold change when compared to the control. Additionally, ICER’s expression was measured in human embryonic kidney cells (HEK-293T) when co-transfected with a plasmid containing ICER and a small interfering RNA (siRNA) using quantitative real time PCR (qPCR) and Western blot. Western blot analysis showed that cells transfected with 20pmol ICER siRNA had a 28% decrease in the ICER protein (p-value= .045). To investigate the double negative feedback loop a GFP reporter assay was conducted to determine if miR-375 binds to ICER’s microRNA recognition element (MRE) in a species-specific way as shown in our sequence alignment data. In our preliminary GFP reporter experiment, data shows there is a 50% decrease between our negative control and the human ICER MRE. Additionally, we will overexpress miR-375 and utilize Western blots and qPCR to investigate its regulation of ICER’s expression. To clarify the species-specificity shown in the sequence alignment potential gene regulation at the full length 3’ Untranslated Region (UTR) of the ICER mRNA will also be observed through a GFP reporter experiment. In summary, we do not have evidence for a negative feedback loop, but our data suggests that the cAMP pathway regulates both miR-375 and ICER.

Poster #: 200
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: American pika, Hybrid zone, Conservation genetics
Project Title: Population Genetics of an American Pika Hybrid Zone
Author List:
Tani, Athena; Undergraduate, Biological Sciences, San José State University, Presenting Author
Makmuri, Kenny; Undergraduate, Biological Sciences, San José State University, Presenting Author
Villaseñor, Monica; Undergraduate, Biological Sciences, San José State University
Castillo Vardaro, Jessica; Biological Sciences, San José State University

Abstract: Genetic diversity is a fundamental component of biodiversity and the basis for evolutionary change by natural selection. Hybrid zones provide a great opportunity to learn how introgression (the mixing of genetic material between previously isolated populations) affects a population’s physiology, behavior, and potential to adapt to a changing environment. For example, introgression may increase genetic diversity, thus increasing the raw material upon which natural selection can act (i.e., increasing adaptive potential). Alternatively, introgression may reduce local adaptation if the resulting phenotypic traits are not as well suited to the local environment as each of the individual populations’ traits (i.e., outbreeding depression). In this context, American pikas are ideal species to study because of their vulnerability to elevated temperatures in some locations and their resilience in others. Preceding studies in our lab group identified a hybrid zone between two subspecies of American pikas (Ochotona princeps) within Rocky Mountain National Park, Colorado; the Northern Rocky Mountain (O. p. princeps) and Southern Rocky Mountain (O. p. saxatilis) subspecies. We further investigated the hybrid zone and determined the geographic extent of contact within Rocky Mountain National Park. Specifically, we extracted DNA from fecal material, performed Polymerase Chain Reaction (PCR) to amplify a region of the mitochondrial Cytochrome B locus, confirmed successful amplification with gel electrophoresis, and compared the resulting sequences to sequences of known origin to determine the subspecies of origin. We sequenced 70 individual pikas, in addition to the 19 included in the original analysis. We determined that the hybrid zone is currently limited to approximately 10 km in the northern part of the national park. Geographic barriers, including glacial valleys and the headwaters of the Colorado River, appear to limit dispersal and in turn gene flow between the two subspecies. Future work will determine the rate and extent of migration and gene flow between the two subspecies, as well as investigate whether there are important behavioral and physiological differences that could potentially restrict gene flow.

Poster #: 201
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Neuroscience, Synapse, Neural Circuit
Project Title: Elucidation of a molecular pathway that underlies synaptic specificity
Author List:
Knitter, Courtney; Undergraduate, Biological Sciences, San José State University, Presenting Author
Ebner, Chase; Undergraduate, Biological Sciences, San José State University, Presenting Author
Varshney, Aruna; Staff, Biological Sciences, San José State University
Siman-Tov, Idan; Staff, Biological Sciences, San José State University
Le, Nghi; Undergraduate, Biological Sciences, San José State University
Coto Villa, Doris; Graduate, Biological Sciences, San José State University
Magallanes, Khristina; Undergraduate, Biological Sciences, San José State University
Pollock, Sierra; Undergraduate, Biological Sciences, San José State University
Bremer, Martina; Biological Sciences, San José State University
VanHoven, Miri; Biological Sciences, San José State University

Abstract: Our nervous system consists of 100 billion neurons connected by a trillion synapses. Correct wiring of neurons is crucial for the proper development of our nervous system. To form neural circuits, neurons must identify their correct synaptic partners among the many neurites in a target region, in a process called synaptic partner recognition (SPR). Elucidating the mechanisms of this critical process may lead to a better understanding of neurological disorders. To visualize synapses in live animals, we utilized the trans-synaptic marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP). Our group previously discovered that the unc-6/Netrin ligand, its receptor unc-40/Deleted in Colorectal Cancer (DCC), and the Receptor Protein Tyrosine Phosphatase (RPTP) clr-1/RPTP act together to mediate SPR. clr-1/RPTPs have extracellular domains similar to the Leukocyte common Antigen-Related protein (LAR) family RPTPs, which have well-studied presynaptic roles in synaptogenesis. We found that unc-40/DCC acts in presynaptic PHB sensory neurons, and unc-6/Netrin and clr-1/RPTP act in postsynaptic AVA interneurons. However, the pathway downstream of clr-1/RPTP in presumptive postsynaptic cells had not yet been elucidated. We hypothesized that syd-2, the C. elegans homolog of the mammalian Liprin-α might act with clr-1/RPTP to direct postsynaptic assembly, as previous work from other groups has demonstrated that Liprin-α acts with other LAR family members in presynaptic assembly. This hypothesis led to two predictions. The first prediction was that PHB-AVA synapses would be reduced in syd-2/Lyprin-α mutants, as they are in clr-1/LAR mutants. Utilizing the NLG-1 GRASP marker, we discovered that PHB-AVA synapses were reduced to 61% of wild-type levels (p<0.01). The second prediction was that if the two genes acted in the same genetic pathway, trans-heterozygotes would have reduced synaptogenesis. Consistent with this, we found that in syd-2/+; clr-1/+ trans-heterozygotes, PHB-AVA synapses were reduced to 54% (p<0.001). These findings strongly support a model in which clr-1/RPTP and syd-2/Lyprin-α act together to promote PHB-AVA synaptogenesis. Future directions include determining if syd-2/Lyprin-α acts in postsynaptic AVA neurons using cell-specific rescue experiments, and testing additional homologs of LAR interactors for roles in SPR. We believe that this is a powerful system in which to understand the postsynaptic roles of LAR family members.

Poster #: 202
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Ethanol, Drosophila, Epigenetics
Project Title: Developmental Ethanol Toxicity in Drosophila is Mediated by Epigenetic Regulation of Gene Expression
Author List:
Mok, Madeleine; Graduate, Biological Sciences, San José State University, Presenting Author
Furtado, Tyra; Undergraduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Biological Sciences, San José State University

Abstract: Fetal alcohol spectrum disorder (FASD) is a neurobehavioral disorder caused by exposure to ethanol during fetal development. FASD can result in a variety of deleterious phenotypes, including developmental delays, cognitive problems, and behavioral problems such as attention deficit hyperactivity disorder, sleep disorders, and feeding problems. Evidence in mammals suggests a role for changes in epigenetic gene regulation in the long-term developmental changes caused by DAE.
We have established the fruit fly Drosophila melanogaster as a model organism for the study of FASD. Using this model, we found that wildtype flies reared in ethanol are slow to age, and this appears to be due to persistent upregulation of the antioxidant genes Superoxide Dismutase 1 (Sod1), Glutathione Synthetase 1 (Gss1) and Catalase (Cat) long after ethanol exposure has ceased. Based on these results, we hypothesized that DAE results in changes in the epigenetic regulation of gene expression. To test this hypothesis, we are testing the effects of mutations that disrupt known ethanol-responsive epigenetic regulatory proteins on sensitivity to DAE. We find that mutation of either the histone deacetylase Sirt1 or the histone methylase Little Imaginal Discs (Lid) causes profound sensitivity to the deleterious effects of DAE.
Finally, we have previously shown that DAE causes downregulation, in larvae, of both the fly insulin Ilp2 as well as the insulin receptor (InR). Reduced insulin signaling is well-known to extend longevity in Drosophila as well as many other model organisms. We therefore asked whether the downregulation of Ilp2 and InR persists into adulthood in ethanol-reared flies. We find that neither InR nor ilp2 expression are changed in adults after DAE. Thus, our evidence demonstrates that DAE does not result in altered epigenetic regulation of insulin pathway genes, but does result in persistent changes in gene expression that are likely due in part to the histone modifiers Sirt1 and Lid. Our next steps will be to test whather mutation of Sirt1 and lid can suppress the DAE-induced slow aging phenotype, to examine the expression of both Sirt1 and Lid in ethanol-reared flies, and to begin to characterize the roles of known Sirt1 and Lid target genes in DAE-induced toxicity.

Poster #: 203
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: scoliosis, genomic, cilium
Project Title: Identification of genes that cause scoliosis
Author List:
Villalobos, Jackelin; Undergraduate, Biology, California State University, Chico, Presenting Author
Ewing, Kelly ; Undergraduate, Biology, California State University, Chico
Vang, John ; Undergraduate, Biology, California State University, Chico, Presenting Author
Coia, Victoria ; Undergraduate, Biology, California State University, Chico
Naruse, Kiyoshi ; National Institute for Basic Biology, Okazaki, Japan
Eveleigh, Robert ; Genome Quebec
Bourque, Guillaume ; Genome Quebec, McGill University
Gorman, Kristen; Biology, California State University, Chico

Abstract: Idiopathic Scoliosis (IS) is a pediatric spinal deformity defined by abnormal three-dimensional curvatures of unknown etiology. The syndrome affects 0.5-10% of the global population, imposing an estimated annual cost of over 3 billion dollars to healthcare in the United States. Although a genetic basis is acknowledged, human studies demonstrate inconsistent inheritance patterns and a complex polygenic architecture. Linkage studies, association studies, and exome studies have failed to elucidate the basis of IS. To gain insights into the basic biology, we use inbred strains of the medaka fish as a model system. Our objective is to identify genes causing spontaneous heritable idiopathic-like curvature in the fish model, in order to direct human studies. We used mapping crosses to identify recessive inheritance, and then genome sequencing to identify regions enriched for curved alleles among curved fish and normal alleles among normal fish. Bioinformatic analyses identified 6 regions containing 1054 genes, of which 338 fit our expected genotypes. Based on hypotheses about the IS biology, we chose 10 genes to survey for differential expression between curved and normal fish. Pooled mRNA from whole bodies was compared and one gene was not expressed among curved. This gene, cMed2, is a part of a highly conserved gene family. To confirm that cMed genes cause curvature, we used CRISPR to disrupt three cMed paralogs in the medaka. We screened over 100 fish that encompassed 6 mutations among the three genes. We found that ~80% of the larvae developed curvature. The curve is identical to the onset phenotype of the spontaneous curve in our inbred lineage, although the CRISPR fish died after the onset of curvature. We used PCR and sequencing of CRISPR targets to confirm that curved fish had the mutation and normal fish did not. We found that while curved fish had the mutation, so did 50% of the normal fish. These results prove that the cMed gene family is capable of causing idiopathic-like spinal curvature, with incomplete penetrance. The cMed genes are characterized for their role in regulation of the actin cytoskeleton. Previous works have demonstrated that this gene family can affect the primary cilium through modifications of the cytoskeleton, and ciliary defects have been recently associated with IS. Hence, our work provides molecular insights into conserved cellular mechanisms causing IS.

Poster #: 204
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Ethanol, Drosophila, Insulin
Project Title: Dietary Changes Exacerbate the Toxic Effects of Ethanol On Development
Author List:
Hoey, Hayley; Undergraduate, Biological Sciences, San José State University, Presenting Author
Vu, Danielle; Undergraduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Biological Sciences, San José State University

Abstract: Fetal alcohol spectrum disorder (FASD) is the leading cause of congenital intellectual disabilities in the world. Despite decades of public awareness campaigns targeted at reducing drinking by pregnant women, the prevalence of FASD remains steady at 2 – 11% of all children worldwide, and there is no approved treatment for FASD. Epidemiological data suggest that public awareness campaigns have reached the limits of their effectiveness, and future efforts towards mitigating the significant personal and societal costs of FASD should focus on treatment, which requires a better understanding of how developmental alcohol exposure exerts its toxic effects.
A growing body of evidence from labs including our own shows that developmental alcohol exposure (DAE) causes long-term changes in insulin signaling, which may lead to insulin resistance and, eventually, diabetes. In our previous work we found that in flies, as in vertebrates, DAE disrupts insulin signaling. We also showed that adult flies exposed during development to ethanol display a significant increase in stored fat, as well as altered expression of genes involved in lipid metabolism. All of these changes are mirrored in mammalian FASD models, where alcohol causes increased adiposity and metabolic syndrome, which appear to be directly mediated by long-term changes in insulin signal transduction, both of which can be exacerbated by a high-fat or high sugar diet.
Here, we tested whether dietary changes could exacerbate the toxic effects of DAE. Ethanol-rearing causes reduced survival and a longer development time, as defined by the time in days it takes for 50% of the total surviving flies to eclose (ET50). We tested the effects of both high fat and high sugar diets on survival and ET50, and found that while a high fat diet alone had no discernible effect on development, it did exacerbate the toxic effects of ethanol on both survival to adulthood and ET50. By contrast, a high sugar diet alone had a small but significant effect on both survival and development time. When high sugar was combined with DAE, we saw a profound synergistic effect on ET50. Thus, we find that dietary changes that are predicted to affect insulin signaling exacerbate the toxic effects of ethanol on development, suggesting the possibility that dietary modifications may be a potential intervention in cases of FASD. We are now examining the effects of dietary changes and ethanol exposure on insulin signaling in developing flies.

