2018 Poster Authors and Abstracts

2018 CSU Biotechnology Symposium
Posters with Author Listings and Abstracts

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Poster #: 1
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: chalcone, soil, microorganisms
Project Title: Effect of Chalcone Combination on Soil and Non-Soil Microorganisms
Author List:
Kendoyan, Sosse; Graduate, Department of Biology, California State University, Fresno, Presenting Author
Shinde, Shantanu; Graduate, Department of Biology, California State University, Fresno, Presenting Author
Calderón-Urrea, Alejandro; Department of Biology, California State University, Fresno
Van Laar, Tricia; Department of Biology, California State University, Fresno

Abstract: Finding an effective nematicide to control plant parasitic nematodes (PPN), such as Meloidogyne incognita, is of paramount importance due to the threat to agricultural production. Nematicides must not only be effective, but environmentally safe. Chalcones are a type of organic molecules that have shown effectiveness in controlling PPN, while being environmentally safe. Our lab identified two specific chalcones useful in controlling PPN, chalcones 17 and 25, which displayed synergistic effectiveness against nematodes. However, the effects of these chalcones have not been tested on soil microorganisms, which are known to have beneficial symbiotic relationships with plants. The goal of this project is to test the effects these chalcones have on the soil microbiota; our hypothesis is that these ch​alcones do not disrupt the soil microbiota. In the first phase of the project, chalcones were tested on individual microorganisms. Serial dilutions of chalcones starting at 2 x 10-3 M were performed on a 96 well plate assay to find the minimum inhibitory concentration of chalcones for seven microorganisms: Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 14579), Listeria monocytogenes (ATCC 7644), Agrobacterium tumefaciens (pGV2260), Saccharomyces cerevisiae (Baker’s Yeast), Trichoderma longibrachiatum (T18648), and Escherichia coli (OP50). The results show that chalcones 17, 25 and mix, are not harmful to the microorganisms tested.
In the second phase of the project, a microbiome analysis will be conducted on soil samples before and after treatment with chalcones. Soil where Meloidogyne incognita infected tomato plant had been grown will be used for these experiments. The microorganisms present in the soil will be detected through 16S and 18S rRNA sequencing. The results will be evaluated quantitatively by comparing the number of detected species present before and after the treatment with chalcones. We are currently conducting these experiments and results will be presented at the symposium.

Poster #: 2
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: Microbial Fuel Cell, Biofuel, Electrogenic Bacteria
Project Title: Ecological Survey of Electrogenic Bacteria
Author List:
Taylor, Zachary; Undergraduate, Biology, California Polytechnic State University, San Luis Obispo, Presenting Author
Sanchez-Avila, Esmeralda; Undergraduate, Engineering, Presenting Author

Abstract: A microbial fuel cell is a bioreactor that utilizes microorganisms to convert chemical energy into electrical energy, facilitated by catalytic materials under anaerobic conditions. Mud can be used as both a fuel source and as an inoculum when it comes to building a microbial fuel cell. For this experiment, fuel cells were created with carbon felt electrodes, wires, and a hacker board outfitted with a capacitor and LED light, all from the MudWatt kit (Magical Microbes, Menlo Park, CA). Mud from eight locations was used as inoculum and nutrient source within each container. Four mud samples came from a saline environment, two samples from a freshwater source, and two samples were made from dry soil. The soil samples were tested for pH, conductivity, moisture content, and carbon/nitrogen content. Voltage output from each fuel cell was measured with a volt-meter and an estimate of power output was made using the MudWatt smart phone app, based on LED blinking rate. Triplicate fuel cells from each soil were monitored over a period of 50 days. Additional experimental fuel cells included autoclaved soil, and freshwater soils with added salt. Saline samples exibited high electrical conductivity (>10 Seimens) correlated to a rapid startup of electrical output. Rapid electrical startup could be replicated by adding exogenous salt to freshwater samples. Autoclaved soils did not produce electrical outputs beyond what could be attributed to soil salt content. Maximum power output was directly corelated to soil carbon content. A compost enriched soil had the greatest carbon content and produced the highest amount of power and voltage for the longest duration. In a confirming experiment, carbon was injected into fuel cells with low carbon content, resulting in a significant increase in electrical output. Future research will focus on identifying electrogenic bacteria in the anodic biofilm microbiome to obtain a better understanding of microbial fuel cell function.

Poster #: 3
Campus: CSU Chico
Poster Category: Agriculture/Biofuels/Environment
Keywords: biodiesel, drywash, coco coir
Project Title: From orchids to biodiesel: coco coir as an effective drywash material for biodiesel fuel
Author List:
Riddell, Meghan; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Ott, Lisa; Chemistry and Biochemistry, California State University, Chico

Abstract: The push for renewable and sustainable energy has continually increased in the recent years. Fossil fuels are being replaced with solar, wind, and other regenerative forms of energy. As such, the production of biodiesel has been increasingly popular among consumers. After being prepared by a simple chemical reaction, biodiesel must be “washed” to remove impurities such as soap and glycerol. These impurities have emulsifying properties that are destructive to the diesel engine. The conventional wash method uses approximately the same amount of water to wash as biodiesel is being produced. This wash process creates an extremely large aqueous waste stream. In order to ensure that biodiesel is a truly sustainable fuel, it is imperative that a dry wash method be employed for the purification of biodiesel. We report here our study of coconut coir as a suitable dry wash material. Using Springboard Biodiesel, LLC’s SpringPro dry wash system as a model, we have produced a sustainable, inexpensive, effective dry wash system with the equal capabilities as industry standard dry wash systems. A coconut coir filtration system is able to remove artificially large quantities of soap, glycerol and methanol from biodiesel, leaving a purified fuel product. This process eliminates aqueous waste generated by the production of biodiesel. The cost associated with a coco fiber column compared to other industry dry wash systems is fractional, making coconut coir sustainable, inexpensive and effective.

Poster #: 4
Campus: CSU Fullerton
Poster Category: Agriculture/Biofuels/Environment
Keywords: Pseudomonas, competition, manganese
Project Title: Pseudomonas putida GB-1: Manganese Coat of Armor
Author List:
Chokry, Jeffrey; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Diamond, Kesean; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Johnson, Hope; Biological Science, California State University, Fullerton

Abstract: Manganese (Mn) is among the most abundant transition metals in earth’s crust and its oxidation to Mn (III & IV) oxides is best facilitated by Mn oxidizing organisms. In addition, the cycling of Mn in the environment has also been tied to the cycling of other elements like carbon, nitrogen, and sulfur, which implicates the importance of these Mn oxidizing organisms in other elemental cycles. The ability to produce Mn oxides from Mn (II) is conserved across diverse phylogenetic lineages and found in many environments, suggesting a physiological purpose for bacterial Mn oxidation. However, the reason for bacterial Mn oxidation is still unclear, but some have suggested the oxides can be used for protection against reactive oxygen species (ROS), protection against UV radiation, and heavy metal resistance. We hypothesize that the extracellular Mn oxide coat created by Pseudomonas putida GB-1 offers physical protection. To do this we performed competition assays between P. putida GB-1, and P. aeruginosa PAO1 with type 6 secretion system (T6SS) in the presence and absence of Mn (II) in the growth media. T6SS utilizes a needle-like apparatus to inject a target bacteria with bacteriolytic proteins which might be hindered by the Mn oxides. Our findings suggest the Mn oxide coat surrounding P. putida GB-1 may be providing a competitive advantage when competed against P. aeruginosa PAO1. Competition assays with a P. putida GB-1 mutant incapable of producing Mn oxides, and a P. aeruginosa PAO1 mutant with a defunct T6SS should confirm the protective role of Mn oxides against T6SS. This study could shed light on a possible purpose for the widespread ability of bacterial Mn oxidation.

Poster #: 5
Campus: Cal Poly Pomona
Poster Category: Agriculture/Biofuels/Environment
Keywords: Animal Science, Monitoring, Ovis aries
Project Title: Commercial tri-axial accelerometry: An economic innovation for animal activity monitoring
Author List:
Pontes, Hailey; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Tran, An; Undergraduate
Brundage, Cord; Animal and Veterinary Sciences, California State Polytechnic University, Pomona

Abstract: In humans “wearable technology” is used to increase the accessibility of biomedical/health information. Tri-axial (3D) accelerometers translate movement in three axes into a composite activity score (AS). This technology provides the human user with an indication of activity level for a given time interval. We tested the function, repeatability and limitations of a commercial 3D-accelerometer (FitBark) as a device for monitoring animal activity in Animal Science Research. Under control conditions we evaluated the sensitivity of the FitBark (n = 4) to manipulation in multiple speeds and axes. Movement in any one axis (50 cm) was consistent and repeatable (0.967 AS +/- 0.135 AS). Movement in multiple axes (2D and 3D orientation) resulted in a proportional increase in AS (2.080 AS +/- 0.194 AS for 2D and 3.099 AS +/- 0.357 AS for 3D). Increases in acceleration were distinguishable over 1 m, but not over a 50 cm course, suggesting bursts of speed may be difficult to identify over short distances. FitBarks were placed concurrently on 5 different locations on four adult sheep. Stationary body locations (neck and thorax) produced lower AS compared to the locomotive part of the body (fore and hind limbs). Animal activity was classified using an observer ethogram every 15 sec for 30 min intervals. The observer classified activity into categories, 1-4 (resting to highly active). This data was compared with FitBark raw minute AS from sheep with collars fitted with 1-2 FitBark devices. FitBark data correlated well with periods of rest and sustained activity. There was minimal inter device variability, and the specificity and sensitivity of the FitBark device ranged from 0.53 to 0.75 in sensitivity, and 0.54 to 0.98 in specificity. The precision ranged from 0.167 to 0.937. Animals monitored for 24 hours (n = 5) demonstrated periods of activity and inactivity consistent with periods for rest and feeding (p < 0.05) . The 24 hours displaced higher activity (AS > 18.54) revolving around feeding times, between 05:30-07:30 and 16:30-17:30, which aligned with observations. Despite some limitations, tri-axial accelerometer are an innovation that can reliably quantify and predict animal activity.

Poster #: 6
Campus: CSU San Marcos
Poster Category: Agriculture/Biofuels/Environment
Keywords: Glutaredoxins, Arabidopsis thaliana, Root growth
Project Title: Functional characterization of the nitrate-induced glutaredoxins in Arabidopsis thaliana
Author List:
Fernandez, Francisco; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Sanchez, Kassandra; Undergraduate, Biological Sciences, California State University San Marcos
Rosas, Miguel; Biological Sciences, California State University San Marcos
Escobar, Matthew; Biological Sciences, California State University San Marcos

Abstract: Glutaredoxins are small redox enzymes that use glutathione as a substrate to reduce disulfide bonds in target proteins. Plants have a far greater number of glutaredoxins than other organisms, mostly due to a unique clade of class III glutaredoxins that are exclusively found in higher plants. Previous studies in our lab demonstrated that a small group of class III glutaredoxin genes are strongly upregulated by nitrate in Arabidopsis thaliana, and that reducing expression of these nitrate-regulated glutaredoxins leads to increased primary root growth. Thus, glutaredoxins appear to link nutrient sensing with plant growth and root system architecture. To further explore this hypothesis, we have generated two groups of transgenic plants that 1) constitutively overexpress several nitrate-induced glutaredoxins and 2) completely inactivate targeted glutaredoxins via CRISPR-Cas9 technology. The coding sequence of the AtGRXS5, AtGRXS6, and AtGRXS8 genes have been sub-cloned into an expression cassette and used to transform A. thaliana. Preliminary results suggest that high-level constitutive overexpression of these genes causes a severe dwarf phenotype in transgenic plant lines, again implicating the nitrate-regulated glutaredoxins as important regulators of plant growth. Work with CRISPR-Cas9 knockout lines is at an earlier stage, but CRISPR vectors targeting AtGRXS6 and the AgGRXS3/4/5/7/8 gene cluster have been constructed and utilized for plant transformation. We are currently screening these transgenic plant lines to identify knockouts in the target gene(s). Because the nitrate-regulated glutaredoxins appear to be important regulators of primary root growth, this work could have significant broader implications related to important agricultural traits such as root depth (drought tolerance) and nitrogen use efficiency. This work was supported by NSF RUI grant 1651584.

Poster #: 7
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: Atribacteria, Thermodesulfobacterium, thermophile
Project Title: Isolation and characterization of a novel Thermodesulfobacterium sp. from laboratory enrichments containing candidate phylum Atribacteria (OP9)
Author List:
Alvarado, Toshio; Graduate, Biology, California State University, San Bernardino, Presenting Author
David, Matthew; Undergraduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Biology, California State University, San Bernardino

Abstract: Atribacteria (OP9) is candidate phylum with no representatives in pure culture, and is found in various anaerobic environments worldwide. “Caldatribacterium”, a thermophilic lineage within the Atribacteria, is currently being maintained in laboratory enrichment cultures with fucose as a carbon source, where “Caldatribacterium” is present at ~95% relative abundance. Metagenomics and 16S rRNA gene tag sequencing techniques showed that the enrichments growing on fucose appear to be a co-culture of “Caldatribacterium” and an uncultivated member of the genus Thermodesulfobacterium, which consists of sulfate-reducing thermophiles. Due to the failed attempts to isolate “Caldatribacterium” by dilution-to-extinction or plating techniques, it has been hypothesized that there is a syntrophic relationship between the two bacteria. To better understand the potential interaction between these two microbes, several isolates of Thermodesuflobacterium were obtained from the fucose enrichments under sulfate-reducing conditions, and one of these isolates was characterized. Genomic comparison of a metagenome bin corresponding to the dominant Thermodesulfobacterium lineage present in the co-cultures to other sequenced members of the genus Thermodesulfobacterium by in silico DNA-DNA hybridization showed percentages lower than 70%, suggesting that the Thermodesulfobacterium in the co-cultures represents a distinct species. The isolate’s 16S rRNA gene sequence is 98.7% identical to other related species, also consistent with it representing a novel species. Temperature and pH ranges for growth were found to be 50˚C to 80˚C, and 5.5 to 8.5, respectively, with a maximum doubling time of ~2 hours. It was found that, out of four different substrates used as an electron acceptor (sulfate, sulfite, thiosulfate, and elemental sulfur), only sulfate was used by the isolate. In addition, out of eleven individual electron donors (hydrogen, formate, lactate, acetate, ethanol, fucose, glucose, xyloglucan, peptone, yeast extract, and casamino acids) tested, only hydrogen, formate, and lactate supported growth. The characterization of Thermodesulfobacterium will help give insight into possible syntrophic interactions between the isolate and “Caldatribacterium”, with Thermodesulfobacterium possibly consuming hydrogen and/or organic acids produced by “Caldatribacterium”, and will also allow for formal proposal for this Thermodesulfobacterium strain as a new species.

Poster #: 8
Campus: CSU Chico
Poster Category: Astrobiology
Keywords: sheathed bacteria, microtubules, microbial genetics
Project Title: Meddling with microtubules: a genetics-based approach for determining the mechanism of sheath production in Leptothrix cholodnii SP-6
Author List:
Stiener, Kirstie; Undergraduate, Department of Biological Sciences, California State University, Chico, Presenting Author
Tamietti, Betsey; Department of Biological Sciences, California State University, Chico
Fleming Nuester, Emily J; Department of Biological Sciences, California State University, Chico

Abstract: Metal-oxidizing bacteria (MOB) such as Leptothrix-Sphaerotilus generate complex surface structures to avoid entombment within an iron or manganese crust and to remain suspended in the water column. Their cellular machinery generates highly ordered organometallic fibrillar microtubule sheaths, which they have been producing for millions if not billions of years. While the structure of these sheaths are now well known to be comprised of long polysaccharide fibrils bonded by amino acids, the assembly machinery and regulation of sheath production in these organisms is not. Determination of sheath-related genes has been complicated due to a lack of a tractable genetic system in these metal-oxidizing bacteria. To establish a genetic system in Leptothrix cholodnii SP-6, we first demonstrated that the cells were sensitive to several antibiotics and were capable of conjugation. We then performed transposon mutagenesis to produce sheath mutants at transposition frequencies of 2.9 x 10-4, similar to what is reported for other organisms. Of the mutants that were produced, sequence similarity analysis determined the disrupted genes to be associated with polysaccharide production (type IV pilus secretion), envelope synthesis (glycosyl-transferase), and chemotaxis (methyl-accepting chemotaxis sensory transducer). Currently, we aim to determine the role of each gene product and their interactions with each other in sheath production by use of mutant reconstruction, complementation assays, and restoration of the wild-type phenotype. From these data we will be able to develop a robust model for sheath formation and assembly

Poster #: 9
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: Methanogen, Hyperthermophile, nitrogen fixation
Project Title: Characterization of nitrogen-fixing hyperthermophilic methanogen Methanocaldococcus strain FS406-22
Author List:
Dimapilis, Joshua; Graduate, Biology, California State University, San Bernardino, Presenting Author
Lie, Thomas; University of Washington, Seattle
Costa, Kyle; California Institute of Technology
Dodsworth, Jeremy; Biology, California State University, San Bernardino

Abstract: Methanocaldococcus FS406-22 is a methanogenic archaeon that was previously isolated from a deep-sea hydrothermal vent. It is capable of nitrogen fixation at 92°C, 28°C higher than the previously known upper temperature limit of biological nitrogen fixation. The initial study reporting strain FS406-22 characterized its growth rates at different temperatures under nitrogen fixing conditions, but a thorough characterization has yet to be performed. This study aims to further characterize FS406-22 using phenotypic tests and comparative genomic analysis, so that it can be formally described as a new taxon.
Growth characterization of FS406-22 included determining pH range, utilization of potential electron donors other than molecular hydrogen, and specific growth rates at different temperatures. These tests performed further physiologically distinguish FS406-22 from other Methanocaldococcus species. Growth with ammonium as a nitrogen source was observed between 60°C and 92°C with optimal growth at 85°C and 87.5°C (doubling time 40 minutes), and growth under nitrogen fixing conditions was not observed above 90°C. This is in contrast to the initial publication describing FS406-22, which observed a maximum growth rate under nitrogen fixing conditions at 92°C and with a fastest doubling time of 3 hours. FS406-22 grew between a pH of 5 and 6.5. In addition to molecular hydrogen, formate is utilized as an electron donor, but growth was not observed with methanol, methylamine, or acetate.
The genome sequence of FS406-22 consists of a 1.76 Mb circular chromosome and a 12.2 kb plasmid. The genome contains 1777 predicted protein-coding genes and 45 RNA-coding genes. The nitrogen fixation (nif) operon lacks a nifN gene present in most other nitrogen fixing organisms, but does contain four hypothetical proteins, three of which are also found in association with nif operons of other thermophilic methanogens. The genome of FS406-22 shares between 28.3-33.5% in silico DNA-DNA cross-hybridization (DDH) to all other closely-related, sequenced members of the genus Methanocaldococcus, below the cutoff delineating distinct species (<70% DDH). Based on these genomic comparisons, 16S rRNA gene sequence phylogenies, and phenotypic characterization, FS406-22 represents a novel species, for which the name “Methanocaldococcus leighii” is proposed. This work paves the way for using this microbe to study of the genetics and biochemistry of nitrogen fixation at high temperatures.

Poster #: 10
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: YdeH, c-di-GMP, feedback inhibition
Project Title: Mutagenic characterization of the noncanonical feedback inhibition site of YdeH
Author List:
Zhou, Yue; Graduate, Biology, California State University, Fresno
Curiel, Joel ; Graduate, Biology, California State University, Fresno, Presenting Author
Montiel, Christian ; Graduate, Biology, California State University, Fresno, Presenting Author
Sulca, Giancarlo ; Undergraduate, Biology, California State University, Fresno
Youn, Hwan; Biology, California State University, Fresno

Abstract: YdeH is one of the nineteen diguanylate cyclases in Escherichia coli which catalyze the synthesis of the bacterial second messenger, cyclic di-GMP (c-di-GMP). The production of c-di-GMP by diguanylate cyclases is under feedback inhibition: when the feedback inhibition site is occupied by c-di-GMP, the enzyme loses the ability to produce c-di-GMP. YdeH feedback inhibition is hypothesized to be weak, as the YdeH motif (197-RDYE-200) deviates from the conserved one, RxxD. In order to analyze the importance of the individual motif residues, we constructed R197A, D198A, Y199A and E200A YdeH mutants and assessed their ability to produce c-di-GMP in vivo. In our E. coli reporter strain, the c-di-GMP produced by wild type YdeH or mutants binds to Clp and blocks the protein from transcriptionally activating the reporter lacZ gene. According to this assay, R197A, D198A and Y199A were indistinguishable from wild type YdeH in terms of c-di-GMP production, suggesting that these residues are not important for feedback inhibition. In contrast, E200A was shown to be superior to wild type YdeH in producing c-di-GMP, and even displayed perturbed growth when overexpressed. It is known that too much c-di-GMP production is toxic to E. coli. This result indicates that E200 is the most important motif residue for the feedback inhibition of YdeH. E200A was then purified through nickel affinity chromatography, and the purified E200A was used for an enzymatic reaction in vitro along with wild type YdeH, and the produced c-di-GMP was estimated via HPLC using a C18 column. Based on this assay, E200A produced c-di-GMP more than wild type YdeH by 1.49-fold, confirming the importance of E200 for feedback inhibition. Further, we made E200D to compare the effectiveness of RxxE with RxxD. E200D and wild type YdeH displayed similar c-di-GMP production, indicating that RxxE is as effective as RxxE in feedback inhibition. Currently, we are randomizing the codon for E200 and screening for mutants with higher c-di-GMP production. This will determine the increase in c-di-GMP production observed in E200A is due to the elimination of glutamate (E), or the introduction of alanine (A), or both. This work demonstrates that altering inhibition site is an effective strategy to engineer YdeH for higher c-di-GMP production.

Poster #: 11
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: site-directed mutagenesis, calcium carbonate crystallization, antifreeze protein
Project Title: Probing the Interactions between Calcium Carbonate Crystals and an Engineered Antifreeze Protein
Author List:
Castellon, Jose; Graduate, Chemistry and Biochemistry, California State University, Los Angeles
Rojas, Fabio; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Kishishita, Audrey; Graduate, Chemistry and Biochemistry, California State University, Los Angeles
Lugo, Joshua; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Wen, Xin; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: Antifreeze proteins (AFP) are highly known for their ability to inhibit ice crystal formation and are characterized by lowering the freezing point of water without changing the melting point. AFPs have been shown to inhibit ice crystal growth by an adsorption inhibition mechanism, in which threonine residues provide the necessary hydrogen bonding groups to interact with the surface structure of the proteins and the water molecules of ice structures. Recently, novel roles for an AFP from the beetles of Tenebrio molitor (TmAFP) in controlling the formation of calcium carbonate crystals have been discovered in our laboratory. Calcium carbonate (CaCO3) is a common scalent. To further understand the interactions between TmAFP and CaCO3 crystals, we designed and prepared a mutant of TmAFP with modified surface charge properties using site-directed mutagenesis. Aspartate residues have been reported to significantly reduce the formation of calcite. The TmAFP mutant, D4, was modified with 4 more aspartate residues that intersperse at an equidistance apart from each other on the surface of the protein. The formation of CaCO3 was studied in the absence and presence of the wild-type as well as the mutant, respectively. The results show that the presence of D4 mutant significantly delays the induction time of CaCO3 crystallization in the tens of nanomolar range and reduces the amounts and sizes of finally achieved CaCO3 solids. The effects of the mutant are stronger than those of the wild-type. These results suggest that the repeat polypeptides may serve as templates for highly efficient antiscalants.

Poster #: 12
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: triosephosphate isomerase, mutagenesis, enzymology
Project Title: Evaluating the Catalytic Role of a Conserved Non-Active Site Residue in Triosephosphate Isomerase
Author List:
Park, Julie Heejin; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Chang, Timothy; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Understanding enzyme function often begins with examination of the active site, but important catalytic roles for residues surrounding the active site have been shown. The active site residues of triosephosphate isomerase (TIM), K12, H95, and E165, have been extensively studied, but the role of the highly conserved E97, situated adjacent to the active site, is not well understood. The proximity of E97 and K12 led to a model where the glutamate positions the active site lysine, and the E97D and E97Q mutations in TIM from Plasmodium falciparum (PfTIM) showed a 100- and 4000-fold decrease in activity, respectively, suggesting an important catalytic role for this residue. In contrast, the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity, challenging E97 as a catalytically critical residue. To investigate further the basis for how the same mutations in essentially superimposable structures give different mutational effects, we studied the effects of mutating E97 in TIM from Trypanosoma brucei brucei (tbbTIM) and Saccharomyces cerevisiae (yTIM). Kinetics, circular dichroism (CD), and fluorescence studies were performed to evaluate the effects of the mutations on enzyme function and structure. A new expression system for generating yTIM mutants was developed to overcome prior challenges in obtaining pure enzymes. Michaelis-Menten kinetics showed the E97D and E97Q mutants led to an ~three-fold decrease in activity, a modest effect compared to the 102-103-fold effect in PfTIM. CD studies showed the mutants contain ɑ-helical contents of 42% (E97D) and 37% (E97Q), similar to the 42% ɑ-helical content in wild type. CD and fluorescence chemical denaturation studies showed the midpoint concentration of denaturant was within 0.2 M for the mutants relative to the corresponding wild-type enzymes, suggesting the mutants have little effect on protein stability. A model for the different functional effects is that variations of the surrounding residues in the TIM isozymes from different organisms allow for such a range of deleterious mutagenic effects. These results highlight the complexity of interactions surrounding the active site. A better understanding of the variations in residues surrounding the active site and their role in positioning residues may aid in the design of enzymes with new functions.
This project was supported by a Single-Investigator Cottrell College Science Award from the Research Corporation for Science Advancement (22489).

Poster #: 13
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Apoptosis, Carbohydrate Metabolism, Bcl-2/xL
Project Title: Effects of high expression levels of anti-apoptic Bcl-2 family proteins on prolymphocytes respiration and lactic fermentation
Author List:
Abed, Ali; Graduate, Chemistry, California State University, Fresno, Presenting Author
Youssef, Elaine; Undergraduate, Biology, California State University, Fresno
Mahmood, Bushra; Graduate, Biology, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno

Abstract: Bcl-2 family proteins are the main mitochondrial regulators of the intrinsic pathway of apoptosis. Many types of blood cancers are associated with high expression levels of Bcl-2 anti-apoptotic proteins such as Bcl-2 or Bcl-xL. Cancer cells also tend to be characterized by high levels of lactic acid fermentation vs. normal cells. This has led to the assumption that cancer cells universally shift their metabolism toward anaerobic respiration (i.e. the Warburg effect). However, several studies also indicate that the rate of aerobic respiration is elevated as well in certain types of tumors such as glioma; suggesting that cancer cells may display elevated rates of both oxidative and non-oxidative types of metabolism. Our hypothesis is that high Bcl-2 and Bcl-xL expression levels are directly associated with metabolic changes observed during cancer. In our study, we used a prolymphocytic murine cell line (FL 5.12) which is a validated model to study transformation processes in blood cancer (Bcl-2-overexpressing FL5.12 cells were shown to induce lymphoma after injection in mouse). FL5.12 cells were cultivated in a media favoring lactic fermentation (i.e. IDMEM supplemented with 25 mM glucose; 25 IDMEM); or a more oxidative type of media containing 5 mM of glucose (i.e DMEM supplemented with 5 mM glucose to mimic normal glycaemia; 5 DMEM). Basal respiration rates of Parental, Bcl-2- and Bcl-xL-overexpressing FL5.12 cells were determined using a Clark-type oxygen electrode (JObasal). Uncoupled (i.e. in the presence of CCCP, JOCCCP) and non-coupled (i.e. in the presence of oligomycin, an inhibitor of the mitochondrial ATP synthase, JOoligo) respiratory rates were also measured. Our results show no significant change in JObasal, JOCCCP, JOoligo or oxidative phosphorylation regime (i.e. [JObasal – JOoligo]/ [JOCCCP – JOoligo]) between the Parental and the Bcl-2/xL-overexpressing FL5.12 cell lines cultivated in either 25 IDMEM or 5 DMEM. However, we observed that Bcl-2 or Bcl-xL overexpression leads to an increase in lactic acid fermentation in both 25 IDMEM and 5 DMEM conditions. Taken together, these results indicate that Bcl-2/xL-overexpression driven increase of lactic acid fermentation in prolymphocytes is not associated to a Warburg-type metabolic shift. These results rather indicate that high Bcl-2/xL expression levels lead to a combined increase of both oxidative and fermentative components of carbohydrate metabolism as previously observed in glioma models.

Poster #: 14
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Cancer , Cell survival, Protein-protein interactions
Project Title: Characterization of GIV-GRP78 interaction during endoplasmic reticulum stress: A promising target to curb cancer cell survival
Author List:
Limso, Clariss; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Finalist
Ngo, Jordan; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Bhandari, Deepali; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Endoplasmic Reticulum (ER) stress occurs when there is an accumulation of misfolded proteins in the ER. Cells facing ER stress initially attempt to restore cellular homeostasis, but if the stress becomes chronic and homeostasis is not achieved within a reasonable timeframe, cells initiate programmed cell death. Cancer cells are able to withstand and survive ER stress better than regular cells. One of the key factors to promote cancer cell survival during ER stress is overexpression of the chaperone Glucose Regulated Protein 78 kD (GRP78). Although it is an ER resident protein, recent studies have shown that when overexpressed, GRP78 can translocate to other cellular locations including the cell surface from where it can activate cytoprotective signals. However, the mechanism by which it does so remains elusive. Our laboratory has recently identified Gα-Interacting Vesicle associated protein (GIV) – a known enhancer of the pro-survival signaling – as a novel binding partner of GRP78. This finding led us to hypothesize that this interaction may be the missing link in understanding the mechanism by which cell surface GRP78 increases cell viability. Because GIV and GRP78 are normally localized in different cellular compartments, it is important to identify the sub-cellular location of this interaction. To look at their subcellular localization, cell lysates of HeLa cells treated with Tunicamycin (an ER stress inducer) were fractionated using differential centrifugation and analyzed by western blotting. Our results show that GIV is present in both membrane and cytosolic fractions during normal as well as ER stress conditions. GRP78, on the other hand, is in the membrane fraction under normal cellular conditions but appears in the cytosolic fraction upon ER stress. Using cell-surface biotinylation experiments, we determined that the cell surface expression of GRP78 is reduced in cells depleted of GIV. Finally, we also mapped the interaction between GIV and GRP78 to the carboxyl terminus domains on both proteins by performing pull-down assays. Together, this work has identified GIV-GRP78 interaction as a potential target to reduce cancer cell viability in the face of ER stress. Our current and future experiments aim at characterizing this interaction further by visualizing its spatiotemporal dynamics using immunofluorescence microscopy and proximity ligation assays.
This project is supported by the NIH grant GM121246 and the NIH MARC program award T34GM008074.

Poster #: 15
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat-shock proteins, Lipids, Mutations
Project Title: HspA1A contains several distinct lipid-binding sites that do not overlap with the chaperone functional sites
Author List:
Daniels, Amanda J; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Smulders, Larissa; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton

Abstract: HspA1A, a stress-inducible seventy-kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cellular survival. In addition to its role in protein homeostasis, HspA1A also localizes at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although these interactions have major physiological consequences their mechanistic details remain elusive and unexplored. We have recently hypothesized that the lipid-binding sites for PS and BMP are distinct and that the lipid-binding and chaperone functions do not overlap. To test these hypotheses, we used a mutation from Tryptophan to Phenylalanine (W90F), which has been described to affect the binding of HspA1A to BMP, and a Lysine to Alanine (K71A), which is known to abolish the hydrolysis of ATP. We first determined whether these mutations alter HspA1A’s stability, intracellular and membrane localization, as well as its ATPase and refolding functions using recombinant proteins and ex vivo experiments. Our results revealed that the W90F mutation does not significantly alter any of the tested functions, but the K71A abolishes both the ATPase and refolding activities, as expected. We then used the lipid vesicle sedimentation (LVS) method and quantified the binding of the wild-type and mutated HspA1A to both PS and BMP. These experiments revealed that the W90F-HspA1A binding to BMP was 50% less than the WT, when 0.4 mM of lipids were used, while the loss-of-binding was only 10% when 1 and 4 mM of lipids were used. Differently, we found that the W90F mutation does not affect the binding to PS under any of the conditions tested. Furthermore, the K71A mutation did not significantly change the binding of HspA1A to PS or BMP under any of the conditions tested. We verified these results using Surface Plasmon Resonance for both mutations and determined that the W90F-HspA1A associates with BMP five times faster and dissociates 18 times faster than the WT. These results reveal that the W90F mutation alters the kinetics of the interaction with BMP, but does not alleviate the binding, while the K71A does not affect the binding to any of the lipids tested. Together these results strongly support the prediction that the lipid-binding sites for PS and BMP are distinct and that the lipid-binding and chaperone functions do not overlap.