Poster #: 205
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Candida albicans, Casein Kinase I, Fusion Gene Analysis
Project Title: Functional analysis between YCK2 and YCK22 in the opportunistic pathogen Candida albicans.
Author List:
Salgado, Katia; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Prieto, Salvador; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Park, Hyunsook; Biological Sciences, California State University, Los Angeles

Abstract: Candida albicans is part of the human microbiome and does not cause any immediate harm to the human host. However, C. albicans can become a life-threatening pathogen that afflicts individuals whose immune systems are compromised. A critical virulence that is associated with the pathogenesis of C. albicans is its ability to undergo morphological switching, which allows the fungi to adapt, invade and colonize a wide range of microenvironments. The morphological transitions are controlled by a series of cell signaling pathways that are not fully understood and the extent to which they are evolutionary conserved is less known. The Casein kinase I (CKI) family, which regulates multiple signaling transduction pathways are highly conserved in most eukaryotic organisms. In C. albicans, there are three CKI paralogs YCK2, YCK22 and HRR25. Our previous studies identified that YCK2 governs the morphology of C. albicans, while further analysis of HRR25 and YCK22 has not been performed. In this study, we aim to understand and characterize the role of the Casein kinase I gene, YCK22 and its functional similarity to YCK2 which has been characterized as a critical player for morphogenesis. These two genes might be functionally related and the overexpression of YCK22 should compensate for the loss of YCK2 function. Using the Gibson Assembly technique, we have generated a heterologous fusion gene containing the YCK2 promoter and the open reading frame of YCK22 and have integrated the fused gene into C. albicans yck2 mutant strain. Transcription of the fusion gene was confirmed by quantitative RT PCR using primers specifically targeting the fusion gene. The expressed fused gene has affected phenotypic changes. Colonies that contained the fused gene appear less wrinkly than the yck2 mutants, suggesting YCK22 could compensate the loss of YCK2 function. Preliminary data suggest that the cells with the fused gene have restored the growth defect of the yck2 mutant strain, indicating that the fused gene has rescued some loss of yck2 function. This finding suggests that YCK22 is functionally related to YCK2 and may play a vital role in regulating the transitional changes from non-invasive yeast form to opportunistic pathogenic hyphal form.

Poster #: 206
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gut microbiome, zebrafish, Next-Generation sequencing
Project Title: Investigating Zebrafish Gut Microbiome Dynamics at two different ages
Author List:
Coulston, John; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Wang, Fang; Biology, California State University, Dominguez Hills

Abstract: Human health heavily depends on our own microbiomes. Imbalances in one’s microbiome have recently been connected to multiple health conditions, such as obesity and diabetes. However, host-microbiome interactions have not been well understood. The zebrafish is a well-established model organism with numerous genetic, molecular, and cellular tools already available, and since zebrafish live for about two to three years in a controlled laboratory environment, we can study and record their microbiome over the course of their entire life. In this study, we utilized DNA metabarcoding and Next Generation Sequencing to isolate microbial DNA and identify which microbial Operational Taxonomic Units (OTUs) are present, as well as their relative proportions. We have collected gut microbiome samples from wildtype zebrafish at two different ages: 4-month old mixed gender fish (the onset of sexual maturity), and 13-month old males and females (mature adults). The microbial DNA was isolated from zebrafish feces and amplified using 16sRNA V4 primers. The DNA was then sequenced using Illumina MiSeq (a Next-Generation Sequencer). We have been using software USEARCH to analyze the sequencing data, and have found that gut microbiomes from 4-month old fish and 13-month old fish contained a noticeably different distribution of OTUs. While the 13-month old male and female zebrafish microbiomes were rather similar. This result suggests that gut microbiomes might vary at different ages, but gender might play a lesser role in microbiome composition. In the future, we will examine samples from 6- and 24-month old wildtype zebrafish to provide a more complete picture of normal gut microbiome dynamics, which will enable further investigations on mechanisms of host-microbiome interactions.

Poster #: 207
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: glyphosate, toxicity, chicken
Project Title: Roundup, a glyphosate-based herbicide, stunts cell proliferation and induces morphological changes in embryonic chicken cell culture
Author List:
Johnson, Amanda; Graduate, Biology, San Francisco State University, Presenting Author
Denetclaw, Wilfred; Biology, San Francisco State University

Abstract: The widespread usage of the herbicide Round Up has become controversial in recent years; due to not only increased crop dependence/resistance but with new scientific literature exposing it’s toxic effects on animal cells. Roundup contains glyphosate as the active ingredient which interferes with aromatic amino acid synthesis through the plant specific shikimate pathway. No toxicity to animals has been declared by the manufacturers of Round Up™ to date, even though it has been recently challenged. Studies now show in animal cell lines the herbicide Round Up™ and pure glyphosate contribute to stunted cell growth, reduced proliferation rates and apoptosis. The mechanism involved may result from the generation of reactive oxygen species (ROS), although this is still under investigation. Animal cell lines with abnormal chromosome numbers, altered biochemistry and loss of cell cycle control have been tested with Round Up™ and glyphosate but have produced contradictory toxicity findings. This problem was addressed by utilizing an embryonic derived finite cell line and a near normal culture of cells, which were incubated with varying concentrations of Round Up™ and glyphosate to support its hypothesized toxicity. Therefore, a 7 day-old chicken embryonic finite cell line was produced in 6-well plates with 160,000 cells/well. They were then incubated in Round Up™ and glyphosate alone and diluted in culture medium with concentrations from 1 ug/ml to 100 ug/ml for 48 hours; after which plates were assessed by hemocytometry and Giemsa staining for growth rates and altered morphology. After 2 days of culture in Round Up™, in comparison to control culture cell numbers, there was a 60% and a 76% decrease in cell numbers in 10 ug/ml and 25 ug/ml Round Up™, respectively. Higher concentrations show even greater cell losses in the ranges of 79% and 93% for Round Up™ for 50 or 100 ug/ml. In 48 hours of culture we determined that cell growth occurred at a rate of 1,354 cells/hour, however in 10 ug/ml Round Up™, the cell loss rate was lower at –1,729 cells/hour and higher with greater concentrations of Round Up™. Giemsa staining showed 6-well plates with estimated confluence of 100% in control wells, 70% in 10 ug/ml and 40% in 50 ug/ml. Furthermore, Round Up™ alterations of COS 7 cell culture the cytoskeleton was monitored by microtubule immunofluorescence after 30 minutes or 1-hour incubation. Control cultures showed many microtubules in a wide array across adherent interphase cells and microtubules newly polymerizing from spindle poles as well as stable structured spindles in mitotic cells. In contrast, Round Up at 10 ug/ml showed severe defects in microtubule structure array and in spindle formation in mitotic cells. Our findings support that Roundup induces cellular toxicity in a chicken finite cell line.

Poster #: 208
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: marine invertebrate, ecological genomics, RNA-sequencing
Project Title: Transcriptome-wide Gene and Protein Expression Differences in the Intertidal Mussel Mytilus californianus Exposed to Field and Lab Treatments
Author List:
Fekete, Florian; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Tanner, Richelle; Washington State University
Dowd, Wesley; Washington State University
Gleason, Lani; Biological Sciences, California State University, Sacramento

Abstract: Although rare, datasets examining both gene and protein expression can increase understanding of organisms’ responses to environmental stressors. Previous work has identified physiological differences in intertidal Mytilus californianus mussels living in unique temperature habitats; however, the gene and protein expression patterns driving these physiological differences have not been investigated. The objectives of this study were 1) to determine whether RNA and protein expression follow similar patterns in M. californianus, 2) to identify genes and proteins that change expression across different field and lab treatments, and 3) to identify genes and proteins whose expression correlates with physiological data. To achieve these aims, we used bioinformatics techniques to analyze RNA-sequencing and proteomics data for ~1500 genes obtained from 49 M. californianus individuals exposed to 5 intertidal and lab conditions that vary in environmental stressors such as temperature. Genes with significantly different RNA and protein expression between treatments were identified using the software packages DESeq2 and EdgeR to address objectives 1 and 2, and the package WGCNA was used to address objective 3. For objective 1, we found that many genes that are differentially expressed at the RNA level are not differentially expressed at the protein level (and vice versa). For objective 2, results indicate that genes related to cilia, motility, and protein folding show differential expression between treatments. No common functions were found in differentially expressed proteins. For objective 3, we found that nucleic acid and ion binding genes and protein folding and proteolysis proteins have expression patterns that correlate with physiological traits such as catalase enzyme activity. Given the unique expression patterns of RNA vs. protein, we conclude that post-transcriptional and/or post-translational processes should be considered when examining M. californianus’ response to environmental differences. In addition, we hypothesize that differential expression of cilia genes are related to a differential need for transporting fluid within M. californianus’ respiratory cavity. Overall, these results provide insight into the genes contributing to physiological differences in M. californianus and can enhance understanding of intertidal invertebrates’ responses to climate change. A CSU Sacramento SURE award and grants to WD provided financial support for this project.

Poster #: 209
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Microbiology, transcriptional fusions
Project Title: The ExoS/ChvI two-component pathway regulates the expression of small RNAs in the nitrogen fixing bacterium Sinorhizobium meliloti
Author List:
Ortega, Jesus; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Chen, Esther; Biological Science, California State University, Fullerton

Abstract: The gram-negative alpha-proteobacterium Sinorhizobium meliloti can inhabit nodules within the roots of Leguminosae plants. S. meliloti provides plant hosts with accessible nitrogen in return for carbon compounds. This interaction has agricultural significance since it can increase crop productivity without environmentally harmful nitrogen-rich fertilizers. S. meliloti employs the ExoS/ChvI two-component system to regulate expression of genes involved in exopolysaccharide production, motility, nutrient utilization, and cell envelope integrity. The histidine kinase ExoS phosphorylates its cognate response regulator ChvI, which then binds to promoter regions and regulates gene expression. Recent work in our laboratory identified small RNA (sRNA) genes as potential ChvI targets. sRNAs often regulate gene expression post-transcriptionally by interacting with mRNAs of target genes. To examine whether ChvI regulates the expression of sRNAs, the upstream region of the SmelA020 and SmelC063 sRNA genes was fused to a β-glucuronidase (GUS) reporter gene. Each construct was integrated into the genome of chvI partial loss-of-function (LOF), chvI gain-of-function (GOF), and wild-type (WT) S. meliloti strains, and the GUS activity of these strains was compared. We found that the promoter-GUS fusion of SmelC063 showed an increase in GUS expression in the LOF chvI mutant compared to WT, but no significant change in the GOF chvI mutant compared to WT. Meanwhile, SmelA020 showed an increase in GUS activity in both chvI mutants compared to WT. These data suggest that ChvI regulates the expression of both SmelC063 and SmelA020. However, since a reciprocal change in GUS expression between the chvI LOF and GOF mutants was not observed for both sRNA genes, additional regulators may influence SmelC063 and SmelA020 expression. Studying sRNA regulation in S. meliloti is important because it may provide insight into how related alpha-proteobacteria regulate gene expression to occupy diverse environments. This work was supported by the CSU Fullerton MARC Program grant (NIH grant 2T34GM008612-23) and by a grant from the CSU Special Fund for Research, Scholarship, and Creative Activity (2018-19).

Poster #: 210
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, stem cells, stress
Project Title: Effect of chronic intermittent stressors on intestinal stem cells in the fruit fly Drosophila melanogaster
Author List:
Castaneda, Jack; Undergraduate, Biology, California State University, Northridge, Presenting Author
Yera, Maria; Undergraduate, Biology, California State University, Northridge, Presenting Author
Khanbabaei, Armen; Graduate, Biology, California State University, Northridge
Rajabi, Alex; Undergraduate, Biology, California State University, Northridge
Khimji, Sailesh; Graduate, Biology, California State University, Northridge
Bhsara, Antonio; Graduate, Biology, California State University, Northridge
Loza-Coll, Mariano; Biology, California State University, Northridge

Abstract: The health of our organs relies on the proper function of resident adult stem cells, whose function is to replace tissue that was lost to injury, disease or normal histological turnover. Given the widely recognized negative effects that chronic stressors can have on our health, we decided to explore whether chronic but intermittent stressors may regulate the function of adult stem cells in vivo. To this end, we use the intestinal stem cells (ISCs) in the posterior midgut of the fruit fly Drosophila melanogaster as our experimental model system. Here we report our findings related to three different experimental paradigms of chronic but intermittent stress: i) ethanol intoxications (10min/day, for 12-14 days); ii) heat shocks (5min at 37C/day, for 12 days) and iii) a pseudo-random combination of thermal shocks (heat or cold, 5min at 4C or 37C/day, respectively), ethanol intoxications (10min/day) and random sleep interruptions (30min every 2-3 hours/night). Using a combination of cell type-specific markers and fluorescence microscopy, we quantified and compared the number, morphology and replicative potential of ISCs and their progeny in stressed flies and controls. There was no significant change on the relative proportion of intestinal progenitors in stressed flies; however, we did observe that chronic ethanol intoxications sporadically caused progenitor nuclei to become slightly larger and rounder than those in control flies. Since those morphological changes could reflect changes to the progenitor identity of these cells, we sought to determine if chronic intermittent stressors could inhibit the response of progenitor cells to over-expression of Unpaired (Upd), a cytokine that triggers the Jak/STAT pathway in Drosophila and serves as a potent proliferative stimulus to ISCs. There was a subtle but reproducible reduction in the number of progenitors that accumulate in response to Upd expression, indicating that chronic stressors impair their ability to respond to mitogenic stimulation in vivo. Further studies using this experimental approach will allow us to exploit the advantages of Drosophila as a model system to investigate the genetic and molecular bases of this potentially novel link between environmental stressors and organ health.

Poster #: 211
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Microsatellite Repeats, Repeat Expansion Disorders, Saccharomyces cerevisiae
Project Title: Analyzing the Chromosomal Rearrangements Caused by Myotonic Dystrophy Type 2 CCTG/CAGG DNA Repeats in Budding Yeast
Author List:
Arguello, Berenice; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Kim, Jane; Biological Sciences, California State University San Marcos, Presenting Author

Abstract: Approximately 30 neurological, neurodegenerative, and developmental diseases are caused by expanded microsatellite repeats. Microsatellites are naturally occurring repetitive DNA sequences within the genome that tend to be one to ten base pairs in length. Friedreich’s Ataxia is a neurodegenerative disorder caused by expanded GAA trinucleotide repeats. Using a budding yeast experimental system, it was previously shown that long GAA repeat tracts cause chromosomal breakage and gross chromosomal rearrangements (GCRs). Myotonic Dystrophy 2 is a distinct repeat expansion disorder caused by long CCTG tetranucleotide repeats. CCTG repeats are not well-characterized, and it is not known whether long CCTG repeats also cause GCRs. We constructed budding yeast strains with CCTG and reverse complementary CAGG repeats to investigate their effect on GCR rates. These DNA repeats were integrated into the LYS2 locus on chromosome V, and then verified using polymerase chain reaction (PCR) and Sanger sequencing. We identified one strain with pre-mutation size repeats, (CCTG)61-int, which had two TTTC interruptions. It is not understood how this occurred, but previous studies have shown that interruptions within microsatellites stabilize the region. Two control length strains of (CCTG)7 and (CAGG)5 were also verified. We performed GCR fluctuation assays to determine the arm loss (GCR) rate among verified strains. (CCTG)7 and (CAGG)4 strains showed elevated rates of arm loss compared to the control (GAA)5 strain. The (CCTG)61-int and (CCTG)7 strains showed similar rates of arm loss (4.98E-08 compared to 5.94E-08 GCRs per cell division and overlapping 95% confidence intervals), suggesting that the interruptions stabilized the longer CCTG repeats. These results demonstrate the feasibility of techniques that can facilitate research into the relationship between microsatellite expansion size and arm loss rate within Myotonic Dystrophy type 2. Strains with longer uninterrupted repeats need to be constructed, and additional analysis with Nanopore sequencing will reveal the types of chromosomal rearrangements caused by CCTG repeats. This research project was done under the guidance of Dr. Jane Kim and was financially supported by the NSF REU program (Award Number 1852189) and the NIH SCORE Grant (SC3GM127198).

Poster #: 212
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Autophagy, CURES, Cell Biology
Project Title: Cellular Resilience During Prolonged Nutrient Deprivation Requires Functional Mitochondria and Autophagy Pathway
Author List:
Mackie, Ariah; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Pope, Amanda; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Gomez, James; Undergraduate, Biological Sciences, Humboldt State University
Silavong, Austin; Undergraduate, Biological Sciences, Humboldt State University
Petrak, Jessica; Undergraduate, Biological Sciences, Humboldt State University
Lenihan, Jake; Undergraduate, Biological Sciences, Humboldt State University
Steele, John; Biological Sciences, Humboldt State University

Abstract: How human cells access resources in response to cellular stress, such as that associated with loss of access to key nutrients, during excessive misfolded protein load, or in response to changes in lysosomal function is a critical question in understanding human disease biology. This study sought to establish a model system to study the viability of cultured healthy human cells in response to prolonged nutrient deprivation, with the goal of understanding which cellular processes are required for survival, and how they might fail in the context of disease.
We treated cultured healthy human embryonic kidney (HEK293t) cells or healthy human neurons under complete or partial nutrient deprivation conditions for 72 hours. We then studied the viability of these cells using multiple assays of cellular and mitochondrial function. We found that cells subjected to deprivation of media components typically found in fetal bovine serum (e.g. lipoproteins, growth factors, etc) ceased dividing and demonstrated an ~10-fold increase in metabolic rate on a cell-per-cell basis (reduction of resazurin to resorufin), by comparison to cells grown in normal growth medium, or deprivation medium that was supplemented with fetal bovine serum. Since we observed similar outcomes with HEK293t cells and human neurons, we focused our study on HEK293t cells.
Next, we treated HEK293t cells with regulators of mitochondrial function, autophagy, cholesterol biosynthesis, protein synthesis, or the ubiquitin proteasome; or we supplemented media components (e.g. cholesterol-lipoprotein complex, bovine serum albumin, squalene, amino acids, etc) in our BIOL 410 Cell Biology laboratory course and are working to independently verify key results. To date, we have performed dose-response experiments with inhibitors of mitochondrial or autophagy function, finding that HEK293t cells in nutrient-deprived conditions are more sensitive to mitochondrial perturbations or loss of the autophagy pathway (e.g. required 5-fold lower concentrations to induce toxicity) by comparison to cells in normal growth conditions treated with the same drugs. Interestingly, cells in prolonged nutrient deprivation conditions were less sensitive to inhibition of the ubiquitin proteasome or protein synthesis, by comparison to cells in normal growth conditions. Our evidence demonstrates that both the autophagy pathway and functional mitochondria are critical to survival of healthy human cells during prolonged nutrient deprivation.

Poster #: 213
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: RNA editing, RNA-SEQ, Chloroplasts
Project Title: Extensive RNA Association with Editing Factor RIP9 Complexes Reveal a Clear Separation between RNA Processing and Translation in Chloroplast
Author List:
Santibanez, Paola; Graduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Ngo, Vihn; Undergraduate, Biology, California State University, Los Angeles, Presenting Author
Hayes, Michael; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: C-to-U RNA editing is a post-transcriptional modification that occurs in chloroplasts and mitochondria of most land plants. RNA editing can recode gene sequences to initiate translation, terminate translation, or repair nucleotides encoded in the plastid genome at the transcript level. RNA editing plays an essential role for organelle function in plants. Discovering the minimal RNA and protein components of RNA editing complexes could lead to the improvement of crops through application of engineered nucleotide changes. Currently, 27 distinct editing sites have been published in chloroplast transcripts of Zea mays. RNA editing complexes (editosomes) in vascular plants require several members of different protein families: PPRs, RIPs, ORRMs, OZs, and DEAH-box helicases. Using size exclusion chromatography (SEC), we determined that RNA editosomes were large, stable, and co-chromatographed with native RNAs. SEC fractions with editing activity were estimated to represent larger native complexes than previous editosome models. We hypothesize that RNA factors might also contribute to the mass and conformation of native editosomes since editing complexes were shown to disassemble after RNase A treatment. We have demonstrated that RNA editing activity for rpoC2 and rps14 transcripts survives immunoprecipitation using an antibody for editing factor RIP9. High-throughput sequencing and application of bioinformatics software (featureCounts, RSEM, IGV, etc.) were used to characterize the general distribution of transcripts within the RIP9 complexes. We have identified two putative editing sites with high percentage levels of C-to-U conversion previously unreported. From RIP-Seq analysis of RIP9 complexes, we observed that RIP9 complexes showed enrichment for mRNAs, tRNAs, and introns sequences. These findings suggest that editing activity precedes several intron splicing steps of mRNA processing. A high enrichment of unprocessed tRNAs also implicates the participation of RIP9 complexes in multiple RNA maturation processes. Moreover, a notable disenrichment of ribosomal RNAs (rRNAs) supports the idea that RIP9 complexes are not associated with polyribosomes. Our data indicate that RIP9 immunoprecipitants may represent a distinct, large complex dedicated to RNA editing, splicing, and tRNA maturation physically separate from chloroplast ribosomes.

Poster #: 214
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: lanthanide, rare earth, methylogroph
Project Title: Isolation and characterization of lanthanide transport suppressor mutations in Methylobacterium extorquens AM1
Author List:
Randall, Kipchoge; Undergraduate, Biological Sciences, San José State University, Presenting Author
Singh, Damini; Undergraduate, Biological Sciences, San José State University, Presenting Author
Clippard, Erik; Graduate, Biological Sciences, San José State University
Cai, James; Undergraduate, Biological Sciences, San José State University
Lien, Nicholas ; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Biological Sciences, San José State University

Abstract: Methylobacterium extorquens is a model organism for the study of methylotrophic bacteria and is being engineered to sustainably recover lanthanide metals from discarded electronics. Recently it was discovered that methylotrophic bacteria use lanthanides as cofactors for methanol oxidation, furthering their environmental and industrial relevance. M. extorquens has two types of methanol dehydrogenase enzymes which oxidize methanol to formaldehyde; MxaF which requires calcium and XoxF which requires lanthanides. Regulation of these enzymes is not well understood. Our previous work identified a cluster of eight genes involved in lanthanide transport and use. Our working model suggests lanthanides are transported through the outer membrane via a TonB-dependent receptor (LutH) and into the cytoplasm through an ABC transport system comprised of LutA, E, and F. Here we show that loss of lutA reduces growth by 88% suggesting important but nonessential role in lanthanide transport. Loss of either lutE or lutF eliminates growth in methanol lanthanum medium, however after 80 hr, suppressor mutations arise. We predict that these suppressor mutations result in expression of mxaF which can oxidize methanol in the absence of lanthanides. To determine if these suppressors are due to loss of gene function, 250 transposon mutants were selected that rescued growth of a lutE mutant on methanol lanthanum medium. Two classes of mutants were isolated; those that had wild-type growth and those that allowed growth but at a reduced rate. Insertion locations were determined using nested PCR and sequence analysis. Suppressors that fully restored growth mapped to lutH, the TonB-dependent receptor. Transcriptional reporter fusions determined that mxaF is expressed when the outer membrane transporter is disrupted suggesting that lanthanide transport into the periplasm is enough to repress expression of the calcium-dependent methanol dehydrogenase. Additional transposons insertions mapped to several transcriptional regulators suggesting they may be involved in repression of mxaF. Mutations in the regulator genes are being reconstructed and growth and expression phenotypes will be tested to determine if the regulator genes are required for repression of mxaF. Results from these studies will increase our knowledge of lanthanide homeostasis which will facilitate strain engineering for biologically-mediated lanthanide recovery. This research was funded by NSF RUMBA and NIH RISE grants.

Poster #: 215
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRP, DNA binding, RNA polymerase recruitment
Project Title: V183 of CRP, a DNA-binding motif residue, has indirect yet high impact on RNA polymerase recruitment
Author List:
Montiel, Christian ; Graduate, Biology, California State University, Fresno, Presenting Author
Rea, Amanda; Graduate, Biology, California State University, Fresno, Presenting Author
Youn, Hwan; Biology, California State University, Fresno

Abstract: Escherichia coli cAMP receptor protein (CRP) requires DNA binding and RNA polymerase recruitment to serve as a transcriptional activator. The CRP F-helix is the recognition helix of the helix-turn-helix DNA-binding motif. On the other hand, CRP possesses surface patches termed activating regions (AR1, AR2, and AR3) which interact with and recruit RNA polymerase. The F-helix and activating regions are separate both functionally and spatially. However, our recent data demonstrated that substitutions at an F-helix residue (Gly184) significantly influenced the transcriptional activation activity by indirectly altering RNA polymerase recruitment. Here, we show another evidence that substitutions at a different F-helix residue (Val183) also influence the transcriptional activation activity and RNA polymerase recruitment. We created and characterized a series of site-directed mutants (V183A, V183I, V183M, and V183F) at position 183. The rank order for the in vivo transcriptional activation activity was V183A > wild type > V183I > V183M = V183F. However, there was no apparent difference in DNA binding. These results suggest that smaller amino acids at position 183 are more conducive to efficient RNA polymerase recruitment. A structural analysis of CRP revealed that a large amino acid at position 183 might restrict the Arg169 and/or Ile172 residues via steric hindrance. Interestingly, Arg169 and Ile172 are near the AR1 residues of CRP, thus directly affecting RNA polymerase recruitment. Our current hypothesis is that a substitution at position 183 affects the position of Arg169 or Ile172 which in turn repositions the AR1 residues, thereby governing RNA polymerase recruitment. We are currently changing the 169 or 172 residue in the above various V183 mutant background to test if an amino acid size compensation can be found between 183 and 169 (or 172) residues. In sum, our data demonstrate that in CRP DNA binding and RNA polymerase recruitment are more intertwined than previously thought.

Poster #: 216
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: sea slugs, barcoding, Maldives
Project Title: Identifying marine snails and slugs from the Maldives with COI barcoding
Author List:
Petrula, Macy; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: The Maldives is a country consisting of over one thousand islands located in the Indian Ocean. This island ecosystem is home to an incredible but understudied diversity of organisms, among which are marine snails and slugs (Gastropoda). Two major lineages are the Vetigastropoda, commonly known as abalones, turban snails, top shells, keyhole limpets, and the Heterobranchia, including nudibranchs, sea slugs, sea hares, bubble snails. In April 2019, over 50 different species of heterobranchs and vetigastropods were collected at the Maldives and brought back to the Giribet lab at Harvard University. At the lab, we barcoded samples from each organism to confirm their identification and to add genetic data to a checklist of photographed specimens. The particular gene we sequenced was the mitochondrial gene cytochrome c oxidase subunit I (COI), and we will deposit the genetic information in the public online database Genbank. This work will help us to recognize cryptic diversity, identify new species, and assist researchers in future studies to better understand, for example, the geographical patterns of population structure and connectivity among these organisms.

Poster #: 217
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: C. elegans, Sleep, Neuroscience
Project Title: Sleep alters the physical architecture of sensory synapses during memory consolidation in C. elegans
Author List:
Farah, Fatima; Graduate, Biological Sciences, San José State University, Presenting Author
Bokka, Anirudh ; Undergraduate, Biological Sciences, San José State University, Presenting Author
Baradwaj, Anjana ; Staff, Biological Sciences, San José State University
Varshney, Aruna; Staff, Biological Sciences, San José State University
Chang, Eric; Undergraduate, Biological Sciences, San José State University
Li, Joy; Graduate, Biological Sciences, San José State University
Benedetti, Kelli ; Graduate, Cell and Tissue Biology, UCSF
Muñoz-Lobato, Fernando ; Postdoc, Cell and Tissue Biology, UCSF
L’Etoile, Noelle ; UCSF
VanHoven, Miri ; Biological Sciences, San José State University

Abstract: Sleep is critical for the consolidation of memory. However, much is unknown about the effects of sleep on specific neuronal connections, though synaptic modulation is thought to underlie learning and memory. We have focused our studies on the AWC olfactory circuit in the nematode C. elegans, which mediates attraction to the attractive odor butanone, which is secreted from a bacterial food source. We hypothesized that we could visualize synaptic changes in animals that acquired a sleep-dependent long-term memory during odor training. Specifically, animals can learn to ignore their innate attraction to butanone if they are trained for three cycles with butanone in the absence of food, then allowed to sleep for two hours. Animals retain this lack of attraction 14 hours later, indicating long-term memory formation in adults that are only approximately 120-hour old. Using the fluorescent split GFP-based trans-synaptic marker Neuroligin 1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP), we tested whether synapses between the AWC olfactory neurons and their primary postsynaptic partners, the AIY interneurons, underwent changes in NLG-1 GRASP fluorescence intensity. We found that in animals that would normally acquire a sleep-dependent memory, those that were trained with butanone in the absence of food, then allowed to sleep on food for two hours, and recovered on food for 14 more hours, AWC-AIY synapses were significantly reduced. However, in sleep-deprived animals that would normally fail to retain a memory, those that were trained with butanone in the absence of food, then deprived of sleep off food for two hours, and recovered on food for 14 more hours, AWC-AIY synapses were not reduced. The reduction in synapses was also not observed in animals trained with a control buffer. The reduction in AWC-AIY synapses could explain the reduced response to butanone at the end of the assay, as these connections have previously been shown to be required for proper chemotaxis to butanone. We propose that this synaptic reduction is the physical trace of the long-term memory, and contribute to the observed change in behavior. By discovering how memory is consolidated during sleep at the level of specific neuronal connections, we hope to set the stage to understand the basic cellular and molecular mechanisms of sleep, and develop more effective treatments for diseases in which memory consolidation is altered, such as Alzheimer’s disease and dementia.