Poster #: 16
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, fluorescence microscopy
Project Title: The necessity of Saw1 in recruiting Rad1-Rad10 to single-strand annealing sites increases as a function of increasing DNA flap length in S. cerevisiae
Author List:
Camberos, Juan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fregoso, Fred; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Odango, Rowen; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge

Abstract: Accumulation of DNA double-strand breaks (DSBs) has cytotoxic consequences including genomic instability and cell death. Cells typically repair DSBs using homologous recombination (HR) or nonhomologous end joining. When a DSB occurs between two DNA repeats, cells may employ one of the HR modes known as single-strand annealing (SSA), a non-conservative repair pathway linked to genome rearrangements and oncogenesis. DSB repair by SSA proceeds through an intermediate bearing 3′ overhanging single-stranded DNA flaps which, in the yeast S. cerevisiae, are cleaved by the Rad1-Rad10 endonuclease. A prior in vitro study showed that Saw1 binds to flap DNAs in a length-dependent fashion. Recent in vivo studies additionally show that Rad1-Rad10 recruitment to short flaps (~10 bases) does not require Saw1, but that recruitment to longer flaps (~500 bases) does require Saw1 in G1 phase cells. Determining the precise flap length triggering Saw1-dependent Rad1-Rad10 recruitment in vivo will aid our understanding of SSA.
To determine the flap length triggering a need for Saw1, specialized fluorescent yeast strains were constructed containing a labeled RAD10 gene (Rad10-YFP) and an inducible DSB site tagged by 224 copies of the TetR-RFP protein (DSB-RFP). The DSB site is flanked by DNA repeats separated by varying distances, which produce flap intermediates during SSA of either 20, 30, or 50 bases. We monitored the recruitment of Rad10-YFP to DSB-RFP sites by counting the percentages of cells containing YFP/RFP colocalized foci and comparing results from strains that were either wild-type in SAW1 (WT) or deleted of SAW1 (saw1del) to determine whether Rad10-YFP recruitment is affected by the absence of SAW1. Microscopy data from the 20-base flap substrate shows an ~50% reduction in the percentage of cells with YFP/RFP colocalized foci in compared to WT, which indicates a partial requirement of Saw1 to recruit Rad1-Rad10 to DSB sites. Results from 30-base flaps show an even more diminished induction compared to 20-base. Results from 50-base flaps show no induction of YFP/RFP foci indicating an absolute requirement for Saw1. Together, these results show a trend confirming that the requirement for Saw1 is a function of flap length and indicating that flaps of ~20 bases trigger a partial requirement for Saw1 that becomes more pronounced in 30-base and absolute in 50-base. These results detail our understanding of how Saw1 recruits Rad1–Rad10 during SSA.

Poster #: 17
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, Fluorescence Microscopy
Project Title: Mechanistic requirements of the single-strand annealing (SSA) pathway in Saccharomyces cerevisiae
Author List:
Guzmán, Jr, Jimmy ; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Darouj, George; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Mardirosian, Melina; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Nalbandyan, Linette; Graduate, Chemistry and Biochemistry, California State University, Northridge
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge

Abstract: Double-strand breaks (DSBs) are repaired by many DSB Repair Pathways including single-strand annealing (SSA), when breaks occur between DNA repeats, and synthesis-dependent strand annealing (SDSA), for breaks without flanking repeats. In S. cerevisiae (baker’s yeast), both pathways require Rad52, Saw1, and Rad1-Rad10 endonuclease, which cleaves DNA flaps to complete repair. It is not clear how distally-situated the DNA repeats can be while engaging SSA instead of SDSA, or which domains of Rad52 and Saw1 are involved. We tested the preference of cells in using SSA over SDSA as a function of the distance between DNA repeats flanking the DSB site and which domains of Rad52 and Saw1 mediate complex formation.
To test the distance-dependence of the preference for SSA, yeast strains were cloned containing two DNA repeats flanking an inducible DSB site that were separated by stretches of 10,000, 1,000, or 20 deoxynucleotides (nt). We labeled the DSB site with red fluorescent protein (DSB-RFP) via constitutive binding of TetR-mRFP to a cassette of nearby TetR DNA binding sites, and Rad1-Rad10 by tagging the RAD10 gene with yellow fluorescent protein (Rad10-YFP). Recruitment of Rad10-YFP to the DSB site during repair was then monitored by formation of colocalized RFP/YFP foci during fluorescence microscopy following DSB induction. To determine if repair had proceeded by SSA or SDSA, strains were either wild-type or deleted of RAD51, as RAD51 is required for SDSA but not SSA. Results showed that at all spacings tested (10,000, 1,000, and 20 nt), wild-type RAD51 was not required to observe RFP/YFP colocalized foci. We conclude that the cell prefers the SSA pathway over SDSA even when DNA repeats are as distally-situated as 10,000 nt.
We cloned plasmids for expression of Maltose Binding Protein-tagged Saw1 and 6X-tandem histidine-tagged Rad52, along with multiple constructs expressing truncated versions of each. The proteins were expressed in E. coli and partially purified by affinity chromatography. We then utilized affinity capture assays to test the physical binding between Saw1 and Rad52 and to elucidate which domains are involved. Western blot results of these assays indicate that there may be a specific, direct interaction between full-length Saw1 and full-length Rad52. Thusfar, preliminary results with truncation mutants do not show specific binding. These results inform us about how the Rad1-Rad10 complex may be recruited to SSA and SDSA repair sites.

Poster #: 18
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: RNA editing, Co-immunoprecipitation, Zea mays
Project Title: The Composition of Active C-to-U RNA Editing Complexes
Author List:
Sandoval, Rafael; Graduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author, Eden Award Finalist

Abstract: C-to-U RNA editing in land plants is a correctional mechanism used to avoid the detrimental effects of T-to-C mutations in the genomes of organelles. Furthermore, it is believed a large RNA editing complex is responsible for the editing observed. Pentatricopeptide Repeat (PPR) proteins are RNA binding proteins (RBP) that provide the specificity for the RNA editing complex. These proteins are composed predominately of serial arrays of the degenerate ~35 amino acid PPR domain, and have recently been discovered to specify their RNA target by using a modular combinatorial code. PPR proteins are not the only proteins involved in C-to-U RNA editing. Known, non-PPR, chloroplast editing factors include Organelle Zinc finger (OZ1), RARE1 Interacting protein 2 (RIP2), RIP9, RIP1, and Organelle RRM 1 (ORRM1). It is not known how these proteins interact with each other mechanistically or if they directly contribute to deaminase activity. Also, it is unknown whether all required members have been identified. This project will investigate the members of the RNA editosome critical for RNA editing activity in the monocot, Zea mays. Here through western blot analysis, we show that editing factor RIP9 co-immunoprecipitates with OTP86, OZ1, and CRR28. Two proteomic analysis techniques: Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI/TOF/TOF) and Multidimensional Protein Identification Technology (MudPIT) corroborate the western blot data while also showing hits for all the putative editing factors listed and proteins not previously demonstrated to interact with editing factors. For example, a chloroplast RNA helicase, ISE2, involved in RNA editing but not previously suspected to form a complex, was specifically enriched in RIP9 pulldown samples after the protein bands were separated by SDS PAGE and submitted for MALDI/TOF/TOF analysis. We also demonstrate the ability to pulldown the active RNA editosome out of an active chloroplast extract using RIP9 as the bait protein.

Poster #: 19
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: heat-shock proteins, lipids, plasma membrane
Project Title: Membrane localization of HspA1A is enabled by the lipid phosphatidylserine
Author List:
Bilog, Andrei; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Mauch, Brandon; Undergraduate, Biochemistry, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton

Abstract: Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for regulating cellular homeostasis. Apart from their indispensable roles in protein homeostasis specific Hsp70s also localize at the plasma membrane (PM). In particular, HspA1A, the major stress-induced Hsp70 in humans and mice, is found at the PM of 90% of human and mouse tumors, a phenomenon that has been directly linked to the severity of the disease. However, it is currently unknown how HspA1A, a protein that lacks membrane localization signals, is translocated and anchored to the PM. We have recently showed that HspA1A interacts with several lipids, including phosphatidylserine (PS), a lipid found primarily in the inner leaflet of the PM during normal cell growth. Taking into account that lipids recruit proteins to the PM, we hypothesized that the interaction of HspA1A with PS allows the chaperone to localize and anchor at the PM. To test this hypothesis, we first determined the translocation pattern of HspA1A in stressed human cells and then determined whether the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, inhibits this translocation by competing for binding to PS. Specifically, Hek and HeLa cells were subjected to mild heat-shock and oxidative stress and the PM-localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. The imaging experiments revealed that HspA1A’s membrane localization increases continuously after heat-shock and reaches a maximal value at 8 hours during recovery. Differently, oxidative stress did not result in membrane localization of the chaperone. In the presence of Lact-C2, however, HspA1A’s membrane localization was minimal and did not show an increase during recovery after heat-shock. These results were verified using cell surface biotinylation. In the latter experiments, Hek cells, after stress were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads, and the biotinylated proteins were analyzed by western blots and quantified using densitometry. Collectively, these results reveal that PM-localization of HspA1A is stress specific and strongly suggest that HspA1A’s membrane localization and anchorage depends on its interaction with PS. This discovery establishes the necessary foundation to elucidate the mechanism that regulates the localization of HspA1A at the PM of several types of cancer cells.

Poster #: 20
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Glutathione, Glyoxalase, Cancer
Project Title: Quantification of reduced and oxidized glutathione in cancer cells by enzymatic kinetic assay
Author List:
Cordova, Benjamin; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Chan, Linda; Undergraduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Matavousian, Arvin; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Mason, James; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Tamae, Daniel; Chemistry & Biochemistry, California State University, Northridge

Abstract: Many tumors are known to rely heavily on glycolysis even in the presence of oxygen. This aerobic glycolysis observed in cancer cells has come to be known as the Warburg effect and is utilized in the clinic for imaging and staging tumors using fluorodeoxyglucose positron emission tomography (FDG-PET). A known by-product of prolonged glycolytic flux is methylglyoxal, a highly reactive electrophile. Methylglyoxal can form adducts with cellular nucleophiles such as amino acids, lipids and nucleic acids, leading to the formation of advanced glycation end products (AGEs). The cell utilizes the highly conserved detoxification system, Glyoxalase 1 and 2 (Glo 1 and 2) to convert methylglyoxal to D-lactate using reduced glutathione as a co-factor. Here, we have utilized a modified form of the Tietze method for the quantification of reduced (GSH) and oxidized glutathione (GSSG) [Ngamchuea, K., et. al., Anal. Chem., 2017]. We have determined baseline levels of GSH concentration in the breast cancer cell lines, MCF-7, MDA-MB-231, and MDA-MB-468. Using 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB) and glutathione reductase, we are able to enzymatically measure GSH as total glutathione. For the quantification of GSSG, we used 1,4 benzoquinone (BQ) to mask GSH, followed by enzymatic kinetic measurement. Baseline levels of total glutathione are as follows, data is from three independent determinations: 16.53 ± 2.96 µM GSH/10^6 cells for the MDA-MB-468 cells, 13.30 ± 2.04 µM GSH/10^6 cells for the MDA-MB-231 cells and 17.83 ± 4.22 µM GSH/10^6 cells for the MCF-7 cells. We have also established inter-day reproducibility of GSH and GSSG standards for this novel assay. The validated method will allow us to interrogate the effects of the Glo 1 inhibitor, S-p-bromobenzyl glutathione cyclopentyl diester (BBGC), with respect to changes in intracellular GSH and GSSG concentrations. This will be informative in our studies to determining whether the Glo 1 and 2 detoxification system may represent a putative target in glycolytic tumors. (This project was supported by a CSUPERB New Investigator Award to Daniel Tamae).

Poster #: 21
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: flavoenzyme, enzymology, Biodesulfurization
Project Title: Investigating Amino Acid Residues in the Active Site of Dibenzothiophene Monooxygenase (DszC)
Author List:
Eberle, Kellee; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Mendez, Gilberto; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Gonzalez-Osorio, Liliana; Graduate, Chemistry and Biochemistry, California State University, Northridge
Jirde, Samatar; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Palfey, Bruce; University of Michigan Ann Arbor, Chemical Biology
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge

Abstract: Fossil fuel combustion releases sulfur dioxide into the atmosphere, which is a harmful air pollutant and precursor of sulfate aerosols that contributes to acid rain. It is necessary to reduce atmospheric sulfur levels to mitigate these effects. A current method, hydrodesulfurization, is costly and inefficient at removing sulfur from complex aromatic compounds. Biodesulfurization is a cost-effective alternative that utilizes bacteria to remove sulfur. Rhodococcus erythropolis uses a four enzyme pathway to remove sulfur from dibenzothiophene. The first enzyme of the pathway, dibenzothiophene monooxygenase (DszC), requires molecular oxygen, reduced flavin, and the formation of an intermediate, C4a-hydroperoxyflavin, to sequentially oxidize dibenzothiophene to dibenzothiophene sulfone. This study aims to elucidate the roles of DszC active site residues in catalysis and binding and to enable rational engineering of DszC for future application in fuel refinement. Active site residue mutants were created using polymerase chain reaction, followed by plasmid DNA isolation and sequence confirmation. Steady state activity assays of DszC, quantitated by reverse-phase HPLC, compared the amount of product formed by the mutants relative to wild type-DszC. The ability of the mutants to form the intermediate was studied using stopped-flow spectrophotometry. In this study, seven active site residue mutants were created and the percent of product formation by each mutant relative to wild type-DszC was: His92Asn (12%), His92Gln (20%), Tyr96Phe (12%), His388Ala (38%), His388Asn (39%), His391Val (0%) and Asp392Ala (3%). These results indicate that all of the residues analyzed are necessary for proper product formation. Additionally, the intermediate formation rate constant for Tyr96Phe (1.03 x 104 M-1 s-1) was similar to wild type-DszC (1.69 x 104 M-1 s-1). These results indicate that Tyr96 is not critical for proper intermediate formation, but has another role in catalysis as evidenced by the low (12%) product formation. His92Asn and Asp392Ala were also analyzed using stopped-flow but showed no intermediate formation, signifying the necessity of these residues in intermediate formation. Overall, these results show the necessity of all the residues presented in product formation and the necessity of residues His92 and Asp392 in intermediate formation. This research was funded by Research Corporation and National Institute of Health.

Poster #: 22
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Glyoxalase, Metabolism, Breast cancer
Project Title: Inhibition of Glyoxalase 1 using S-p-bromobenzyl glutathione cyclopentyl diester (BBGC) results in reduced cellular metabolism in cancer cells
Author List:
Ahmad, Saadman; Graduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Cordova, Benjamin; Graduate, Chemistry & Biochemistry, California State University, Northridge
Gardon, Nicole; Undergraduate, Chemistry & Biochemistry, California State University, Northridge, Presenting Author
Bueno, Lisa; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Cambronero, Elisa; Undergraduate, Chemistry & Biochemistry, California State University, Northridge
Tamae, Daniel; Chemistry & Biochemistry, California State University, Northridge

Abstract: Breast cancer is the second leading cause of death in the United States. In 2017, of the 1.6 million new cases of breast cancer, 15 to 20 percent will be diagnosed with the triple negative breast cancer (TNBC) subtype. Patients with TNBC have a poorer prognosis as their tumors lack the estrogen receptor (ER), progesterone receptor (PR) and the Her2/neu receptor which are targets of current therapies. TNBC tumors have been shown to be readily imaged using fluorodeoxyglucose positron emission tomography (FDG-PET) as they consume high amounts of glucose relative to normal tissue. High levels of glycolysis result in elevated levels of methylglyoxal (MG), a toxic by-product of glycolysis. MG is an electrophile and reacts with cellular nucleophiles including, amino acids, lipids and nucleic acids. The highly-conserved, glutathione dependent glyoxalase detoxification pathway is over-expressed in glycolytic cells to convert MG to D-lactate. The main objective of our study is to determine the effect of Glyoxalase 1 (Glo 1) inhibition as a putative target in the therapeutically intractable TNBC. To address this, we are using S-p-bromobenzylglutathione cyclopentyl diester (BBGC) to inhibit Glo 1 and we are measuring Glo 1 expression in the TNBC cell lines, MDA-MB-468 and MDA-MB-231, we are also using the non-TNBC cell line, MCF-7. Using the MTT assay, which measures cell metabolism as a surrogate of cell proliferation, we have constructed dose response curves to determine the IC50 values in the cell lines. The IC50 values from three independent determinations of the Glo 1 inhibitor, BBGC are as follows: 14.59 μM for MCF-7 cells, 19.54 μM for MBA-MB-231 cells and 11.72 μM for MDA-MB-468 cells. Using Western blots, we have confirmed that Glo 1 is expressed in MCF-7, MDA-MB-468 and MDA-MB-231 cell lines. Taken together, we are establishing that metabolically targeting cancer cells via Glo 1 inhibition may be a viable therapeutic target in TNBC and other glycolytic tumors. (This project was supported by a CSUPERB New Investigator Award to Daniel Tamae).

Poster #: 23
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Mucin, Cancer, immunotherapy
Project Title: Humanization of a MUC16 Specific Monoclonal Antibody for the Treatment of Pancreatic Cancer
Author List:
White, Brandy; Graduate, Chemistry, California State University, Fresno, Presenting Author, Eden Award Nominee
Brooks, Teresa; Chemistry, California State University, Fresno
Ayala, Demi; Undergraduate, Chemistry, California State University, Fresno
Brooks, Cory; Chemistry, California State University, Fresno

Abstract: Pancreatic cancer is the third leading cause of cancer death in the USA. Owing to a lack of effective treatments, this disease has a 5-year survival rate of only 8%. Therapeutic antibodies have had spectacular clinical success treating several cancers, raising the hope of developing antibody immunotherapies for pancreatic cancer. MUC16 is a mucin family protein overexpressed on pancreatic cancer cells. MUC16 is known to mediate the migration, proliferation and metastases of tumor cells. The role of MUC16 in tumor growth and metastases suggests that it is an ideal target for antibody immunotherapy. A recently discovered mouse monoclonal antibody (mAb) that binds MUC16 was found to reduce tumor metastases in animal models and may function by inhibiting the growth factor function of MUC16. However, mouse antibodies have limited use in the clinic, as humans mount an immune response against the foreign protein treatment. To reduce the immunogenicity of the MUC16 specific mAb, we have humanized the antibody by grafting the antigen binding loops from the mouse mAb onto a human scaffold. The mouse antibody variable region was sequenced and the structural properties of the antigen binding loops predicted. Compatible human antibody germline gene segments were identified based on predicted structural similarity to the mouse antibody. Chimeric and humanized antibodies were produced by transient transfection in CHO cells and purified. The mouse mAb was produced and purified from hybridoma cells. The binding of the three antibodies to a recombinant MUC16 fragment was assessed using ELISA. All three antibodies bound to MUC16 with similar high affinity indicating that the antigen binding loop graft was effective and that a fully functional, humanized anti-MUC16 therapeutic antibody had been produced. The results presented here pave the way for clinical trails to assess the therapeutic efficacy of the antibody for the treatment of pancreatic cancer. This work is funded by a private research grant from Quest Pharmatech Inc.

Poster #: 24
Campus: Sonoma State University
Poster Category: Biochemistry
Keywords: H2S, Polysulfide, Persulfide
Project Title: The Reaction of Hydrogen Sulfide with Disulfides: Formation of a Stable Trisulfide and Implications to Biological Systems
Author List:
Bianco, Christopher; Johns Hopkins University
Goddu, Robert; Graduate, Biology, Sonoma State University, Presenting Author
Akaike, Takaaki; Tohoku University
Lin, Joseph; Biology, Sonoma State University
Fukuto, Jon; Chemistry, Sonoma State University

Abstract: Hydrogen sulfide (H2S) has long been implicated as a modulator of numerous physiologic processes ranging from dilation of blood vessels to inflammation within tissues, however the biochemical mechanisms associated with these outcomes have not been elucidated. Recently, it has been hypothesized that some of the effects attributed directly to H2S may instead be due to the presence of hydropersulfides (RSSH) and other polysufide species since hydropersulfides and polysulfide species can be in equilibrium with H2S. Importantly, it is already known that hydropersulfides and other polysufide species such as glutathione trisulfide (GSSSG) and cysteine trisulfide (Cys-SSS-Cys) exist in cells at biologically relevant concentrations.
In this study, we identify trisulfides as a stable component of the complex equilibrium between H2S, oxidized thiols, and hydropersulfides. We show that reacting H2S with oxidized thiols such as glutathione (GSSG) or 2-hydroxyethyl disulfide (2-HED) result in a second equilibrium that produces the respective oxidized trisulfide species (RSSSR). These species are much more stable and less reactive than their hydropersulfide counterparts. Interestingly, when cells are treated with extracellular cysteine trisulfide (Cys-SSS-Cys), intracellular cysteine hydropersulfide (Cys-SSH) levels increase dramatically. This is not the case when cells are treated with cystine (Cys-SS-Cys). These data together lead us to hypothesize that relatively stable trisulfide species may function as a labile cellular pool for use in the production of hydropersulfides and that a cellular enzymes such as cystathionine λ-lyase (CSE) may be important to produce hydropersulfides on demand.

Poster #: 25
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: West Nile virus, Medicinal chemistry, serine protease
Project Title: Optimization of a Lead Compound Discovered by High Throughput Screening for Inhibition of the West Nile Virus NS2b-NS3 Serine Protease
Author List:
Martinez, Anastasia; Graduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Finalist
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton

Abstract: West Nile Virus (WNV) is a flavivirus that is transmitted to humans via mosquitoes with most infections being asymptomatic, however 20% progress into more severe symptoms or detrimental neurological diseases. Currently, there are no vaccines or antiviral therapeutics for WNV infections. The RNA is directly translated into the viral polyprotein in the cellular endoplasmic reticulum. The NS2B-NS3 serine protease cleaves the viral polyprotein at multiple positions to allow viral replication in the cell. The enzymatic activity of the NS2B-NS3 protease is required for viral replication and any disruption of enzyme activity would directly affect replication and viral infection. The NS2B-NS3 protease is therapeutic target for the treatment of WNV infections. Our laboratory has completed a high throughput screening campaign for WNV NS2B-NS3 protease inhibitors. An FDA approved drug for the treatment of chronic asthma, zafirlukast, was identified with 83% inhibition of the protease at a concentration of 60 μM. The structure of zafirlukast contains an indole core with cyclopentyl carbamate attached at the 5-position and at the β-position two aromatic rings connected by a sulfonamide linker. It is hypothesized that by decreasing the molecular appendages the inhibitor activity for the N2B-NS3 protease could be increased. We report the structure activity relationship study of zarfirlukast to discover essential structural elements required for NS2B-NS3 inhibition with the goal of designing potent inhibitors. Through chemical synthesis we explored the structural elements around the 5-nitroindole core by varying substituents in three positions: the 5-position, the nitrogen of the indole ring, and the β-position. The cyclopentyl carbamate substituent residing on the 5-position of the indole ring was replaced with a nitro-, amino-, or acetylamide, or removed altogether. Inhibition of the protease was significantly decreased with every modification. However, replacing the cyclopentyl with a phenyl group slightly increased inhibition from 83% to 92% at 60 μM. Substituting the nitrogen methyl group of the indole with hydrogen decreased inhibitor activity of the compound by half. However, moving the entire group attached at the β-position to the nitrogen of the indole resulted in similar inhibition to the parent. These results indicated groups attached to the indole are required for inhibition and future compounds will be modified at the indole core.

Poster #: 26
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Medicinal Chemistry, Protease inhibitor, Neurotoxin
Project Title: Amino Acid Profiling of Peptidomimetic Inhibitors against the Botulinum Neurotoxin
Author List:
Amezuca, Martin; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Cruz, Ricardo; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton, Presenting Author

Abstract: The botulinum neurotoxin serotype A (BoNT/A), a byproduct of the Clostridium botulinum bacteria, is responsible for causing the paralytic disease, Botulism. BoNT/A has a lethal dose of 1.3 ng/kg of body weight in humans, making it the world’s most lethal toxin. Its potency, ease of extraction, and reproducibility raises concern the toxin may be weaponized and used in bioterrorism. The therapeutics currently available to treat the disease are not viable for large scale infections, resulting in mass casualties in the case of an attack. Therefore, efforts to develop alternative therapeutics are essential.
The toxin is a dimer composed of a heavy chain (HC) and a light chain (LC). The HC binds to neuronal cells and aids the LC into the cytosol of the cell. Once in the cytosol the LC, a zinc metalloprotease, enzymatically cleaves SNARE proteins resulting in termination of neurotransmission. This process is irreversible resulting in long recovery times or even death.
Our laboratory focuses on the development and synthesis of small molecules as a therapeutic countermeasure against BoNT/A LC. Previously, our laboratory discovered a promising small molecule inhibitor consisting of hydroxamic acid, Isoleucine, sulfonamide, and 4-chlorobiphenyl. We hypothesized that increasing the number of amino acids in the scaffold would increase the inhibitor’s affinity to the BoNT/A LC thereby providing a potential novel therapeutic for the treatment of botulism. The purpose of our research is to synthesize and optimize lead molecules capable of inhibiting BoNT/A LC via an amino acid profiling approach to determine the optimal sequence for inhibition. Using a solid phase synthesis, various amino acid sequences were coupled onto a solid support to create a diverse library of dipeptides with 4-chlorobiphenyl sulfonyl chloride coupled to the N-terminus of the peptides. The molecules were cleaved from the solid support to yield the hydroxamic acid on the C-terminus. An enzymatic assay was employed to evaluate the molecules ability to inhibit the BoNT/LC. In our efforts, we discovered that amino acids with hydrophobic side chains resulted in good inhibition with five small molecules displaying 84% or greater inhibition at an inhibitor concentration of 15 µM. Based on these results we synthesized a novel molecule that exhibited 75% inhibition of the BoNT/A LC at an inhibitor concentration of 0.5 µM. These results will aid in the design of potent inhibitors for the BoNT/A LC.

Poster #: 27
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Medicinal Chemistry, Neurotoxin , Metalloprotease Inhibitor
Project Title: Investigating peptidomimetic scaffolds for inhibition of the botulinum neurotoxin light chain
Author List:
Palomino, Alejandra; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton, Presenting Author
Rodriquez-Beltran, Sandra; Undergraduate, Chemisry & Biochemistry, California State University, Fullerton, Presenting Author
Morales, Elizabeth; Undergraduate, Chemistry & Biochemistry, California State University, Fullerton
Salzameda, Nicholas; Chemistry & Biochemistry, California State University, Fullerton

Abstract: The botulinum neurotoxin (BoNT) a lethal protein produced by Clostridium botulinum causes the deadly paralytic disease botulism. The BoNT is categorized as a bioterrorist agent due to its ease of production and high toxicity that could be dispersed to large populations. The neurotoxin consists of a heavy (HC) and light chain (LC) that work together to cause muscle paralysis, which can lead to death. Once humans are infected with the toxin the HC attaches to nerve cells and injects the LC into the cytosol. The LC is a zinc metalloprotease that cleaves SNARE proteins inside nerve cells. Once SNARE proteins are cleaved neurotransmission is halted resulting in flaccid paralysis, which is irreversible and in severe cases results in long recovery times or death. An attractive therapeutic treatment for botulism is small molecules that can disrupt the enzymatic activity of the LC. This would provide an excellent therapeutic counteragent for mass distribution in the event of a BoNT bioterrorist attack.
We have identified a molecular scaffold that inhibits the BoNT/LC. The scaffold is centered around amino acids functionalized with a hydroxamic acid on the C-terminus, coupled to a biphenyl aromatic ring via a sulfonamide bond on the N-terminus. Our current inhibitor activity study is on the importance of the sulfonamide linker on the N-terminus and amino acid stereochemistry. The compounds were synthesized through a 3 step synthesis. Various bromine substituted benzoic acids or sulfonyl chlorides were coupled to the amino acid. A Suzuki coupling with aromatic boronic acids gave the aromatic ring system and hydrolysis of the C-terminal gave the hydroxamic acid. A variety of molecules were synthesized that contained sulfonamide or amide linker with both L and D-amino acids.
The molecules were evaluated for BoNT/LC inhibition via an enzymatic assay. Based on the assay results compounds with the sulfonamide linker displayed better inhibition with D-amino acids while the amide linker had better inhibition with the native L-amino acids. The best inhibitor was L-isoleucine coupled via an amide linker with an IC50 of 15 μM. The results indicate the orientation and molecular shape of the scaffold that is controlled by both amino acid stereochemistry and the amide or sulfonamide linker is critical for inhibition. The L-amino acids and amide linker gave improved inhibition because it better mimicked a peptide binding to the active site of the BoNT/LC.

Poster #: 28
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: CRP, DNA binding, F-helix
Project Title: Examining the importance of CRP’s F-helix flexibility in DNA binding and transcriptional activation
Author List:
Mosharaf, Pegah ; Graduate, Biology, California State University, Fresno, Presenting Author
Venkatesh Babu, Gokhul ; Graduate, Biology, California State University, Fresno, Presenting Author
Sangha, Navjot ; Undergraduate, Biology, California State University, Fresno
Manzula, Alisa; Undergraduate, Biology, California State University, Fresno
Youn, Hwan; Biology, California State University, Fresno

Abstract: The Escherichia coli cyclic cAMP receptor protein (CRP) binds DNA via F-helix, the latter helix of the “helix-turn-helix”. The F-helix is composed of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184 and Arg185), and we recently found that Gly184 is another critical F-helix residue in addition to Arg180, Glu181 and Arg185 in the DNA-binding and transcriptional activation functions of CRP. However, glycine does not have any side chain, so the molecular basis for the Gly184 requirement is unclear. Under the hypothesis that Gly184 provides flexibility to the F-helix and that flexibility is critical for the function, we tested whether such flexibility is specific to position 184 or can be furnished by other F-helix residue positions. To test this, we introduced glycine at each one of the positions 180, 181, 182, 183, and 185 replacing the original residues in the G184S background strain and in wild type CRP background strain. G184S is missing the required Gly184 and displays only some CRP activity. If a wild type-level CRP activity is detected from any of the CRP mutants, it then indicates that flexibility can also be provided by other F-helix positions. Based on the in vivo transcriptional activity, none of the mutants showed wild type-level CRP activity. Following explanations are possible for this result. (i) Flexibility is not the critical property of Gly184 for CRP activity. (ii) Flexibility is the critical property but is specific to position 184. (iii) The original amino acids have a critical function, so cannot be replaced by glycine. We are currently analyzing the criticalness of other F-helix amino acids by constructing and characterizing site-directed mutants.

Poster #: 29
Campus: CSU East Bay
Poster Category: Biochemistry
Keywords: biochemistry, high density lipoprotein, fluorescence
Project Title: An assay to monitor apolipoprotein A-I exchange on reconstituted HDL particles in the forward and reverse directions simultaneously
Author List:
Borja, Mark; Chemistry & Biochemistry, California State University, East Bay, Presenting Author
Oda, Michael; Children’s Hospital Oakland Research Institute

Abstract: Objective: Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), spontaneously exchanges between HDL-bound and lipid-free states. HDL-apoA-I exchange (HAE) is typically measured in a single direction (e.g., lipid-free to lipid-bound or lipid-bound to lipid-free). In this study, we present a method to examine both the lipid-free to bound and lipid-bound to free transition on reconstituted HDL (rHDL) particles in a single assay.
Method and Results: Recombinant human apoA-I modified with single cysteine mutations were expressed in bacteria and labeled with Alexa 488 and Alexa 647 fluorophores. ApoA-IAlexa647 was used to synthesize rHDL particles of defined sizes with phosphatidyl choline (POPC) and cholesterol. By varying the protein:lipid ratio, rHDL of 17.0, 12.2, 9.6, 8.4 and 7.8 nm were obtained. These particles were incubated with excess lipid-free apoA-IAlexa488 (10:1 lipid-free to lipid-bound excess) to initiate exchange of apoA-I. Exchange was monitored using non-denaturing gradient gel electrophoresis to separate HDL particles from lipid-free apoA-I. Gels were imaged using FITC and Cy5 filters. The extent of apoA-IAlexa488 binding and apoA-IAlexa647 released from HDL was determined by densitometry. There was no residual fluorescence from apoA-IAlexa488 with the Cy5 filter or apoA-IAlexa647 with the FITC filter. Preliminary results with rHDL of different sizes indicate that apoA-IAlexa488 may bind rHDL more quickly than apoA-IAlexa647 is released. Conclusions: ApoA-I exchange can be successfully measured in both the lipid-free to bound and bound-to free directions using fluorescent apoA-I probes. This method will be useful in studying the mechanism of HAE and the role of HDL particle size and composition on apoA-I conformational dynamics.