Poster #: 218
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sexual differentiation, Apoptosis, Gene expression
Project Title: Sex Differences in Apoptosis-related Gene Expression in the Developing Mouse Cortex and Hippocampus
Author List:
Behrend, Anna; Graduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Meza, Edward; Undergraduate, Department of Biological Sciences, California State University, Long Beach
Duenas, Nikolas; Undergraduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Department of Biological Sciences, California State University, Long Beach

Abstract: Sexual differentiation of the brain is the developmental process by which discrete neural circuits underlying sexually dimorphic behaviors form. Sexual differentiation of non-gonadal tissues, including the brain, is largely influenced by perinatal rises in testosterone secreted by the developing testes via the activation of androgen and/or estrogen receptors. The cerebral cortex and hippocampus are known to play an important role in cognitive functions and social behaviors, many of which are sexually dimorphic. However, the identity of the molecular elements responsible for the functional differences between the sexes in these two brain regions remains ambiguous. We have recently found that during this critical developmental period at postnatal (PN) days 0 and 7, the male and female mouse cortex and hippocampus differentially express the Rbm48 gene. The neural function of the Rbm48 gene-encoded protein is unclear, but RBM48 knockdown increases apoptosis in human cancer cell lines. Since sex differences in cell number in several dimorphic brain nuclei is created by apoptosis, we hypothesize that androgen-regulated, differential expression of Rbm48 between the sexes might control brain sexual differentiation via regulation of apoptosis in a sex-specific manner. To test our hypothesis, we used RT-qPCR to measure mRNA levels of three major apoptotic genes¬ – Bax, Bcl2, and Bcl2l1 – in the developing mouse cortex and hippocampus at PN0, PN7, PN14 and PN21 (N=8 per sex per age). Bcl2 and Bcl2l1 are anti-apoptotic genes while Bax is a pro-apoptotic gene. We found a sex difference in expression of Bax (p=0.022), not Bcl2 or Bcl2l1, with higher levels in males than females at PN0 and PN7. Regarding the effect of age, a significant increase in levels of Bcl2 and Bcl2l1 mRNA was observed from PN7 to PN14 (p=0.001 and <0.001). In contrast, Bax expression was reduced at PN21 as compared to PN7 (p=0.001). Our data demonstrate that increased Bax expression in the male cortex/hippocampus is closely associated with a male-biased decrease in Rbm48 levels, suggesting that Bax might be the downstream target of Rbm48, which induces sex differences in apoptosis in response to perinatal testosterone. To further investigate this, we will be conducting immunohistochemistry to detect the cells expressing activated caspase-3, the marker and final executor of cell apoptosis, in the developing mouse cortex and hippocampus.

Poster #: 219
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: bacteria, long-term culture, evolution
Project Title: Identifying Genes That Play a Role in Long-Term Stationary Phase in Escherichia coli
Author List:
Martinez, Erik; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Gutierrez, Raul; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Kram, Karin; Biology, California State University, Dominguez Hills

Abstract: Escherichia coli can adapt to live in an environment without any additional nutrients for long periods of time. When E. coli grows in an environment where nutrients are scarce, they enter long term stationary phase (LTSP). We can use this phase in the lab as a proxy for a more natural environment and observe how cells adapt to these conditions. It is not yet known which genes may be important for survival or adaptation in this phase. In order to identify nonessential genes that play a role in survival during LTSP, we competed cells in the KEIO collection with wild type (WT) E. coli cells. The KEIO collection is a set of E. coli strains, each with a single-gene deletion of a nonessential gene, representing all non-essential genes – a total of 3985 strains. We performed competitions with WT cells during ten days of incubation, into LTSP. We observed cell growth after one, five, and ten days of incubation. After screening the entire collection, we identified 101 gene deletions that affected the cell’s ability to compete with WT. 29 strains with deletions had an advantage over WT cells, whereas the remaining 72 strains had a disadvantage when competed against the WT. While some of the identified strains did not show a difference in growth once competitions were in tubes versus the 96-well plates used for screening, we were able to confirm that some of the strains did have a difference in growth compared to WT cells. Several strains, including those missing sucC (encodes enzyme involved in central metabolism), dgkA (encodes an enzyme involved in lipid biosynthesis), mprA (produces a regulator involved in antibiotic resistance and efflux), cpxA (encodes a sensor kinase that responds to cell envelope stress) or hns (encodes a gene encoding that regulates stress-related genes by condensing DNA) were outcompeted by WT cells during LTSP, indicating that these genes are essential for survival in long-term cultures. Overall, we have identified several genes that are important for survival in LTSP, and can begin to determine why these genes play a role in survival into LTSP by determining their function in long-term cultures.

Poster #: 220
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR, Hematopoietic Stem Cell, Histone deactylase inhibitors
Project Title: Histone Deacetylase Inhibitors enhance CRISPR-Cas9 cutting efficiency in both lineages of the hematopoietic stem cell
Author List:
Björnson, Ymer M.; Graduate, Biological Sciences, San José State University, Presenting Author
Castaneda, Guadalupe; Undergraduate, Biological Sciences, San José State University, Presenting Author
Huang, Codey Y.; Undergraduate, Biological Sciences, San José State University
Yamamoto, Emiko; Undergraduate, Biological Sciences, San José State University
Johnston, Jennifer M.; Biological Sciences, San José State University

Abstract: The CRISPR/Cas9 technology is a prominent genome-editing tool as an adaptable nuclease capable of producing a double strand break at almost any genomic loci. However, the modification of hematopoietic stem and progenitor cells (HSPCs) via the homology directed repair (HDR) pathway is still inefficient. We hypothesize that histone deacetylase inhibitors (HDACi), such as Valproic Acid (VPA) and Sodium Butyrate (NaB), could enhance HDR efficiency by increasing the accessibility of the genome-editing machinery by preventing DNA from wrapping tightly around histones. To address the potential utilization of HDACi therapeutically, we began by assessing the effect of VPA and NaB on cell viability. We previously demonstrated no statistically significant effect on cell growth or viability in a myeloid-erythroid leukemic cell line (K562 cells) at concentrations as high as 50mM. However, K562 cells only represent one lineage of the HSPC progeny. Therefore, we treated an immortalized T lymphocyte cell line (Jurkat cells), representative of the lymphoid lineage, with either VPA or NaB. No effect on the growth pattern or viability of Jurkat cells was evident at concentrations as high as 5mM VPA (p = 0.2310) and NaB (p = 0.170). However, Jurkat cells appear to be more susceptible to both VPA and NaB as compared to K562 cells in that 50mM did significantly effect the growth pattern of Jurkat cells (p = 0.0008 for VPA and p = 0.0006 For NaB). Therefore, in order to assess the availability of the CRISPR-Cas9 complex to the genome of Jurkat cells, we treated cells with a lower concentration (0.005mM) of VPA or NaB 24 hours prior to delivery of a Cas9 expression plasmid containing a guide RNA specific to the vWF locus. The cutting efficiency of six different guide RNAs were assessed by a TIDE analysis in which insertions and/or deletions are identified as evidence of Cas9 directed cleavage. An increase in cutting efficiency for several guide RNAs was observed in both VPA and NaB treated cells. Specifically, a 5-fold enhancement was observed upon treatment with NaB with the -36 vWF guide RNA. In addition, a comparable enhancement was observed with the +29 vWF guide with a 5.20% cutting efficiency before treatment and a 23% efficiency following treatment with NaB. The increase in CRISPR cutting efficiency observed in Jurkat cells confirms an enhancement seen in the myeloid K562 cell line treated similarly. Currently, a second locus (the RhD locus) is being assessed for an enhancement in CRISPR cutting efficiency in the lymphoid Jurkat cell. If the same trend follows, one can conclude that treatment with HDACi can be expanded to virtually any genome editing experiment utilizing the CRISPR-Cas9 system to enhance cutting and thus therapeutic gene editing.

Poster #: 221
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Androgen receptor, Social behavior, Sexual differentiation
Project Title: Investigating the Role of Androgen Receptor in Social Behavior and its Underlying Neural Substrates in Mice
Author List:
Leung, Darren; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Lee, Junho; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Wong-Hazehara, Kylie; Undergraduate, Biological Sciences, California State University, Long Beach
Padilla, David; Undergraduate, Biological Sciences, California State University, Long Beach
Chavarria, Roxana; Undergraduate, Biological Sciences, California State University, Long Beach
Zaldana, Aileen; Undergraduate, Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Biological Sciences, California State University, Long Beach

Abstract: Sexual differentiation of the brain is the process of development by which discrete neural circuits underlying sexually dimorphic behaviors form. The perinatal rise in testosterone plays a critical role in the organization of sexually dimorphic social behaviors, such as copulation and aggression, but the role of androgen receptor (AR) in the process is less clear. We hypothesize that perinatal activation of AR might masculinize the neural circuits underlying social behaviors, resulting in sex differences in behavior. To test our hypothesis, we conducted tests of sociability and social novelty preference in adult male mice carrying the testicular feminization mutation (Tfm, n=3) as well as their wild-type (WT) male (n=10) and female (n=9) littermates. Sociability was assessed via time a subject mouse spent investigating a wire-mesh cup containing an unfamiliar intact female conspecific compared to an empty cup. For social novelty, a subject mouse was similarly observed for time spent with a confined novel mouse versus a familiar one. When intact females were used as the social stimuli, a significant difference in sociability was noted among the three groups (p= 0.029) with WT males showing a stronger preference than females. Although the difference was not significant, Tfm males displayed a trend of female-like sociability. Additionally, there was no significant difference in social novelty among WT and Tfm mice (p= 0.169), but a trend of spending more time with the novel social stimulus than the familiar one was observed in WT males than WT females and Tfm males. Likely due to the small sample size (n=3), we found an insignificant trend of female-like sociability and social novelty behaviors in Tfm males. In summary, our data support that social behavior is sexually dimorphic, and in part, AR potentially influences sex differences in behaviors and possibly in the underlying neural circuits. In the future studies, we will use immunochemical detection of c-Fos to detect behavior-evoked activations in the brain regions responsible for sex differences in social behavior.

Poster #: 222
Campus: CSU East Bay
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Toxoplasma gondii, Developmental regulation, starch metabolism
Project Title: Contribution of Developmentally Regulated Metabolic Enzymes to Stage Conversion During Toxoplasma Pathogenesis
Author List:
Quach, Emily ; Graduate, Biology, California State University, East Bay, Presenting Author
Moline, Ariel; Undergraduate, Biology, California State University, East Bay, Presenting Author
Guiton, Pascale; Biology, California State University, East Bay

Abstract: Toxoplasma gondii is a ubiquitous protozoan responsible for toxoplasmosis in humans. A critical stage in Toxoplasma pathogenesis involves the interconversion of two asexual forms: tachyzoites and bradyzoites. This differentiation is crucial for dissemination, immune evasion, and persistence in the host. Yet, we know very little about the mechanisms underlying this pathogenic process. Comparative transcriptomic and proteomic analyses revealed that Toxoplasma possesses stage-specific metabolic requirements that enable the parasite to survive and to be efficiently transmitted between hosts. Electron micrographs of bradyzoites revealed the presence of numerous starch granules that are largely absent in their tachyzoite counterparts. These granules are rich in amylopectin and were implicated in tissue cyst formation in chronically infected individuals. Specifically, how amylopectin accumulates and is metabolized in bradyzoites is not well understood. Several developmentally-regulated metabolic enzymes have been associated with differentiation, including enolase 1 and lactate dehydrogenase 2; however, many glycolytic enzymes remain understudied. We postulate that the loss of these stage-specific enzymes will hinder Toxoplasma differentiation by disrupting amylopectin metabolism.
Glucosephosphate mutase (gpm1) is one of 234 developmentally regulated enzymes in Toxoplasma. This glycolytic enzyme has a 6-fold upregulation in bradyzoites relative to tachyzoites in vivo. GPM1 catalyzes the interconversion of glucose-6-phosphate to glucose-1-phosphate during gluconeogenesis. To understand how GPM1 contributes to amylopectin accumulation in Toxoplasma, we used the CRISPR/Cas9 gene-editing system to disrupt gpm1. This mutation did not affect tachyzoite growth in vitro in the presence of glucose. In glucose-free media; however, the mutant displayed a significantly higher replication rate compared to wildtype parasite. The mutant did not lose its ability to produce cysts in vitro. Toxoplasma possesses a homolog of gpm1, gpm2. We reasoned that GPM2 may be compensating for the loss of GPM1. We are currently investigating the transcriptional regulation on gpm2 in the gpm1 mutant and in wildtype using quantitative RT-PCR. These enzymes may represent novel targets for much-needed therapeutics against this incurable disease, should they be critical for Toxoplasma differentiation.
Funding: CSUPERB New Investigator grant, CSUEB Faculty Support Grant and CSR Supply grant

Poster #: 223
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: gene regulation, promoter, bioinformatics
Project Title: Characterization of the Mitotic Arrest Deficient-like 1 Binding Protein Proximal Promoter
Author List:
Billoo, Rabia; Undergraduate, Biology, California State University, Northridge, Presenting Author
Salazar, Jonathan; Graduate, Biology, California State University, Northridge, Presenting Author
Malone, Cindy; Biology, California State University, Northridge

Abstract: Mitotic Arrest Deficient-like 1 Binding Protein is a gene that encodes the MAD2L1-binding protein. MAD2L1BP is vital for proper cellular development and may be a component of the spindle-assembly checkpoint that prevents the initiation of anaphase until all chromosomes are correctly aligned during metaphase. Accurate regulation of the MAD2L1BP gene is essential for proper cellular development. Promoter characterization will identify the regulatory elements driving transcription of MAD2L1BP. The putative promoter region was isolated, PCR amplified and sub-cloned into a luciferase reporter vector. Sequential 5’ deletions were constructed based on bioinformatics data identifying consensus transcription factor binding sites. The deletion constructs were transiently transfected into HEK293T cells and luciferase assays were performed. We identified the proximal promoter region of the MAD2L1BP gene by the high promoter activity found within approximately 500 base pairs of the Genbank-assigned transcription start site. Within this region, our sequence analysis found c-Ets-1, SP-1, and XBP-1 consensus transcription factor binding sites by utilizing several web-based algorithms. We determined that these transcription factor consensus sequences were evolutionary conserved across multiple species in this defined MAD2L1BP gene proximal promoter region via multiple sequence alignment analysis. We performed PCR by exclusion to eliminate each of these evolutionarily conserved consensus transcription factor binding sites, alone and in combination, within our defined MAD2L1BP gene proximal promoter region. Transient transfections of these deletion constructs is currently underway. We hypothesize that at least one of the conserved transcription factor binding sites will be important for activity of the defined MAD2L1BP gene proximal promoter. Future experiments using chromatin immunoprecipitation and/or EMSA will confirm that the transcription factors important for activity do in fact bind to the consensus sites. Ultimately, characterization of this gene’s promoter region and the regulatory elements driving transcription will help us understand how the MAD2L1BP gene can become dysregulated.
This work was supported by NIH BUILD PODER 8TL4GM118977-02, CIRM Bridges TB-01183 Educational Enhancement Funds.

Poster #: 224
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Stem Cells, Neurodegeneration, Gene expression
Project Title: Characterizing gene expression patterns in human induced pluripotent stem cell derived adhered and neural sphere culture systems.
Author List:
Anthoney, Kyle; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Schoeffler, Jamie; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Brown, Danielle; Undergraduate, Biological Sciences, Humboldt State University
Nisson, Haley; Undergraduate, Biological Sciences, Humboldt State University
Pope, Amanda; Undergraduate, Biological Sciences, Humboldt State University
Chung, Kevin; Undergraduate, Biological Sciences, Humboldt State University
Jones, Emma; Undergraduate, Biological Sciences, Humboldt State University
Quinn, Patrick; Undergraduate, Biological Sciences, Humboldt State University
Coblentz, Azariah; Undergraduate, Chemistry, Humboldt State University
Steele, John; Biological Sciences, Humboldt State University

Abstract: The goal of our research program is to model and understand the molecular pathogenesis of both common (e.g. Alzheimer’s disease) and rare (e.g. progressive supranuclear palsy) neurodegenerative diseases using cultured human neural cell types that are affected by those diseases. In order to model these diseases, we study neural cell types made from patient-derived human induced pluripotent stem cells (iPSCs). One major hurdle in the study of human neurodegeneration is that monolayer (2-D) culture systems do not accurately represent the complex 3-dimensional (3-D) growth and cell types found in the human brain. Traditionally, we have used 2-D pure neuron cultures or pure astrocyte cultures, or co-culture methods, to study the individual contributions of these cell types to disease processes. However, these methods are negatively impacted by very different medium and growth conditions of each cell type, which typically are cell-type exclusive (e.g. reduced viability of both cell types in co-cultures).
Recent studies suggest that 3-D neural sphere cultures more accurately recapitulate the natural environment of neurons and non-neuronal cell types, including cortical-like layering and 3-D interactions of multiple cell types, along with improved viability of these cells. Therefore, we sought to study the gene expression patterns of neural cell types from traditional 2-D vs 3-D neural sphere cultures. First, we developed a panel of probes to analyze gene expression profiles (40 genes differentially expressed in relevant cell types) for 2-D vs 3-D cultures for 2 independent cell lines, representing healthy controls or tauopathy disease. We analyzed these data for differences in expression between healthy and disease models, as well as differences from neural progenitor cells to differentiated (28 days differentiation) cell types. We further developed five different cell type specific fluorescent promoter-reporters (EEF1A1 – all cells; GFAP – astrocytes; CNP – oligodendrocytes; SYN1 – mature neurons; PRNP – neurons), which were stably integrated into the genomes of these cell lines at the AAVS1 safe harbor locus using CRISPR-based gene editing methods. We used these reporters to confirm expression in these cell types across the same differentiation period. Finally, we are treating healthy and disease cells with regulators of key pathways (e.g. autophagy, ubiquitin proteasome, etc) to understand how these pathways relate to cell viability in each model system.

Poster #: 225
Campus: San Francisco State University
Poster Category:
Keywords: Nitric Oxide, Heart loop Morphology, Chicken
Project Title: Nitric Oxide Signaling as a Regulator of Heart Loop Morphology in the Chicken Embryo
Author List:
Pereira, Ashley; Graduate, Cell and Molecular Biology, San Francisco State University, Presenting Author
Denetclaw, Wilfred; Cell and Molecular Biology, San Francisco State University

Abstract: As in all vertebrates, the first organ to develop is the heart and an early indicator of a properly forming heart is a dextral C-shaped heart loop. In the chicken embryo cardiogenesis begins in bilateral heart fields within the splanchnic mesoderm closely associated with the endoderm. Both are created by the migration of cardiac progenitors from the anterior third region of the primitive streak at gastrulation (HH3-4 stage embryos).The progenitors migrate lateral and anterior in the cranial mesoderm to be in parallel with the embryo midline and slightly anterior to Hensen’s node.There an oblong region of cardiac progenitors is formed creating the bilateral heart fields which consist of the first heart fields (FHF) and secondary heart fields (SHF). Endocardial tubes then form showing cellular cardiac differentiation and progression of the nascent heart that forms in an anterior-posterior axis in the embryo as a heart tube at HH10 stage embryo which loops at HH13-15. When calcium levels are elevated on the right side or blocked on the left side of Hensen’s Node, both result in 30% of embryos with situs inversus (reversal of the heart loop). Nitric oxide (NO) formation also depends on elevation of calcium and can promote cardiomyocyte proliferation. However the role of Nitric Oxide (NO) in regulation of heart looping is unknown. Our objective for this study was to investigate NO signaling effects on early heart tube looping as a regulated process of morphogenesis in the chicken embryo. We hypothesize NO involvement in cardiac looping may be by its ability to regulate cardiomyocyte proliferation and under absence of NO may cause a looping defect due to absence of sufficient cardiogenic cells. To investigate, acrylic beads soaked in L-NAME, a competitive inhibitor of nitric oxide synthase(NOS), or DETA-NONATE, an NO donor, was placed to the right side of Hensen’s Node to block or elevate NO signaling. Our findings show 80% abnormal heart tube formation with situs ambiguous, a range of morphological heart looping defects, when NO signaling is disrupted. These results suggests that the calcium elevation on the right side functions in the activation of NOS for NO production and signaling, possibly by its canonical pathway, to regulate in the normal heart loop. NO signaling in early embryo development is not well understood and it’s role in cardiomyocyte proliferation may also need to be studied to fully understand cardiogenesis. NSF DBI-1548297

Poster #: 226
Campus: San Diego State University
Poster Category: Other
Keywords: NADH, Coenzyme regeneration, Electrochemistry
Project Title: Facile and Selective Electrochemical Coenzyme Regeneration on Metal Nanoparticle Decorated Carbon Electrodes
Author List:
Williams, Nicholas ; Graduate, Department of Chemistry , San Diego State University, Presenting Author
Goodman, Ryan; Undergraduate, Chemistry, San Diego State University, Presenting Author
Hahn, Karley; Undergraduate, Chemistry, San Diego State University
Li , Zhida; Graduate, Chemistry, San Diego State University
Gu, Jing ; Chemistry, San Diego State University

Abstract: Various strategies exist to address the increasing demand for energy while considering climate issues, such as increased utilization of renewable energy sources, conversion of greenhouse gases to fuels, and increasing efficiency of pre-existing energy consuming processes. The latter two methodologies can be accomplished with cell free enzymatic cascade systems which can convert CO2 into useful value-added organic molecules. Body temperature cell free enzymatic systems could replace some traditional energy intensive processes which use very high temperature and pressure along with expensive catalysts, like the Formox process for formaldehyde production. Enzyme cascades require coenzymes as sacrificial reagents, but they tend to be very expensive and must be recycled to make such a process economically feasible for the industrial utilization. To further the field of coenzyme regeneration, this study focuses on the direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD) to its reduced form (NADH). Direct electrochemical reduction of NAD tends to produce the enzymatically inactive isomer of NADH or NAD dimers, as such several transition metal nanoparticles were screened as potential catalysts, which are expected to facilitate the concerted proton two-electron transfer to NAD+ to selectively produce the active NADH isomer. Ni(0) nanoparticles (NPs) were found to be the best candidate due to their favorable catalytic properties, facile synthesis, and low toxicity. The Ni(0) NPs were drop cast over conductive carbon fiber paper electrodes with loading densities from 0 mg to 1 mg per cm2. Using a potential as low as -0.8 V vs Ag/AgCl NAD+ was reduced to NADH with a faradaic efficiency of 6%. The faradaic efficiency was found to increase with decreasing catalyst loading. Selectivity and efficiency were monitored using nuclear magnetic resonance spectroscopy and UV-Vis spectroscopy respectively. The metal modified substrates were durable, usable for successive tests with little change when viewed by SEM imaging. Further work will entail on improving the efficiency and use a semiconductor photoelectrode for photoelectrochemical reduction of NAD into NADH. These studies were done in collaboration with Dr. Xiaowen Chen of the National Renewable Energy Lab at the biomass lab, with the thanks of SDSU and ARCS for funding.

Poster #: 227
Campus: CSU Sacramento
Poster Category: Other
Keywords: Rheology, Complex Materials, Food Science
Project Title: It’s All About the Mouthfeel – A Rheological Study of Complex Food Materials
Author List:
Carnahan, Christopher; Undergraduate, Physics, California State University, Sacramento, Presenting Author
Jensen, Mikkel; Physics, California State University, Sacramento
Morris, Eliza; Physics, California State University, Sacramento

Abstract: Rheology is the science of quantifying the physical characteristics of viscous fluids and elastic solids. It has been used in the development of high-tech materials such as medical implants and antifouling coatings on ships. More recently, as our country moves toward a more plant-based diet, there has been an increase use of rheology for analyzing the texture of meat or cheese replacements. The goal of this project was to obtain rheological data and generate a model to quantify the physical characteristics of two soft materials with properties similar to those used in the replacement industry. The first material, ketchup, which is an excellent room temperature stable model material for yogurts, was probed using a creep and recovery test. Using this test, we were able to determine the elastic and viscous properties in the form of recoverable and lost strain. Ketchup, as it turns out, can be modeled as a Bingham plastic. The Bingham plastic model describes materials that exhibit a linear stress vs strain rate curve once an initial “yield” stress has been reached. The second material, gelatin, is much stiffer, more like meat substitutes than ketchup or yogurt. We analyzed two different gelatin concentrations, measuring the difference in their storage moduli during gelation in a time sweep experiment, which provided a baseline for their stiffness in equilibrium. We then performed stress ramps, repeatedly shearing and releasing the material in a pattern similar to chewing. We found that a damped oscillation occurred when a stress was applied or released. We modeled the gelatin oscillation to an underdamped oscillation model, which we was then adapted to include an additional linear displacement, in order to account for the continued flow deformation of the sample as the stress is applied. This equation is derived from Maxwell’s model of oscillation. In the study of ketchup, we found that, following the Bingham plastic model, it displayed a yield strength of approximately 10Pa. For the gelatin, we found that the amplitude of the oscillations, for our higher concentration, increased with applied stress at a rate of ~.67% per Pa at 4C and 0.12% per Pa at 25C. Using these models we predict the elasticity, viscosity, and subsequently, texture of ketchup and gelatin. In the future this work will be expanded to a more generalized model that can be applied to other materials, such as artificial yogurts and meats.

Poster #: 228
Campus: CSU Sacramento
Poster Category: Other
Keywords: Algae, Rheology, Optical tweezers
Project Title: Construction of Optical Tweezers for Microrheological Study of Algae and Bacteria in the American River
Author List:
Cavanaugh, Jeff P. S.; Undergraduate, Physics and Astronomy, California State University, Sacramento, Presenting Author
Castaneda, Francisco; Undergraduate, Biological Sciences, California State University, Sacramento
Morris, Eliza; Physics and Astronomy, California State University, Sacramento
Jensen, Mikkel Herholdt; Physics and Astronomy, California State University, Sacramento

Abstract: Algae and bacteria present a public health crisis in Sacramento’s American River. Harmful toxic bacteria and dangerous bacteria like E.Coli have been found in high levels in the river and the causes of their growth and propagation are not yet known. Public health officials are looking for ways to cull the populations of these harmful microorganisms and prevent their growth. Purpose: In order to find better ways to reduce the populations of harmful bacteria and algae and prevent future blooms of harmful microorganisms, we set out to characterize the local mechanical properties of algal and bacterial gels. To do this we needed an instrument to measure the viscoelastic properties of these microbiological structures. This type of study is called Microrheology. Methods: We designed and built an optical trap to test algal-bacterial composites and characterize their individual and codependent behavior. Using a 900mW (class IV) near infrared (976nm) laser to optically trap and manipulate a small dielectric bead inside of a sample of gel, we observe its response to applied forces. Our laser tweezers use piezo electric motors to move a sample on the scale of nanometers in order to perturb the sample’s microstructures. Using a high resolution Back Focal Plane position detector, this system detects the position of the silica bead with respect to the laser position and allows us to determine the complex mechanical behavior of a given biological sample. Results: We were successful in designing and constructing the optical tweezers system and have acquired microrheological data on a range of viscoelastic materials. For a 2um diameter silica bead we produced trap stiffnesses up to 0.2 pN/nm. We have observed that fibrous algae can entangle and, in combination with bacteria growth in a river environment, can form a gel-like substrate whose behavior is viscoelastic with a strong viscous component. Because of the heterogeneity of these materials, further study with more comprehensive data is important for understanding these microbiomes and the affect they have on bacterial and algal growth. This mechanical characterization can provide physical information for proper design of treatment materials that can infiltrate and fight bacterial-algal gels in the American River. In future work, we hope to study whether algal-bacterial composites’ mechanical properties vary with river depth or if their properties vary strictly based on the availability of nutrients.

Poster #: 229
Campus: San Diego State University
Poster Category: Other
Keywords: PROTAC, EGFR kinase, medicinal chemistry
Project Title: Using PROTACs to counter EGFR resistance mechanisms in lung cancers
Author List:
Johnson, Ronnesha; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Dimacali, Chalmer; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Toenjes, Sean; Graduate, Chemistry and Biochemistry, San Diego State University
Piedrafita, Javier; Chemistry and Biochemistry, San Diego State University
Gustafson, Jeffrey; Chemistry and Biochemistry, San Diego State University

Abstract: Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is activated by miscellaneous extracellular ligands to complete cellular activities such as cell survival, proliferation, development and growth. Hyperactivation of EGFR occurs through mutations and leads to various diseases including non-small cell lung carcinoma, therefore a large scientific effort has been geared to the impediment of mutated EGFR signalling, which has led to 3 generations of EGFR inhibitors. The first generation EGFR was met by T790M gatekeeper mutation which called for a second generation of inhibitors targeting Cys797. However this class of inhibitors possessed severe side-effects caused by off-target wild-type (WT) EGFR inhibition which led to a third generation.
These third generation inhibitors are competitive ATP inhibitors that rely on covalently targeting Cys797. They are quite potent and possess favorable kinetics, but they are met with resistance when Cys797 mutates to a less nucleophilic serine. To begin generating the fourth generation inhibitor, rather than utilizing typical covalent inhibition like the previous generations, we decided to develop a series of EGFR PROTACs. PROTACS are molecules that recruit E3 ubiquitin ligase to a targeted protein (EGFR), causing ubiquitination followed by proteasomal degradation of the desired protein. To induce specific binding of PROTACs to EGFR, we utilize atropisomerism to control the conformation of the molecule by creating steric hindrance about the chiral axis. This will create ideal binding conditions for C797S mutant EGFR compared to WT EGFR. To access atropisomerically pure inhibitor, we have developed an enatioselective synthetic route that allows us to obtain gram quantities of PROTAC product. Thus far we have synthesized and tested a few PROTACS via this route, all with low micromolar degradation IC50s. Using the facile synthetic approach coupled with established PROTAC cell based assays, we plan to optimize the current PROTACs and generate a couple lead compounds with sub micromolar activity in lung cancer cells.