Poster #: 30
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Biochemistry, DNA Repair, Molecular Cloning
Project Title: Construction of plasmids to make a specialized yeast strain to investigate timing of Saw1 protein recruitment in single-strand annealing
Author List:
Alon, Alexander; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Rakibova, Yulduz; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Chemistry and Biochemistry, California State University, Northridge

Abstract: DNA damaging agents such as ionizing radiation can lead to double-strand breaks (DSBs) and if left unrepaired may result in cancer. Living systems are equipped with DNA repair pathways that alleviate such damage as it occurs. One mode of repair is single-strand annealing (SSA), a non-conservative pathway that utilizes DNA repeats flanking the DSB site. DSBs are resected 5’ to 3’ exposing complementary strands of the repeats which become annealed by the protein Rad52, generating overhanging 3´ flaps deriving from the DNA originally located between the repeats. Flap cleavage is achieved by the Rad1-Rad10 endonuclease which is recruited by the protein Saw1. The point-in-time at which Saw1 recruits Rad1-Rad10 is not known but likely occurs following annealing. To investigate this question, we are constructing a specialized yeast strain containing two inducible and fluorescently tagged DSB sites to monitor SSA by fluorescence microscopy. Simultaneous induction of both DSB sites will lead to repair by SSA that results in convergence of the two fluorescent signals during annealing, so Saw1 arrival can be pegged to the annealing step.
The first project toward this goal has been to construct DNA plasmids enabling modification of the yeast genome with the features described above. The first plasmid is designed to integrate one of the inducible DSB sites and one of the DNA repeats. Plasmid 1 was constructed by ligating into vector “pLAY500”: 1) a HYG marker from plasmid “pAAH1”, 2) a cassette containing the HIS3 marker situated next to an HO endonuclease restriction site, and 3) flanking yeast DNA sequences generated by Polymerase Chain Reaction (PCR). The desired recombinant plasmid product was confirmed by PCR screens and DNA sequencing, but contained mutations. We corrected these by site-directed mutagenesis which was confirmed by DNA sequencing. The second plasmid is designed to remove an unwanted HO endonuclease site present in the target yeast strain by replacing this portion of the yeast chromosome with DNA sequence lacking the HO endonuclease cassette. Making Plasmid 2 entailed PCR amplification of several DNA fragments which were then linked to each other by adaptamer-mediated PCR. The resulting linked PCR product was ligated into the pGEMT-Easy vector and E. coli transformants were screened. Plasmids 1 and 2 will enable creation of the specialized yeast strain for fluorescence microscopy to elucidate the timing of the end-processing steps in SSA.

Poster #: 31
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: YdeH, dimerization, chimeric protein approach
Project Title: Designing chimeric YdeH proteins to increase c-di-GMP production
Author List:
Chand, Shristi; Graduate, Biology, California State University, Fresno, Presenting Author
Gala, Akshay; Graduate, Biology, California State University, Fresno, Presenting Author
Youn, Hwan; Biology, California State University, Fresno

Abstract: As chemical production of the c-di-GMP is time consuming and complex, enzymatic production of c-di-GMP is pursued. This work concerns YdeH an Escherichia coli diguanylate cyclase which synthesizes c-di-GMP from GTP. YdeH needs to dimerize to be functionally active. We therefore hypothesize that if the dimerization of YdeH increases by any means, that will increase c-di-GMP production. Under this hypothesis, a chimeric YdeH protein was made by fusing catalytic domain of YdeH to a strong dimerization motif, the dimerization domain of GCN4, a representative leucine zipper protein, generating a chimeric protein termed dGCN4-linker-cYdeH. Then, in vivo assay screening for c-di-GMP production was conducted to compare dGCN4-linker-cYdeH with wild type YdeH. Both proteins yielded the same color in our E. coli reporter strain, indicating that dGCN4-linker-cYdeH is as functional as wild type YdeH. Then, purification of the wild type YdeH and dGCN4-linker-cYdeH was performed at a large scale. After purified proteins were run through an SDS gel to confirm the purity, in vitro enzyme reactions were performed for c-di-GMP production, and the produced c-di-GMP was estimated by HPLC using a C18 reverse phase column. The HPLC results contradicted the in-vivo assay results. Unlike wild type YdeH, dGCN4-linker-cYdeH did not show observable c-di-GMP production activity. At present, we do not know exactly what made the chimeric protein less effective in vitro. We are currently lengthening the leucine zipper motif by one or two heptad repeats to improve dimerization and therefore c-di-GMP production.

Poster #: 32
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: enzymology, kinetics, desulfurization
Project Title: Investigating the kinetics, mechanism, and reaction pathway of a biodesulfurizing enzyme from Rhodococcus erythropolis, dibenzothiophene monooxygenase
Author List:
Jirde, Samatar; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Méndez, Gilberto; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Gonzalez-Osorio, Liliana; Graduate, Chemistry and Biochemistry, California State University, Northridge
Eberle, Kellee; Graduate, Chemistry and Biochemistry, California State University, Northridge
Palfey, Bruce; University of Michigan Ann Arbor
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge

Abstract: Fossil fuel combustion is a major source of sulfur dioxide, dangerous to respiratory health and the environment. Sulfur removal from fossil fuels eases these effects, however, the current method, hydrodesulfurization, requires costly high temperatures and pressures. The bacterial species Rhodococcus erythropolis uses a four-step enzymatic process, known as biodesulfurization, to remove sulfur in fuel compounds. The first enzyme in the pathway, dibenzothiophene monooxygenase (DszC), uses reduced flavin and molecular oxygen to generate a flavin intermediate that oxygenates its substrate, dibenzothiophene, to dibenzothiophene sulfoxide, then dibenzothiophene sulfone. Our objectives are to understand DszC’s enzymatic mechanism and steps of the catalytic pathway to engineer DszC to accept various organosulfur compounds and increase catalytic efficiency. Towards our goals, we have purified active DszC; determined steady-state kinetic parameters of DszC as a function of dibenzothiophene, dibenzothiophene sulfoxide, and flavin substrates; performed steady-state experiments to describe DszC’s enzymatic mechanism; and used transient-state kinetics to ascertain the effects of dibenzothiophene and age times on flavin binding. Using affinity chromatography, we purified DszC at 0.5 gram/liter of culture in its native tetrameric state. Reverse-phase HPLC experiments showed DszC had faster product turnover using dibenzothiophene sulfoxide as substrate versus dibenzothiophene (11.8 x 10-3 s-1 vs. 4.84 x 10-3 s-1). HPLC assays also demonstrated that having equimolar flavin and DszC resulted in a faster turnover rate than using excess flavin (4.84 x 10-3 s-1 vs. 1.49 x 10-3 s-1). Varying flavin and dibenzothiophene concentrations under steady-state conditions indicated DszC likely forms a ternary complex with the flavin intermediate and dibenzothiophene to facilitate oxygenation. Using stopped-flow spectrophotometry, we observed fast flavin binding (<1 second) and that having equimolar dibenzothiophene and DszC increased intermediate formation rates. Dibenzothiophene pre-incubation with DszC, however, decreased intermediate formation rates, suggesting reduced flavin is the initial substrate to bind to DszC. These results provided us kinetic and mechanistic insights towards understanding the full catalytic pathway of DszC to support our goals of engineering a robust and efficient DszC enzyme for industrial applications. We thank the NIH and Research Corporation for funding.

Poster #: 33
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Beta-Methylamino-L-alanine, L-serine, Amyotrophic lateral sclerosis-Parkinsonism dementia complex
Project Title: The Role of Beta-methylamino-L-alanine and L-serine In Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex
Author List:
Rivera, Lemuel Vince; Undergraduate, Chemistry , California State University, Fresno, Presenting Author
Goto, Joy J.; Chemistry , California State University, Fresno

Abstract: Beta-Methylamino-L-alanine (BMAA), a neurotoxin produced by cyanobacteria, is part of the etiology of Amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC) which is a set of neurodegenerative symptoms including myopathy, tremors, and dementia. The abrogating role L-serine plays on the effects of BMAA in the short term locomotor tests have previously been demonstrated in our lab. We hypothesize the molecular mechanism could be a misincorporation of BMAA for L-serine. In this investigation, BMAA’s crippling effects and L-serine’s capacity for treatment in a longer term were analyzed through monitoring the circadian rhythm of Drosophila melanogaster (fruit flies).

Methods: Drosophila Activity Monitors (DAM2, Trikinetics) are utilized to measure the activity level of 64 individual fruit flies (gender and age-matched). IR sensors within these monitors quantify the activity level through the number of times the fly passes by the sensor. The control group are fed with 5% sucrose and 2% agar solution while the treated groups include 7 mM BMAA, 7 mM L-serine and 7 mM BMAA + L-serine. Data was measured over a 10-day period (12:12 hr light:dark cycle at 22 OC).

The activity peak differences between control vs. BMAA, show that BMAA only fed flies had a higher level of activity over the initial three days but gradually lessened over the full 10-day period. On the other hand, flies co-fed with L-serine and BMAA show a similar activity to the control, demonstrating L-serine abrogates the effects of BMAA alone. The L-serine only had no effect on activity. We hypothesize that BMAA initiates a hyperactive state which is supported by electrophysiology data of the AMPA/NMDA dependent glutamate receptors in the fruit fly flight muscles. The L-serine is hypothesized to compete with BMAA to reduce the latent protein misincorporation by BMAA. The DAM fruit fly activity data corroborates our findings and demonstrates the long-term effects of co-feeding L-serine and BMAA.
Acknowledgements: This project was funded by the Fresno State Undergraduate Research Grant Award, and the College of Science & Mathematics Faculty Sponsored Student Research Award.

Poster #: 34
Campus: San Francisco State University
Poster Category: Biochemistry
Keywords: Biocatalysis, Thioesters, Dehydrogenase
Project Title: Biocatalytic Synthesis of Thioesters by Phenylacetaldehyde Dehydrogenase
Author List:
Alvarez, Mariah; Undergraduate, Chemistry & Biochemistry, San Francisco State University, Presenting Author
Silva, Stephanie; Undergraduate, Chemistry & Biochemistry, San Francisco State University, Presenting Author

Abstract: Intro- Aldehyde dehydrogenases (ALDH’s) are found in all organisms where they are responsible for cellular metabolism and detoxification. Phenylacetalaldehyde dehydrogenase (NPADH) catalyzes the NAD+-dependent oxidation of phenylacetalaldehyde (PAL) to phenylacetic acid (PAA) in the styrene metabolic pathway of Pseudomonas bacteria. We recently discovered that NPADH can catalyze the synthesis of thioesters from aldehydes by using thiols as co-substrates.
Objective- To study NPADH as a biocatalyst for the synthesis of thioesters, which can serve as building blocks for the production of pharmaceuticals and other compounds of biotechnological value.
Methods- Using a stopped-flow, NPADH was reacted with various thiols, NAD+, and PAL. The production of NADH was monitored by its absorbance at 340 nm. This provided information about pre-steady state reduction of NAD+ by hydride-transfer and the steady state synthesis of thioester products by transthioesterification. NPADH was found to react with mono- and dithiol substrates. Data were plotted and fit using KaleidaGraph nonlinear least squares curve-fitting software.
Results- In general, thiol substrates were found to accelerate the reaction of NPADH as the rate-limiting step changed from hydrolysis to transthioesterification. It was found that the reduction of NAD+ by hydride-transfer was not influenced by thiols. Thiols were found to react with hyperbolic saturation kinetics with apparent Km values ranging from 0.1 mM to 3.3 mM. NPADH has a greater specificity for thiols functionalized with neutral R-groups: 2-mercaptoethanol, N-acetylcysteineamine, and DTT with kcat/Km ranging from 1.9-7.0 mM-1min-1 and lower specificity for thiols with charged R-groups such as mercaptoacetic acid (kcat/Km = 0.1 mM-1min-1 ).
Conclusion and Future Directions- In this study we evaluate a new thioester synthetic reaction for NPADH, an enzyme whose normal function is the oxidation of aldehydes to acids. Thioesters are valuable intermediates for the synthesis of an array of compounds. One of the most important findings was the reaction between NPADH and SNAC, a coenzyme-A surrogate. Future studies will focus on the preparation of a library of SNAC thioesters, which can be used as substrates by N-acyltransferases to synthesize amide bonds. Joining the activity of NPADH with N-acyltransferases enzymes will provide a synthetic route to antibiotics and pharmaceuticals using aldehydes and amines as starting materials.

Poster #: 35
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Inhibitor Cysteine Knot (ICK), Lipid Bilayer, Microscopy
Project Title: The Interaction of Inhibitor Cysteine Knot (ICK) Neurotoxins with Model Biological Membranes
Author List:
Thai, Rich; Graduate, Department of Chemistry and Biochemsitry, San Diego State University, Presenting Author
Polido, Geraldine ; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Ortega, Paola; Undergraduate, Department of Chemistry and Biochemsitry, San Diego State University
Holland, Gregory; Department of Chemistry and Biochemistry, San Diego State University

Abstract: Neurotoxins from spider venom have garnered considerable attention in physiological and pharmacological research because of their ability to modulate a variety of ion channels that are involved in cell signaling and pain. Due to their high stability and high affinity toward ion channels, our research group is interested in investigating the interactions between a class of inhibitor cysteine knot (ICK) peptides and model lipid bilayers with physical characterization methods including Differential Scanning Calorimetry (DSC), Transmission Electron Microscopy (TEM) and Optical Microscopy. Specifically, we have isolated GsAF2 from the Chilean Rose tarantula (Grammostola rosea) due to its high relative abundance in native venom and purified the peptide with High Performance Liquid Chromatography (HPLC). GsAF2 is known to selectively inhibit sodium and mechanosensitive ion channels by inducing stress to the lipid membrane in contrast to interacting with the ion channel protein directly as is the case for other ion channel modulators. To investigate this unique interaction mechanism without the negative effects of image staining, we utilized electroformation to assemble giant unilamellar vesicles (GUV) composed of dimyristoylphosphatidylcholine (DMPC) allowing us to image the ICK-lipid bilayer interactions under a traditional phase contrast optical microscope. To supplement the change in morphology of the lipid membrane determined with optical imaging, we are probing the phase transition temperatures and enthalpies for DMPC bilayers as a function of toxin concentration with DSC. We find that the ICK peptides lyses the DMPC lipid bilayer forming elongated, cylindrical vesicles and ultimately isotropic micelles as the toxin concentration exceeds 1 mmol . In addition to imaging methods, we are probing these interactions at a molecular level with Solid-State Nuclear Magnetic Resonance (SSNMR). This work will help elucidate the interaction between ICK toxins and lipid bilayers.
Funding from CSUPERB, San Diego State University (SDSU) College of Sciences, and the President’s Leadership Fund (PLF) at SDSU is greatly acknowledged.

Poster #: 36
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: Spider Silk, Nuclear Magnetic Resonance (NMR), Dynamic Light Scattering
Project Title: The Structure, Dynamics and Supramolecular Organization of Glandular Spider Silk Proteins
Author List:
Stengel, Dillan; Graduate, Department of Chemsitry and Biochemistry, San Diego State University, Presenting Author
Onofrei, David ; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Department of Chemistry and Biochemistry, San Diego State University

Abstract: Spider silk fibers spun from the major ampullate (Ma) gland are amazing biological polymers that display mechanical properties (strength and toughness) that rival steel, Kevlar and other man-made materials. The Ma gland contains two spidroin (Sp) proteins, MaSp1 and MaSp2. These proteins are intrinsically disordered (unstructured) containing nonrepetitive hydrophilic termini and a primarily hydrophobic repetitive core. The Holland Lab is currently investigating the molecular structure and organization of these glandular proteins using nuclear magnetic resonance spectroscopy (NMR), dynamic light scattering (DLS) and cryo-electron microscopy (EM) techniques. Diffusion NMR results indicate that diffusion of MaSp proteins is restricted to a ~300 nm length scale in vitro in the gland. This size, also observed with DLS, further indicates that glandular silk proteins associate to form micelle-like structures whose hydrophilic ends are solvent exposed with their hydrophobic core buried in the micelle interior. DLS has revealed the monomeric MaSp protein size to be between 20-50 nm in diameter depending on concentration and other experimental conditions. Diffusion NMR indicates a protein diameter between 38 and 44 nm for the monomer in rough agreement with DLS. To correctly determine the protein diameter, protein size as a function of pH was examined using DLS and shown to produce a constant diameter of ~50 nm. This size is consistently observed at low pH, and across a range of protein concentrations. We will propose a potential molecular model for spider silk spinning where a decrease in pH joins the termini of the MaSp proteins while they are still organized in micellar structures. As the silk protein micelle moves into the duct, sodium is removed and the protein strands are aligned due to shear flow while, water is displaced. The displacement of water allows the silk protein oligomers to assemble and form the well-defined crystalline β-sheet regions observed in spider silk fibers.
Funding from the Department of Defense Air Force of Scientific Research (DOD-AFOSR) FA9550-17-1-0282, Defense University Research Instrumentation Program (DURIP)-DOD-AFOSR FA9550-17-1-0409, CSUPERB and the San Diego State University College of Sciences is greatly acknowledged.

Poster #: 37
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: flavoenzymes, transient state kinetics, enzymology
Project Title: Biochemical characterization of DszD, the flavin reductase involved in bacterial biodesulfurization
Author List:
Mendez, Gilberto; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Jirde, Samatar; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Madha, Bilal; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Vey, Jessica; Chemistry and Biochemistry, California State University, Northridge

Abstract: Dibenzothiophene (DBT) is a compound found in crude oil that upon combustion releases sulfur dioxide. The current chemical process used to remove sulfur from crude oil, hydrodesulfurization (HDS), is inefficient because it fails to work effectively on heterocyclic organosulfur compounds, such as DBT. Biodesulfurization (BDS) is a promising alternative to HDS for industrial sulfur removal. Microorganisms, like the bacterium Rhodococcus erythropolis, utilize BDS enzymes to remove sulfur from these organosulfur compounds while retaining the hydrocarbon structure of the fossil fuel components. In our work, we focus on the flavin reductase from the 4S BDS pathway, dibenzothiophene flavin reductase (DszD) from R. erythropolis, which catalyzes the reduction of flavin for subsequent use of dibenzothiophene monooxygenases DszC and DszA. This project entails the use of biochemical and structural experimentation to fully comprehend catalysis of DszD. DszD has been successfully overexpressed and purified to homogeneity from E. coli. Kinetic parameters have been determined using steady-state and transient-state kinetics. We are currently looking at binding interactions between DszD and its substrate, DBT. Our preliminary experiments suggest that DBT may be acting as an effector; which means that DBT is selectively binding to DszD and regulating its biological activity. We plan to follow similar experiments with other sulfur-containing compounds found in crude oil to see there is a direct interaction between substrate and DszD. Our current work will ultimately propel our research towards the long-term goal of improving the BDS pathway for widespread industrial use.

Poster #: 38
Campus: San José State University
Poster Category: Biochemistry
Keywords: calorimetry, thermophoresis, hydration
Project Title: Biophysical Approaches for Studying Desolvation Energy in DNA:DNA Binding Systems
Author List:
Harmon, Caroline; Graduate, Chemistry, San José State University, Presenting Author
Rangel, Juan; Undergraduate, Chemistry, San José State University
Trinh, Christopher; Undergraduate, Chemistry, San José State University
Eggers, Daryl; Chemistry, San José State University

Abstract: Hydration spheres, which consist of the first few layers of water around a molecule, have unique water behavior compared to the disorder of the bulk water phase. These hydration spheres have been shown to be crucial for structural and conformational stability, yet their effect on binding association has been largely ignored. For a 1:1 binding system, we propose to append the classical Gibbs-free energy equation with a term for the desolvation energy which is weighted by the concentration of complex formed at equilibrium. Having a stronger grasp on how desolvation energy affects binding will ultimately aid studies from drug development and metabolism to interpretation of structural models and enzyme activity. To test our equation on DNA hybridization, we have studied small oligonucleotides that follow Watson-Crick base pairing using two different methods. We used (1) Isothermal Titration Calorimetry (ITC); a well-established binding method that measures the heat from direct binding interactions as one reactant is titrated into the system, and (2) MicroScale Thermophoresis (MST); a new method that directly measures the movement of a fluorescently-labeled molecule as heat is applied. When conducting a binding assay, changes in thermophoresis can be translated into a binding curve if the response of the unbound state is different from the bound state. We studied both methods at varying reactant concentrations to obtain the desolvation energy for more than one pair of complementary DNA oligos. In each case, as the concentration of bound DNA increased, we found the binding affinity weakened linearly in terms of the quantity –RTln(K), with R2 > 0.90 and R2> 0.80 for ITC and MST, respectively. Thus, we conclude that the desolvation energy has a negative impact on DNA:DNA binding and, interestingly, this energy is orders of magnitude larger than that of our published model system, chelation of calcium(II) by EDTA. The larger energy value for DNA:DNA binding is not too surprising given (a) the much larger surface area of the DNA binding interface relative to EDTA and calcium, and (b) recent reports of a water “spine” in the minor groove of the DNA double helix. Acknowledgement: This project was funded by a NIH R15 award to DKE, #GM110654.

Poster #: 39
Campus: San Francisco State University
Poster Category: Biochemistry
Keywords: Styrene Monooxygenase, Flavoprotein, Anisotropy
Project Title: Subunit Interactions in the Mechanism of SMO, a Biocatalyst for Green Chemistry
Author List:
Chen, Patrick; Undergraduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Chan, Jennifer; Undergraduate, Biology, San Francisco State University, Presenting Author

Abstract: Styrene monooxygenase (SMO), a FAD-dependent two-component flavoprotein composed of reductase (SMOB) and epoxidase (SMOA), is targeted as a biocatalyst due to its ability to form enantiopure epoxides from a range of styrene derivatives. SMO can produce chiral oxides in 99% enantiomeric excess in the aqueous phase and outperforms the best currently available organic and organometallic synthetic catalysts in reaction rate and efficiency. These features identify SMO as a valuable enzyme for green chemistry that can be used in the synthesis of products ranging from pharmaceuticals to plastics.
One of the outstanding challenges encountered in the development of SMO as scalable biocatalysis is the two-component structure of this enzyme and the associated requirement of coenzyme recycling. Several approaches have been evaluated to optimize reaction conditions including use of surrogates for the flavin and pyridine nucleotide coenzymes required in these reactions. Each of these steps has contributed to the development of SMO and related flavin monooxygenases as biocatalysts, but the nature of the protein-protein and protein-subunit interactions has remained elusive. 4-hydroxybenzoate-3-hydroxylase (PDB id: 1PBE), a similar structure to SMO, reveals an alternative subunit interface (FAD-exchange state) that could provide SMOB direct access to the active site of SMOA. We propose that in catalysis the binding energy of reduced FAD is coupled to the conversion of SMOA from apo-resting state to an FAD-exchange state.
To evaluate this hypothesis of alternate subunit interfaces in catalysis, structure files of SMOA subunits were submitted to the ClusPro 2.0 protein interactions server. Inspection of the resulting models indicate that lysine residues sequestered in the interface of the resting-state subunits are exposed in the exchange-state. After anaerobic labeling of SMOA(FADred) with the lysine reactive probe, Dansyl-Cl, followed by FAD-reoxidation, the enzyme retains activity, but binds tightly to FADox. This feature is consistent with the exchange-state of SMOA, which is postulated to bind FADox with high affinity relative to the resting state. By this procedure we have disrupted the resting-state interface of SMOA and trapped the enzyme in the FAD-exchange state. A structural model of the FAD-exchange complex supported by fluorescence anisotropy measurements will be presented.

Poster #: 40
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: apolipoprotein, protein biochemistry, chimera
Project Title: The C-terminal domain of apolipoproteins AI and E confer structural and functional properties to apolipophorin III
Author List:
Horn, James; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Kakutani, Leesa; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Narayanaswami, Vasanthy; Chemistry and Biochemistry, California State University, Long Beach
Weers, Paul; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Apolipoproteins (apo) AI and E are exchangeable apolipoproteins critical in lipoprotein metabolism and cholesterol homeostasis. Each protein is composed of an N-terminal (NT) helix bundle domain and a smaller less structured C-terminal (CT) domain. The CT domains of apoA-I and apoE are thought regulate many properties of the protein including initiating lipid binding, oligomerization, and interactions with key lipid transporters. A pair of chimeric apolipoproteins was generated by attaching the CT domain of apoAI (residues 179-243) or apoE (residues 201-299) to the CT of apolipophorin III (apoLp-III), a monomeric one-domain model apolipoprotein. The combination of apoLp-III and the CT domains of apoAI and apoE yielded two novel apolipoproteins featuring a helical NT domain and less structured CT domain. Furthermore, the apoLp-III domain of each chimera was modified prevent lipid binding through introduction of Cys residues at residue 20 and 149. This locks the apoLp-III domain in a “closed” conformation, unable to undergo a conformational change needed to interact with lipids. The engineered proteins apoLp-III/CT-apoAI and apoLp-III/CT-apoE had similar α-helical contents compared to their parent proteins. Fluorescence analysis with 8-anilinonaphthalene-1-sulfonic acid, a dye which fluoresces upon binding hydrophobic patches in proteins, showed that each chimera contained more hydrophobic surface area than apoLp-III alone indicating that the chimeras more readily bind to lipid. Crosslinking studies in the presence of dimethylsuberimidate showed that each chimera forms oligomers in similar patterns to the parent protein while apoLp-III remained monomeric in the presence of crosslinker. Furthermore, the capacity for the chimera protein to efflux cholesterol from J774 macrophages was measured; treatment with either chimera resulted in greater efflux than apoLp-III similar to the parent proteins with apoLpIII/CT-apoE having higher efflux capacity than apoE. These results show that oligomerization and cholesterol efflux reside in the CT-domain of apoAI and apoE and can be transferred to another apolipoprotein. Because the CT-domains of apoAI and apoE are less structured it may indicate that increase in α-helical content upon binding lipid may entropically drive this process.
This research was supported by NIGMS of NIH under award numbers GM089564, 8UL1GM118979-02, 8TL4GM118980-02, and 8RL5GM118978-02.

Poster #: 41
Campus: Sonoma State University
Poster Category: Bioengineering
Keywords: Incentive Spirometer, Remote Sensing, Data Logger
Project Title: Designing a Low-Cost Detachable Sensing System for Incentive Spirometers
Author List:
Roohian, Hassan Ali; Undergraduate, Engineering Science , Sonoma State University, Presenting Author

Abstract: Approximately, 12 Medicare patients suffering from pneumonia are readmitted every hour across the U.S. High rates of readmission jeopardize patient’s health and increase the treatment cost. Thus, hospitals constantly seek more effective approaches to prevent respiratory conditions.
Many hospitals use incentive spirometer (IS) devices as a low-cost alternative approach to prevent respiratory conditions. A disposable manual IS consists of an inhalation nozzle and an air chamber that holds a moving plunger indicating the volume of air the patient breaths in.
The PROBLEM with such IS devices is that someone must read and manually monitor the plunger’s movement. Electronic spirometers are expensive and designed for personal use.
The GOAL of this project is to automate the measurement process by designing a low-cost detachable sensing system for a disposable IS to digitally record and display patient’s breathing exercises. The key challenge in designing the detachable system is to choose a low-power, compact proximity sensing device (PSD) that can remotely monitor plunger’s movement without modifying the original IS structure.
In our METHOD, we characterized 4 PSDs, each using a different sensing technology: visible light beam, infrared, ultrasonic, and time-of-flight (ToF). To characterize each sensor, we developed a test-setup to hold each sensor under IS’s air chamber in exactly the same position. Using the test-setup, we recorded the power usage and data reported from each PSD while manually moving the plunger under different ambient lighting conditions, temperature, nominal input voltage, and capturing range. Our test RESULTS indicated that the ToF sensor exhibited 96% accuracy in terms of recording the plunger’s position. Our tests also showed that ToF’s measurements were highly stable under different test conditions. The ToF’s overall superior linear performance is attributed to its narrow light source. However, according to our tests, ToF uses slightly more power, reducing the system lifetime by 12%. Our test results confirmed that ultrasonic waves, infrared, and visible beams passing through the IS’s thick plastic air chamber tend to bounce back significantly. Therefore, these sensors on average exhibited about 17% lower accuracy and lower stability in terms of recording the plunger’s position. Our NEXT STEP is to build 5 systems using ToF for clinical testing by our collaborators at the Health Sciences at the Univ. of Oklahoma.

Poster #: 42
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: Spider Silk, Silica Nanoparticles, Biomaterials
Project Title: Synthesis and Characterization of Spider Silk-Silica Nanocomposite Biomaterials
Author List:
Pahlavanneshan, Mahsa; Graduate, Department of Chemistry and Biochemistry, San Diego State University, Presenting Author
Thai, Rich; Graduate, Department of Chemistry and Biochemistry, San Diego State University
Spurrier, Dani ; Undergraduate, Department of Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Department of Chemistry and Biochemistry, San Diego State University

Abstract: We aim to investigate the adsorption and binding of spider silk mini spidroin peptide mimics on Mesoporous Silica Nanoparticles (MSNs). Dragline spider silk (major ampullate) fibers are comprised of two proteins, major ampullate spidroin 1 and 2 (MaSp1 and 2). These silk proteins have highly repetitive core sequences consisting of certain consensus motifs, mainly poly(Ala), Poly(Gly-Ala), Poly(Gly-Gly-X) and Gly-Pro-Gly-X-X. This study is focused on synthesizing and characterizing the spider silk peptide mimics (Ala)n and Gly-Ala-Gly-(Ala)5-Gly-Ala-Gly variants that comprise the region that forms the spider silk’s β-sheet core and hypothesized to impart spider silk its remarkable strength. The various peptides are being synthesized using a CEM High Efficiency Solid Phase Peptide Synthesizer (HE-SPPS), purified using reverse phase High Performance Liquid Chromatography (HPLC), Flash Chromatography, and Thin-Layer Chromatography (TLC), and structurally characterized with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and solution Nuclear Magnetic Resonance (NMR) spectroscopy. MSNs are also being synthesized via sol-gel methods, and characterized using Transmission and Scanning Electron Microscopy (TEM and SEM), Thermogravimetric Analysis (TGA), InfraRed (IR), Solid State Nuclear Magnetic Resonance (SSNMR) spectroscopy, X-ray Diffraction (XRD), and Brunauer–Emmett–Teller (BET). We have successfully synthesized and characterized both the silk peptide mimics and silica MSNs and will report on these results. The long-term goal of the project is to interface the spider silk peptide mimics with silica nanoparticles to produce spider silk-silica nanocomposite materials. We have found in our laboratory that some types of silica surfaces can effectively catalyze peptide thermal condensation reactions. We are testing whether this chemistry can be used to cross-link and assemble spider silk-silica nanocomposites. If successful, one can envision utilizing such a nanocomposite biomaterial for hard tissue repair (e.g. bone and teeth).
Funding from CSUPERB, San Diego State University (SDSU) College of Sciences, and the President’s Leadership Fund (PLF) at SDSU is greatly acknowledged.

Poster #: 43
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: gait rehabilitation, orthotics, six-bar linkage
Project Title: Building a Knee-Ankle-Foot (KAFO) Lower Leg Orthotic for Restoration of Normal Physiological Gait
Author List:
Ghosh, Shramana; Graduate, University of California, Irvine
Masone, Kevin; Graduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Robson, Nina; Mechanical Engineering, California State University, Fullerton, Presenting Author

Abstract: The aim of this research is to design a lower-leg orthotic device based on a single degree of freedom multi-loop linkage to facilitate task-specific repetitive lower-extremity physical training, as well as assist people with temporary limited mobility in walking. We hypothesize that devices that closely mimic natural human walking motion will be able to provide more desirable design solutions, such as comfort, stability and safety among others. The first prototype of the device was a hands-free mobility device that incorporated a multi-axis knee consisting of a four-bar linkage. By comparing the performance of the multi-axis knee device with that of a commercially available stiff knee crutch substitute, we found that a user with our four-bar knee device shows an increased stride length as compared to the stiff knee crutch substitute. In addition, the test subjects reported increased strain in the non-supported leg as well as reduced stability while walking with the multi-axis device.
In the second iteration, a six-bar linkage that coordinates the angular motion of the knee and the ankle was selected, so as to guide the foot though the natural walking trajectory. The natural trajectory was specified by using motion capture data from a healthy human subject walking on a treadmill. The linkage synthesis equations were solved using polynomial homotopy methods. Six linkages were found that move smoothly through the specified natural trajectory, and had pivot positions that can be easily located and attached to the human lower extremity. The most anthropomorphically looking design was selected for manufacturing the second device prototype. Testing the device on human subjects revealed that it can mimic knee motion satisfactorily, however large deviations were found at the ankle joint.
In order to obtain more accurate ankle motion and end-effector trajectory, additional criteria such as stricter compliance with the desired trajectory and singularity avoidance, that the linkage comprising the device must satisfy were identified and incorporated into the synthesis process as optimization problem with penalty functions. Three new design candidates were obtained and the candidate that has the least deviations from the desired trajectory was selected for the design of the third prototype device. The new prototype will be tested on a human subject to quantify its performance as compared to the desired performance using motion capture, force-plate and electromyogram data.