Poster #: 230
Campus: San José State University
Poster Category: Product-focused Innovation
Keywords: prosthetic, 3D printing, pediatric
Project Title: Developing a repeatable manufacturing process for fabricating a life-like and prosthetic hand for infants
Author List:
Hooda, Shruti; Undergraduate, Biomedical Engineering, San José State University, Presenting Author
Leineweber, Matthew; Biomedical Engineering, San José State University

Abstract: The overarching goal of this research is to develop a life-like and durable prosthetic hand with grasping capabilities, catering specifically to infants from 3 to 6 months of age. Currently, there are no commercially available prostheses that combine aesthetic appearance, small size, and grasping function for so young an age. This project is specifically developing a low-cost and repeatable 3D printing process to fabricate customized prosthetic hands for infants with congenital upper limb deficiency. Our novel process uses 3D printing to create customized negative molds, into which silicone elastomers can be cast to create soft, flexible, and anatomically accurate prostheses. Growth rate during infancy is fairly quick, which is why 3D scanning and printing offers a low-cost and accessible method of printing new hands when needed. The method of this study began with developing a prosthetic design through the 3D modelling software, SolidWorks. Through this software, we more specifically developed a negative mold of the infant hand by subtracting the existing hand model from a rectangular part model, splitting the result to reveal the cavities of the prosthetic creating the mold. After designing the negative mold, we used the (Prusa i3 Mk2) 3-D printer to print the mold with PLA (polylactic acid) polymer. As part of the molding process, a wireframe skeleton is inserted into the silicone to provide structural support and grasping capabilities to the prosthetic. Ongoing work is exploring the use of 3D scanning to digitally capture the true anatomy of an individual infant hand. Then we can follow the same modeling process as explained above to create customized devices. The principal setback with this method is that the time duration for scanning makes it difficult to keep an infant still enough to ensure accurate scanning. The progress we have made in developing this new 3D-printing and molding process brings us closer to achieving a low-cost, lifelike, and functional prosthetic hand for infants that can be customized for individual patients within the clinic. This research was funded by the 2019 CSUPERB President’s Scholars Commission Award.

Poster #: 231
Campus: San José State University
Poster Category: Product-focused Innovation
Keywords: capacitive sensor, dehydration, geriatric
Project Title: Capacitive sensor, the potential for its use in the monitoring of hydration in organic tissue
Author List:
Mai, Vinh; Graduate, Biomedical Engineering, San José State University, Presenting Author
Saini, Verndeep; Graduate, Biomedical Engineering, San José State University, Presenting Author
Leineweber, Matthew; Biomedical Engineering, San José State University

Abstract: The prevalence of dehydration among seniors residing in long term care facilities is estimated to be between 40% to 60%. Complications associated with dehydration include increased risk for infections and poor quality of life. Current system for the early detection of dehydration in long term care settings have traditionally been relying on a combination of staff vigilance and blood draws, thereby requiring significant investment of valuable resources. The need for low cost alternative method of hydration monitoring is warranted as staffing challenges and invasive clinical blood tests remain barriers to early detection. Our project aims to develop a noninvasive sensor capable of quickly measuring changes in hydration level in organic tissue. We created a low-power proof-of-concept sensor using a capacitive circuit in conjunction with an Arduino Nano, and tested the sensor on porcine skin, which served as an analog to human skin. The sensor measured the transient time between the send pin and the receive pin to build up to a threshold voltage. When a sample made contact with the sensor, it exerted its influence to the transient time due to the inherent dielectric constant native to the tissue, in particular, the influence of the tissue’s water content. Using the oven drying method, porcine skin samples were weighed and dehydrated over time to achieve varying degrees of moisture content. At each weighing increment, the samples were measured by the sensor to track changes in the water composition versus sensor output. Result: When examining the data, we found a positive correlation (R2 = .93) between the porcine skin weight change and the sensor output, suggesting that our sensor can feasibly be used to track changes in hydration level. Future goals of this project include tuning the sensor and circuit parameters to calibrate for human use, testing on human subjects against the current gold standard clinical methods for further validation, and developing this sensor further into a novel product.

Poster #: 232
Campus: CSU Bakersfield
Poster Category: Product-focused Innovation
Keywords: diatoms, biofuels, waste water
Project Title: Oil field-produced water sustains microalgae growth and influences cellular lipid content amenable for biofuel production
Author List:
Gillard, Jeroen; Biology, California State University, Bakersfield, Presenting Author
Contreras, Javier ; Undergraduate, California State University, Bakersfield, Presenting Author
Contreras, Gabriela ; Undergraduate, California State University, Bakersfield
Hernandez, Alex; Undergraduate, California State University, Bakersfield
Francis, Isolde; Biology, California State University, Bakersfield
Cabrales, Luis; Physics and Engineering, California State University, Bakersfield

Abstract: The agricultural industry of the San Joaquin Valley (SJV) is challenged by a supply of fresh water to irrigate crop fields. With rising atmospheric carbon emissions and associated climate change events, these challenges are expected to intensify. In the southern SJV, substantial amounts of fresh water are used for the extraction of fossil fuels. At least a quarter of this oil field-produced water (PW) remains unused in disposal wells. Microalgae are a diverse group of photosynthetic microbes that could potentially remediate this PW through nutrient assimilation and absorption of salts, metal(loid)s and hydrocarbons. In addition, because some species can accumulate up to 60% of their dry mass as neutral lipids, these water sources could be used for the production of biofuels. Here, we collected and chemically characterized two different sources of oil field-PW. Both PW sources were used for the cultivation of the marine diatom Phaeodactylum tricornutum. We assessed i) the growth potential of both media, ii) the maximum photosynthetic capacity of the cells, iii) the cellular lipid index, and iv) the differential gene expression dynamics of cells grown in one of the selected media. Compared to control conditions, these experiments showed that P. tricornutum grew to similar densities in the stationary phase of PW cultures. Furthermore, by comparisons with control media of identical salinity, we found that the lipid index and growth characteristics of P. tricornutum were mainly affected by the salinity of the PW media and not the chemical constituents of the PW medium. Finally, our transcriptome analysis showed that the expression of large sets of genes were differentially regulated by the PW medium. For example, we show that genes involved in diatom cell division and metabolism are impacted by the PW constituents. In conclusion, this research shows that the diatom P. tricornutum exhibits an exceptional tolerance toward the chemical constituents of oil field PW, and it opens a pathway to the genetic engineering of this model diatom species to maximize lipid production and remediation of these valuable water sources.

Poster #: 233
Campus: CSU San Marcos
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: retina, stem cells, optimization
Project Title: OPTIMIZING A CONE RICH POPULATION FROM HUMAN PLURIPOTENT STEM CELLS
Author List:
Martin, Heather; Undergraduate, Biology, California State University San Marcos, Presenting Author
Diaz, Emily; UCSD
Ogata, Anna; UCSD
Mothe, Bianca; Biology, California State University San Marcos
Wahlin, Karl; UCSD

Abstract: Millions of Americas suffer from some form of retinal degeneration (RD). While these can be the result of inherited mutations, the vast majority, such as in age-related macular degeneration (AMD), have a cause that is less well understood. As our population ages, there is a need for better cell-based models to study RD and potential therapies. The discovery of pluripotent stem cells and new methods to differentiate them into a variety of cell types and tissue makes them well suited to address these needs. Using CRISPR-Cas9 gene editing, we were able to create cell type specific fluorescent reporters that allow tracking of retinal development in both 2D and 3D cultures. Our current protocol produces 3D laminar retinal structures (organoids) containing all the respective neuronal cell types typical of the human retina. Like human retinas, these organoids are generally rod rich, and although they do contain cones, they lack a dense cone rich structure resembling the macula. A cell was engineered to incorporate a fluorescent reporter into the SIX6 gene—an early eyefield marker—and CRX—a gene associated with the production of photoreceptors. Using this dual fluorescent reporter system, we were able to track target gene expression in real-time. A previous study has suggested that human recombinant COCO pushes iPSCs grown in 2D towards a blue-cone specific fate through Wnt, BMP, and TGFβ inhibition (Zhou et al., 2015). First, confluent cells were differentiated in 2D cultures either in a standard control medium, or a control medium that had been enhanced with COCO, FGF-2, and IGF (CI)—both described by the previous study. Unlike the Zhou et al. study, our cultures did not show an increase in photoreceptor precursor cells—higher expression of SIX6/CRX—as compared to the control (SIX6 p = 0.1449; CRX p 0.8612). This could be the result of excessive cell death seen in wells treated with the CI medium; SIX6 is density dependent and much expression was lost as the cell density decreased. We then tried to introduce the CI medium at different time points to see if introduction at later points was less toxic for the cells. While a trend was seen suggesting that later introduction might cause less cell stress, due to variability between replicates these results were not statistically significant (SIX6 p = 0.240; CRX = 0.2390). Further efforts in successfully reproducing and translating the COCO study to organoids would allow for a more human like model for study in RD.

Poster #: 234
Campus: CSU Northridge
Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM)
Keywords: cerebral organoid , hPSCs, TGF-β
Project Title: The Transcriptomic State of Human Pluripotent Stem Cells Predicts Successful Cerebral Organoid Differentiation
Author List:
Turcios, Felix; Undergraduate, Biology, California State University, Northridge, Presenting Author
Malone, Cindy; Biology, California State University, Northridge
Novitch, Bennet; UCLA Neurology, California State University, Northridge

Abstract: Defects in brain development underlie many neurological diseases; yet, the critical mechanisms are not understood. Efforts to investigate these mechanisms have traditionally utilized animal models. However, the human brain has many distinct features that are not seen in other species. Increasing evidence suggests that these human-specific features may be impacted by neurological disease, illustrating the need for human-specific models. One such model involves directing human pluripotent stem cells (hPSCs) to form brain-like structures termed organoids. Cerebral organoids recapitulate many aspects of the developing human fetal brain including the formation of neural progenitor regions and cortical layers. Our lab previously developed a reproducible and efficient protocol to generate cerebral organoids. Nevertheless, we have found that successful cerebral organoid formation is strongly influenced by the conditions under which hPSCs are maintained prior to differentiation. Variabilities arise due to batch differences in mouse embryonic fibroblast feeders and defined media components, both of which are critical for organoid formation. Here, we define the transcriptomic differences within hPSC maintained under different conditions and find that successful organoid development correlates with enriched expression of genes associated with TGF-β superfamily signaling and naïve pluripotency. We further show that hPSC maintained in the presence of specific combinations of TGF-β superfamily proteins exhibit elevated expression of naïve pluripotency genes and enhanced capacity to form cerebral organoids and other forebrain structures. Together, our studies define optimal organoid differentiation conditions suitable for human neurological disease modeling and drug discovery.

Poster #: 235
Campus: CSU Fullerton
Poster Category: Proteins (Include Proteomics)
Keywords: zinc transporter, membrane protein, transition metal
Project Title: Transmembrane 163 (TMEM163) protein interacts with distinct ZNT efflux transporters
Author List:
Escobar, Adrian; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Styrpejko, Daniel; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Ali, Saima; Staff, Biological Science, California State University, Fullerton
Cuajungco, Math; Biological Science, California State University, Fullerton

Abstract: Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. The CDF family consists of species-conserved members that include 10 mammalian zinc transporters called ZNT (i.e. ZNT1-ZNT10 efflux proteins). Based on our current findings, we hypothesize that TMEM163 is a potential interaction partner of at least one of the ZNT proteins, namely ZNT3 and ZNT4. Both ZNT3 and ZNT4 localize to synaptic vesicles and lysosomal compartments, respectively, where TMEM163 happens to be situated as well. To show that TMEM163 interacts with either ZNT3 or ZNT4, we cloned their cDNAs into mammalian expression constructs containing either Myc-DDK or HA peptide tag. Using HEK-293 cells, we individually transfected TMEM163, ZNT3, and ZNT4 constructs as negative controls, while we co-transfected TMEM163 and ZNT3, or TMEM163 and ZNT4. As a positive control, we co-transfected HA-tagged TMEM163 with a Myc-DDK-tagged TMEM163 construct in HEK-293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co-immunoprecipitation assay using either anti-HA- or anti-DDK-bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163 physically bound ZNT3 or ZNT4 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc-specific fluorescence dyes, Fluozin-3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single- and co-transfection of TMEM163, ZNT3, and ZNT4 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163, ZNT3, and ZNT4 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163 proteins varied slightly in magnitude when compared with heterodimers of TMEM163 and ZNT3, or TMEM163 and ZNT4 proteins. These results suggest that the interaction between TMEM163 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of TMEM163 protein. Overall, our investigations showed for the first time that TMEM163 forms functional heterodimers with ZNT3 and ZNT4 proteins. Thus, TMEM163 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types. This work is funded by NIH R15 NS101594.

Poster #: 236
Campus: San José State University
Poster Category: Proteins (Include Proteomics)
Keywords: sirtuin, ovaries, mammary gland
Project Title: Expression of sirtuin 4 in mammary glands and ovaries reveals a potential role in female development
Author List:
Llaguno, Fiara; Undergraduate, Department of Biological Sciences, San José State University, Presenting Author
Nguyen, Tina; Undergraduate, Department of Biological Sciences, San José State University, Presenting Author
Lin, Zhihong; Duke Molecular Physiology Institute
Hirschey, Matthew; Duke Molecular Physiology Institute
Huynh, Frank; Department of Biological Sciences, San José State University

Abstract: Sirtuins are a family of seven proteins that contribute to maintaining homeostasis and a healthy lifespan. In particular, sirtuin 4 (SIRT4), is located in the mitochondria and has been identified as a regulator of nutrient metabolism. However, compared to the other sirtuins, relatively little is known about the physiological roles of SIRT4. We recently discovered a novel role for SIRT4 in mammary gland development. Nearly all pups born to female mice lacking SIRT4 (SIRT4KO mice) died two days after birth. This was due to the mothers’ low milk production, caused by impaired mammary gland development in SIRT4KO females during puberty. Interestingly, treatment with the ovarian hormones, 17-beta-estradiol and progesterone, rescues this phenotype and normalizes mammary gland development. These observations led us to hypothesize that SIRT4 may play a role in regulating 17-beta-estradiol and progesterone signaling in mammary glands or in regulating 17-beta-estradiol and progesterone secretion from ovaries. To test this hypothesis, our goal was to first determine whether SIRT4 protein is expressed in mammary glands and ovaries. Methods: Applying basic mouse anatomy and organ dissection techniques, we extracted proteins from the mammary glands and ovaries of wildtype mice. Then, we performed Western blots to determine levels of SIRT4 protein expression in these tissues. Results: SIRT4 was expressed in mammary glands of wildtype mice, suggesting that SIRT4 may directly contribute towards the healthy development of mammary glands. SIRT4 was also expressed at high levels in ovaries compared to other tissues, especially when normalized to mitochondrial proteins. This indicates that SIRT4 could also play an important role in normal ovarian function. Conclusions: Overall, our data infer that SIRT4 is potentially a key protein, regulating both 17-beta-estradiol and progesterone signaling in mammary glands and secretion in ovaries. Since SIRT4 is already known to be a regulator of nutrient metabolism, our latest data highly suggest that SIRT4 is an important mediator between nutritional status and female development.
This work was supported by a San Jose State University Undergraduate Research Grant.

Poster #: 237
Campus: CSU Long Beach
Poster Category: Proteins (Include Proteomics)
Keywords: Proteomics, Sexual Dimorphism, Brain research
Project Title: Proteomic Analyses of Amygdala in Juvenile Male, Female and Testicular-feminized Mice
Author List:
Soriano, Jennifer; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author, Nagel Award Nominee
Tasi, Houng-Wei; Biological Sciences, California State University, Long Beach
Lee, YuanYu; Biological Sciences, California State University, Long Beach

Abstract: Androgens play a critical role in sexual differentiation of non-gonadal tissues, including the brain. Among many brain regions, the amygdala is responsible for autonomic and behavioral responses associated with fear and fear conditioning, and some functions and structures of the amygdala are found to be sexually dimorphic, which is largely regulated by perinatal rises in testosterone via the activation of androgen receptor (AR). AR is a ligand-activated transcription factor with the capability to regulate the expression of different proteins through the influence on transcription. However, the molecular mechanisms underlying AR-mediated sexual dimorphism in the amygdala remain unclear. Thus, we hypothesized that AR might govern a network of sex-dimorphically expressed proteins, which constitute the regulatory mechanisms for sexual differentiation of the developing amygdala. To address this, we used global proteomics approach to identify proteins differentially expressed in the amygdala of wild-type male, wild-type female and testicular-feminized mutant (Tfm) male mice, lacking functional AR, during early development. Protein samples were extracted from the amygdala of male, female and Tfm mice collected at postnatal day 21 (n=3 per group) and then separated by 2D-gel electrophoresis. These gels were imaged and were quantified using Progenesis. A total of 300 protein spots were recognized to express in the amygdala, and 31 of them displayed a fold-change of 1.5 or greater between the sexes. One AR-dependent, sexually dimorphic spot (~25 kDa and pI= 6.5; male = 1, Tfm = 1.5, and female = 1.2 folds) was excised, followed by in-gel digestion and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for protein identification. Mass spectrometric analysis revealed 14-3-3 theta as one of the candidates present in that protein spot. Next, we performed STRING analysis to search for potential interactions between AR and 14-3-3 theta. Although no interaction between AR and 14-3-3 theta was found, previous studies showed androgen regulated expression of other 14-3-3 protein family members at the mRNA and protein levels. Prior to uncover the biological relevance of 14-3-3 theta in sexual dimorphism in the amygdala, we will use immunoblotting to confirm the sex difference in 14-3-3 theta protein expression and AR regulation of its expression in the developing mouse amygdala of male, female, and Tfm mice.

Poster #: 238
Campus: CSU Chico
Poster Category: Synthetic Chemistry
Keywords: organic synthesis, pesticides, nematodes
Project Title: Library Synthesis of Nematocides: The Coupled Chalcones
Author List:
DuBose, Austin; Undergraduate, Chemistry, California State University, Chico, Presenting Author
Kim, Minjoo; Undergraduate, Chemistry, California State University, Chico
Nusser, Brandon; Undergraduate, Chemistry, California State University, Chico
Calderón-Urrea, Alejandro; Biology, California State University, Fresno
Arpin, Carolynn; Chemistry and Biochemistry, California State University, Chico

Abstract: Nematodes are small round worms that annually destroy over $80 billion dollars’ worth of agricultural industry worldwide. Certain pesticides that were previously used for this issue have been banned due to their environmental and human health hazards. There has yet to be another safe and successful pesticide to combat nematodes. In pursuit to develop a new nematocide, the lab of Dr. Alejandro Calderón-Urrea at CSU Fresno screened over 100 different chalcones to find three that worked well at exterminating Caenorhabditis elegans (a free-living nematode) and Meloidogyne incognita (a plant parasitic nematode). Intriguingly, our collaborators found that subjecting the nematodes to these three chalcones together had a synergistic rather than an additive effect. They also tested the effects of the chalcones on beneficial soil organisms and concluded that they were safe for use. Our hypothesis is that linking two chalcones together to form one compound will enhance this synergy and thus reduce the amount of pesticide required to save our agriculture. We have therefore identified and successfully carried out an efficient synthesis of combining two chalcones. Our route utilizes only four reaction steps, it is modular, and it is accommodating of various linkers between the chalcones. Our work has resulted in the library synthesis of nine different coupled chalcone compounds that are currently being assessed by our collaborators for their nematocidal activity. We are also currently pursuing other libraries that differ in position of linkage on the chalcones as well as linking other small molecules that are known nematocides.

Poster #: 239
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: catalysis, enzyme mimic, selective oxidation
Project Title: Targeting Enzyme-Like Catalysis Using a Synthetic Polymer to Mimic the Secondary Coordination Sphere of an Enzyme
Author List:
Seymour-Cozzini, Thomas; Undergraduate, Chemistry, San José State University, Presenting Author
Tafuri, Victoria; Undergraduate, Chemistry, San José State University
Navarro, David; Undergraduate, Chemistry, San José State University
Ordona, Harris; Undergraduate, Chemistry, San José State University
Radlauer, Madalyn; Chemistry, San José State University

Abstract: Natural enzymes facilitate impressive reactivity and selectivity, but synthetic mimicry of enzyme activity is a continued challenge. Success holds the potential for wide-ranging applications in medicine, agriculture, fuel production, and beyond. For example, particulate methane monooxygenase (pMMO), a bacterial enzyme, has the ability to convert methane into methanol. Methane is a potent greenhouse gas, a common industrial and natural byproduct, and a poor fuel compared to other hydrocarbons because of low energy density. Methanol, on the other hand, is a versatile liquid, useful as solvent, fuel, and reagent. Unfortunately, methanotrophic bacteria are difficult to farm, so synthetic alternatives for this reaction must be found. To this end, we synthesized N,N-(2-pyridylmethyl)-1,3-diamino-2-propanol (1) by imine condensation of 2-pyridine carboxaldehyde and 1,3-diamino-2-propanol, followed by reduction with sodium borohydride to form the primary ligand precursor of a dicopper complex. For incorporation into a polymer framework that mimics the secondary coordination sphere around the pMMO active site, an alkenylated version of the same ligand precursor was produced. 6-Bromo-2-pyridine carboxaldehyde was vinylated via Stille coupling prior to condensation and reduction to yield N,N-(6-ethenyl-2-pyridylmethyl)-1,3-diamine-2-ol (2), enabling thiol-ene click chemistry with a polymer precursor.
The catalytic activity of the non-alkenylated dicopper complex was benchmarked in kinetic studies of the oxidation of tert-butylcatechol using UV-vis spectrometry. With synthesis methods determined and baseline catalytic functionality demonstrated, syntheses were scaled up for purification and characterization. It was determined that the products of the Stille coupling could be purified on a silica gel plug, and (2) must be stored over BHT in amber vials. Purification of (1) by column chromatography was abandoned in favor of solubility-based methods currently under development, as (1) decomposes on silica gel. All products are characterized with proton nuclear magnetic resonance and infrared spectroscopy. The catalytic oxidation of tert-butylcatechol will be repeated using the dicopper complexes from metalation of ligand precursors (1) and (2). Future work includes attachment of (2) to polymer precursors via thiol-ene chemistry and testing of the polymer-bound catalysts. We acknowledge San Jose State University and CSUPERB for funding our work.

Poster #: 240
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: Flavone, medicinal chemistry, anticancer agents
Project Title: Luteolin derivatives selectively suppress androgen receptor-positive LNCaP prostate cancer cell proliferation
Author List:
Rajaram, Pravien; Graduate, Chemistry, California State University, Fresno, Presenting Author
Olveda, Nicholas; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Chemistry, California State University, Fresno
Muthima, Kevin; Graduate, Chemistry, California State University, Fresno
Chen, Qiao-Hong; Chemistry, California State University, Fresno

Abstract: Prostate cancer is a global issue with high prevalence in the U.S., South America, Central and South Africa, and Australia. Current treatments for advanced stages of prostate cancer often results in recurrence. Luteolin is a dietary flavone ubiquitously distributed in several of vegetables and fruits, as well as traditional medicines. Luteolin has been demonstrated to have capability in suppressing prostate cancer cell proliferation in cell models and in reducing tumor size in mice models. However, the therapeutic potential of luteolin is limited by its moderate potency. To alleviate this drawback, ten new 7-O-substituted-3′,4′,5-O-tertramethylluteolins have been designed and synthesized from commercially available hesperidin through a four-step procedure, including oxidation, global methylation, glucoside hydrolysis, O-alkylation, and N-alkylation. Among them, six derivatives have so far been evaluated for in vitro potency against both androgen receptor-positive LNCaP cells and androgen receptor-null DU145 and PC-3 cell lines. Luteolin and enzalutamide (a current leading prostate cancer therapy) were used as positive controls. The following conclusions can be drawn from our current data from the WST-1 cell proliferation essay: (1) modification on 7-OH of trimethylluteolin can significantly improve its antiproliferative potency; (2) 7-O-bromopropyl-3′,4′,5-O-tertramethylluteolin was established as the optimal compound that is more effective towards androgen receptor-positive LNCaP cells than androgen receptor-null cell models; and (3) the optimal derivative exhibits greater efficacy than enzalutamide in the LNCaP cell model. We are grateful to CSU-Fresno for an Undergraduate Research Grant to N. Olveda and to CSU-Fresno Bridges to Doctorate Program for the support to K. Muthima.

Poster #: 241
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: fluorescence, organic chemistry, nucleic acids
Project Title: Spectroscopic Properties of Fluorescent 8-DEA-tC DNA-RNA Heteroduplexes
Author List:
Ceja, Jesús; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Turner, Marc; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Kim, Grace; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Purse, Byron; Department of Chemistry and Biochemistry, San Diego State University

Abstract: Fluorescence in situ hybridization (FISH) has been an essential method for detecting specific sequences in biological samples, including specific mRNA, genomic DNA, or cloning vectors. This method can however yield false positives by hybridizing to similar sequences that may differ by 1-2 bases, yet exhibit similar thermostability as if perfectly complemented, highlighting the major limitation of FISH in that fluorescence is the direct result of preferential binding, not necessarily sequence recognition. Our lab’s nucleoside analogue 8-diethylamino-tricyclic cytidine (8-DEA-tC) can potentially discriminate sequences more accurately than current hybridization methods. 8-DEA-tC has low intrinsic fluorescence (Φem = 0.006), however the emission can increase up to 0.12 when 8-DEA-tC pairs with guanosine in double-stranded DNA (dsDNA) . The fluorescence turn-on does not occur when 8-DEA-tC is mismatched with A, which may enable the use of 8-DEA-tC in higher fidelity probes.
In this current work, we examine the fluorescent properties of 8-DEA-tC DNA-RNA heteroduplexes. The fluorescence turn-on is greater with RNA as compared with DNA when using otherwise identical sequences. By testing eight sequences, we found GXC is the highest performing sequence and CXT is the lowest performing. GXC exhibited the largest Φem of 0.22 while the other sequences had Φem values from 0.10-0.17. CXT experienced an average fluorescence turn-on of less than 10% and results showed it had the lowest turn-on in fluorescence from single-stranded DNA. AXA experienced the largest turn-on comparing ssDNA and DNA-RNA, while CXA had the largest turn-on relative to the dsDNA. CD spectra indicates that the 8-DEA-tC DNA-RNA duplexes adopt the A-form conformation, thus having a compact helical structure whose base pairs are not perpendicular to the helical axis as in B-DNA, resulting in different π overlap and solvation of the exposed edges of the bases.

Poster #: 242
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: gold catalysis, Friedel-Crafts, microwave
Project Title: Microwave-Assisted Gold-Catalyzed Reaction of Benzylic Alcohols: Substrate Scope and Mechanistic Investigations
Author List:
Alvarez, Araceli; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Melanese, Amanda; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Nominee
Iafe, Robert; Chemistry& Biochemistry, California State University San Marcos

Abstract: Syntheses of 1,1-diarylalkanes have attracted significant interest within the past decades. The 1,1-diarylalkane moiety is a prevalent constituent of medicinally relevant compounds, supramolecular systems, and synthetic receptors. Cyclic derivatives of these molecules—fluorenes—have unique properties in materials science, pharmaceuticals, and organic synthesis. The Friedel–Crafts (F.-C.) alkylation reaction represents one of the most accessible and widely used methods to obtain these compounds. F.-C. alkylation strategies have recently been utilized as key C–C bond-forming steps in the total synthesis of bioactive compounds. During a prior investigation using gold(I) salts to catalyze etherification reactions, our lab discovered that phenols react via a F.-C.-like reaction mechanism instead of the etherification pathway. To our satisfaction, we were successfully able to cleanly promote the F.-C.-like reactivity using microwave conditions to afford substituted 1,1-diarylalkanes in high yield and regioselectivity. We have further expanded this methodology to nucleophiles including anisole, p-methylanisole, p-tert-butylphenol, sesamol, and heteroaromatic compounds including 1,2,3-benzotriazole and benzimidazole. While examples of gold(III)-catalyzed F.-C. reactions are well-known, a microwave assisted methodology using gold(I) salts is currently unprecedented in the literature. A long-term objective of this project is the synthesis of bioactive natural products and other building blocks useful in the synthesis of bioactive molecules.
Results: The gold-catalyzed F.-C.-like reaction has been optimized using methylanisole as the nucleophile, phenylethanol as the electrophile, gold(I)/silver(I) salts (5 mol%), and elevated temperatures to afford 99% of the target 1,1-diarylethane. A substrate scope for this reaction was explored using the following nucleophiles: anisole, p-methylanisole, sesamol, p-tert-butylphenol, 1,2,3-benzotriazole, and benzimidazole. Excellent yields (34–99%) were obtained when electronics were varied on the electrophile. Deuterium labeling experiments show no proton scrambling at the benzylic position. Secondary kinetic isotope effect experiments support rehybridization of Csp3 to Csp2 in the rate-determining step. Hammett plot analyses reveal a buildup of positive charge in the transition state.
This work was supported by NSF grant 1566124, ACS Division of Organic Chemistry, and TRiO McNair Program.