Poster #: 44
Campus: CSU Sacramento
Poster Category: Bioengineering
Keywords: diabetes, foot ulcers, temperature monitoring
Project Title: Temperature Monitoring Footwear: Detecting Early Signs of Diabetes-related Complications
Author List:
Bachinsky, Alex; Undergraduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Lazarev, Vadim; Undergraduate, Electrical and Electronic Engineering, California State University, Sacramento
Moody, Erik; Undergraduate, Electrical and Electronic Engineering, California State University, Sacramento
Muslih, Khalid; Undergraduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Smith, Warren; Electrical and Electronic Engineering, California State University, Sacramento

Abstract: Twenty-nine million people in the United States (9.3 percent) have diabetes, and 1.7 million people aged 20 years or older were diagnosed with diabetes in 2012. Experts suggest that around 10 percent of people with diabetes develop foot ulcers at some point in their life. While early detection is crucial to mitigate the injury and suffering caused by foot ulceration, this often does not occur, because many diabetics also experience loss of sensation due to nerve damage (peripheral neuropathy). This means that problems can quickly escalate in severity and may require amputation or even become life threatening. The objective of this project is to develop an in-shoe monitor of temperature at several locations on each foot. A temperature difference of 2-3 degrees between corresponding locations on the left and right feet could serve as an early sign of inflammation, which is indicative of infection. This information will be valuable for medical intervention. We use small, low-power thermistors that are suitable for placement at sites in the shoe insole. We use small Bluetooth Low Energy (BLE) chips in the left and right shoes, one we call Central and the other Peripheral. The shoe with the Peripheral BLE chip also contains a compact mobile telephony (GSM) module. The BLE chips sample the thermistor temperature values, and the Central chip wirelessly communicates with the Peripheral chip to allow comparison of temperatures between the left and right feet. If there are signs of inflammation, the GSM module allows a physician to be notified. Our system operates on a thin, light lithium ion polymer power cell, and we recharge it via an intelligent USB charger. To supplement the power cell, we use small, thin-film solar panels connected to ultra-low power energy-harvesting converters. To date, we have tested our system of temperature sampling and wireless comparison between feet and using the GSM module for one pair of thermistors. We now will expand the system to six pairs of thermistors and completely interface all the system components to develop a final fully-functioning, power-efficient product. We also will embed our system components into actual therapeutic shoes.

Poster #: 45
Campus: San José State University
Poster Category: Bioengineering
Keywords: Rare earth elements, adaptive evolution, Methylobacterium extorquens
Project Title: Evolution of a Methylotrophic Extremophile for Biorecycling Rare Earth Metals from Electronic Waste
Author List:
Grace, Joseph; Graduate, Biological Sciences, San José State University, Presenting Author
Jacob, Mariam; Undergraduate, Biological Sciences, San José State University, Presenting Author
Hecker, Kathleen; Undergraduate, Biological Sciences, San José State University
Hoeber, Caitlin; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Biological Sciences, San José State University

Abstract: Methylobacterium extorquens is a methylotrophic bacterium capable of using single carbon chemicals like methanol as a sole source of carbon and energy. In 2011, it was shown that methanol oxidation could be carried out by methanol dehydrogenase enzymes that require rare earth elements (RREs) as cofactors. Prior to this discovery, there was no known inherent use of REEs in any biological pathway. REEs are vital to many technologies which facilitate our society such as electronics and transportation. The last domestic REE mine went bankrupt in 2015 due to costly and environmentally destructive mining practices making the U.S. dependent on foreign nations for REE supply. We are developing M. extorquens as a platform for recovering REEs from waste materials such as discarded electronics and fly ash, a coal combustion byproduct. We previously showed that M. extorquens can obtain REEs from mining ores and computer hard drive magnets. Using growth curve analysis monitoring CFU/mL, we show that 0.5% fly ash and pulverized smartphone powder can also serve as sources of REEs for M. extorquens and support REE-dependent growth. However, when supplied in the growth media at concentrations of 5% or higher, these sources are toxic and growth is inhibited. For M. extorquens to be a commercially viable REE recovery platform, a resistant derivative needs to be developed while retaining the cell’s ability to uptake REEs. To generate resistant strains, an adaptive evolution approach was taken. Strains were exposed to UVC and challenged with increasing concentrations of blended phone in an iterative process. After four rounds of adaptive evolution, strains achieved ~20-fold resistance to blended phone. Ongoing work continues these adaptive evolution experiments to increase resistance further. Eventually, comparative genomics on resistant strains will be used to identify mutations leading to resistance. This work will promote the commercialization of our existing biological REE recycling platform thereby decreasing U.S. reliance on foreign REE suppliers. This work was supported by a SJSU graduate research fellowship.

Poster #: 46
Campus: San Francisco State University
Poster Category: Bioengineering
Keywords: Electromyography (EMG), pattern recognition, mobile and cloud computing
Project Title: Integrating Mobile and Cloud Computing for Electromyography (EMG)-based Gesture Recognition
Author List:
David, Alexander; Graduate, School of Engineering, San Francisco State University, Presenting Author
Zirbel, Chloe; Undergraduate, School of Engineering, San Francisco State University, Presenting Author
Chang-Kam, Kattia ; Cañada College
Abad, Karina ; Cañada College
Colin, Ricardo ; Cañada College
Malloy, Cameron ; Cañada College
Tolentino, Charles ; Cañada College
Zhang, Xiaorong; School of Engineering, San Francisco State University

Abstract: Electromyography (EMG) is a technique for measuring the electrical activity generated by skeletal muscles. These EMG signals contain neural information that can be recorded and evaluated to discern movement intentions. EMG pattern recognition (PR) is an intelligent method for deciphering neuromuscular information from EMG signals to identify users’ intended movements. EMG PR has great potential to provide efficient and natural human-machine interaction in a wide variety of applications such as neural-controlled prostheses, assistive robots, and virtual input devices. To apply EMG PR-controlled systems in practice, these systems must be portable, real-time, and robust. However, existing platforms for developing EMG PR algorithms are typically based on MATLAB and running on PCs, which prohibits their widespread to real applications. This project aims to develop a low-cost, portable, and flexible research platform for developing EMG PR-based real-time systems. The platform provides real-time processing speed and sufficient storage capacity for computationally complex EMG PR algorithms by integrating mobile and cloud computing techniques. Specifically, a mobile Android application has been developed which provides easy interface with a commercial EMG sensing armband Myo (Thalmic Labs) and a modular software engine seamlessly integrating a variety of signal processing modules, from data acquisition through pattern recognition, to real-time evaluation and control. In addition, an interface between the Android application and the Amazon Web Services Cloud Server has been built which allows real-time cloud computing and storage. Real-time experiments have been conducted on 10 able-bodied subjects for recognizing eight hand gestures. Four EMG PR algorithms have been evaluated including Linear Discriminant Analysis, Logistic Regression, Decision Tree and, K-Nearest Neighbor (KNN). The pattern classification accuracies averaged across all subjects were over 95% for all four PR algorithms. The results from a post-experiment user survey also indicated that the subjects found the developed application responsive, aesthetic, and easy to use. Extensive analysis has been ongoing to evaluate the relations between algorithm complexity, data size, computing power of mobile devices and the cloud, as well as mobile-cloud communication latency.
This project was supported by the US Department of Education (MSEIP P120A150014) and CSUPERB.

Poster #: 47
Campus: Sonoma State University
Poster Category: Bioengineering
Keywords: Microbial Fuel Cells, Sensor, Wireless
Project Title: Wireless Data Transmission Using Microbial Fuel Cells
Author List:
Gonsalves, John; Undergraduate, Engineering Science, Sonoma State University, Presenting Author
Young, Grant; Undergraduate, Engineering Science, Sonoma State University, Presenting Author

Abstract: Power generated by microbial life is largely an underutilized source of green energy. The GOAL of this project was to harvest and deliver a sufficient amount of power from garden soil samples assembled into Microbial Fuel Cells (MFC) in order to transmit a byte of data between two previously design low-power sensor nodes.
As part of our research METHOD, we “first” measured the open-circuit voltage (OCV) of different MFS cell-configurations. For each MFC cell we placed a graphite cloth (the anode) at the button of the cell covered with garden soil and a copper wire (the cathode). We then measured OCV of 6, 14, and 28 cells connected together in series. Next, we connected each multi-cell MFC to a 2-ohm load and measured the produced power under different soil moisture conditions (10%-79%). In the “second” part of our research, we connected each multi-cell MFC to a 1F supper capacitor (SC). Once the SC was charged, the power was directed to the booster circuitry (BC) to provide 5V. We tested two different SCs (Glassy carbon and EDLC Gold both from Panasonic) and BCs (FemtoGen and SparkFun LiPower) to identify the best circuit combination in order to deliver the maximum power at 5V for the longest discharge period.
Our RESULTS indicated that with about 30% soil moisture, the OCV for a single-cell MFC reached 13 mV. For 6, 14, and 28 cell-configurations the measured OCV was 30mV, 160 mV, and 210 mV, respectively. In our tests the Glassy carbon SC exhibited very short discharge time (20 micro-sec.), whereas the EDLC Gold offered about twice the discharge time. Comparing FemtoGen and SparkFun LiPower modules, we observed that the FemtoGen booster module yielded about ¼ of power generated by LiPower. We then connected the MFC, EDLC Gold, and LiPower booster together and measured the output power as we increased the number of cells in the MFC. For 6, 14, and 28 cell-configurations we measured 8mW, 36 mW, and 56 mW, respectively. For each case we used a 2-ohm load. The recharge time for the 28-cell configuration from 12% to 99% of supply voltage was measured to be 26 seconds, which is relatively fast. Our tests confirmed that the 28-cell MFC using the LiPower and EDLC capacitor can generate maximum power for transmitting a single byte wirelessly. Our NEXT STEP is to compare discharging period of Aerogel SCs and Li-ion batteries in order to be able to transmit multiple bytes between the sensor nodes.

Poster #: 48
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: Neuralprosthetic, Neural Probes, BCI – Brain Computer Interface
Project Title: Novel MEMS Pattern Transfer Technology for Origami-Styled 3D Integrated Intracortical/Epicortical Neural Probes
Author List:
Cea, Claudia; Graduate, Mechanical Engineering , San Diego State University, Presenting Author
Gautam , Abhivyakti; Graduate, Mechanical Engineering , San Diego State University, Presenting Author
Kodira-Cariappa, Brinda ; Graduate, Mechanical Engineering , San Diego State University

Abstract: A new class of novel 3-D origami-style neural probes with a combination of surface and penetrating electrodes is presented in this study. These devices allow for the simultaneous recording of electrophysiological signal from both the brain surface (ECoG) and at varying depths (single neuron) opening up new opportunities for fundamental investigation of complex electrical signaling involved in complex tasks. The microelectrode arrays (MEAs) are fabricated from lithographically patterned glassy carbon (GC) integrated into a flexible polymer substrate. The superiority of GC over thin-film metals such as platinum microelectrodes with regard to corrosion-resistance is demonstrated using Tafel extrapolation method. The corrosion rates were compared between the two types of microelectrodes in a phosphate buffered solution, a pseudo-physiological solution that simulates the composition of body fluids. On the basis of preliminary experiments conducted, platinum exhibited corrosion rate approximately ten times that of GC electrodes, hence justifying GC to be a preferred choice of material. Additionally, both in-vitro and in-vivo characterizations of the devices were performed. The electrochemical in-vitro characterization was carried out in a 0.9% Sodium chloride aqueous solution through electrochemical impedance spectroscopy (EIS). This is implemented by superimposing a sine wave onto the open circuit potential in the frequency range of 1 to 105 Hz . The in-vivo characterization was accomplished by acute experiments, where the device was implanted into multiple rats and intracortical spikes at different depths and surface evocated potentials were documented. We plan on further expanding the capability of our devices by replacing the polymeric insulating layer with a patternable elastomer layer. We are investigating the use of Polydimethylsiloxane as a potential material for this purpose. This would make our devices be not only flexible but stretchable which could decrease the variation of impedance after implantation due to the conformal ability of a stretchable device. The electrodes can further be enhanced by treating the surface of the GC electrode Physically and Electrochemically to perpetuate its hydrophilic properties for a prolonged duration.
This work was supported by National Science Foundation [Grant number EEC-1028725] under the ERC program.

Poster #: 49
Campus: CSU Fullerton
Poster Category: Bioengineering
Keywords: mirror neuron, physical therapy of post-stroke patients, customer discovery
Project Title: Customer Discovery Interviews for the Augmented Reality Wearable Device (ARWED) used in the Physical Therapy of Post-Stroke Patients
Author List:
Mocanu, Iulian; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Tang, Wesley; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
Robson, Nina; Mechanical Engineering, California State University, Fullerton

Abstract: Retraining the motor system of subjects suffering partial loss of motor ability due to stroke is a task that presents a number of difficulties. In many cases, only partial success is accomplished after long training sessions. The discovery of mirror neurons has led to extensive research that has revealed a neural network that supports humans’ ability to learn through action imitation and observation. Currently the training of individuals that has suffered from stroke is done using a box with mirrors. While the mirror therapy is suggested to be a simple, inexpensive and patient-directed treatment that may improve upper-extremity function for stroke patients, it has been shown that the patients find it boring and the nurses cannot accurately detect and measure the patients’ progress to propose appropriate training, specific to each patient.
The aim of this research is the marketing of the developed by our team ARWED that creates a virtual reality environment for the human arm in order to increase the success of training of post-stroke patients. Compared to the current mirror-box therapy, we believe, that the ARWED will help physical therapists improve the rehabilitation process of post-stroke patients by providing a reliable tracking of the patients’ progress, customized virtual environment for entertainment, and stimulating protocols.
During the 2017 CSU I-Corps Summer Sprint course, our team conducted more than 30 customer discovery interviews. The interviews revealed the following major lessons learned and future team activities: (i) gain a clear idea on the measures of clinical improvement, i.e. cost and time effectiveness, as well as patient satisfaction with using the ARWED in comparison with the standard mirror-box therapy; (ii) consider private facilities for pilot testing in parallel with bigger hospitals and outpatient centers; (iii) put efforts into selling or renting the device to patients for home use; (iv) gain a better understanding of Food and Drug Administration (FDA) Regulatory Compliance.
Based on our team’s arc of learning at the CSU I-Corps, final reports and reviews from the evaluation panels during the final presentation meeting, the CSU I-Corps teaching team recommended our team to NSF for the National Team’s program. At the 30th Annual CSU Biotechnology symposium we are planning to present our results from the customer interviews conducted, our experience as part of the 2017 CSU I-Corps cohort and outline some future directions.

Poster #: 50
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: VNP20009, cytolethal distending toxin, triple negative breast cancer
Project Title: Tumor-Targeted Salmonella VNP20009 Expressing Cytolethal Distending Toxin are Cytotoxic Toward Triple Negative Breast Cancer Cells
Author List:
Carrafa, Jamie; Graduate, Biology, California State University, Northridge, Presenting Author
Vincent, Lena; Graduate, Biology, California State University, Northridge
Bermudes, David; Biology, California State University, Northridge

Abstract: Attenuated Salmonella VNP20009 preferentially replicate within the tumors of both mice and humans. However, in human clinical studies where this bacterium was demonstrated to be safe, the presence of the bacteria in tumors did not result in an anti-tumor response. In vitro, VNP20009 culture supernatants have negligible ability to kill cancer cells. In order to enhance the tumor cell killing ability of VNP20009, we engineered VNP20009 to express the Salmonella paratyphi A cytolethal distending toxin (CLDT), which is not naturally occurring in VNP20009. Culture supernatants of VNP20009 expressing the CLDT operon were highly cytotoxic toward MDA-MB-468 triple negative breast cancer cells as measured by the methylthiazol diphenyltetrazolum (MTT) mitochondrial viability assay. We further investigated CLDT C-terminal fusions that have the potential to confer tumor-cell specificity through tumor-selective augmentation of apoptosis. We found that altering the position of the C-terminal cysteine diminished the cell killing ability of CLDT when making fusions to the canary virus proapoptotic protein apoptin, and fragments of apoptin. The highest activity of C-terminal fusions was obtained with fusion to the promyelocytic leukemia (PML) interacting fragment of apoptin, amino acids 82-121, which partially retained cell killing ability of the CLDT. We have also disrupted the bipartite nuclear localization signal (NLS) of CLDT and are assessing the tumor cell specificity of the CLDT-PML fusion in an NLS(-) CLDT background.

Poster #: 51
Campus: CSU Sacramento
Poster Category: Bioengineering
Keywords: therapeutic footwear, patient compliance, wearable monitor
Project Title: An In-Shoe Compliance Monitor for Therapeutic Footwear
Author List:
Estepanian, Sevak; Graduate, Electrical and Electronic Engineering, California State University, Sacramento, Presenting Author
Smith, Warren; Electrical and Electronic Engineering, California State University, Sacramento
Thomas, Douglas; Electrical and Electronic Engineering, California State University, Sacramento
Sobel, Robert; Pedorthic Footcare Association

Abstract: Therapeutic footwear often is prescribed to treat patients with diabetes. If the therapeutic footwear is not worn as requested, the effectiveness of the therapy is compromised, and the cost is not justified. We aim to develop a miniature, low-cost, single-use in-shoe monitor to allow the clinician to track the patient’s compliance in using the footwear. The monitor should not require any attention by the patient or interfere with the patient’s experience wearing the shoe. In order to meet the size constraints, we had to identify components that could provide the needed operating power and robust functionality for up to 8 weeks of unattended monitoring. A communication capability between the monitor and the clinician’s computer for data transfer also is necessary. We identified and utilized a low-cost, ultra-low-power microcontroller. According to the microcontroller specifications, a small coin cell should be able to provide sufficient power for the desired monitoring period. We chose a thin force sensitive resistor (FSR) placed on the underside of the insole to verify shoe usage by sensing the pressure of the patient’s foot. We developed a software algorithm to process and store the usage data into the memory of the monitor. The algorithm identifies and stores usage data in 15-min. epochs for the data acquisition period. It also provides the clinician with a summary of the total number of hours of daily usage. For each 15-min. epoch that the shoe is in use, the algorithm also categorizes and stores the patient’s level of physical activity as low, moderate, or vigorous. Our preliminary experimental results confirm that a small coin cell can power the device for more than 8 weeks and indicate that the designed algorithm successfully captures whether the shoe is worn, as well as the level of physical activity of the user. Our next steps are to develop appropriate packaging of the monitor components for in-shoe use and to fully test the monitor’s functionality, robustness, and patient comfort.
Acknowledgement: This research was funded in part by the Pedorthic Footcare Association.

Poster #: 52
Campus: San José State University
Poster Category: Clinical
Keywords: clinical nutrition, tube feeding,
Project Title: A Comparison Between Volume-Based And Rate-Based Tube Feeding Methods In Intensive Care Unit Patients
Author List:
Swiatlo, Travis; Graduate, Nutrition, Food Science, and Packaging, San José State University, Presenting Author
Berta, Janine; Stanford Health Care
Mauldin, Kasuen; Nutrition, Food Science, and Packaging, San José State University, Presenting Author

Abstract: Adequate nutrition support (defined as patients receiving ≥ 80% of estimated energy requirements (EER)) in the intensive care unit (ICU) setting is important in preventing and treating malnutrition and improving safety and clinical outcomes. In the conventional method of rate-based tube feeding (RBTF), patients are prescribed a constant goal rate of tube feeding based on daily caloric needs; a set rate that does not change to compensate for any feeding interruptions. In contrast, per the volume-based tube feeding (VBTF) protocol, the hourly infusion rate can be increased (max 150 mL/hour) to make up for feeding deficits incurred due to feeding interruptions, thus ensuring patients receive the targeted 24-hour volume. We hypothesize that patients on the VBTF protocol will be more likely to receive ≥ 80% of EER and will have fewer incidences of adverse events (hyperglycemia, hypoglycemia, emesis, diarrhea, and/or high gastric output). Data was collected from medical charts of patients admitted to Stanford Health Care (SHC) adult ICUs from April 2017 – September 2017. Patients must be ≥18 years of age, admitted to a SHC ICU and on tube feeding within the first 7 days of ICU stay. Patient demographics (gender, age, BMI), adverse events, and percent goal volume per tube feeding day were collected. For the VBTF group (n=29), there were 55% females, average age 61.4 ± 15.8 years, and average body mass index (BMI) was 28.4 ± 6.9 kg/m2. For the RBTF group (n=68), there were 37% females, average age 60.5 ± 16.4 years, and average BMI was 28.3 ± 6.4 kg/m2. Regarding adequate nutrition as defined by patients receiving at least 80% of goal volume or EER, 86.2% of VBTF patients received adequate nutrition and on average 90.5 ± 0.1% of EER per day on tube feeding. In contrast, only 45.6% of RBTF patients received adequate nutrition and 74.1 ± 0.2% of EER per day on tube feeding. Higher incidences of high gastric output, hyper- and hypoglycemia, and ileus were associated with RBTF, while VBTF had higher incidences of diarrhea and emesis. On average, 17.8% of VBTF and 18.6% of RBTF patients experienced an adverse event. This study provides evidence for clinicians to be more proactive and aggressive in providing tube feeding in the ICU setting without increased risk to patients.

Poster #: 53
Campus: Cal Poly San Luis Obispo
Poster Category: Clinical
Keywords: Probiotics, Gene expression, Cytotoxicity
Project Title: Probing human intestinal epithelial cell responses to commercial probiotic strains
Author List:
Jifcu, Radu; Undergraduate, Biological Sciences , California Polytechnic State University, San Luis Obispo, Presenting Author
Johnson, Taylor; Undergraduate, Wine and Viticulture, California Polytechnic State University, San Luis Obispo, Presenting Author
Yeung, Marie; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Over the past decade, interest in the use of microbes for human health advancement and remediation has grown. Probiotics, or “good bacteria”, may be consumed to confer health benefits to the host, ranging from inhibiting colonization of foodborne pathogens, to modulating immune responses. Previous studies have shown that probiotic benefits are strain specific, meaning that research must be done on individual strains to evaluate their probiotic potential. This study focused on three commercial probiotic strains, BW12, BW13, and BW14, and their relationship to human health using a cell culture model. The objectives were to measure the cytotoxicity and characterize the cytokine gene expression of Caco-2 cells upon exposure to the probiotics. Probiotics were added to Caco-2 cells at multiplicity of infection (MOI) of 100:1. At 24 h post-exposure, the % cytotoxicity of Caco-2 cells were calculated based on the amount of released lactate dehydrogenase from the treated cells, relative to the maximal release following a chemical cell lysis treatment. Our results show that untreated Caco-2 cells yield ~5% cytotoxicity while BW12, BW13, and BW14 yielded 4.0±1.8%, 2.2±1.3%, and 3.9±2.6% cytotoxicity, respectively. This implies the presence of these strains conferred a protective effect to the host. RT-qPCR array was used to measure expression of 84 cytokine genes, in addition to 5 housekeeping genes and 7 controls. Preliminary data shows that major anti-inflammatory cytokines (e.g., IL-10) did not show significant changes (<2-fold) in treated Caco-2 cells. IL-17F and IL-27 were highly upregulated (>20 fold), while most other cytokines are either unchanged or downregulated. IL-17F is considered a pro-inflammatory cytokine, while IL-27 can be involved in both pro- and anti-inflammatory responses. Increased understanding of strain-specific probiotic effects may help us develop new methods for combating pathogens beyond common remediation such as antibiotics. Funding for this study is provided by BiOWiSH and the Cal Poly Biological Sciences Department.

Poster #: 54
Campus: Cal Poly Pomona
Poster Category: Clinical
Keywords: Rabbit, Sedation , Animal Science
Project Title: Trazodone HCl provides sedation and improves handling in rabbits (Oryctolagus cuniculus)
Author List:
Kuhlman, Kierra; Undergraduate, Animal and Veterinary Sciences, California State Polytechnic University, Pomona, Presenting Author
Fukushima, Courtney ; Undergraduate, Animal and Veterinary Sciences, California State Polytechnic University, Pomona, Presenting Author
Brundage, Cord; Animal and Veterinary Sciences, California State Polytechnic University, Pomona

Abstract: The unique metabolism and handling requirements of rabbits (Oryctolagus cuniculus) makes sedation a particular challenge. There are limited drugs available that can safely decrease the risk of harm or stress in handling and housing laboratory rabbits. Trazodone hydrochloride (TZN), a selective serotonin and antagonist and serotonin reuptake inhibitor, has been useful in sedating anxious behavior in humans as well as anxiety and aggression in dogs and cats for clinical purposes, but has yet to be used in rabbits. We evaluated the use of TZN as an innovative alternative sedative option in laboratory rabbits. Veterinary examinations were performed on 15 adult female rabbits prior to receiving TZN (10 mg/kg, 20 mg/kg, 100 mg/kg by mouth) or a placebo. Each rabbit was given a score based on their levels of tension, struggle, aggressive behavior, and overall tractability during the examination. In this double blind placebo controlled randomized cross-over study the rabbits were also recorded/monitored with an ethogram for a three hour period after treatment was administered, followed by a re-examination and scoring at the end of the three hours. Rabbits that received TZN showed a reduction in the levels of tension, struggling, aggression, and overall handling tractability compared to rabbits receiving the placebo (P < 0.05). TZN decreased the level of tension (26%), struggling (38%), aggressiveness (81%), overall tractability (19%), and complete a composite handling score (34%). Rabbits that received placebo had minimal decreases in tension (4%), tractability (8%), handling composite score (3%), and increased struggling by 5%. These results suggest that TZN may be an appropriate sedative/anti-anxyolitic option for laboratory rabbits during handling or veterinary examinations.

Poster #: 55
Campus: CSU Fullerton
Poster Category: Clinical
Keywords: antibiotic resistance, ESKAPE, ionophore
Project Title: Phenotypic conversion to antibioticPhenotypic c susceptibility induced by clioquinol, an ionophore used to treat cancer and neurodegenerative diseases
Author List:
Magallón, Jesús; Graduate, Biology, California State University, Fullerton, Presenting Author, Eden Award Nominee
Chiem, Kevin; Graduate, Biology, California State University, Fullerton
Hue, Fong; Graduate, Biology, California State University, Fullerton
Tolmasky, Marcelo; Biology, California State University, Fullerton

Abstract: The dangerous increase in multidrug resistant bacteria seriously compromises our ability to treat infections and complicates medical and dental procedures that require preventive treatment. Aminoglycosides are powerful tools against Gram-negative infections. However, the dissemination of aminoglycoside modifying enzymes (AMEs) that confer resistance are rendering them less effective. The aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6’)-Ib] is one of the most common AMEs and mediates inactivation of the aminoglycoside by acetylation. As a consequence, numerous infections and hospital outbreaks results in an elevated number of fatalities. We recently showed that Zn(II) ions inhibit enzymatic acetylation by AAC(6’)-Ib in vitro, and in complex with the ionophore pyrithione causes phenotypic conversion to susceptibility in otherwise resistant Gram-negative bacteria. Clioquinol (5-chloro-7-iodo-8-quinolinol) is an ionophore and metal chelator with moderate affinity to zinc ions that is currently studied as a treatment of cancer and neurogenerative diseases. The objective of this study is to evaluate the efficacy of clioquinol in combination with zinc as an inhibitor of the resistance to amikacin mediated by AAC(6’)-Ib. Acinetobacter baumannii A155 (Ab) and Escherichia coli TOP10(pNW1) (Ec) were used in this study as both carry the aac(6’)-Ib gene. In vitro acetylation activity was assessed using the 5,5′-dithiobis-nitrobenzoic acid spectroscopic method. In cellulo activity of zinc complexed to clioquinol was assessed culturing the cells in 100-µL Mueller-Hinton broth containing 0.1% dimethyl sulfoxide and various concentrations of amikacin, zinc chloride, and clioquinol added in microtiter plates using BioTek Synergy 5 microplate reader. Cultures were carried out for 20 h at 37°C with shaking and the OD600 of the cultures was measured every 20 minutes. In vitro inhibition assays using purified AAC(6’)-Ib showed that enzymatic acetylation of amikacin and other aminoglycosides was strongly inhibited in the presence of Zn(II)-clioquinol complexes. Ab and Ec exhibited robust growth in the presence of 8 µg/mL amikacin. However, when 5 µM clioquinol and 25 µM zinc chloride were present in addition to amikacin neither strain was able to grow. We conclude that combinations of Zn(II) and ionophores are firm candidates as inhibitors of AMEs that could be sued as adjuvants in combination with aminoglycosides in the treatment of multidrug resistant infections.

Poster #: 56
Campus: San Francisco State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: electrostatic interaction, decarboxylase, density functional theory
Project Title: Role of electrostatic interaction in ligand recognition by orotidine-5’-monophosphate decarboxylase
Author List:
Lee, Jesi; Graduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author, Eden Award Nominee
Gokey, Trevor; Graduate, Chemistry and Biochemistry, San Francisco State University
Guliaev, Anton; Chemistry and Biochemistry, San Francisco State University

Abstract: The calculation of electrostatic potential (EP) maps on enzyme active sites provides pivotal information regarding ligand affinity and specificity. Here we probed the electrostatic interactions between the orotidine-5’-phosphate decarboxylase (ODCase) active site and the substrate orotidine-5’-monophosphate (OMP), the product uridine-5’-monophosphate (UMP), and two potential anticancer therapeutic inhibitors biturate-5’-monophophsate (BMP) and xanthosine-5′-monophsophate (XMP) using density functional theory calculations. BMP is the strongest known inhibitor to date. The charge distribution of XMP and BMP shared clear similarities with the substrate OMP than the product UMP. We propose that the resemblance of the EP maps between XMP, BMP, and OMP is likely to account for the ability of XMP and BMP to effectively bind to ODCase active site and act as potent inhibitors. Using molecular dynamics simulations, we observed that the binding of an inhibitor induced changes in the enzyme active site leading to a distribution of an additional positive charge not observed in the apo form simulations. In addition, upon BMP binding, the inhibitor was stabilized by hydrogen bond interactions between S154 residues of a loop (residues 151-165) adjacent to the active site and N3 and O4 atoms of hydroxyuridine ring. Based on the EP map calculations, these atoms have the largest negative potentials compare to other ligands tested in this work. The S154/BMP interactions also reduced flexibility of the loop which helped to form a well-defined binding cavity around the substrate. Our calculations show that the ability of BMP to enhance the formation of a positive charge at enzyme active site combined with the interaction with S154 explains why BMP is such a potent inhibitor. This work shows how the design of specific electrostatics interactions could improve the ligand binding and will aid in the development of future therapeutic inhibitors.

Poster #: 57
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: RNA-Seq, zinc, ZNT3
Project Title: Transcriptomic analysis of brain tissue RNA from Mucolipidosis IV knockout mice
Author List:
Chacon, Jonathan A; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Rosas, Lauren; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Behseta, Sam; Mathematics, California State University, Fullerton
McEligot, Archana; Health Science, California State University, Fullerton
Cuajungco, Math P; Biological Science, California State University, Fullerton

Abstract: Mucolipidosis IV (MLIV) is an autosomal recessive disorder that is characterized with neuronal and retinal degeneration. MLIV is caused by loss of function mutations in the human Mucolipin-1 (MCOLN1) gene. Our laboratory has previously reported abnormal zinc levels in MLIV patient fibroblasts, human MCOLN1-knockdown cells, and Trpml1-knockout (KO) mouse brain tissues. These observations indicate that the loss of cellular zinc homeostasis could play an underlying role in MLIV pathogenesis. To further examine the mechanisms behind zinc dyshomeostasis in MLIV, we analyzed the brain transcriptome of three Mcoln1 KO and three wild-type (WT) littermate control mice using RNA sequencing (RNA-seq). Transcriptomic analysis of Mcoln1 expression between biological replicates verified the KO from WT mice. Gene ontology data showed significant differential gene expression in the category of “Zinc Ion Transmembrane Transporter Activity.” Gene cluster analyses of zinc transport and zinc buffering genes showed distinct reduction in transcript levels of solute carrier 30a3 (Slc30a3; also known as zinc transporter ZnT3) and transmembrane (Tmem)-163 genes in KO mice. Both ZnT3 and Tmem163 proteins play a role in zinc transport within neuronal synaptic vesicles. Comparison of RNA-seq data between individual assembly and combined assembly of KO and WT sequencing reads showed distinct transcript level variability that could be explained by inter-subject differences. Validation using real-time quantitative polymerase chain reaction (qPCR) confirmed this effect. Nevertheless, both RNA-Seq and qPCR results consistently showed a decrease in ZnT3 transcript levels in KO mice compared to WT control mice. These data imply that abnormal ZnT3 expression could be a mitigating factor for the observed zinc dyshomeostasis in MLIV cells. Overall, our findings suggest that the loss of human TRPML1 function perturbs the expression of specific genes necessary for intracellular zinc homeostasis. Future studies will investigate whether the reduced ZnT3 transcript levels reflect decreased protein levels in MLIV KO mice. This work was funded by NIH R15 NS101594 and NIH R25 MD010397.