Poster #: 243
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: benzofurans, gold(I) catalysis, heterocycles
Project Title: Gold(I)–NHC-catalyzed synthesis of benzofurans via migratory cyclization of 2-alkynyl aryl ethers
Author List:
Lichtenstein, Dana; Undergraduate, Biology, California State University, Fresno, Presenting Author
Dillon, Christopher; Graduate, Chemistry, California State University, Fresno, Presenting Author
Le, Quang; Graduate, Chemistry, California State University, Fresno
Muchalski, Hubert; Chemistry, California State University, Fresno

Abstract: Benzofuran is a heterocyclic compounds made out of fused benzene and furan rings. It is present in numerous bioactive natural products, polymers, and pharmaceuticals. Thus, benzofurans are considered privileged molecular scaffolds in drug discovery and medicinal chemistry. Currently, clinically-approved drugs containing the benzofuran core include Amiodarone (irregular heartbeat and tachycardia), Vilazodone (anxiety and depression), and Naloxone (opioid antagonist) among others. There is considerable interest in research focusing on new methodologies for the synthesis of benzofurans. Catalytic methods that aim to minimize the use of stoichiometric reagents or harsh reaction conditions are of particular interest. In 2018, we published an efficient method for gold(I)-catalyzed cyclization of 2-alkynyl thioethers that generated benzothiophenes. However, this catalyst did not promote transformation of 2-alkynyl ethers to benzofurans. The aim of this research project was to discover and develop a reaction that would deliver benzofurans in a similar manner. We discovered the gold(I)–NHC dimer catalyst outperforms other gold(I) phosphine complexes that we tested and that an electron-rich ether is necessary for the reaction to take place at a reasonable rate. The reaction proceeds smoothly at room temperature and requires very small amounts of the catalyst. Our method tolerates a wide range of functional groups with various steric and electronic properties. We will present the details of reaction optimization and discuss of the possible reaction mechanism, as it differs significantly from the mechanism we observed in benzothiophene chemistry.

Poster #: 244
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: C-H Functionalization, Lewis Base, Peptide Stapling
Project Title: Selective C-H Functionalization of Arenes and Heterocycles for Biological Applications
Author List:
Dinh, Andrew; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Brown, Zach; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Vaidya, Sagar; Postdoc, Department of Chemistry and Biochemistry, San Diego State University
Saputra, Mirza; Postdoc, Department of Chemistry and Biochemistry, San Diego State University
Gustafson, Jeffrey; Department of Chemistry and Biochemistry, San Diego State University

Abstract: Aryl chlorides are numerous in a wide variety of small molecules, especially prevalent in pharmaceuticals as synthetic handles for late-stage functionalization and for improvement in bioavailability. As such, there is a comprehensive library of methodologies to access aryl chlorides, with electrophilic aromatic substitution (SEAr) in the forefront. However, one of the main shortcomings of SEAr is the innate electronic properties of the substrate leading to a mixture of undesired regioisomers. Therefore, it becomes a synthetic challenge to access the less favored regioisomers when the substrate has a strong activating electronic preference.
In 2015, we disclosed that sulfur-derived Lewis bases such as triphenylphosphine sulfide can catalyze the chlorination of arenes and heterocycles under mild conditions through activation of N-chlorosuccinimide. One of the shortcomings was that it did not address the issue of regioselectivity. Hence in 2016, we developed a regiocontrolled EAS chlorination and bromination of phenols, which highlighted various Lewis Base catalysts that could reverse or enhance the innate regioselective nature of the phenol. Despite these results, we found limitations in reactivity and substrate scope for less electron-rich heteroarenes.
To solve these problems, we embarked on mechanistic studies to better understand how our Lewis base catalysts function. Our initial in silico findings predicted our catalyst can interact with the chlorine atom through a halenium adduct, implying that catalyst structure can control regioselectivity. Additionally, monitoring reactions via nuclear magnetic resonance (NMR) spectroscopy and 1H kinetic experiments revealed the formation of unique catalytic byproducts for both triphenylphosphine and Nagasawa’s bis-thiourea in the presence of N-chlorosuccinimide, which can heavily impact the rate and regioselectivity of SEAr halogenation. Based on these results, we then developed new selenium Lewis base catalysts derived from 1-indanol that could enhance the initial rate of reaction while imparting comparable regioselective control on phenol without degradation over time. Additionally, this catalyst can selectively functionalize a variety of electrophiles, including the sulfenylation and trifluoromethylthiolation of arenes, expanding the breadth of our general methodology to biological applications such as peptide stapling.

Poster #: 245
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: photoredox, thiolation, C-H functionalization
Project Title: Photocatalytic Oxidative C-H Thiolation: Synthesis of Benzothiazoles and Sulfenylated Indoles
Author List:
Dinh, Andrew; Graduate, Chemistry and Biochemistry, San Diego State University
Nguyen, Ashley; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Millan Aceves, Ernesto; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Albright, Samuel; Undergraduate, Chemistry and Biochemistry, San Diego State University
Cedano, Mario; Graduate, Chemistry and Biochemistry, San Diego State University
Smith, Diane; Chemistry and Biochemistry, San Diego State University
Gustafson, Jeffrey; Chemistry and Biochemistry, San Diego State University

Abstract: A variety of sulfur containing scaffolds widely exists in both natural products and pharmaceuticals. There are many synthetic routes that have been developed to synthesize aryl sulfides from aryl halides, commonly using various sulfenylating reagents like N-thiosuccinimide. These methods result in a milder yet efficient conversion of simple substituted arenes to aryl sulfides, however sulfenylation via photoredox catalysis is not as widely explored. The use of photoredox catalysis allows for the generation of a more reactive radical intermediate that can be employed to generate a C-X bond, or C-S in this case.
We report studies on the photocatalytic formation of C-S bonds to form benzothiazoles via an intramolecular cyclization and sulfenylated indoles via an intermolecular reaction. Cyclic voltammetry (CV) and density functional theory studies (DFT) suggest that benzothiazole formation proceeds via a mechanism that involves an electrophilic sulfur radical, while the indole sulfenylation likely proceeds via a nucleophilic sulfur radical adding into a radical cationic indole. These conditions were successfully extended to several thiobenzamides and indole substrates.

Poster #: 246
Campus: Cal Poly Pomona
Poster Category: Synthetic Chemistry
Keywords: Bioinorganic, X-ray Absorption Spectroscopy, Nitrosyls
Project Title: Synthesis of Nickel Nitrosyl Complexes for Understanding Biological NO Reduction
Author List:
Zhang, Zijie; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author, Nagel Award Nominee
Stieber, S. Chantal E.; Chemistry & Biochemistry, California State Polytechnic University, Pomona

Abstract: Activation of small molecules like CO, N2 or NO is could harness new chemistry with underutilized starting materials and offer insight towards biological reduction mechanisms. NO is reduced by enzymes, but the redox activity of the metal and NO makes the characterization of the metal nitrosyl complexes complicated. The fundamental interactions between metals and NO could be elucidated with metal nitrosyl model complexes which are more easily studied than metal centers in enzymes. Nickel nitrosyl complexes with bidentate N-heterocyclic carbene ligands were synthesized to examine the effect of strongly sigma donating NHC ligands on the electronics of the nickel-NO bond. The ligand precursor mesityl bis(imidazolium) salt (1,1′-di(mesityl)-3,3′-methylene-diimidazolium dibromide) was synthesized by having 1-(2,4,6-trimethylphenyl)-1H-imidazole) react with dibromomethane, which was characterized by 1H NMR spectroscopy. The salt was deprotonated with potassium bis(trimethylsilyl)amide (KHMDS) in THF to generate the bidentate carbene ligand. A new metal complex, (MesNHC2Me)Ni(COD), {1,1′-di(mesityl)-3,3′-methylenediimidazolin-2,2′-diylidene}nickel(0)cyclooctadiene}, was synthesized with the free carbene ligand and bis(1,5-cyclooctadiene)nickel(0), which was structurally characterized. A new Ni-NO complex [(MesNHC2Me)Ni(NO)][BF4] was synthesized with (MesNHC2Me)Ni(COD) and one equivalent of [NO][BF4]. The presence of a new Ni-NO bond and a BF41- counterion was demonstrated by infrared spectroscopy, which establishes a new example of a three-coordinate nickel nitrosyl complex with strongly sigma donating ligands. This Ni-NO complex was then functionalized by reacting with triphenylsilane and sodiumtriethylborohydride, to adding an H atom to the N-O bond. All complexes were characterized by X-ray absorption spectroscopy, supporting the proposed reactivity, and establishing rising edge features for NO complexes. These reactions suggest the possibility for NO activation at nickel centers and provide spectroscopic information for metal-NO complexes that could help characterize metals in biological systems. This work was supported by a CSUPERB New Investigator Grant and NSF CAREER Award (1847926).

Poster #: 247
Campus: San Jos​é State University
Poster Category: Synthetic Chemistry
Keywords: polymer purification, enzyme mimic, single chain nanoparticle
Project Title: Harnessing enzyme-like reactivity in a scalable synthetic system: Assembling chemical building blocks to mimic the environment around an active site
Author List:
Tien, Derscene; Undergraduate, Chemical Engineering , San José State University, Presenting Author
Gupta, Vanshika; Undergraduate, Chemistry, San José State University
Nguyen, Khanh ; Undergraduate, Chemical Engineering, San José State University
Radlauer, Madalyn; Chemistry, San José State University

Abstract: Enzymes are natural catalysts that lower the energy barriers of chemical reactions. Many of them have transition metals as active sites, however, it has been shown that without the appropriate secondary coordination sphere of surrounding substrates bound to the metal, the selectivity and reactivity are lost. Hence, in building enzyme mimics, we focus on preparing an appropriate secondary structure around the primary coordination sphere of the metal-containing synthetic active site. With the goal of making a single chain nanoparticle (SCNP) structure constructed from an ABA-type triblock polymer, we set forward to obtain more pure samples of our polymers of tert-butyl acrylate, methyl acrylate, 2-hydroxyethyl acrylate and N-isopropylacrylamide. Revisiting the polymerization process, the large focus for us this summer was to ensure that we could obtain pure samples of our polymers, as our initial testing phase of our proposed polymerization processes and that of aminolysis were determined to be effective. Because the solvents used in literature are highly toxic, we aimed to minimize their use to reduce the safety hazard. After a series of trials, we formulated a three-step process for polymer purification. After polymerization via reversible addition-fragmentation chain transfer, the Schlenk line was used to remove the majority of our dimethylformamide or 1,4 dioxane, the polymer was redissolved in a lower boiling point and less toxic solvent, and multiple precipitations were run before drying the polymers in a vacuum oven. Following analysis utilizing nuclear magnetic resonance spectroscopy and size exclusion chromatography, we were able to confirm the purity of our products. We also successfully synthesized various purified block polymers using this procedure. The next step in SCNP synthesis is aminolysis of the polymers, but literature procedures using cyclohexylamine were only successful with a couple of our polymer precursors. We will continue to optimize the aminolysis procedure for all of our polymer precursors. Meanwhile, we will proceed to the thiol-ene click chemistry reaction, which will allow the attachment of our polymers to the synthetic active site. By mimicking these secondary coordination sphere interactions, we aim to reach a higher level of selectivity and reactivity than simply focusing on the metal center itself. We would like to thank San José State University and CSUPERB for funding.

Poster #: 248
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Atropisomer, Kinase inhibitor, Medicinal Chemistry
Project Title: Modifying Ibrutinib’s conformational space to create selective BTK inhibitors
Author List:
Albright, Samuel; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Hazin, Ramsey; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Toenjes, Sean; Graduate, Chemistry and Biochemistry, San Diego State University
Ortiz, Toni; Chemistry and Biochemistry, San Diego State University
Gustafson, Jeffrey; Chemistry and Biochemistry, San Diego State University

Abstract: Kinases are proteins that control biochemical pathways and if mutated or overexpressed, they can lead to serious diseases such as cancer. Therefore, many medicinal chemists focus on designing kinase inhibitors that attenuate the unregulated signaling. However, due to many kinases having very similar active binding sites, many inhibitors have off-target inhibition which can lead to undesirable side effects.
Modern small molecule inhibitors contain many bi-aryl bonds, leading to instances of atropisomerism. Atropisomerism is a form of chirality that arises from hindered rotation about a bi-aryl bond. Another consequence of hindered bond rotation is a narrow range of low energy dihedral angles accessible to the molecule. We hypothesize that kinase inhibitors bind their target at specific dihedral angles depending on the kinase, and by controlling the dihedral angle of a kinase inhibitor, we can increase the selectivity of the inhibitor to its target.
We wanted to apply this hypothesis to design an inhibitor of a specific kinase, Bruton’s tyrosine kinase (BTK). BTK is a kinase involved in B cell development and is a major player in types of leukemia and lymphoma. This has lead to several FDA-approved BTK inhibitor drugs, including Ibrutinib, a potent inhibitor that covalently targets a cysteine residue in the binding site of BTK to irreversibly bind the kinase. Unfortunately, Ibrutinib strongly binds to other kinases with similar cysteine residues, causing it to be fairly unselective. To test our hypothesis, we designed several analogs of Ibrutinib, all of which hinder the rotation of the main biaryl bond and narrow the dihedral window. We found that with our lead compound, which added two bulky methyl groups, we gained selectivity towards BTK compared to Ibrutinib, despite a small loss of potency. We also noticed through computational studies, that introducing the methyl groups causes steric strain between the methyl and the acrylamide, the motif that targets the cysteine. This causes the acrylamide to face away from the cysteine, greatly reducing its ability to form the covalent bond. To solve this issue, we inverted the point chiral center from (R) to (S) forcing the acrylamide to face towards the cysteine. Our hypothesis was proven accurate when the (S) analog displayed a 10x increase in potency towards BTK. Our future goal is to create more modifications to the Ibrutinib scaffold to increase potency while maintaining selectivity.