Poster #: 58
Campus: CSU Northridge
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: antibiotics, antimicrobial peptides, Escherichia coli
Project Title: Combination Therapies with Antimicrobial Peptide LL-37 and Conventional Antibiotics
Author List:
Siavoshi, Mehrnaz; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Prokopczuk, Federico; Undergraduate, Biology, California State University, Northridge
Del Rosario, Nathan; Undergraduate, Biology, California State University, Northridge
Abasi, Lannah; Undergraduate, Chemistry and Biochemistry, California State University, Northridge

Abstract: The emergence of antibiotic resistance is one of the largest problems in current public health. A potential solution is combination therapy, where two or more drugs are combined to fight resistant bacteria. In this work, we evaluate harnessing the power of antimicrobial peptides (AMPs), which are part of the innate immunity of virtually all eukaryotic life. The evolutionary stability of AMPs hints that it is difficult for bacteria to develop resistance against AMPs’ mechanism of action, which is based on membrane perturbation. We conducted a battery of combination therapy tests to determine the outcome of combining AMPs with more than 20 conventional antibiotics representing a range of drug classes. To this end, we employed a serial dilution method to test the effect of combining varying dosages of AMPs and antibiotics on growth of Escherichia coli cultures. A robust statistical analysis of the experimental data allows us to quantify growth rates and any extended delay in initial growth to discern if and to what extent any interactions occur. We discovered that AMPs’ and antibiotics’ action influence each other. Certain drugs, like chloramphenicol, experienced heightened antibacterial activity while other drugs, like streptomycin, were suppressed. Ultimately, our results give insight into how antibiotic effectivity in a clinical setting can be impacted by these naturally occurring biomolecules.

Poster #: 59
Campus: San José State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: sequence entropy, conformational switch, acetyltransferases
Project Title: Sequence Entropy and Flexibility Descriptors Identify a Possible Conformational Switch in Allosteric Dodecameric Polyamine Acetyltransferase SpeG
Author List:
Huynh, Angelina; Graduate, Chemistry, San José State University, Presenting Author
Veeraklaew, Mild; Graduate, Biomedical, Chemical and Materials Engineering, San José State University, Presenting Author
Oribello, Jonathan; Chemistry, San José State University
Montermoso, Saira; Chemistry, San José State University
Kuhn, Misty; Chemistry and Biochemistry, San Francisco State University
Lustig, Brooke; Chemistry, San José State University

Abstract: The spermine/spermidine N-acetyltransferase SpeG is an enzyme thought to play an important role in regulating the formation of biofilms by acetylating polyamines. Protein structural classification shows that SpeG belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily but features unique structural characteristics. For instance, SpeG assembles into dodecamers formed by two stacked hexamers, while most GNAT proteins act as monomers or dimers. Additionally, a novel polyamine allosteric binding site was identified that may act as a conformational switch. Our comparison of secondary structures, alpha carbon distances, packing density, and sequence entropy in Vibrio cholerae SpeG structures strongly suggests a connection between ligand binding and conformational variation, notably for an allosteric domain containing residues 15-52. In aggregate, the 6095 residues in the full set of structures examined exhibit similar correlation plots of sequence entropy versus flexibility (here inverse packing density) to earlier large-scale learning sets. However, the key allosteric sub-region 25-35, first identified by sequence conservation (as indicated by sequence entropy) as well as secondary structure and residue inter-chain distance variability, shows a unique correlation plot signal. Interestingly, the Lobanov-Galzitskaya disorder propensity remains consistent with the aggregate correlation plot for that same 25-35 region. This suggests sequence entropy may be a unique identifier in regards to a particular switch type.
Further, different bound ligand combinations exhibit distinct entropy correlation plot behavior that may explain the unique nature of the earlier sequence conservation results. Here we identified a confirmatory conservation signal via sequence entropy correlated with the structural signals shown to be consistent with a switch for region 25-35. Our computational analysis suggests further exploration of the properties of such a switch by site-directed mutagenesis studies. In the future, we hope to expand this analysis to other appropriate protein structures, including SpeG from other organisms as well as other large complexes without complete high-resolution structural information.
Acknowledgements: NSF Grant 1626645 (to BL) and CSUPERB New Investigator Grant (to MLK).

Poster #: 60
Campus: CSU Sacramento
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: enediyne cyclization, density functional theory (DFT), anticancer
Project Title: Using Steric Effects to Promote the Cyclization of Angularly Benzannelated Enediynes
Author List:
Bohne, Bryan; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Keel, Terell; Undergraduate, Chemistry, California State University, Sacramento
Daly, Morgan; Undergraduate, Chemistry, California State University, Sacramento
Gherman, Benjamin; Chemistry, California State University, Sacramento
Spence, John; Chemistry, California State University, Sacramento

Abstract: Enediynes are organic compounds capable of cyclizing to form diradicals, which can abstract hydrogen atoms in DNA. Therefore, enediynes can be used as antitumor prodrugs due to their ability to cause DNA cleavage and kill cancerous cells in the body. In previous research, it was found that angularly benzannelated enediynes with naphthalene or quinoxaline supporting groups exhibited improved Bergman (C1-C6) cyclization reactivity due to significant aromaticity gain upon cyclization. The addition of sterically bulky substituents at the alkyne termini of the enediynes led to competitive Schreiner-Pascal (C1-C5) cyclization. In this research, we examine how the placement of bulky substituents (e.g., methyl or t-butyl groups) on the naphthalene and quinoxaline supporting groups can be used to further increase the cyclization reactivity of angularly benzannelated enediynes. Density Functional Theory (DFT) calculations were used to determine the cyclization activation and reaction energies along both C1-C6 and C1-C5 reaction pathways for the angularly benzannelated enediynes. The full set of compounds investigated included both naphthalene and quinoxaline supporting groups bearing substituents proximal to the enediyne unit with terminal, phenyl, and methoxyphenyl-substituted alkynes. In addition, nucleus-independent chemical shift (NICS) calculations were carried out to measure the aromaticity in the newly formed rings from the cyclization reactions. Calculations indicated that overall cyclization reactivity of the angularly benzannelated enediynes were improved when bulky substituents were included on the naphthalene and quinoxaline supporting groups, as evidenced by marked decreases in activation energies and reaction energies for both C1-C5 and C1-C6 reaction pathways. The energetic effect is consistent among the enediynes with and without terminal alkyne substituents. The improved reactivity can be traced to geometric distortion in the reactant caused by the bulky supporting group substituent and favorable interaction between the supporting group substituent and the diradical centers in the cyclized products.

Poster #: 61
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: escherichia coli, T4 phage, agent-based model
Project Title: T4 bacteriophage and E. coli encounter rates in mucus: A computational approach
Author List:
Joiner, Kevin; Graduate, Mathematics & Statistics, San Diego State University, Presenting Author
Luque, Antoni; Mathematics & Statistics, San Diego State University, Presenting Author

Abstract: Bacteriophages are viruses that infect bacteria and represent a promising strategy to fight antibiotic-resistant pathogens. Unfortunately, the understanding on how phages interact with bacteria in mucus—a substance which coats the organs in many living systems—is limited and experimentally challenging. The goal of our research is to develop a computational framework to provide a rapid and systematic platform to screen for ideal viruses to fight pathogenic bacteria. For the project, we conduct ‘in-silico’ phage-bacteria-mucus experiments to investigate how phage and bacteria diffusion effect their encounter rates in 0% and 1% mucin solutions. We use T4 bacteriophage and its host, Escherichia coli, as model species. The motion of T4 phage is captured using a continuous time random walk which recovers the subdiffusion of T4 due to its “sticky” interaction with mucus. The motion of E. coli is modeled to reproduce its characteristic run-and-tumble stages. Our T4 model is calibrated using empirical data obtained from the Viral Information Institute at San Diego State University. Simulation results are compared with standard analytical diffusion models. Our simulation data, in agreement with analytical models, reveal concentrated doses of T4 phage released near E. coli, are sustained longer when the phage sub-diffuse and the bacteria tumble frequently. However, we observe once conditions relax into steady state, spatially uniform concentrations, T4 and E. coli encounter rates have little dependence on whether the phage diffuses normally or sub-diffusively. These results suggest phage subdiffusion elicits a transient, short term, benefit to increase its effectiveness to fight pathogenic bacteria.

Poster #: 62
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: phylogeny, extinction, mammal
Project Title: Phylogenetic Assessments for California’s Highest Risk Mammals
Author List:
Jorgensen, Christopher; Graduate, Biology, California State University, Fresno, Presenting Author, Eden Award Nominee
Reece, Joshua; Biology, California State University, Fresno

Abstract: Anthropogenic impacts have significantly altered global biodiversity over the past 200 years, and these environmental implications have drawn attention to the need for conservation biology. Conservation Biology is crisis-driven, and utilizes emerging interdisciplinary techniques to assess and make policies on environmental protection. Traditionally, conservation biology has investigated how ecology is altered by and responds to human influences. Recently, phylogenetics has been implemented into this field to quantify evolutionary value as a metric for how conservation priorities are set. Phylogenies are instrumental for predicting the impacts of anthropogenically-selective extinctions, because unlike natural extinctions, these are highly non-random. Phylogenetically, mammals are one of the most well studied groups of animals, making them an ideal system for making conservation policies that reflect evolutionary diversity. California is well known for its robust mammalian diversity and multifarious habitats, making it an ideal setting for this project. We utilized previously published molecular phylogenetic data, and the conservation status of California’s mammal species. We used these data to investigate the relationships between extinction risk and phylogeny using the statistical analysis program R. Specifically, we measured the potential loss of phylogenetic diversity and disparity by calculating the percentage decrease in phylogenetic diversity (PD) and Net Relatedness Index (NRI) when all species and subspecies with elevated extinction risk are pruned from the California mammal tree of life. The proportion of the total decrease in PD and NRI are greatest when both threatened, and endangered species are categorized together. However, even when both threatened and endangered species are removed from the tree, there is less than a ten percent decrease in either phylogenetic diversity or disparity. Our results provide a novel understanding of the future phylogenetic losses of California’s native mammalian tree of life. Simultaneously, our methodology further legitimizes the use of computational phylogenetics in conservation practices.

Poster #: 63
Campus: CSU Dominguez Hills
Poster Category: Diagnostics/Imaging/Analytical
Keywords: pharmacokinetics , biosensor, drug monitoring
Project Title: Comparison of sex-specific pharmacokinetics using a novel aptamer-based biosensor in vivo.
Author List:
Vieira, Philip; Psychology, California State University, Dominguez Hills
Shin, Christina; Graduate, UCSB Psychological & Brain Sciences
Arroyo, Netzahualcóyotl; Postdoc, UCSB Chemistry
Gabriella, Ivett; Graduate, Psychology, California State University, Dominguez Hills, Presenting Author
Plaxco, Kevin; UCSB Chemistry
Kippin, Tod; UCSB Psychological & Brain Sciences

Abstract: Atypical patient populations, such as the elderly, infants and children, pregnant women, and individuals receiving chemotherapy or antiretroviral therapy may have unique responses to drug therapy, which may result in adverse reactions or ineffective treatment. Furthermore, sex-specific measurements of pharmacokinetics may have different dynamics. Current laboratory measures of drug quantification rely on chromatography, mass spectroscopy, and immunoassays, none of which are direct in vivo measurements of drug pharmacokinetics. The present study used a novel electrochemical aptamer-based biosensor capable of high precision (seconds resolution), long term (across 6 hours) in vivo monitoring of drug concentration in anesthetized male and female rats to measure individual variability and sex-specific differences in pharmacokinetic response to the aminoglycoside tobramycin. The hypothesis predicted significant differences between males and females in drug adsorption, elimination, area under the concentration-time curve (AUC), and maximum concentration measured at one point in time during the recording (CMAX). Tobramycin, was administered through intramuscular or intravenous (via jugular vein catheter) injections. The design included one intramuscular injection group (22 total, 7 female), and another intravenous injection group (18 total, 8 female). One way-MANOVA statistical analysis was employed to assess preliminary results. After winsorizing outliers in both groups, and log transformations of adsorption, elimination, and AUC variables in the intramuscular group, two MANOVA analyses were performed to assess the effects of sex on drug adsorption, elimination, AUC, and CMAX. The analysis showed near significant effect in the intramuscular group (F=2.59, Wilks’ Lambda p=.074), and in the intravenous group (F=2.974, Wilks’ Lambda p=.06) as well. These preliminary results warrant further investigation to determine the impact of sex on individual drug pharmacokinetics. This study helps to further validate this novel biosensor technology for use in therapeutic drug monitoring and may have implications on future patient-specific drug dosing (i.e, personalized medicine). This work was supported by grants from the NIH, W.M. Keck Foundation, and CSU Dominguez Hills.

Poster #: 64
Campus: Sonoma State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: convolutional neural network, Computed tomography, interstitial lung disease
Project Title: Using pre-trained convolutional neural networks to classify interstitial lung diseases in chest computed tomography scans
Author List:
Granados, Joseph; Undergraduate, Mathematics, Sonoma State University, Presenting Author
Gill, Gurman; Computer Science, Sonoma State University, Presenting Author

Abstract: Chest computed tomography (CT) scans are widely used for automatic detection and classification of Interstitial lung disease (ILD) using computer-aided diagnostic (CAD) systems. The goal of these CAD systems is to reduce the time taken for and to optimize the diagnostic decisions made by radiologists. Convolutional neural networks (CNN) have been shown to be extremely effective at visual classification tasks. They comprise of successive “layers” of neurons that learn to respond to salient image features such as color, texture, edges that are combined in later layers to detect global concepts such as faces, blood vessels, etc. However, CNNs require large, labeled datasets in order to tune millions of parameters. Medical datasets large enough to train a CNN from scratch are not readily available. In this work, we explore the feasibility of using CNNs that have been fully trained with a non-medical dataset to classify ILDs in CT scan patches using “transfer learning”: a technique where features identified by the pre-trained CNN are then used to train a new CAD system. The CNN model architecture we used is called InceptionV3 (Google Inc.) which has been pre-trained on a ImageNet dataset consisting of 1.2 million images belonging to 1000 non-medical categories such as “bus” or “cat”. The dataset used for “transfer learning” and evaluation is the Talisman Test Suite comprising of 14594 CT scan patches with 407 emphysema, 2962 fibrosis, 2226 ground-glass, 3011 healthy, and 5988 micronodules across 85 patients. To evaluate the model, we used Leave One Patient Out cross validation, where for each patient in the dataset we classify the patient’s CT scan patches using a model trained with data from all the other patients. We then calculate an average F1 score across all patients, which is a measurement that gives us a better idea of performance when evaluating unbalanced data. The best results (average F1 score: 0.8308 | standard deviation: 0.0594) came from extracting features from an intermediate layer of the InceptionV3 model. Compared with results from the last layer (average F1 score: 0.8085 | standard deviation: 0.0599), this indicates that early layers in the InceptionV3 network trained on the ImageNet dataset can identify features that are feasible for classifying ILD patches. We struggled to classify emphysema (average recall of 0.59) but it is underrepresented in this dataset and our future work involves using data augmentation techniques to address this.

Poster #: 65
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Parkinson’s disease , alpha-synuclein , scanning ion conductance microscopy
Project Title: Real-time determination of the disruption and pore formation of cell membranes by aggregated alpha-synuclein using scanning ion conductance microscopy
Author List:
Wong Su, Stephanie; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Chieng, Andy; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Wang, Yixian ; Chemistry and Biochemistry , California State University, Los Angeles

Abstract: Parkinson’s disease (PD) is recognized as the second most common neurodegenerative disorder that causes patients’ motor skills to deteriorate and has affected approximately one million people in the United States alone. A large body of evidence has suggested that deposition of aggregated alpha-synuclein (α-Syn), an abundant brain protein near presynaptic termini, in intracellular protein inclusions (Lewy bodies) results in neuronal cell damage and ultimately contributes to the progression of PD. However, the exact mechanism is still unclear. One hypothesis is that α-Syn aggregates disrupt the cell membranes by forming pores that lead to permeabilization of lipid bilayers, eventually causing cell death. To demonstrate this, we have been using scanning ion conductance microscopy (SICM) to continuously monitor the morphological changes of SH-SY5Y neuroblastoma cells (a cell line commonly used to mimic neuronal cells for the studies of neurological disorders) in the presence of α-Syn aggregates. Dramatic disruption of the cell membranes has been observed in real time after adding α-Syn aggregates to the culturing media. The next step is to further improve the spatial resolution for monitoring the possible pore formation on the membrane. If the pore-forming hypothesis involving a certain form of α-Syn (e.g. oligomers) is demonstrated, our data can help guide the development of drugs that inhibit or reverse the α-Syn aggregation and thus prevent the progression of the disease. Such information will facilitate the ultimate development of the PD treatment modality. This work is supported by the CSULA Startup Fund and CSUPERB New Investigator Grant.

Poster #: 66
Campus: CSU San Marcos
Poster Category: Diagnostics/Imaging/Analytical
Keywords: imaging device, clinical research, human factors
Project Title: Human Factors in Operating Room Technology: Image Guidance Navigation Surgery
Author List:
Shweikeh, Faris; Biological Sciences and Biotechnology , California State University San Marcos, Presenting Author

Abstract: BACKGROUND: Spine surgery has seen tremendous changes over the last decade. One of the advances has been use of image-guidance or computer-assistance in the spine surgery operating room. This technology is in the form of an intraoperative computed-tomography (CT) scanner (i.e. O-Arm by Medtronic). The impression of this technology is variable; while some surgeons utilize the device, others do not. It is very likely that human factors, such as the multiple dimensions of human-system interactions, underlie ease of use as well deterrents and we seek to identify them in this study.
METHODS: Forty spinal operations that incorporated the O-Arm at a certain point during surgery were observed and disruptions to the flow were recorded. Flow disruptions (FDs) are derangements within the natural sequence of events of a procedure that potentially jeopardize safety or efficiency. They were classified into: coordination problems, equipment issues, training interruptions, external disturbances, and decision making. These FDs were assessed during 3 phases of the operation: pre-imaging, during imaging, and post-imaging. Duration of each phase was also taken into account as well as experience of the surgeon and the OR team, and patient age and BMI.
RESULTS: A total of 530 FDs were observed, with a mean of 14.7 (95% CI 11.93 – 17.47) per case. The most FD were encountered during screw placement, with 38% attributed to coordination problems, 30% to equipment issues, and another 30% among the rest. Coordination FDs occurred mostly in the pre-imaging and spin (imaging) phases, while equipment problems predominated during screw placement (in the post-imaging phase). Using flow disruption rates during the first spin, a linear regression suggests a baseline FD rate of 13.8 FD/ Hour, reduced by 5.91 FD/hr (p=0.0001) with an expert surgeon & novice IGS tech, by 6.47 FD/hr (p<0.0001) with an expert IGS Tech and novice surgeon, and by 8.38 FD/hr (p=0.0063) if both are experts. This model accounts for 39% of the total variation in flow disruption rate.
CONCLUSIONS: Coordination and equipment flow disruptions were the greatest source of disruption. Most FDs occurred during screw placement phase. These disruptions underscore how spinal navigation is a “multi-systems, multi-persons” event. Each “Phase” of Image Guidance Technology has specific flow disruption characteristics. Having an expert surgeon and expert IGS technician is critical for a smooth procedure.

Poster #: 67
Campus: CSU Chico
Poster Category: Disease (Pathogens)
Keywords: avian influenza, waterfowl, virology
Project Title: Isolation and characterization of avian influenza viruses circulating in migratory waterfowl in Northern California
Author List:
Bianchini, Elizabeth; Graduate, Dept. of Biological Sciences, California State University, Chico, Presenting Author
Barragan Trejo, Analucia; Undergraduate
Bogiatto, Jay; Dept. of Biological Sciences, California State University, Chico
Donatello, Robin; Dept. of Mathematics and Statistics, California State University, Chico
Plancarte, Magdalena; UC Davis
Boyce, Walter ; UC Davis
Cline, Troy; Dept. of Biological Sciences

Abstract: Recent human infections with influenza viruses of avian origin highlight the need for continued surveillance of avian influenza viruses in waterfowl with the goal of detecting virus strains that are adapted for infection of mammals. In 2014, highly pathogenic avian influenza (HPAI) H5N8 first detected in South Korea entered North America through the Pacific Flyway, a major migratory route for waterfowl and caused outbreaks in poultry in the United States. California’s Sacramento Valley provides wintering habitat for 44% of the birds that use the flyway. As such, the Sacramento Valley serves as an important site from which avian influenza viruses may be isolated. We collected cloacal swabs from 1,434 hunter-killed ducks across three hunting seasons at different locations in the Sacramento Valley. Seventeen species were represented with a slight male sex bias. The presence of influenza viruses in cloacal swabs was determined by PCR for the matrix gene. PCR positive swabs were inoculated into embryonated chicken eggs for virus isolation and subtyping. Overall, the PCR positive rate was 13%. We observed a significantly higher positive rate in 2015-2016 (19.9%), a phenomenon that may be related to overcrowding on wetlands due to drought conditions. Northern shovelers had a statistically higher carriage rate relative to other species. Chi-squared tests of equal proportions of positive rate between groups were used for statistical analyses. Among PCR positive samples that yielded egg isolates, diverse HA and NA subtypes were represented. Of particular interest, we detected HPAI H5 influenza viruses by PCR but were unable to retrieve an egg isolate. Three H7N3 isolates were obtained and, given recent human infections with H7 viruses, were characterized with respect to in vitro replication kinetics in mammalian tissue culture. All three H7 viruses were capable of replication in mammalian cell culture at levels similar to a human H1N1 virus.

Poster #: 68
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Acineterbacter baumannii, Transformation , Anitbiotic resistance
Project Title: Effect of antibiotics on natural transformation in Acenitobacter baumannii
Author List:
Martinez, Jasmine; Undergraduate, Biological Science , California State University, Fullerton, Presenting Author
Quinn, Brettni; Graduate, Biological Science , California State University, Fullerton
Liu, Christine ; Undergraduate, Biological Science, California State University, Fullerton
Nguyen, Meaghan; Undergraduate, Biological Science , California State University, Fullerton
Ramirez , Maria Soledad; Biological Science , California State University, Fullerton

Abstract: Acinetobacter baumannii, a member of the highly resistant ESKAPE pathogens, has emerged over the last few decades as a severe nosocomial pathogen due to its ability to resist desiccation and nutrient starvation and obtain novel resistance genes. Horizontal genetic transfer (HGT) plays a key role in the evolution and adaptability of bacterial species as their capability to acquire and incorporate DNA into their genome allows them to evolve and survive in hostile environments. However, as little is known about how competence is induced in A. baumannii, we focused on identifying additional inducers of competence for natural transformation in this species.
Several kanamycin susceptible A. baumannii strains (A118, ATCC 17978, ATCC 19606 and A42) were challenged by sub-inhibitory concentrations of antibiotics (mitomycin C (MMC), nalidixic acid (NAL), meropenem (MEM)). Briefly, transformation assays using plasmid DNA (pDSredAAC, conferring kanamycin resistance(KanR)) or genomic DNA (from strain A. baumannii 144, known to carry a KanR determinant) were performed. Transformation events were scored by counting KanR colonies, while total CFUs were assessed by plating serial dilutions on LB agar.
MMC statistically significantly (P-value < 0.05) increased transformation frequencies in strain ATCC 17978 by 19.83 and 13.04-fold and in strain ATCC 19606 by 2.18 and 3.03-fold when transformed with plasmid and genomic DNA, respectively. NAL increased (P-value < 0.05) transformation frequencies in strain A42, transformed with plasmid DNA, by 3.44-fold while MEM increased (P-value < 0.05) transformation frequencies in A42, transformed with genomic DNA, and ATCC 17978, transformed with plasmid DNA, by 2.33-fold and 12.12, respectively. While MMC, NAL and MEM did not have a statistically significant effect on transformation frequencies with other strains and DNA combinations, transformation frequencies did increase in all cases.
Our results show that antibiotics, that are present in hospital setting as well in the environment, can induce competence in A. baumannii.

Poster #: 69
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: hemolysis, staphylococcus aureus, acinetobacter
Project Title: Interactions between Staphylococcus aureus and Acinetobacter spp. strains: Using bacteria to stop superbugs.
Author List:
Fung, Sammie; Undergraduate, Biology, California State University, Fullerton, Presenting Author
Ramirez, Maria; Biology, California State University, Fullerton

Abstract: Although bacteria are commonly studied in isolation, research has shown that bacteria often act in communities. In humans, infections are often polymicrobial, with multiple bacterial species found together sharing the same infection site. Descriptions of co-infections are well documented, and in some cases, these polymicrobial infections increase morbidity and mortality. This project aims to observe how two distantly human pathogens belonging to the group of “ESKAPE” species can work cooperatively or competitively to affect their virulence, measured through hemolysis assays.
Six different strains, two Staphylococcus aureus (LS1 and USA300) and four Acinetobacter spp. (A118, A42, A23, and A47) strains were used in this study. Hemolytic assays were performed by growing the bacterial strains in rabbit blood with and without the presence of cell-free conditioned media (CFCM) of distantly related strains. Optical density of the supernatant at 540 nm and 570 nm was used as a surrogate marker for hemolytic activity. Assays were incubated for 12 hours.
When grown alone, Acinetobacter A47 demonstrated moderate hemolytic activity. However, in the presence of S. aureus CFCM, Acinetobacter A47 hemolytic activity was found to be over 200% greater than when incubated alone. This increase in hemolytic activity was observed as early as 3 hours after incubation, suggesting a significant collaborative interaction between the two strains. On the contrary, S. aureus USA300 showed reduced hemolytic activity when grown in the presence of A. baumannii A118 (p < 0.05), suggesting a competitive interaction between the two strains. Hemolytic activity of S. aureus LS1 did not change when grown with A. baumannii A118.
USA300 is a virulent strain of methicillin-resistant S. aureus responsible for rapidly progressing and often fatal disease in humans. Our results suggest that competitive interactions between specific pairs of bacterial strains may reduce activity of virulent strains. Additional experiments have been planned to identify interactions between other strains that may exhibit similar collaborative or competitive interactions. Microbes mainly exist as members of a polymicrobial community—a fact that is still greatly underappreciated. By studying how different bacterial interactions can alter the pathogenic outcome, we can learn to leverage certain interactions to better fight infections and gain better insight into the fight against antibiotic resistance.

Poster #: 70
Campus: CSU Chico
Poster Category: Disease (Pathogens)
Keywords: influenza virus, macrophage, interferon gamma
Project Title: Highly Pathogenic Avian Influenza Virus H5N1 Restricts Interferon-γ Responses in Macrophages in a Replication-Dependent Manner
Author List:
Beck, Donald; Graduate, California State University, Chico, Presenting Author
Sekar, Keerthana;
Cline, Troy; Faculty

Abstract: Highly pathogenic avian influenza (HPAI) viruses continue to cause human infections with a reported mortality rate of ~50%. However, the mechanism of enhanced disease severity is not fully understood. Through the production of antiviral cytokines and phagocytosis of dead/infected cells, macrophages play a critical role in protection against influenza virus infection. On the other hand, macrophages have been implicated in exacerbated disease following H5N1 infection via a dysregulation of macrophage antiviral responses. H5N1 influenza viruses are unique in their ability to replicate in some macrophage models, a feature that maps to the viral hemagglutinin (HA) gene. We hypothesize that H5N1 influenza viruses alter critical macrophage functions in a replication-dependent manner. We investigated the impact of influenza virus replication on macrophage phagocytosis by infecting RAW264.7 macrophages with live/UV-inactivated H1N1 influenza virus A/California/04/2009 (CA/09; unable to replicate in macrophages) or live/UV-inactivated CA/09 expressing an H5 HA gene (CA/09-H5 HA; productively replicates in macrophages). Phagocytosis of a non-opsonized, FITC-tagged E. coli K12 particle was determined using the Vybrant Phagocytosis Assay kit. While macrophage phagocytic capacity was unaffected by CA/09 infection, a significant reduction in phagocytic capacity occurred in CA/09-H5 HA-infected macrophages. Further, macrophage phagocytic capacity was also decreased in CA/09-H5 HA-infected cells that were stimulated with IFN-γ, suggesting that an H5 influenza virus can inhibit the stimulatory effect of IFN-γ on macrophages. In both cases, inhibition of phagocytosis was dependent on virus replication as infection with a UV-inactivated H5 virus rescued the phagocytic response. While phosphorylation of STAT1 (as measured by western blot) in response to IFN-γ was not inhibited by CA/09-H5 HA infection, this virus did block expression of the IFN-γ-responsive gene IRF-1 in macrophages in a replication-dependent manner. These data demonstrate that H5N1 influenza viruses decrease phagocytic capacity and IFN-γ responsiveness of macrophages in a replication-dependent manner. This suggests that macrophage dysfunction during severe H5N1 disease may be an effect of the unique ability of these viruses to productively replicate in macrophages.

Poster #: 71
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: diabetes, candidiasis, liposomal amphotericin B
Project Title: Efficacy, Drug Biodistribution and Tissue Cytokine Levels Associated with AmBisome Treatment of Systemic Candidiasis in Mice with Type 2 Diabetes
Author List:
Simargi, Shirleen; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Olson, Jon; Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract:
Background: Obesity is a strong risk factor for type 2 diabetes (T2DM) and excess fat negatively impacts immune function, making diabetic individuals more susceptible to infections, such as systemic candidiasis. This study examined the efficacy, biodistribution and cytokine responses of T2DM mice systemically infected with Candida albicans and treated with liposomal amphotericin B (AmBisome, AmBi).
Methods: ICR male mice were maintained on a high (60%) fat diet for 4 weeks and given a chemical induction of nicotinamide (60mg/kg) and streptozotocin (100 mg/kg), intraperitoneally, to induce T2DM by d14 post-induction based on mean blood glucose levels of >373 mg/dL. Mice (n=13/gp) were challenged intravenously (IV) with C. albicans (ATCC #62342) (3.2×106 cells/mouse) and 24h later, treated IV for 6 days with 3, 5 or 7.5 mg/kg AmBi or 5% dextrose in water (D5W). Tissues (kidneys, liver, spleen, pancreas, brain, and fat) were collected d7 post-challenge (n=6/gp), homogenized and plated on Sabouraud’s agar to determine fungal burden and AmBi tissue concentrations by bioassay. Tissue cytokine levels were quantified using a multi-bead assay. Mice (n=7/gp) were monitored for morbidity to d21.
Results: AmBi at all doses was effective in treating T2DM mice with systemic candidiasis based on 100% survival versus (vs) 0% survival for D5W (p≤0.0001), lower disease signs vs D5W (p≤0.0033) and significantly less weight loss for 3 and 7.5 mg/kg vs D5W (p≤0.0411 and p≤0.0260, respectively). Yeast burden was significantly reduced in all tissues vs D5W (p≤0.01) and yeast were undetectable in spleen, liver, and pancreas. Drug concentration varied depending on the tissues (high to low: spleen>liver > kidneys= pancreas> brain>fat) with drug concentrations in kidneys, spleen, and liver significantly higher at 7.5mg/kg compared to lower doses. Proinflammatory cytokines IL-6, IL-1-alpha and IL-1-beta in the kidneys were significantly lower with AmBi versus D5W (p<0.01) and TNF-alpha and IL-10 in liver were significantly lower with AmBi versus D5W (p<0.03 and p< 0.01, respectively)
Conclusions: AmBi at a dose as low as 3 mg/kg, was able to significantly reduce systemic candidiasis in T2DM mice. The drug was able to penetrate into many tissues including the pancreas and the fat. AmBi also lowered the proinflammatory cytokine levels in the infected tissues compared to non-drug treated mice, indicating that AmBi was able to reduce potentially damaging cytokine responses.

Poster #: 72
Campus: CSU San Marcos
Poster Category: Disease (Pathogens)
Keywords: Breast cancer, Gene copy number, LEFTY
Project Title: Implication of LEFTY and BMP7 Amplifications in Breast Cancer
Author List:
Sanchez, Kassandra; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Zabala, Maider; Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University
Heitink, Luuk ; Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University
Escobar, Matthew; Biological Sciences, California State University San Marcos
Clarke, Michael; Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University

Abstract: Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of death in this population. Thus, it is crucial to find further insights into aberrantly activated pathways that could elucidate the identification of novel therapeutic targets. One pathway linked with the development of breast cancer is the transforming growth factor-β (TGF-β) pathway. Among its functions, it regulates cell proliferation and differentiation in development. Recently, we found that LEFTY endows proliferative advantage to mammary epithelial cells whereas BMP7 has opposite effects. As cancer could be considered as an abnormal proliferation of cells, we wanted to investigate whether LEFTY and BMP7 may play a role in breast cancer tumorigenesis. First, we interrogated human breast cancer genome datasets (METABRIC and TCGA; 2867 patients) and found that 24.5% of patients showed an increase in copy number (CN) of the LEFTY and/or BMP7 genes. Only 1.2% of patients displayed amplification of both genes, indicating these alterations are mutually exclusive. We then validated the existence of an increase in CN in breast cancer patient-derived xenograft (PDX) mouse models and corroborated its association with mRNA expression levels of both genes. When LEFTY transcript levels were decreased using short hairpin RNAs in our PDX models, we observed a significant reduction in tumor growth. These findings corroborate that LEFTY confers a proliferative advantage also to breast cancer cells. Using a reporter assay for BMP7 signaling, we elucidated that LEFTY opposed BMP7 through pSMAD1/5/8 signaling pathway. Our results reveal how LEFTY and BMP7 may contribute to breast cancer formation and elucidates novel targets for future breast cancer therapies.

Poster #: 73
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: delta-9-tetrahydrocannabinoid, Candida albicans, Cytokines
Project Title: Chronic ∆9-tetrahydrocannabinol treatment alters mouse splenocyte cytokine response against systemic Candida albicans infection
Author List:
Vitharanage, Hansini; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author, Eden Award Finalist
Buckley, Nancy; Biological Sciences, California State Polytechnic University, Pomona

Abstract: ∆9-tetrahydrocannabinol (THC), a constituent of Marijuana is known to suppress the resistance to many microbial infections, but the effects of THC on fungal infections are unclear. Since marijuana use is becoming more popular, and the opportunistic fungus, Candida albicans (C. albicans) cause severe systemic infections, investigating the immunomodulation by THC against C. albicans is imperative. Our laboratory previously found that chronic THC treatment decreased the survival during a secondary C. albicans infection in immune competent female mice. The spleen plays a crucial role in host immune responses against systemic microbial infections, and the spleen is populated with numerous immune cells. Therefore, we investigated the in vivo effects of chronic THC treatment on cytokine production from splenocytes derived from mice infected with C. albicans. For our studies, we used two infection models, an acute and a secondary systemic C. albicans infection model. For both studies, c57BL/6 female mice were treated with vehicle (ethanol, cremophor, saline (1:1:18)) or THC in vehicle (16mg/kg/mouse, intraperitoneal (IP) injection) 4 days a week, for three weeks (experimental days 1-18). For the acute infection model, the mice were infected with C. albicans (5×105 cells/mouse, IV) on day 19. For the secondary infection model, the mice were given a priming dose of C. albicans (0.75×105 cells/mouse, IV) on day 2 and a challenge dose (5×105 yeast cells/mouse, IV) on day 19. On day 22, spleens were harvested from mice (n=5) to establish a splenocyte culture. Splenocytes were treated with Concanavalin A (ConA, 5µg/ml) or heat killed C. albicans (HKCa) (6.25×106 yeast cells/ml). The splenocytes were then incubated at 37oC for 48h, and cell supernatants were collected to analyze secreted cytokines (Interleukin 12(p40) (IL 12(p40), IL-6, IL-17A, Interferon-gamma (IFN-γ), and Tumor Necrosis Factor-α (TNF-α)). In both infection models, HKCa-induced IL-12(p40), IFN-γ, and TNF-α and ConA-induced IL-12(p40) and IL-6 levels were lower in the THC group. In the acute infection model, ConA-induced IL-17A and TNF-α levels were lower in the THC group. In the secondary infection model, HKCa stimulated IL-6 was lower in the THC group. These results strongly suggest that in vivo administration of THC modulates splenocyte function which alters splenic immune response against C. albicans.

Poster #: 74
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: carbepenem-resistance, Ochrobactrum anthropi, emerging pathogens
Project Title: Characterization of carbapenem-resistant (CR) Ochrobactrum anthropi strains involved in an outbreak
Author List:
Fernandez, Jennifer; Graduate, Biological Science, California State University, Fullerton, Presenting Author, Eden Award Nominee
Hernandez, Marlene ; Undergraduate, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Montaña, Sabrina; Instituto de Microbiología (IMPaM), Facultad de Medicina, Universidad de Buenos Aires, Instituto de Microbiología (IMPaM), Facultad de Medicina, Universidad de Buenos Aires
Almuzara, Marisa; Laboratorio de Bacteriología, Hospital Interzonal de Agudos Eva Perón, San Martín, Buenos Aires, A
Ramirez, Maria Soledad; Biological Science, California State University, Fullerton

Abstract: In the last years the use of technologies in the microbiology diagnostic laboratory have contributed to the increase in identification of uncommon pathogens. Ochrobactrum anthropi is an emerging pathogen that was lately recognized as an opportunistic nosocomial pathogen. In 2016 an outbreak caused by O. anthropi occurred in a hospital. Surprisingly the four isolates involved in the outbreak were CR.
The aim of this project was to characterize the strains involved in the outbreak and identify the resistant determinants that can explain the multidrug-resistance (MDR) phenotype observed. Four isolates were recovered from the blood sample of four patients. The isolates were identified as O. anthropi by 16s rDNA sequencing. Susceptibility tests were performed and PCR reactions for various carbapenemases were carried. OD-PCR was used to determine their genetic relationship. In addition, the draft genome sequence of one of the strains (OA 107383) was obtained with Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPADES and RAST was used to predict the open reading frames (ORF). Further genomic analysis was carried out using ARG-ANNOT, ISFinder, and PHAST.
All isolates were MDR and EDTA synergies exhibited positive results, suggesting the presence of a MBL in each isolate. However, PCR reaction for blaIMP, blaVIM, blaKPC and blaNDM were negative. The same pattern of bands was obtained by OD-PCR suggesting an epidemiological relationship. The whole genome sequencing of OA 107383 was performed and its draft genome consists of 4,792,719-bp sequences. By RAST server, 4554 protein-coding genes were predicted. Sequence analysis of the genome identified the presence of multiple resistance genes including various class C β-lactamases, and most importantly, a metal-depended hydrolase, phnP. Moreover, genes coding for efflux pumps and two intact phages were also found.
To our knowledge, this is the first description of an outbreak cause by carbepenem-resistance O. anthropi. We have identified different β-lactamases genes in the genome of O. anthropi as well as various efflux pumps. The presence of these determinants could explain the observed resistance phenotype. We also highlight the importance of the isolation of uncommon MDR pathogens from clinical specimens since they can serve as reservoir of resistance determinants.

Poster #: 75
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Acinetobacter baumannii, siderophore biosynthesis, markerless gene knock-out
Project Title: Markerless gene knock-outs for the genes from two iron siderophore biosynthesis gene clusters in hypervirulent Acinetobacter baumannii LAC-4
Author List:
Viramontes, Yesica; Undergraduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Ewing, Peter; Department of Biological Sciences, California State University, Los Angeles
Xu, Howard; Department of Biological Sciences, California State University, Los Angeles

Abstract: Acinetobacter baumannii is a Gram negative, multi-drug resistant (MDR) pathogen gaining prevalence in hospital intensive care units. Due to the MDR nature of clinical isolates of this bacterium and a scarce pipeline of new antibiotics in development, the therapeutic options to treat infections caused by this bacterium are increasingly limited. Targeting disease-causing capabilities of bacterial pathogens has emerged as a novel approach to combating bacterial pathogens. Iron-dependency has been a known feature of bacterial pathogens. Therefore, bacterial factors involved iron acquisition are potential targets for discovering anti-virulence therapeutics. In this report, a molecular genetics approach was employed to determine the role of genes in siderophore biosynthetic pathways in the pathogenesis of A. baumannii LAC-4 strain, a clinical isolate found to be hypervirulent to mice. Specifically, the three linked genes coding for the ABC transporter proteins (named SBC) of the putative actinebactin siderophore biosynthetic pathway and the tonB gene of the putative hydroxamate siderophore biosynthetic pathway were separately deleted from the LAC-4 chromosome. The gene knock-out (KO) strategy relies on the homologous recombination between the chromosome and introduced KO construct. The upstream and downstream regions of the gene(s) of interest and a gene coding of apramycin resistance were amplified, linked via overlap extension PCR and then cloned into a suicide plasmid pMo130. The recombinant plasmid containing the KO construct was transformed into LAC-4 and apramycin resistance colonies were obtained. Aided by the xylE gene encoded on the plasmid, transformants with yellow color after catechol spray were verified as a result of a single cross-over recombination between the chromosome and the upstream or downstream homologous region of the KO construct. After the initial first crossover event, the co-integrate (mutant) cells were passaged in sucrose containing medium via sacB-based counterselection to enrich cells that underwent a second crossover event. Characterization of apramycin sensitive mutants, subsequent PCR screens and DNA sequencing of deletion junction confirmed that SBC and tonB markerless KO mutants of LAC-4 were generated. These mutants will be tested for changes in virulence in Galleria mellonella survival assays, in comparison to LAC-4 wild-type cells. Grant support from CSUPERB’s Research and Development grant program is acknowledged.

Poster #: 76
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Candida, Pseudomonas, biofilm
Project Title: Antifungal Drug Penetration of Bacterial Biofilm Kills Yeast Cells and Yeast Pseudohyphae in In Vitro Co-Infection Model
Author List:
Slarve, Matthew; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Olson, Jon; Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract:
Background: Cystic Fibrosis is a genetic disease that is characterized by a dampening of the innate immune response and a coagulation of mucus in the lungs creating a perfect environment for biofilm formation in the lung alveoli by the bacteria Pseudomonas aeruginosa. Additionally, invasive fungi (ie: Candida albicans or Aspergillus fumigatus) can colonize the tissues below the alveolar membrane, resulting in a bacterial/fungal co-infection. The bacterial biofilm in the alveoli may prevent passage of antifungal drugs to the fungi growing below the alveolar membrane. Treatment of these coinfections needs to eliminate the fungi and bacteria and if the antifungal drug can be administered as an aerosol, it could possibly penetrate through the P.aeruginosa biofilm.
Methods: In this study we used an in vitro transwell model to observe the ability of the antifungal drug AmBisome (liposomal amphotericin B) to penetrate through a P. aeruginosa biofilm on the transwell membrane, and kill the fungi below the membrane. The alveolar membrane on which the bacteria grows in vivo is simulated by the porous polystyrene transwell membrane in vitro. C. albicans was dispensed as yeast cells (4 X 10ex3 cells) or yeast pseudohyphae (yeast cells grown for 72h at 370C) into the bottom of a transwell, and the P.aeruginosa was allowed to develop a biofilm on the surface of the transwell membranes. AmBisome was then dispensed (16 or 32 μg/mL) over the transwell membrane on top of the bacterial biofilm, and allowed to incubate for 24h (yeast cells) or 72h (pseudohyphae) at 370C. The fluid in the bottom of the transwell was then diluted, and plated on Sabouraud’s agar to determine the concentration of viable yeast and AmBisome concentration was determined by bioassay.
Results: The P. aeruginosa required about 24h to form a complete biofilm which was detected by using a periodic acid Schiff stain. AmBisome (16 or 32 μg/mL) penetrated through the bacterial biofilm resulting in 2 μg/mL or 4 μg/mL, respectively, in the bottom of the transwell. This amount of drug killed 100% of the C.albicans yeast cells and 97% of C.albicans pseudohyphae in 24h.
Conclusions: These in vitro results show promise for using the AmBisome to treat bacterial/fungal coinfections in the lungs. Topical application of AmBisome in the transwell system was an effective method for delivering the drug through the bacterial membrane biofilm to kill the yeast growing below it.

Poster #: 77
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Biofilm, Ex vivo model, Wound healing
Project Title: Amelioration of Bacterial Biofilm Formation by Topical Administration of Adrenergic Antagonists to Improve Wound Healing in a Human Skin Explant Model
Author List:
Tran, Jennifer; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Nguyen, Gloria; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: Chronic wounds account for a large portion of health related problems and pose significant costs to the clinical care system. Persistent bacterial infections impede the treatment and healing of chronic wounds by forming multi-species communities called biofilms. Biofilm formation is highly regulated and culminates in the production of a self-initiated extracellular matrix (ECM), and these population-dense communities prolong tissue inflammation and provide resistance to antibiotics. Thus, a critical barrier to wound healing is the paucity of non-antibiotic therapeutics that inhibit microbial growth and prevent biofilm development. Prior work from our lab demonstrated that levels of the catecholamine (CAT) neuroendocrine transmitter epinephrine (EPI) are increased in wound tissues and that, in lab test tube and coverslip assays, similar concentrations augmented growth rates and biofilm biomasses of prominent skin pathogens. Here we developed a translation model for evaluating microbial mechanisms of prolonged wound chronicity using human skin explants obtained from the UC Davis Medical Center. Our results suggest that biopsy punch-induced wound tissues infected with lab grown clinical isolates of biofilm-forming Staphylococcus aureus and Pseudomonas aeruginosa are impaired for healing and express pro-inflammatory mediators as shown experimentally by hematoxylin and eosin staining and blinded scoring for stratum cornea degradation and multiplex quantitative real-time PCR for cytokines and reparative growth and keratinocyte migratory factors, respectively. Furthermore, we used crystal violet staining, confocal laser scanning microscopy for ECM-bound sugars, and next generation RNA sequencing technologies to determine that EPI and norepinephrine (NE) augmented species-specific transcriptional pathways involved in quorum sensing, adhesion, and exopolysaccharide synthesis to modulate bacterial growth and biofilm biomass, and that these genotypic and phenotypic associations with bacterial pathogenicity were ameliorated by topical addition of the adrenergic receptor antagonists timolol and propranolol. Collectively, these data establish a novel ex vivo model for determining mechanisms by which persistent bacterial infections impair wound healing and, by showing that CAT-mediated enhancements to growth and biofilm by skin pathogens can be reversed to alleviate wound chronicity, suggest a novel non-antibiotic therapeutic: translational CAT bio-inhibitors.

Poster #: 78
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: liposomes, influenza, vaccine
Project Title: Liposomal Vaccines with Adjuvants but No Influenza Proteins Protect Swiss Webster Mice Against Influenza Challenge
Author List:
Faneuff, Eden; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Introduction: Commercial influenza vaccines depend upon stimulation of the adaptive immune response to viral proteins that mutate year to year. We have been investigating a liposomal vaccine containing only adjuvants to stimulate an innate immune response against influenza which does not require activation of the adaptive immune response directed against influenza proteins.
Methods: In a previous study in our laboratory, we observed that a protective innate immune response in inbred BALB/c female mice against H1N1 influenza was generated by a comaleimide (CMI) liposomal vaccine (VesiVax®, Molecular Express Inc.) containing the immune stimulating TLR2 agonist, Pam3CAG administered intranasally (IN) 4 and 2 days pre-challenge (d-4/d-2). In this study, we used the same CMI liposomes (L) containing Pam3CAG and administered them IN (40 μL/day) d-4/d-2, d-2, or d-2(PBS) to female outbred Swiss Webster mice [20 mice/group (gp)]. D0 mice (n=5/gp) were euthanized and BAL and lungs collected to determine cytokine production by a multi-bead assay. D0, other mice (n=10/gp) were challenged IN with 40 μL of 10XLD50 H1N1 and monitored for morbidity 2X/day to d26. D5 post-challenge, mice (n=5/gp) were euthanized and lungs collected for viral burden using a foci assay and to analyze cytokine production.
Results: Survival was significantly better for mice given L-Pam3CAG d-2 (70%) compared to mice given L-Pam3CAG d-4, d-2, (10%) or PBS (20%) (p= 0.008 and p= 0.02, respectively). Disease signs and weight loss data paralleled survival with L-Pam3CAG d-2 producing the lowest disease scores and weight loss compared to the other groups. On d0 there were elevated levels of many of the pro-inflammatory cytokines in the BAL and lungs of mice given the d-2 treatment, versus mice given d-4/d-2 or PBS treatments. Between d0 and d5 post-challenge, pro-inflammatory cytokine levels in lungs decreased with d-2 treatment but not with d-4/d-2 or PBS treatment. Viral burden was significantly higher in the d-4/d-2 and PBS groups compared with the d-2 group (p= 0.016 and p=0.008, respectively).
Conclusions: Liposomes containing only the adjuvant Pam3CAG given IN d-2 produced significantly prolonged survival, less morbidity and lower lung viral burden compared to liposomes given IN -4/-2. The elevated levels of pro-inflammatory cytokines observed on the day of challenge following d-2 treatment are indicative of an enhanced innate immune response which decreased as the infection resolved.

Poster #: 79
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: resistome, microbiota, american crow
Project Title: Characterization of the resistome of the Corvus branchyrhyncos microbiota by functional antibiotic screening
Author List:
Nelson, R. Lee; Graduate, Biology, California State University, Fresno, Presenting Author, Eden Award Nominee
Castro, Michael; Undergraduate, Biology, California State University, Fresno
Constable, John; Biology, California State University, Fresno
Van Laar, Tricia; Biology, California State University, Fresno

Abstract: The Centers for Disease Control and Prevention (CDC) estimates that over 2 million people per year acquire antibiotic resistant infections, with a mortality of approximately 23,000 annually. With the increasing number of antibiotic resistant (AR) organisms being identified in human-associated environments, these numbers are likely to go up.
This study focuses on the biotic reservoir Corvus branchyrhyncos, the American Crow. Crows are well adapted to living closely with humans in urban areas, roost in large numbers, and have been observed feeding at waste-treatment facilities and on human refuse. These characteristics make them likely to encounter AR enteric pathogens and deposit them in anthropogenic settings as they roost.
We hypothesize the Fresno and Davis crow microbiota will have some differences in their resistomes but will share similarities in antibiotics used in agriculture as farming is substantial in both cities economies.
Feces were collected from the ground below the roosts and the DNA was extracted using the MoBio Soil Kit. We have submitted the DNA for 16S rDNA and fungal ITS-2 sequencing to characterize the diversity of the microbiota. Resistome characterization is being performed through generation of a metagenomic library. Briefly, we are performing selective enzyme digestion of the DNA extracted from the crow feces and ligating into plasmids. The plasmids are then transformed into E. coli and grown in the presence of antibiotics. Inserts conferring resistance to E. coli will be sequenced to determine their relatedness to known AR genes.
We have already identified multiple antibiotics in which bacteria from crow fecal samples grew readily. These drugs included Vancomycin, Meropenem, Erythromycin, Hygromycin and others. Based on 16S rDNA sequencing, we have identified isolates from genera causing human disease (Enterococcus, Staphylococcus, Pantoea, Bacillus, and Klebsiella) displaying AR. The generation of our metagenomic library will give a more complete picture of AR in the crow gut and could illustrate the potential for spread of these genes within the crow microbiota. Our comparison of samples from different locations in California will also serve to highlight if varying environmental pressures can alter the microbiota and resistome of the highly cosmopolitan species.

Poster #: 80
Campus: San José State University
Poster Category: Disease (Pathogens)
Keywords: obesity, Motor Reflex, Electrophysiology
Project Title: Diet-induced obesity increases H-reflex excitability in adult mice of both sexes.
Author List:
Nguyen, Gerard; Graduate, San José State University, Presenting Author
Haile, Mulatwa; Undergraduate, San José State University
Putnam, Shea; Graduate, San José State University
Nguyen, Jenny; Undergraduate, San José State University, Presenting Author
Wilkinson, Katherine; San José State University

Abstract: Obesity is a metabolic condition characterized by excess adipose tissue and chronic inflammation. It is associated with deficits in balance and motor control leading to an increased risk of falling and fall related injuries. These deficits may be partially due to alterations in the muscle stretch reflex, which helps correct motor movements and respond to uncertain terrain. Muscle stretch is sensed by muscle spindle afferents, which then synapse onto alpha motor neurons and leads to reflex contraction. We tested the hypothesis that spinal cord integration of sensory information from muscle spindle afferents is altered in a mouse model of diet-induced obesity. Changes in spinal cord integration was measured using the Hoffman’s or H-reflex, the electrical analog of the muscle stretch reflex. Following electrical stimulation of the sciatic nerve, two waves of muscle contraction are recorded. The M wave is caused by direct excitation of motor neuron axons and the longer latency H wave is caused by reflex activation of motor neurons by muscle spindle afferent axons. Adult mice of both sexes were fed either a control diet (CD, 10% kcal fat; n=16 M, 8 F) or high-fat diet (HFD, 60% kcal fat; n=15 M, 9 F) for 10 weeks. We measured three properties of the H-reflex as indicators of excitability: (1) H latency, the time from stimulus to the peak of the H wave; (2) Hmax/Mmax, the percent of motor neurons capable of being recruited from stimulus of Group Ia afferents; and (3) rate-dependent depression (RDD), the decrease in H-reflex amplitude with high-frequency (5Hz) stimulations. There were no significant differences in H latency (p=0.558). However, the HFD group showed a significant increase in Hmax/Mmax (p=0.013) in both sexes (p=0.768). High fat feeding also decreased the degree of RDD (p=0.001) in both sexes (p=0.797). These results suggest that obesity increases spinal cord sensitivity to signals from the muscle spindle afferents. Future studies will determine whether these changes in spinal cord excitability translate into changes in the strength of the muscle stretch reflex and/or balance.

Poster #: 81
Campus: CSU Los Angeles
Poster Category: Disease (Pathogens)
Keywords: Defensins, Tuberculosis, Innate defense
Project Title: Effects of Antimicrobial Peptides Against Mycobacterium smegmatis
Author List:
Berton, Roger; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author, Eden Award Finalist
Beadell, Brent; Undergraduate, Biological Sciences, California State University, Los Angeles
Powell, Tyler; Undergraduate, Biological Sciences, California State University, Los Angeles
Lu, Wuyuan; Institute of Human Virology, University of Maryland School of Medicine, Baltimore
Porter, Edith; Biological Sciences, California State University, Los Angeles

Abstract: Background: Mycobacterium tuberculosis (Mtb) is the main infectious agent responsible for tuberculosis and, like other species of the genus Mycobacterium, is an acid-fast aerobic rod shaped bacterium with a lipid-rich cell wall. Tuberculosis causes over 10 million illnesses worldwide annually. Multidrug resistance of Mtb and lack of a protective vaccine in the adult pose a major threat to the public. Our laboratory is interested in exploring whether the innate mucosal host response may be exploited in novel approaches to combat tuberculosis. As part of the innate immune response epithelial cells lining the mucosal surfaces of the airways and resident or rapidly recruited phagocytes release antimicrobial peptides (AMPs; e.g., LL-37 and defensins) and antimicrobial lipids (AMLs; e.g., cholesteryl esters). AMPs are cationic with activity against a wide range of microorganisms. Cytokines secreted by T helper cells, effector cells of the adaptive immune system, also induce the production of these antimicrobials. We hypothesize that AMPs and AMLs, due to their cationic and hydrophobic properties, act synergistically against mycobacteria. Objective: In the present study we wished to determine the effective antimycobacterial concentration of selected AMPs for future synergism studies. Using the avirulent M. smegmatis (Ms) as a model organism for Mtb and a microtiter based metabolic assay we determined effective concentrations of LL-37 and the defensins HNP1, HBD1, HBD2, and HBD3. Methods: Ms was incubated in Middlebrook 7H9 in the presence and absence of AMP (0.3125 to 5 microM final concentration or solvent control) for up to 48 h at 37 °C. Metabolic activity was assessed by resazurin reduction at 3 h intervals. Results: HNP1 and HBD2 dose dependently inhibited Ms metabolic activity at all concentrations tested (p < 0.001), while LL-37, HBD1, and HBD3 had no effects on Ms metabolic activity. Extended susceptibility testing revealed that HBD2 was effective at concentration as low as 20 nM. Furthermore, L- and D-forms of HNP1 and HBD2 showed comparable activity suggesting that these AMPs cause unspecific membrane disruption rather than a receptor mediated lesion. Synergism testing of AMPs and AMLs are now under way. Conclusion: This research may generate important preliminary data that could inform future novel vaccine and drug design against M. tuberculosis. Acknowledgements: NIH 1SC1GB096916 and Biological Sciences FaStA Funds to EP.

Poster #: 82
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Wnt catenin, proliferation, mitochondria
Project Title: Mitochondrial Uncouplers and Protonophores Decrease β-catenin Expression and Reduce HeLa Cell Proliferation
Author List:
Nguyen, Alan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Salhotra, Ankit; Undergraduate, Biological Science, California State University, Fullerton
Patel, Nilay; Biological Science, California State University, Fullerton

Abstract: The canonical Wnt/b-catenin pathway is one of the main biological pathways that mediates cell proliferation. Many small molecules have been screened by the scientific community to identify novel compounds that can attenuate the canonical Wnt pathway. Niclosamide is an FDA approved anthelmintic compound that is able to reduce both b-catenin levels and cell proliferation, and has been tested as a chemotherapeutic agent in clinical trials. However, niclosamide is also a known mitochondrial uncoupler. To our knowledge, other mitochondrial uncouplers and protonophores that disrupt the mitochondrial membrane potential have not been evaluated for their effects on the Wnt pathway or proliferation. Our results show that all of the mitochondrial uncouplers/protonophores tested – CCCP, DCCD, FCCP, 2,-4 dinitrophenol, and niclosamide – decrease cell proliferation and b-catenin levels in human HeLa cells in a dose-dependent manner. Our results show that the doses yielding approximately 50% reduction in cell proliferation for a given drug also dramatically reduced b-catenin protein levels. All compounds except FCCP also reduced b-catenin mRNA levels, which shows that b-catenin gene expression is affected at both transcriptional and post-transcriptional levels. Since mitochondrial uncouplers and protonophores would affect almost all cell types, we suggest that the use of compounds like niclosamide in cancer studies should be approached with caution.

Poster #: 83
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: neural crest, Cadherin-11, apoptosis
Project Title: Cadherin-11 is required for neural crest specification and survival
Author List:
Camacho, Alberto; Graduate, Biology, California State University, Northridge
Manohar, Subrajaa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal ; Biology, California State University, Northridge

Abstract: The neural crest (NC) is an embryonic population of stem-like cells that are responsible for forming various tissues in vertebrate embryos including craniofacial bone and cartilage, pigment cells, and the peripheral nervous system. NC cells are multipotent ectoderm-derived progenitor cells that begin as epithelial cells in the neural tube, but detach and migrate throughout the body after undergoing an epithelial to mesenchymal transition (EMT). These cells express a variety of cell adhesion molecules, such as cadherin proteins, that control their specification, EMT, and migration. In our study, we identify the role of Cadherin-11 (Cad11) in early chicken NC development. Cad11 is a type II cadherin protein that is crucial for NC cell migration in amphibian embryos, and has also been implicated in cancer cell survival, proliferation, and migration. Using immunohistochemistry in chicken embryos, we distinguished the spatiotemporal localization of Cad11 protein in multiple stages to determine where and when it is functioning during embryonic development. Our data shows that Cad11 has previously unreported dynamic expression, which co-localizes with Sox2 in neural progenitor cells in early embryos. Then, as NC cells are specified in the dorsal neural tube and begin to undergo EMT, Cad11 becomes restricted to premigratory and migratory NC cells and is down regulated in the neural tube. Electroporation of a translation-blocking Cad11 morpholino at gastrula stages demonstrates that Cad11 is required for NC specification. Loss of Cad11 is followed by a loss of Pax7 and Sox9 positive NC cells in the dorsal neural tube, but has no effect on more ventral neural tube cells marked by Sox2. We also determined that Cad11 is necessary for NC cell survival because after Cad11 knockdown, markers of apoptosis (programmed-cell death) increased in the dorsal neural tube. These findings show that Cad11 is required for normal NC development, and may increase our understanding of early NC-related developmental defects.

Poster #: 84
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, courtship, histamine
Project Title: Histamine regulates courtship initiation in Drosophila melanogaster
Author List:
Tran, Cac; Graduate, Biological Sciences, San José State University, Presenting Author
Zaki, Sadaf; Graduate, Biological Sciences, San José State University
French, Rachael; Biological Sciences, San José State University

Abstract: Understanding how genes direct neurodevelopment, which in turn specifies particular behaviors, is one of the main goals of neuroscience. Drosophila melanogaster is an excellent model for courtship behavior research. Male Drosophila perform an elaborate courtship ritual that consists of a series of stereotyped behaviors that must be performed correctly and in the right order in order to ensure female receptivity. These behaviors, while complex, are innate – male flies are born knowing how to perform them – but can be modified through environmental interactions (for example, rejection by the female). In addition, all of the steps of the ritual are established and regulated by the behavioral sex determination gene fruitless (fru). Thus, this set of behaviors provides an idea model for the study of genetic and neural programming of behaviors.
We previously identified the gene Trapped in endoderm 1 (Tre1) as regulated by fru and required for normal courtship behavior. Tre1was previously known for its function in cell migration and cell polarity determination in the developing nervous system. Loss-of-function of Tre1 results in an unusual courtship phenotype: male flies mutant for Tre1 initiate courtship on average twice as fast as wild type males. In addition, a subset of Tre1 mutant males fail to initiate courtship at all, indicating a complex requirement for this gene in courtship behavior.
Tre1 encodes an orphan G-protein coupled receptor (GPCR) which shares sequence homology with vertebrate histamine receptors; therefore, we tested histamine as a candidate ligand for Tre1. We found that mutations in histidine decarboxylase mutants, which prevent the conversion of histidine to histamine, displayed rapid courtship initiation similar to that seen in Tre1 mutant flies. This demonstrates that histamine is required for normal courtship behavior, and suggests that histamine is the likely ligand of Tre1. If true, this would be a novel and exciting result, as there are no known metabotropic histamine receptors in insects.
We are currently feeding metabotropic histamine receptor antagonists (antihistamines) to wild type males in order to confirm that histamine regulates courtship initiation via a metabotropic receptor. We will present the results of these experiments (as well as those described above).

Poster #: 85
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Antibiotic Resistance, Metabolomics, Escherichia coli
Project Title: Deletion of the AcrAB-TolC Multidrug Efflux Pump in Escherichia coli Leads to the Intracellular Accumulation of Gluconeogenesis Intermediates
Author List:
Ramos, Karen; Undergraduate, Biology, California State University, Northridge, Presenting Author
Cauilan, Allea; Undergraduate, Biology, California State University, Northridge, Presenting Author
Ruiz, Cristian; Biology, California State University, Northridge

Abstract: Antibiotic resistance is a major challenge to public health worldwide. AcrAB-TolC is the main multidrug efflux pump in Escherichia coli and other Enterobacteriaceae. Previous findings suggest that, beyond effluxing antibiotics out of the cell, the AcrAB-TolC pump may also be involved in the efflux of intracellular metabolites. To identify these metabolites, we used untargeted metabolomics to compare the intracellular metabolites of wild type, ΔacrB and ΔtolC Escherichia coli strains grown in EZ Rich Defined media. Five replicates of each strain were analyzed and the intracellular metabolites were identified and quantified using gas chromatography and time of flight mass spectrometry. We found that inactivation of the AcrAB-TolC pump by deleting the acrB gene caused an intracellular accumulation of some metabolites associated with the gluconeogenesis pathway. These metabolites include malic acid, oxalic acid, and fumaric acid which increased 1.6–2.5-fold in the ΔacrB mutant compared to the wild type (P < 0.05). The acrB mutant also showed a 1.3–1.6-fold accumulation of polar and charged amino acids like lysine, aspartic acid, proline, cysteine, glutamic acid, and asparagine (P < 0.05). Deletion of tolC, which inactivates AcrAB-TolC and eight other efflux pumps, also caused an intracellular accumulation of gluconeogenesis intermediates. These results suggest that efflux pumps hold a major role in cell metabolism. Current experiments on the metabolites identified in this study include the use of Protein Thermal Shift assays to explore ligand to AcrB binding. We also plan to use mutants from the Keio Collection to determine the effect of the gluconeogenesis pathway on AcrAB expression. These and other future experiments will contribute to determine the endogenous substrates of the AcrAB-TolC pump, and how this pump and other TolC- dependent pumps affect cell metabolism. The authors would like to thank CSUPERB for supporting our research.

Poster #: 86
Campus: CSU Channel Islands
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Cannabidiol, breast cancer, HER2
Project Title: Cannabidiol Decreases Proliferation and Increases the G1 Phase of Breast Cancer Cells Overexpressing Human Epidermal Growth Factor-2
Author List:
Bucci, Brianna; Graduate, Biology, California State University Channel Islands, Presenting Author
McCoy, Melissa; Biology, California State University Channel Islands, Presenting Author
Kniseley, Atherton; Graduate, Biology, California State University Channel Islands, Presenting Author

Abstract: Human epidermal growth factor receptors are a family of transmembrane receptor proteins that are expressed in both cancer and normal tissues. Human epidermal growth factor-2 (HER2/ErbB2/neu/p185) is a tyrosine kinase which has been found to be overexpressed in 20-30% of human breast cancer cell lines. This overexpression has been linked to tumor cell proliferation, metastasis and cancer progression. Tumors with overexpression of HER2 have been shown to be more aggressive and consequently provide a worse prognosis for patients.
The endocannabinoid system has been receiving large amounts of interest in its applications as possible therapeutic agents in cancer treatments. These antitumor effects are due to the inhibition of tumor growth via cell-cycle arrest/apoptosis and the reduction in neovascularization and metastases. Previous research in other breast cancer cell lines has shown anti-tumor effects in the presence of cannabinoid treatments, but SKBR-3 cell lines overexpressing HER2 have yet to be examined.
This study involves the investigation of the effects of different concentrations of cannabidiol (CBD), a non-psychoactive component of the endocannabinoid system, have on the behavior of SKBR-3 breast cancer cell lines overexpressing HER2 and normal human foreskin fibroblasts (HFF-1). Using MTS assay proliferation techniques, it was demonstrated that the addition of 20uM CBD, decreases the proliferation of SKBR3 cells but does not affect the proliferation of HFF-1 cells. In addition, when SKBR-3 cells are treated with 20uM CBD, there is an increase in the percentage of cells in the G1 phase of the cell cycle as well as a decreasing percentage of cells in the S and G2/M phase. DNA content was analyzed using propidium iodide (PI) solution and Millipore easyCyte flow cytometer. Our results conclude that CBD interferes with the normal proliferation and cell cycle of SKBR-3 cells but does not manipulate the proliferation of HFF-1 cells. Future works will include the investigation of expression of HER2 in the presence of CBD, along with utilizing a human breast cancer PCR array to determine the expression of key genes commonly involved in breast cancers in the presence of CBD.

Poster #: 87
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: cadherin, ectoderm, cell fate
Project Title: N-cadherin expression alters neuroectodermal cell fate choices during avian embryonic develop-ment
Author List:
Sorrells Smith, Lisa; Graduate, Biology, California State University, Northridge, Presenting Author, Eden Award Nominee
Rogers, Crystal; Biology, California State University, Northridge

Abstract: Early developmental processes are tightly regulated and require tissues to segregate into three germ lay-ers: ectoderm, mesoderm and endoderm during gastrulation. After these tissues are specified, each germ layer is further determined, eventually differentiating into all the cells and tissues that will make up the adult organism. We are interested in the process that controls the specification and differentiation of ectodermal tissues. Ectodermal cells become the neural tube, non-neural ectoderm (skin and sensory placodes), and neural crest cells, which become the craniofacial bone and cartilage and the peripheral nervous system. Little is known about the molecules that regulate the cell fate choices of ectodermal derivatives. However, previous studies have shown that cell adhesion through cadherin proteins is im-portant for the process. Neural cadherin (Ncad) is a calcium dependent cell-cell adhesion molecule and is required for the development of neural ectoderm, however its role in ectodermal cell fate specification remains understudied. To identify the role of Ncad in ectodermal fate choice, we performed in vivo loss of function experiments in chicken embryos using a translation blocking morpholino. In vivo microinjec-tion of the morpholino followed by whole mount immunohistochemistry produced embryos with abnor-mal ectodermal derivative specification; we hypothesize that without Ncad, the ectodermal cells maintain a progenitor-like fate rather than differentiating. Loss of Ncad produces an increase in proliferation marked by PH3, an increase in non-neural ectoderm marked by Ecad, a reduction of neural crest marked by Snail2 and Pax7 and a loss of neural progenitor cells marked by Pax2. These results identify a previ-ously unknown role played by Ncad during early development by showing that it influences early tissue specification. Recognition of the expanded role that Ncad plays may have implications for other aspects of development and disease that involve Ncad such as the formation of the heart and gut and cancer formation and metastasis.

Poster #: 88
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: proprioception, electrophysiology, muscle spindle afferent
Project Title: The effects of reactive oxygen species on muscle spindle afferent activity
Author List:
Hill, Megason; Undergraduate, Biological Sciences, San José State University, Presenting Author
Hui, Sam; Undergraduate, San José State University, Presenting Author
Kwon, Anthony; Graduate, San José State University
Wilkinson, Katherine; Biological Sciences, San José State University

Abstract: Proprioception is the ability of a body to determine its position in space and is crucial to be able to perform complex motor tasks. Muscle spindle afferents (MSAs) are sensory neurons which relay information about changes in muscle length to the nervous system to allow completion of these tasks. Fatigue from exercise is linked to deficits in MSA function. During exercise, reactive oxygen species (ROS) are released as byproducts of metabolism, which could be the cause of changes to MSA function. However, whether ROS act centrally or at the MSA peripheral endings is unknown. We theorized that ROS would increase responsiveness to stretch by acting on MSA receptor endings. A ROS, hydrogen peroxide, was added to an in vitro bath of oxygenated synthetic interstitial fluid (SIF), to mimic physiological conditions, with a dissected extensor digitorum longus (EDL) muscle and the innervating sciatic nerve. The EDL was subject to different stretches and vibrations in the SIF, and then subject to the same stretches and vibrations in the presence of 1mM or 10mM hydrogen peroxide. Induced stretches on the EDL consisted of ramps held at a 5% increase of physiological muscle length whereas high frequency vibrations would consist of 9 second vibrations of 50 um amplitude at 50Hz and 100Hz. MSA firing rates without the drug were then compared against the conditions with hydrogen peroxide. Hydrogen peroxide exposure for 1mM resulted in an increased stretch response in about half of the MSAs tested and did not alter the activity of the other half (n=10; 23 ± 13% increase , n=7; 1.5 ± 2.8%). Exposure with 10mM caused an overall increase in stretch response, partially caused by activating formerly silent afferents (n=10; 220%). MSA response to vibration was unchanged at 1 mM and increased in 10 mM hydrogen peroxide (n=10; 72%). These results support our hypothesis that hydrogen peroxide can act on muscle spindle afferent receptor endings and increase responsiveness to stretch. Exercise released ROS likely contribute to impaired proprioception following fatiguing exercise. Future studies will determine whether other exercise released substances, like bradykinin and IL-6, can also modify MSA function.

Poster #: 89
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Built Environment, Microbiology, FACS
Project Title: FACS analysis of microbial community dynamics on building materials
Author List:
Tandon , Ruby ; Graduate, Biology , San Diego State University, Presenting Author
Kelley , Scott T; Biology , San Diego State University

Abstract: Indoor environments are replete with microbial communities (virus, bacteria and fungi). These communities are knowns to vary in abundance, composition and diversity depending on material type, moisture levels and occupants. Previous research from our laboratory shows that microbial growth varies significantly based on building material type particularly when materials have high amounts of moisture. In the current study, we extended our analysis of the dynamics of microbial growth and metabolic on building materials by using flow cytometry based method to differentiate between viable and non-viable bacterial cells over time. We also developed and standardized measurements of fungal biomass by staining fungal cells and subsequently imaging using epifluorescence microscopy. Preliminary results from four indoor materials (carpet, ceiling, plywood and fiberboard) showed that the materials start with low viable:non-viable ratio, which increases upon wetting. Interestingly, the number of non-viable cells also increased over time suggesting that there is a continuous turnover of bacterial cells, as various bacterial species compete for space and resources. Carpet materials in particular had high levels of bacterial growth and a much higher ratio of viable to non-viable cells than the other materials. We also demonstrated that we can use Fluorescence Assisted Cell Sorting (FACS) to differentially sort viable and non-viable cell populations, whose constituents can later be identified using culture-independent sequencing methods. Having developed these method, we next plan to investigate these community-based interactions by conducting a large scale study for different types of building materials using the multi-technique approach (flow cytometry, microscopy and 16S and ITS sequencing) described above. Overall, this study has important implications for buildings with leaks, in flood-prone regions, spaces with prolonged damp conditions like bathrooms and their impact on human health.

Poster #: 90
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Molting, chemosensation, parasitism
Project Title: Examination of a Dauer-Specific Molting Defect in the Nematode Pristionchus pacificus
Author List:
Carstensen, Heather; Undergraduate, Biology, California State University, Northridge, Presenting Author
Villalon, Reinard; Undergraduate, Biology, California State University, Northridge, Presenting Author

Abstract: Parasitic nematodes pose an enormous burden to human health, crops, and livestock. The nematode Pristionchus pacificus has significant similarities to parasitic nematodes that allow researchers to investigate parasitic characteristics in a tractable organism. These features include interaction with beetle hosts and retention of an extra cuticle layer in the long-living “dauer” stage, similar to the infective stage of many parasitic nematodes. To investigate P. pacificus dauer larvae, we characterized a dauer-constitutive mutant allele, tu391.
Extensive characterization of tu391 suggest not only a defect in regulating dauer entry, but also inability to properly retain the outer cuticle. To investigate possible causes of the inappropriate dauer formation, we looked for defects in the amphid sensilla, which are chemosensory organs responsible for controlling commitment to the dauer stage by sensing the dauer pheromone. We found tu391 amphid neurons are defective in the uptake of the live dye, DiI, suggesting there may be functional defects in the amphid glia or neurons that contribute to the tu391 phenotypes. By incubating tu391 nematodes at different temperatures, then scoring for presence of dauer larvae versus the equivalent J3 larval stage, we found that higher temperatures increased dauer formation frequency. Interestingly, nearly half of the dauers formed at 20°C were trapped within a completely loose outer cuticle that hindered their locomotion, but very few exhibit this phenotype at 25°C. The dauer formation and cuticle defects thus respond to changes in temperature differently.
With defects in the amphid sensilla, tu391 may not properly sense other odors including signals from their host beetles. Exposure to the beetle sex pheromone causes dauer paralysis and a mutation in the gene obi-1 results in hypersensitivity at lower pheromone concentration. We found that the tu391 mutation suppressed obi-1 hypersensitivity to the beetle pheromone, since tu391;obi-1 double mutants lack hypersensitivity exhibited by the obi-1 mutants. Understanding the function of tu391 and its role in obi-1 hypersensitivity will help determine how P. pacificus responds to host odors overall.
Further investigation into P. pacificus dauer larvae characteristics will help researchers further understand analogous features in the infective stages of parasitic nematodes. This may ultimately lead to innovative ways to prevent costly parasitic infections of crops and livestock.

Poster #: 91
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Mutant complementation, shuttle vector, virulence factor
Project Title: Complete sequencing of pWH1266 and its use as an expression shuttle vector for complementation of KO mutants of Acinetobacter baumannii LAC-4
Author List:
Madrid, Athena; Graduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Ewing, Peter; Department of Biological Sciences, California State University, Los Angeles
Xu, Howard; Department of Biological Sciences, California State University, Los Angeles

Abstract: Acinetobacter baumannii is a Gram-negative bacterium that has become increasingly multi-drug resistant (MDR) and is known for becoming a common cause of nosocomial infections in intensive care units. One clinical isolate obtained from a Los Angeles County Hospital, named LAC-4, was found to be hypervirulent to mice. Previous work from our laboratory has generated unmarked knockout (KO) mutants of putative virulence genes in this bacterium in order to elucidate their role in LAC-4’s hypervirulence. However, techniques to complement KO mutants are not well established for MDR strains of A. baumannii, such as LAC-4. Here we report the development of the E. coli/A. baumannii shuttle vector pWH1266 as a potential tool for complementation of LAC-4 KO mutants. To accomplish this, we sequenced unknown regions of the plasmid by primer walking with Sanger sequencing and characterized a potential promoter of the plasmid. Primers were first designed based on the known regions of the plasmid. The generated sequences were analyzed and new primers were designed based on the new sequences. The steps were repeated until all previously unknown regions of the plasmid were sequenced. Additionally, we cloned the reporter gene xylE, which codes for catechol-2-3-dioxygenase, downstream of the putative promoter on the vector to confirm that there is a strong constitutive promoter on the plasmid. The results showed both E. coli and A. baumannii LAC-4 robustly express XylE reporter enzyme. To obtain proof of concept, we cloned the hemO gene (coding for heme oxygenase) of LAC-4 into pWH1266 and transformed the recombinant plasmid via electroporation into A. baumannii LAC4 ΔhemO strain. The complemented strain’s virulence was then compared to both wild-type LAC-4 and LAC-4 ΔhemO in Galleria mellonella survival assays. Our results showed that the complemented strain’s phenotype was comparable to the wild-type A. baumannii LAC-4, indicating successful re-expression. In conclusion, the development of the shuttle vector pWH1266 as a tool to construct complementation strains will facilitate the studies of the mechanisms of pathogenesis in MDR A. baumannii strains. Grant support from CSUPERB’s Research and Development grant program is acknowledged.

Poster #: 92
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: 16S rRNA, polycystic ovary syndrome, hyperandrogenism
Project Title: Decreased Diversity of the Gut Microbiome in Women with Polycystic Ovary Syndrome Is Correlated with Hyperandrogenism
Author List:
Torres, Pedro J.; Graduate, Biology, San Diego State University, Presenting Author
Siakowska, M.; Graduate, oznan University of Medical Sciences,Poland
Banaszewska, Beata; oznan University of Medical Sciences,Poland
Duleba, Antoni; Dept. of Reproductive Medicine, University of California, San Diego
Thackray, Varykina; Dept. of Reproductive Medicine, University of California, San Diego
Kelley, Scott T.; Biology, San Diego State University

Abstract: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-aged women. In addition to anovulation and hyperandrogenism, a majority of women with PCOS have metabolic abnormalities that result in an increased risk of type 2 diabetes and cardiovascular disease. Although androgen excess is considered to be a cornerstone in the pathology of PCOS, the etiology of this disorder remains elusive. Studies have demonstrated that changes in the gut microbiome are associated with metabolic disorders such as obesity and type 2 diabetes, and that the gut microbiome may play a causal role in these disorders. Recent studies in Caucasian and Han Chinese women reported a significant decrease in gut bacterial alpha diversity (species richness) in women with PCOS compared with healthy women and we also observed a decrease in gut alpha diversity in a hyperandrogenic, letrozole-induced PCOS mouse model compared with placebo controls. Here, we investigated whether changes in alpha diversity in women with PCOS correlated with specific endocrine parameters. We also asked whether there was a significant change in the diversity of the gut microbiome in women with polycystic ovarian morphology (PCOM) that lack the other diagnostic criteria of PCOS. Rectal swabs and clinical data were collected from Caucasian patients recruited from the Infertility Clinic at Poznan University of Medical Sciences. Gut microbial diversity profiles of healthy women (n=48), women with PCOM (n=42), and women diagnosed with PCOS using the Rotterdam criteria (n=73) were determined by next-generation sequencing of bacterial 16S ribosomal RNA genes and bioinformatics analysis using the QIIME pipeline. Our results showed that women with PCOS had a significant decrease in overall species richness and phylogenetic diversity compared with healthy women. Women with PCOM also had a change in gut microbial diversity that was intermediate between that of healthy women and women with PCOS. Multiple regression analysis of diagnostic and physiological parameters showed that hyperandrogenism had the highest correlation with alpha diversity. Additional single factor linear regression analyses found a significant negative correlation between alpha diversity and both total testosterone levels and hirsuitism, while no correlations were observed with age or BMI. Altogether, these results suggest that hyperandrogenism may play a critical role in altering the gut microbiome in women with PCOS.

Poster #: 93
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: speciation, experimental evolution, epistasis
Project Title: Genetic effects on fitness recovery of dysfunctional hybrids
Author List:
Jorgensen, Chris; Graduate, Biology, California State University, Fresno, Presenting Author
Fita, Lesly; Undergraduate, Biology, California State University, Fresno
Pavic, Boris; Undergraduate, Biology, California State University, Fresno
Ross, Joseph; Biology, California State University, Fresno

Abstract: One challenge in genetics is identifying how and what genetic differences between two populations of the same species, when combined in a hybrid offspring, interact to cause the offspring to be less healthy than its parents. Accumulation of sufficient numbers or severities of such interactions can lead to complete hybrid dysfunction: sterility or lethality, which are hallmarks of speciation. An outstanding question in speciation genetics is the extent to which species boundaries can be penetrated. We previously gathered empirical evidence that limitations on inter-population hybridization are weak in the nematode Caenorhabitis briggsae, a close relative of C. elegans. Both species are model organisms for genetics research, particularly because of their small and fully sequenced genomes, short generation times, and ability to self-reproduce. We now report the creation of panels of hybrids between wild populations called AF16 and HK104. We designed these hybrids to combine the nuclear genome of one strain with the mitochondrial genome of the other, providing the genetic basis for reduced hybrid fertility. To address the question of whether these hybrids would remain less fit, we allowed each hybrid line to grow in parallel and determined fecundity every fifth generation. We observed no initial change in fitness in hybrids with AF16 paternal heritage and an early increase in fitness for strains with HK104 paternal heritage. Later results show there has been a slight increase of HK104 fitness for paternal heritage. As for AF16 hybrids, a decrease in fitness was observed. In conclusion, after initially experiencing hybrid dysfunction, the fitness trajectories of different strains do vary based, in part, on parentage. This finding of asymmetry in fitness recovery following hybrid dysfunction suggests the possibility that some genetic variants unique to each parental population might facilitate or inhibit hybrid fitness. Further exploration of the genetic basis for this observation will be important in understanding how species boundaries form and how penetrable they are.

Poster #: 94
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer, PEAK1, Fibroblasts
Project Title: Novel Tumor-Promoting Functions of PEAK1 Kinase in the Fibroblast Compartment of Breast Cancer Stroma
Author List:
Hamalian, Sarkis; Graduate, Biology, California State University, Northridge, Presenting Author
Molnar, Justin; Graduate, Biology, California State University, Northridge
Agajanian, Megan; Graduate; Department of Surgery at the Hospital of the University of Pennsylvania, Biology, California State University, Northridge
Tchou, Julie; University of Pennsylvania, Biology, California State University, Northridge
Kelber, Jonathan; Biology, California State University, Northridge

Abstract: In solid malignancies, such as breast cancer, tumor cells interact with their surrounding microenvironment (ME), and it is well-accepted that these interactions promote tumor progression and resistance to available therapies. The breast cancer microenvironment (BCME) is composed of multiple cell types including, but not limited to, mesenchymal stem cells (MSCs), immune cells, adipocytes, endothelial cells, and cancer associated fibroblasts (CAFs) as well as the tumor cells themselves. Paracrine and juxtracrine signaling mechanisms between these cells have been demonstrated to mediate the tumor-promoting/supportive effects of the BCME. Among the various tumorigenic factors present in the BCME, we decided to study PEAK1 (Pseudopodium-Enriched Atypical Kinase 1) because it is a cytoskeleton-associated kinase expressed in fibroblasts and is upregulated in the stroma of breast cancer patients with recurrent disease. We hypothesized that PEAK1 expression in the fibroblast compartment of the BCME promotes tumor cell proliferation/survival, metastasis and therapy resistance. We report that PEAK1 is expressed in patient-derived CAFs, with CAFs from all breast cancer subtypes (i.e., ER/PR+, HER2+ and triple-negative)expressing high levels of PEAK1 protein and RNA. Conditioned media treatment from these CAFs increased BC proliferation, migration and therapy resistance in vitro. Furthermore, co-xenografting PEAK1hi CAFs with BCCs significantly increased primary tumor weight. In parallel with PEAK1 loss-of-function studies in CAFs, we have generated PEAK1 knockdown derivatives of MSCs to evaluate the tumor-promoting functions of PEAK1 in another fibroblast-like cell type found in the BCME. Notably, blocking PEAK1 expression/function does not affect MSC proliferation/survival. Current work is aimed at evaluating whether PEAK1 knockdown in CAFs and/or MSCs affects breast cancer cell growth/metastasis or breast cancer cell responses to targeted therapies, and whether any tumor-promoting functions of PEAK1 in fibroblast cell types is mediated by paracrine or juxtacrine signaling. The long-term objective of this work is to characterize novel approaches for targeting the tumor-supportive microenvironment that will increase the efficacy of existing therapies and block breast cancer progression.

Poster #: 95
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Environmental Pollution, Calcium Signaling, Hypothalamus
Project Title: Pollutant-Induced Changes in the Ryanodine Receptor and the L-Type Voltage Gated Ca2+ Channel Alter DREAM-Mediated Gene Transcription
Author List:
Schuko, Nicolette; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Lepe, Javier; Undergraduate, Biological Sciences, California State University, Long Beach
Bhandari, Deepali; Chemistry, California State University, Long Beach
Holland, Erika; Biological Sciences, California State University, Long Beach

Abstract: Environmental pollutants, including non-coplanar polychlorinated biphenyl (ncPCB) congeners and triclosan, cause cellular Ca2+ signal disruption (CSD) by altering the activity of the ryanodine receptor (RyR) or the L-type voltage gated Ca2+ channels (CaV1). These channels are important to numerous Ca2+ signaling pathways within the cell but the extent to which CSD through RyR and CaV1 contribute to altered cellular function is unclear. We investigated whether CSD can alter gene transcription normally regulated by the Ca2+-sensitive transcriptional repressor DREAM (downstream regulator element antagonistic modulator). This research utilized GT1-7 hypothalamic neuronal cells or TαT1 thyrotrophic cells to assess whether CSD alters transcription of gonadotropin releasing hormone (GnRH) or thyroid-stimulating hormone (TSH), respectively, which are regulated by DREAM. Cells were exposed to varying concentrations of each pollutant for multiple time-periods, and GnRH or TSH levels assessed using qPCR. GT1-7 cells exposed to the potent RyR activator PCB95 did not alter GnRH mRNA expression, which was supported by low RyR basal gene expression in the cell line. GT1-7 cells exposed to triclosan, a CaV1 inhibitor, decreased GnRH transcription in a dose-dependent manner after just three hours. When CaV1 channels are inhibited or when intracellular Ca2+ concentrations are decreased, DREAM remains bound to DNA, repressing transcription, which is consistent with reduced GnRH transcription in the presence of triclosan. This work helps address whether CSD is contributing to alterations in DREAM-mediated transcription, which has been implicated in the function of the digestive system, central nervous system, and tied to pain reception, learning and memory and thyroid-gland health.

Poster #: 96
Campus: CSU Fresno
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: phylogenetic comparative methods, hagfish, habitat depth
Project Title: A slimy problem: body size and shape evolution in hagfish
Author List:
Reece, Joshua; Biology, California State University, Fresno, Presenting Author

Abstract: Body shape and size are two of the most prominent axes of the phenotype. In marine fish, several ecological and evolutionary trends describe patterns of evolution in body shape and size. Two such trends are that fish species in deeper environments tend to be 1) larger and 2) more elongate. The association of body size and depth is termed Heincke’s Law, while the association between body shape and depth is more variable and not formalized as a law. Comparative phylogenetics provides a framework for assessing the degree to which traits displayed by species reflect shared ancestry more or less than random variation or differences in ecology. Here, we use a phylogeny of 30 species of hagfish to evaluate phylogenetic signal for maximum body length and for body shape, assessed as standard length divided by maximum width/height. We also evaluate the influence of habitat depth on either body shape or size, and use phylogenetically corrected regression to test for any associations between body shape and size. We find that Heincke’s Law does not explain variation in body size in hagfish, but there is a weak influence of phylogeny on body size. Surprisingly, body shape also appears to evolve independent of depth, phylogeny, or maximum body size. Hagfish represent a sister group to all vertebrates, and thus the patterns of morphological evolution in this group are useful for reference relative to vertebrate size and shape evolution. Despite occupying every ocean basin, existing at a variety of depths, and displaying diversity in body shape and size, hagfish appear to have evolved that variation either as a random process or through adaptation to ecological forces not captured by habitat depth. This is surprising given patterns of size and shape evolution in most marine fishes.

Poster #: 97
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Mucolipidosis type IV, lysosomes, gene complementation therapy
Project Title: Induction of endogenous Mucolipin-2 (MCOLN2) gene expression in human glial and neuronal cell lines using transcription factor PAX5
Author List:
Rosas, Lauren; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Cuajungco, Math P; Biological Science, California State University, Fullerton

Abstract: Mutations or deletions in the Mucolipin-1 (MCOLN1) gene, which encodes the Transient Receptor potential Mucolipin-1 (TRPML1) protein, is known to cause the neurodegenerative disease Mucolipidosis type IV (MLIV). MLIV is manifested by motor problems, cognitive dysfunction, low gastric acid production, cataract, and blindness caused by retinal cell death. Other members of the Mucolipin family consist TRPML2 and -3 proteins, which are encoded by MCOLN2, and -3 genes, respectively. The TRPMLs serve as non-selective cation channels located within endosomes and lysosomes. Previous research indicates that MCOLN1 and MCOLN2 share a high degree of homology. Thus, we hypothesize that the MCOLN2 could substitute for the loss of functional MCOLN1 in MLIV, which could potentially rescue the disease phenotype. To this end, we screened and discovered PAX5 as the transcription factor responsible for MCOLN2 expression. We also showed that its over-expression parallels an increase in MCOLN2 transcripts in cultured human embryonic kidney 293 and human H4 neuroglioma cell lines. In order to study the potential therapeutic effect of MCOLN2 complementation in MLIV, we must first show that it is present in the brain and that PAX5 could induce or increase its expression. Using standard reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR), we found that endogenous MCOLN2 transcripts are detected in human SH-SY5Y neuroblastoma and H4 neuroglioma cell lines, albeit at very low levels. This result adds to current knowledge that MCOLN2 has tissue-specific expression with little to no detectable transcripts in brain tissue; however, it also suggests that we could use PAX5 to influence its expression level in neurons and glial cells. Next, we transduced SH-SY5Y neuroblastoma cells with either PAX5 cDNA-expressing adenovirus or lentivirus. Our RT-PCR and real-time qPCR analyses show for the first time that endogenous MCOLN2 transcripts are markedly increased in neuroblastoma cells at 48 and 72 hours post-transduction with either PAX5-expressing adenovirus or lentivirus. Thus, the current study opens the possibility for gene complementation to rescue the cellular phenotype in MLIV disease. Future studies will focus on showing that PAX5-mediated increase in endogenous TRPML2 protein is detected by Western blot, and that elevated TRPML2 causes a reversal of brain pathology using a mouse model for MLIV. This work was funded by NIH R15 NS101594.

Poster #: 98
Campus: CSU San Marcos
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Arabidopsis thaliana, Reactive oxygen species, mitochondria
Project Title: Defining the role of the Arabidopsis thaliana alternative oxidase gene family in mitochondrial production of reactive oxygen species
Author List:
Caldwell, Maylin; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Allen, Nick; Graduate, Biological Sciences, California State University San Marcos
Escobar, Matthew; Biological Sciences, California State University San Marcos

Abstract: The mitochondrial electron transport chain (ETC) plays a central role in the bioenergetics of all eukaryotic cells. The ETC is also a major source of reactive oxygen species (ROS), a byproduct of electron transport. In humans, ROS-associated damage has been linked to aging and neurodegenerative disorders, and it is important to understand how ROS production is managed by different organisms. Plants have several “alternative” pathways for electron flow in their ETC which are not found in animals. This research focuses specifically on characterizing the function of the alternative oxidases (AOX), which have been proposed to help minimize the production of ROS in plants. To better understand the connections between AOX and ROS production in plants, we generated a series of transgenic Arabidopsis thaliana plants using two RNA interference vectors designed to silence the entire AOX gene family (five genes). Real time RT-PCR analyses demonstrated significantly reduced levels of AOX transcripts in several transgenic plant lines, and in vivo respiratory assays showed significantly reduced AOX capacity in those same lines compared to wild-type. We then used these elite transgenic lines to gain insight into how decreasing expression of alternative oxidases affects ROS levels and ROS-associated damage in plants. Hydrogen peroxide, a type of ROS, was quantified in plant leaf extracts, and significantly higher levels were measured in AOX-silenced plants than in wild-type plants. Lipid peroxidation, a type of oxidative damage, similarly showed elevated levels in AOX-silenced plants. Overall, these findings suggest that AOX does play an important role in managing ROS levels and ROS-related damage in plants.

Poster #: 99
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: blood, zebrafish, stem cells
Project Title: ccl44 depletion negatively affects vertebrate hematopoiesis
Author List:
Laurie, Payton; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Harris, Elena; Computer Science, California State University, Chico
Stachura, David; Biological Sciences, California State University, Chico

Abstract: Hematopoiesis is a complex and highly regulated system where hematopoietic stem and progenitor cells (HSPCs) differentiate into the numerous types of cells that make up the hematopoietic system. Understanding the genetic and molecular pathways involved is an important step to treating many diseases associated with the blood system. Due to the conservation of this system and many of its genes in vertebrates we utilize zebrafish (Danio rerio) as a model organism. Three stromal cell lines from known sites of zebrafish hematopoiesis were generated and shown to expand hematopoietic cells when plated on these stromal cells. With the use of RNA sequencing we compared genetic expression of these three stromal cell lines and generated a list of 447 genes that we believe are important regulatory factors in the hematopoietic system. A highly expressed transcript from these areas was chemokine (C-C motif) ligand 44 (ccl44). In order to test its effect on hematopoiesis I performed knockdown experiments using morpholinos (MO). Transgenic zebrafish lines with fluorescently labelled myeloid and erythroid cells were injected with ccl44 MO and a decrease in those cell lineages was observed with flow-cytometry and fluorescence microscopy. These results indicate that ccl44 is an important gene in normal vertebrate hematopoiesis. Further examination of ccl44’s effects on the hematopoietic system could have clinical importance for HSPC expansion and treatment of diseases like anemia and leukemia.

Poster #: 100
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer, Premetastatic Niche, Macrophages
Project Title: High-Grade Breast Cancer Cells Elicit Pro-Tumorigenic Responses in Non-Tumor Cell Types Found within the Premetastatic Niche
Author List:
Meade, Kayla; Graduate, Biology, California State University, Northridge, Presenting Author
Aguayo, Analine; Undergraduate, Biology , California State University, Northridge, Presenting Author
Kelber, Jonathan; Biology, California State University, Northridge

Abstract: Metastatic spread of solid tumors within the breast accounts for over 95% of cancer-related deaths. However, before cancer cells can survive in these “foreign” microenvironments, these new tissues must be primed and acquire tumor-permissive properties. It is understood that the primary tumor can communicate with the premetastatic niche through the release of factors that can recruit cells, such as mesenchymal stem cells (MSCs) and macrophages (MACs), to this new environment. Within the premetastatic niche, however, very little is known about whether MSCs and MACs communicate with one another and if secretomes from high-grade breast cancer cells (BCCs) promote this communication. Understanding these events may lead to therapeutic interventions to target these cancer-permissive characteristics within the pre-metastatic niche. To address this unmet challenge, we use in vitro and in vivo methods with high-/low-grade mouse/human BCCs to evaluate how secreted factors produced from these tumor cells affect MACs and MSCs, and whether tumor-educated MSCs can elicit tumor-promoting functions of MACs. We determined that the secretomes of high-grade mouse BCCs increase MSC and MAC viability, as well as MAC cell spreading and motility. Furthermore, we observed that high-grade BCC secretomes enable MACs to adopt an anti-inflammatory, pro-tumorigenic gene expression profile. Finally, we found that naïve MSCs increase MAC viability, however do not have an effect on MAC polarization. Experiments are in progress to analyze lung, liver and brain tissue from mice that received chronic intraperitoneal injections of media conditioned from these high-/low-grade mouse BCCs. While there were no noticeable gross or histological changes in these tissues between the two recipient groups, IHC/IF staining is currently being performed to evaluate the infiltration of and polarization of resident MACs in these tissues.

Poster #: 101
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, leukemia, drug development
Project Title: Analysis of novel inhibitors of the GRB2 SH2 domain that decrease proliferation in chronic myeloid leukemia
Author List:
Rodriquez, Sofia; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Aquiar, Stephanie; Undergraduate, Biological Sciences, California State University, Chico
Hanson, Tina; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Arpin, Carolynn; Chemistry, California State University, Chico
Stachura, David; Biological Sciences, California State University, Chico

Abstract: Chronic myeloid leukemia (CML) is a disease affecting the normal growth of myeloid cells in the blood and bone marrow caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1). Once transcribed, this fusion protein, known as BCR-ABL, causes an over-proliferation of myeloid cells. Downstream of BCR-ABL is growth receptor bound protein-2 (GRB2), an intracellular adapter protein involved in cellular growth and differentiation. BCR-ABL binds with the SRC homology-2 (SH2) domain of GRB2 accelerating leukemic transformation. We created and tested four novel SH2 antagonists (NHD2-15B, NHD2-92, NHD2-107, and NHD2-114) in an enzyme-linked immunosorbent assay (ELISA) to determine if the drugs outcompeted GRB2’s natural ligand. We then tested the drugs’ effects on the growth of K562 cells, a BCR-ABL+ immortalized myelogenous leukemia cell line, and found significant growth reduction after 48 hrs. An apoptosis/necrosis assay was performed to elucidate if the growth reduction was due to drug activity or toxicity, and each antagonist then was tested on developing zebrafish to further validate the toxicity of these molecules. These assays indicated that our SH2 antagonists antagonize GRB2 and kill CML cells, bringing us closer to determining a key mechanism in CML oncogenesis and developing targeted therapies for this disease.

Poster #: 102
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: dyrk1a, crispr, zebrafish
Project Title: Analysis of Dyrk1a in Zebrafish Craniofacial Development
Author List:
Godinez, Eduardo; Graduate, California State University, Los Angeles, Presenting Author
Medina, Eric; Graduate, California State University, Los Angeles, Presenting Author
Alvarado, Estibaliz; Graduate, California State University, Los Angeles
Sinclair, Jason; NIH/NHGRI
Burgess, Shawn; NIH/NHGRI
Nissen, Robert; California State University, Los Angeles

Abstract: The dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk) gene family is a highly conserved group of protein kinases. Dyrk1a is implicated in various processes including neuronal proliferation, differentiation, and craniofacial development. While overexpression of Dyrk1a is associated with Down Syndrome and Alzheimer’s disease, Dyrk1a haploinsufficiency causes microcephaly in humans. While it is known that the mouse Dyrk1a-/- mutant is an early embryonic lethal, detailed studies of craniofacial development have not been completed in the mouse knockout model. This may be due to the difficulty inherent in working with very early developmental stages in the mouse model compounded by additional challenges associated with Dyrk1a-/+ haploinsufficiency. Therefore, we are creating a zebrafish model for the study of Dyrk1a in early development. Notably, while human and mouse have only a single Dyrk1a gene, the zebrafish has two paralogous family members, dyrk1aa and dyrk1ab. Here, we present progress towards the creation and characterization of dyrk1aa and dyrk1ab mutants. Zebrafish founders were mutagenized by microinjection of gRNAs and Cas9 mRNA, raised to adults, and outcrossed to wildtype. We then screened the resultant F1 animals by PCR to identify carriers for mutant alleles of dyrk1aa and dyrk1ab. To date, we recovered seven different alleles for dyrk1aa and five different alleles for dyrk1ab. We further outcrossed select alleles for expansion to an F2 generation. For example, dyrk1aa-csu27 is a 2bp deletion predicted to cause reading frame-shift and dyrk1aa-csu31 is a 68bp deletion that removes a 3’-splice site. Both are predicted to yield loss-of-function for dyrk1aa. Likewise, dyrk1ab-csu37 is a 9bp deletion plus 29bp insertion while dyrk1ab-csu40 is a 15bp deletion plus 2bp insertion. Both alleles are predicted to cause reading frame-shift and loss-of-function. Further breedings are underway and we will present progress on the molecular characterization of the mutant alleles as well as phenotyping single and double mutant zebrafish embryos. Specifically, we hypothesize that dyrk1aa-/-; dyrk1ab-/- embryos will present general growth retardation and craniofacial development defects. This research will improve our understanding of the functions of dyrk1aa and dyrk1ab in early craniofacial development with potential implications for therapeutic interventions. This work was supported by a grant from the NIH.

Poster #: 103
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: rare earth elements, methanol dehydrogenase, Methylobacterium extorquens
Project Title: A Mutation in the MxbD Sensor Kinase Eliminates the Need for XoxF in Regulating Methanol Dehydrogenase Expression in Methylobacterium extorquens
Author List:
Luong, Bang; Undergraduate, Biological Sciences, San José State University, Presenting Author
Skovran, Elizabeth; Biological Sciences, San José State University

Abstract: Methylobacterium extorquens is a methylotrophic bacterium that uses single carbon compounds like methanol for growth. Our lab is engineering M. extorquens to recycle rare earth elements (REEs) from electronic waste to create a sustainable domestic REE supply. REEs are essential for our current technologies as components of our computers, smart phones, and hybrid car batteries. To create an efficient REE recovery platform, we need to better understand how the enzymes that sense, use, and respond to REEs are regulated. We previously showed that there are two two-component systems (MxcQE and MxbDM) required for expression of the Ca2+-dependent methanol dehydrogenase (MeDH), mxaF, and for repression of the REE-dependent MeDH, xoxF. This differential regulation is termed the REE-switch. Additionally, we showed that XoxF is required by the cell for two separate processes: 1) as a REE-dependent MeDH and 2) as an activator of mxaF expression and repressor of xoxF expression. Here we show that the need for XoxF in activation of mxaF expression can be bypassed by a second site suppressor mutation which spontaneously arises in a xoxF mutant in the absence of REE. Using growth curve analysis and transcriptional reporter fusion assays, we show that expression of mxaF is restored and methanol growth now occurs in the suppressor strains. Three genes encoding senor kinases were targeted as candidates for harboring this mutation. MxbD and MxcQ were chosen as they are known sensors kinases that regulate mxaF and xoxF expression. A third sensor kinase was chosen due to its similarity to a regulator reported to control expression of xoxF in Methylmicrobium buryatense. These sensor kinase genes were amplified from two xoxF suppressors and cloned into an expression vector. Complementation tests using xoxF sensor kinase double mutants revealed that only mxbD cloned from the xoxF suppressor could restore growth to strains lacking xoxF. Sequence analysis of the xoxF suppressors identified the insertion of an additional proline residue resulting in three sequential prolines in the suppressor strains. These results suggest that adding an extra proline at this position in MxbD may eliminate the need for XoxF in sensing REE presence by activating the MxbM response regulator. This work adds new information to the complex regulatory network that controls the REE switch lending new insight as to how M. extorquens senses REEs and controls gene expression accordingly.

Poster #: 104
Campus: CSU Los Angeles
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, dyrk1a, wdr68
Project Title: Wdr68/Dcaf7 is required for expression of the Down Syndrome kinase Dyrk1a
Author List:
Salven, David; Undergraduate, California State University, Los Angeles, Presenting Author
Zitser, David; Undergraduate, California State University, Los Angeles, Presenting Author
Xu, Jenna; Graduate, California State University, Los Angeles
Yousefelahiyeh, Mina; Graduate, California State University, Los Angeles
Yu, Yang; Graduate, California State University, Los Angeles
Alvarado, Estibaliz; Graduate, California State University, Los Angeles
Nissen, Robert; California State University, Los Angeles

Abstract: Overexpressed Dyrk1a contributes to the neurodevelopmental defects in Down Syndrome and Alzheimer’s disease. Wdr68 regulates lower jaw development and binds Dyrk1a. We previously generated Wdr68 gene deletion (Δwdr68) cells and non-target (NT) control cells using CRISPR/Cas9 gene targeting in mouse C2C12 cells. Here, we report that these Δwdr68 cells also lack Dyrk1a protein relative to NT controls. We then generated Δwdr68 human HeLa cell sublines and found they also lack Dyrk1a relative to NT controls. Thus, Wdr68 is required for Dyrk1a in both mouse and human cells. We also generated mouse C2C12 Δdyrk1a sublines and found near normal levels of Wdr68 expression. Thus, the requirement is unidirectional (Wdr68→Dyrk1a). Dyrk1a is a multi-functional protein. As a nuclear-localized RNApII CTD-kinase, it acts as a transcriptional co-activator via a Dyrk1a-response element. We tested a Luciferase reporter containing five copies of the Dyrk1a-response element and found its activity to be reduced in both mouse and human Δwdr68 cells relative to NT controls. Thus, Wdr68 is required for Dyrk1a activity in mouse and human cells. We then used qRT-PCR analysis to assess mRNA expression levels for Dyrk1a. We readily detected mRNA for Dyrk1a in mouse NT cells and found that it was not reduced in Δwdr68 cells. Thus, Dyrk1a is transcribed normally in Δwdr68 sublines relative to NT controls. Consistent with a Wdr68→Dyrk1a relationship, we also found that overexpression of a GFP-Wdr68 fusion protein restored Dyrk1a expression in Δwdr68 cells and increased Dyrk1a level in NT control cells. In contrast, overexpression of a GFP-Dyrk1a fusion protein did not alter Wdr68 levels in NT control cells. Taken together, these findings demonstrate that Wdr68 is required for Dyrk1a expression via a post-transcriptional mechanism. Wdr68 may represent a novel therapeutic target for the modulation of Dyrk1a activity relevant to Down Syndrome and Alzheimer’s disease.
To further probe the mechanism by which Wdr68 regulates Dyrk1a, we will also present progress on experiments using protease inhibitors to explore whether Wdr68 protects Dyrk1a from proteolytic destruction. Intriguingly, we also isolated an internal deletion fragment of Wdr68, Wdr68-Δex2, that appears capable of stabilizing Dyrk1a. We will present progress on testing it in Δwdr68 sublines for stabilization of Dyrk1a and zebrafish embryos for supporting craniofacial development. This work was supported by the NIH.

Poster #: 105
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetobacter, copy number variation, gene editing
Project Title: Construction of single gene knockouts using PCR splicing and investigations into gene amplification mechanisms
Author List:
Clayton, Shannon; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Cabral III, Samuel; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: Our goal is to elucidate the mechanisms of gene amplification, universal mutations central to tumorigenesis and play key roles in increasing virulence in pathogens and the evolution of new genes. The cellular components involved in forming and collapsing these important genomic rearrangements remain unclear for all organisms. To identify the specific gene products implicated in forming gene amplifications, we used a model system in the genetically tractable bacteria Acinetobacter baylyi, where exclusively gene amplification mutants spontaneously arise under selective benzoate minimum media. Here we describe the adoption of a new multi-step PCR splicing technique used to create hybrid PCR fragments. These hybrid fragments were transformed into the naturally competent Acinetobacter baylyi parent strain, where they underwent allelic replacement to construct site-specific single gene knockout mutants. Using this new gene editing technique, we constructed and verified several strains containing null mutations within targeted single genes. For this project, we knocked out genes hypothesized, but not yet directly demonstrated, to be involved in gene amplification formation, such as genes annotated to play roles in recombination or DNA repair. We systematically tested each of these single gene knockout strains for their ability to undergo gene amplification mutations. Interestingly, one gene, yqgF, was found to decrease gene amplification mutant frequencies by a striking 50-fold, suggesting it plays an important role in forming gene amplifications. The function of YqgF, which is essential in E. coli but not essential in Acinetobacter, is not yet known, but recently has been proposed to function in opposition of a resolvase called RuvC. Based on these results we propose a new model for gene amplification formation involving YqgF-stimulated unequal crossing-over. These studies were supported by the Summer Undergraduate Research Experience (SURE) award from Sacramento State, and through the CSU-LSAMP under a grant from the National Science Foundation (HRD-1302863) and the Chancellor’s Office of the California State University.

Poster #: 106
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetobacter, gene amplification, stress-induced mutagenesis
Project Title: Nonselective Pre-growth Conditions Determine Gene Amplification Mutation Frequencies in Acinetobacter baylyi
Author List:
Cabral III, Samuel; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Villegas, Nathalie; Undergraduate, Biological Sciences, California State University, Sacramento

Abstract: Gene amplifications are mutations central to cancer formation and increased antibiotic resistance in pathogens. Recent studies have suggested these mutations are stimulated by “stress” imposed by nutrient starvation. To study gene amplification we used a model system in the bacteria Acinetobacter baylyi, where exclusively cat gene amplification mutant colonies continually accrue under prolonged exposure to selective minimum benzoate media plates. While previous experiments have found cat gene amplification mutants arise at a low rate of 1 mutant per 10+10 parent cells in 14 days, we describe here a new parent strain and optimized pre-growth conditions that yield a 2,000-fold higher gene amplification mutant frequency (2 mutants per 10+7 parent cells in 14 days), thereby increasing the efficiency of this model system. Independent mutants were analyzed and verified by qPCR to carry between 3 and 30 copies of the cat genes. According to the stress-induced model, gene amplification mutants are stimulated under selective nutrient starvation (benzoate media) and not during non-selective pre-growth (minimum or rich media). We tested this hypothesis by analyzing the effect of varying pre-growth conditions on mutation rates. For this purpose, the parent strain was grown under different pre-growth conditions, including various growth temperatures (22, 30, and 37°C) and non-selective media (minimum media with either succinate or pyruvate as a single carbon source, and two types of rich media, Luria Broth and Nutrient Broth). Our results show gene amplification mutant frequencies were increased up to 30-fold in populations pre-grown in rich media compared to minimal media. Gene amplification mutant frequencies were 4-fold higher in cultures pre-grown in minimal succinate at 22°C, compared to 30°C and 37°C. Based on these results, we propose an alternative hypothesis where gene amplification formation is an un-induced slow, sequential, multi-step process where partial gene amplification intermediates (i.e. duplications, triplications, etc.) form during non-selective growth and slowly amplify under selection. These studies were supported by the CSU-LSAMP under a grant from the National Science Foundation (HRD-1302863) and the Chancellor’s Office of the California State University.

Poster #: 107
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: microRNA, diabetes, cell proliferation
Project Title: miR-375 stimulates pancreatic alpha cell proliferation but keeps metabolism in check as glucose concentration rises
Author List:
Prator, Grace; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
French, Melissa; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Diaz, Pablo; Graduate, Biological Sciences, California State University, Chico
Keller, David; Biological Sciences, California State University, Chico

Abstract: MicroRNA-375 (miR-375) is a small non-coding RNA that suppresses insulin secretion in pancreatic beta cells and is important for development of pancreatic alpha and beta cells. In type 2 diabetes miR-375 overexpression is correlated with an increase in alpha cells and a reduction in beta cells. Based on this, we hypothesize that miR-375 will have a positive role in alpha cell proliferation. We find that when transfected with a miR-375 inhibitor, mouse alpha TC1-6 cell proliferation is decreased by 40% (p < 0.01). However, we observed no significant change when mouse MIN6 beta cells were similarly transfected. We also find that miR-375 keeps alpha cell metabolism and glucagon secretion in check as glucose concentration rises. When transfected with a miR-375 inhibitor, alpha TC1-6 cell but not MIN6 cell metabolism is enhanced by 40% at 24 mM glucose (p < 0.05), and glucagon secretion goes up 38% (p < 0.05). Our data supports a model in which miR-375 inhibits catabolic processes such as metabolism, but stimulates anabolic processes such as cell proliferation when nutrient levels are high. We suggest that miR-375 is sensitive to the energy level in the alpha cell and adjusts cell outcomes based on available resources.

Poster #: 108
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Sinorhizobium meliloti, Regulation, small untranslated RNAs
Project Title: Determining if the SmelC023 and SmelC775 Genes are Transcriptional Targets of the Response Regulator ChvI
Author List:
McMillan, Taren; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Dela Cruz, Francesca; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Alcala, Elizabeth; Undergraduate, Biological Science, California State University, Fullerton
Segovia, Denice; Undergraduate, Biological Science, California State University, Fullerton
Chen, Esther; Biological Science, California State University, Fullerton

Abstract: Nitrogen fixation occurs during the symbiosis between the bacterium Sinorhizobium meliloti and its host plant Medicago sativa. During nitrogen fixation, the bacteria converts dinitrogen from the atmosphere into ammonia, a form of nitrogen that plants can metabolize for growth. In return, S. meliloti receives dicarboxylic acids from the plant. The ExoS/ChvI two-component signaling pathway is necessary for this symbiotic relationship to begin and is known to regulate many genes, including genes involved in exopolysaccharide production. The periplasmic histidine kinase ExoS autophosphorylates in response to a signal, then phosphorylates the response regulator ChvI to regulate specific downstream genes. We hypothesize that some of the genes regulated by ChvI encode small untranslated RNAs (sRNAs), which can act as posttranscriptional regulators of gene expression. The aim of this research is to determine whether the sRNA genes SmelC775 and SmelC023 are transcriptional targets of ChvI. To measure gene expression, the promoter region of each gene was fused to the GUS reporter gene in S. meliloti. Each promoter-GUS fusion was introduced into chvI gain-of-function mutant, chvI partial loss-of-function mutant, and wild-type strains. We observed an increase in SmelC775 gene expression in the chvI gain-of-function mutant, and a decrease in SmelC775 gene expression in the chvI partial loss-of-function mutant, compared to in the wildtype strain. For SmelC023, however, we observed an increase in expression in the chvI gain-of-function mutant, but no significant difference in expression in the chvI partial loss-of-function mutant, compared to in the wildtype strain. Thus, we conclude that SmelC775 is a transcriptional target of ChvI. We also conclude that SmelC023 may be a transcriptional target of ChvI since its expression was increased in the chvI gain-of-function mutant, although we did not detect a decrease in expression in the chvI partial loss-of-function mutant compared to wildtype. In future experiments, we will use bioinformatics to identify complementary mRNA transcripts that may represent regulatory targets of these sRNAs, to gain insight into how sRNAs regulate gene expression during symbiosis.

Poster #: 109
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: lanthanide, siderophore, Methylobacterium extorquens
Project Title: Identification and Characterization of Genes Involved in Lanthanide Acquisition and Transport in Methylobacterium extorquens
Author List:
Coakley, Aeowynn; Undergraduate, Biological Sciences, San José State University, Presenting Author
Subuyuj, Gabriel; Undergraduate, Biological Sciences, San José State University
Roszczenko, Paula; Michigan State University, Microbiology and Molecular Genetics
Martinez-Gomez, N. Cecilia ; Michigan State University, Microbiology and Molecular Genetics
Skovran, Elizabeth; Biological Sciences, San José State University

Abstract: Lanthanide metals (Ln) are integral to our electronic technologies. However, Ln mining is caustic and expensive. We are developing Methylobacterium extorquens as a bacterial platform to recover Ln from electronic waste to create a domestic sustainable supply of Ln. M. extorquens obtains Ln for use as a cofactor during methanol oxidation. To increase Ln extraction yields, a better understanding of how Ln are acquired and transported is needed. Transposon mutagenesis identified 3 transporters as essential for Ln-dependent methanol growth. Mutations in these genes were reconstructed and their requirements verified using growth curve analysis. While these transporters may be required for Ln transport, the phenotypes of these transporter mutants are also consistent with a role in export of PQQ (coenzyme for methanol oxidation) into the periplasmic space or transport of formaldehyde into the cytoplasm. Formaldehyde accumulation in spent medium from the transporter mutants was measured using the NASH Assay. None showed a significant difference from wild type eliminating formaldehyde transport as a likely function for the transporters. Growth rescue by PQQ was tested to determine if these transporters are involved in PQQ export but PQQ did not allow growth. Future work will test Ln transport directly using ICP-MS. Transposon mutagenesis also identified a TonB-dependent receptor as required for Ln-dependent growth suggesting that Ln may be acquired by secretion of siderophores, small metal chelating complexes. Consistent with this hypothesis, we show that when cells are grown with limiting Ln, metabolites exhibiting a spectroscopic peak at 360 nm are excreted into the growth medium. Liquid chromatography was used to purify these metabolites and ICP-MS and fluorescent shift analysis confirmed that these metabolites contain Ln. To identify the genes responsible for production of these Ln chelating complexes, a comparative genomics approach was used. 11 genes were targeted for mutagenesis based on similarity to siderophore synthesis genes in other organisms. Future work will focus on completing construction of mutations in putative siderophore synthesis genes and testing if loss of these genes disrupts Ln acquisition using growth curve analysis, dye-based siderophore chelation assays, and spectroscopic detection of the Ln chelating complex. This work will identify engineering targets to increase Ln recovery from electronic waste.

Poster #: 110
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zinc transporter, Mucolipidosis type IV, TRPML1
Project Title: Functional characterization of single nucleotide polymorphisms in the TMEM163 gene
Author List:
Sanchez, Vanessa; Undergraduate, Biological Science, California State University, Fresno, Presenting Author
Ali, Saima; Biological Science, California State University, Fullerton
Cuajungco, Math P; Biological Science, California State University, Fullerton

Abstract: We previously identified Transmembrane 163 protein (TMEM163) through a yeast two-hybrid screen in search of interaction partners for Mucolipin-1 (TRPML1) ion channel. We found both proteins to co-localize in the plasma membrane and lysosomes. The function of TMEM163 has yet to be fully elucidated. However, evidence suggests that it transports zinc and current data in our laboratory indicate that TMEM163 is a zinc influxer. To study its function and how it might be involved in human diseases like Mucolipidosis type IV (MLIV), we used the National Center for Biotechnology Information single nucleotide polymorphism (SNP) database to study specific nucleotide sequence variations within the TMEM163 gene. We identified SNPs that produce amino acid substitution when compared with wild-type (WT) TMEM163 protein sequence. We then used bioinformatics to predict the topology of TMEM163 protein, and found that some SNP-associated amino acid changes are located in areas of TMEM163 that could affect post-translational modification and change its structure and function. Thus, we hypothesize that SNPs that result in amino acid substitution dramatically alters the zinc transport function of TMEM163. To accomplish this study, we performed site-directed mutagenesis using In-Fusion homologous recombination cloning technique to systematically replace specific nucleotides corresponding to the SNPs located within or in proximity of N- and C-termini, as well as TMD-1 to TMD-6. Upon sequence verification, we tested the effect of each SNP-associated mutant by transfecting them into cultured HeLa cells. Twenty-four hours post transfection, the cells were exposed to 100 micromolar zinc chloride and assayed for intracellular zinc transport using FluoZin-3, a high affinity zinc-specific fluorescence dye. We observed that the SNP-associated mutants have marked decrease in intracellular zinc fluorescence following exposure to exogenous zinc. The data further confirmed that TMEM163 is a zinc influx transporter. Overall, these observations indicate that certain SNP-associated amino acid substitution located within critical areas of TMEM163 protein results in abnormal zinc transport. Future investigations on the role of TMEM163-TRPML1 protein interaction is necessary to understand our previous findings of zinc dyshomeostasis in MLIV cells. This work was funded by CSUPERB Research and Development Grant 2016 and NIH R15 NS101594.

Poster #: 111
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, alcohol, Alzheimer
Project Title: Mutations that cause Alzheimer Disease-like pathology in flies also confer sensitivity to developmental alcohol exposure
Author List:
Darwish, Nahed; Graduate, Biological Sciences, San José State University, Presenting Author
Belhorma, Khaoula; Graduate, Biological Sciences, San José State University, Presenting Author
French, Rachael; Biological Sciences, San José State University

Abstract: Every year 40,000 children are born with fetal alcohol spectrum disorder (FASD) as a result of excessive maternal alcohol consumption during pregnancy. These children often go on to develop neurodegeneration similar to that observed in Alzheimer Disease (AD).
We have established Drosophila melanogaster as a model organism for the study of FASD, and Drosophila have also been used as a model for the study of AD. We recovered an allele of dementin (dmtn), the Drosophila ortholog of TMCC2, from a genetic screen for mutations conveying sensitivity to developmental alcohol exposure.
TMCC2 is a human protein that physically interacts with amyloid beta precursor protein (APP), a key protein in AD pathology, and mutations in Dmtn cause abnormal metabolism of the fly homolog of APP (APP-like, APPL) and neurodegeneration in flies (Hopkins, 2013).
We have tested 3 additional loss of function alleles of dmnt for ethanol sensitivity, and find that all three reduce survival in ethanol by 36 to 72% relative to wild type controls under the same conditions. In addition, these mutant alleles result in a profound increase in development time, further demonstrating that dmtn is necessary for the developmental response to ethanol. In addition, quantitative reverse-transcriptase mediated PCR (RT-qPCR) analysis demonstrates that dmtn transcription is decreased an average of 64% in larvae reared in ethanol, indicating that dmtn expression is disrupted by ethanol.
Because of Dmnt’s interaction with APPL, we are now testing nervous system function in both wild type and dmnt animals reared in ethanol. We are performing negative geotaxis (climbing) assays on ethanol-reared animals to test nervous system function, and our preliminary data indicate the ethanol-rearing may reduce the animal’s ability to climb, another phenotype shared in common with dmnt and appl mutant animals. We are now beginning to examine the ultrastructure of the brain, using the GAL4/UAS gene expression system to drive expression of Green Fluorescent Protein (GFP) in brain regions known to be involved in the regulation of climbing and locomotion. We will present the results of these investigations.

Poster #: 112
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Neural crest, Cadherin-7, Specification
Project Title: Analysis of Cadherin-7 in cranial neural crest specification
Author List:
Javidan, Aria; Undergraduate, Biology, California State University, Northridge, Presenting Author
Hyde-Bozer, Dustin; Undergraduate, Biology, California State University, Northridge, Presenting Author
Rogers, Crystal ; Biology, California State University, Northridge

Abstract: Cadherin proteins play a significant role in the processes governing cell adhesion, migration, specification and differentiation of many cell types in developing embryos. Here, we focus on the role of cadherins in the development of neural crest cells, a stem-like cell type that can become craniofacial cartilage and bone, pigment cells and the peripheral nervous system. Cadherin-7 (Cad7) is a type II cadherin, which is a calcium-dependent, cell-cell adhesion molecule that has been implicated as a regulator of neural crest migration and neurogenesis in the developing brain and spinal cord. Using whole mount immunohistochemistry (IHC) in chicken embryos, we first characterized the spatiotemporal localization of Cad7. Expression analysis shows that prior to the departure of neural crest cells, Cad7 is expressed in the developing neural tube and premigratory neural crest cells. We performed gain and loss of function experiments to identify the function of Cad7 in neural crest specification. Electroporation of either a Cad7 translation-blocking morpholino or full-length Cad7 DNA in chick embryos was followed by IHC for neural crest markers, HNK1, Pax7, and Sox9. We also tested the effects of Cad7 perturbation on the cell proliferation marker, PH3, and programmed cell death using Caspase. Alteration of Cad7 revealed inhibition of neural crest specification marked by Pax7 and Sox9 when compared to control treatments suggesting that the level of Cad7 in premigratory cells is tightly controlled to allow normal neural crest cell development. Preliminary data also indicates that the loss of Cad7 has little effect on cell proliferation or cell death, which suggests a direct role for Cad7 in neural crest formation. These results provide new insight into the important role that Cad7 plays in neural crest specification. In the future, we hope to identify the mechanism by which Cad7 acts on neural crest cells.

Poster #: 113
Campus: CSU Sacramento
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Acinetobacter, gene amplification, stress-induced mutagenesis
Project Title: Replica-printing experiments demonstrate gene amplification mutations are present before selective nutrient-starvation stress
Author List:
McPherson, Eric; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Herrmann, Jennifer; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: Gene amplifications are mutations implicated in many human disorders, particularly cancer, where they promote tumorigenesis and chemotherapy resistance. Recent studies have suggested these mutations are induced by “stress” imposed during growth-limiting environments. An alternative hypothesis proposes gene amplifications are merely selected by such environments since they provide a fitness advantage allowing the mutant cells to overcome the growth-limitation and proliferate. We tested these two hypotheses using a model system in the genetically tractable bacteria Acinetobacter baylyi, where exclusively gene amplification mutants are selected and continually accumulate under prolonged growth-limiting selective benzoate minimum media exposure. Replica-printing experiments were performed to determine whether the gene amplification mutants arise either prior or during the selective-stress exposure. For these experiments, over 1,000 colonies of the parent strain were cultivated on non-selective rich media agar plates, where growth does not require gene amplification. These non-selective plates were incubated at 30C for 16 days at which point they were replica-printed to at least three selective benzoate agar plates along with a nonselective rich media master plate. Our results show gene amplification mutant colonies continually arose on the selective benzoate agar plates, increasing in number over several days of incubation. Interestingly, these mutant colonies arose in the same positions on the benzoate replica-plates, strongly suggesting the mutants were pre-existing before selective stress was imposed. These results corroborate with several other independent experiments performed by our lab group, supporting the alternative hypothesis that gene amplification mutations and/or their intermediates are present prior to selection, and thus are not stress-induced by the growth-limiting selective media. These studies were supported by the Albert Delisle Scholarship for Undergraduate Research.

Poster #: 114
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, alcohol, NPF
Project Title: Alcohol exposure during development reduces food reward in Drosophila melanogaster
Author List:
Guevara, Amanda; Biological Sciences, San José State University, Presenting Author
Urbina, Brianna; 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 collection disorders that are due to developmental alcohol exposure (DAE). In both flies and mammals DAE can cause developmental delay, decreased survival, and neurobehavioral changes including feeding abnormalities and sleep disorders. Despite decades of research, the mechanisms of ethanol’s effects on molecular and neurological pathways are not well understood. In addition, public health campaigns have reached the limits of their effectiveness, and the proportion of women who drink while pregnant has remained steady for at least the last 15 years. We aim for a greater understanding of the mechanisms underlying ethanol’s developmental toxicity in order to enable the establishment of treatments for FASD.
We have established Drosophila melanogaster as a genetic model organism to study FASD and here we have used it to investigate feeding abnormalities induced by alcohol exposure. Using food labeled with blue dye, we have shown that exposure to ethanol during development reduces both meal size and the motivation to eat, and that this effect persists to adulthood.
Neuropeptide F (NPF) is a peptide hormone that is homologous to Neuropeptide Y in mammals, and has been shown to regulate reward-mediated behaviors including feeding, mating, and ethanol-seeking behavior. NPF was therefore a strong candidate target for DAE’s effects on feeding behavior. We tested flies mutant for NPF signal transduction and found that partial loss of NPF signaling causes an enhancement of the feeding deficiencies seen in ethanol-reared animals. Strikingly, however, we find that complete loss of function of the NPF receptor (NPFR), in combination with DAE, is developmentally near-lethal. This is unexpected, because, without ethanol, loss of NPFR has no effect on developmental survival, and NPF signaling has not been shown to affect survival in any organism studied to date.
We hypothesize that NPFR null animals die of insufficient feeding – that is, they starve to death. Our data show that these animals die late in the first larval instar, and that feeding of first-instar NPFR null animals is severely reduced by DAE.
Finally, we show that DAE results in increased NPF expression in 3rd instar larval brains. We propose that DAE reduces feeding by way of an as-yet-unidentified target, and that NPF is upregulated as a compensatory response, leading to starvation in animals mutant for NPF that are reared in ethanol.

Poster #: 115
Campus: CSU Chico
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: blood, stem cells, zebrafish
Project Title: Characterization of rapunzel5 in Blood Formation
Author List:
Pulido, Omar; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Harris, Elena; Computer Science, California State University, Chico
Stachura, David; Biological Sciences, California State University, Chico

Abstract: Due to their optical transparency and external development, the zebrafish is an excellent vertebrate model to study blood formation (hematopoiesis). They have analogous sites of blood formation, differentiation, and expansion, and have remarkably similar blood cells when compared to humans. Hematopoiesis occurs through the stepwise maturation of hematopoietic stem cells (HSCs) into lineage-restricted progenitor cells. The hematopoietic system is tightly orchestrated by protein signaling molecules, many of which have yet to be fully characterized. We collaborated with computer scientists to discover zebrafish genetic transcripts present in cells that support hematopoiesis to identify specific genes that are involved in the production and differentiation of HSCs. From this analysis, we generated a dataset containing the top hundred most highly expressed genes in these hematopoietic tissues, sixteen of which have an undetermined function. I chose to examine rapunzel 5 (rpz5), a gene largely uncharacterized in the literature. rpz5 is suspected to be a secreted protein essential for normal hematopoiesis and bone development. Data from my experiments indicate that genetic depletion of rpz5 results in irregular hematopoiesis, causing circulation defects, blood clotting, and decreased red blood cell production. We predict that rpz5 is essential for normal blood cell production, and performing gain- and loss-of-function experiments will confirm this. By disturbing normal gene expression in the animal model, we are able to expand our understanding of the organization and molecular regulation of the human hematopoietic system. Gaining a comprehensive understanding of the signaling molecules governing HSC proliferation and differentiation could lead to the development of therapeutics that more effectively treat hematopoietic malignancies and other cancers.

Poster #: 116
Campus: CSU Chico
Poster Category: Other
Keywords: metal-organic frameworks, endocrine-disrupting chemicals, carcinogens
Project Title: Sponges for Decontaminating Water with Endocrine-Disrupting Chemicals and Carcinogens
Author List:
Yoon, Sungwon; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
So, Monica ; Chemistry and Biochemistry, California State University, Chico

Abstract: Contaminated water with high levels of persistent organic pollutants (POPs), such as endocrine disrupting chemicals (EDCs) and carcinogens, is becoming a huge threat to the health of humans and marine animals. If ingested, POPs are accumulated in the fatty tissue or organs of animals, which can lead to serious health issues involving disruption of the endocrine system, cancer, genetic defects, and weakened immune system. Thus, water filtration using porous materials becomes necessary in our society. Previous literature showed the vital role of hydrogen bonding in the adsorption of contaminants in porous materials but the types of materials used are limited. In this research, we synthesized two porous metal-organic frameworks (MOFs), UiO-66 and NH2-UiO-66, as powders, which will act as a control and the variable material respectively. We confirmed their crystallinity, functional groups, and porosity using powder X-ray diffraction (PXRD), Fourier-transform infrared spectroscopy (FTIR) and isotherms, respectively. In addition, we will test the filtration efficiency of each MOF by monitoring changes in mass adsorption of contaminants using quartz crystal microbalance (QCM). The results of this research will help us develop more efficient water filtration membranes by understanding the hydrogen bonding within MOFs.

Poster #: 117
Campus: CSU Fullerton
Poster Category: Other
Keywords: Wheelchair, Sip-and-Puff , Autonomous Navigation
Project Title: Autonomous Wheelchair with a Sip-and-Puff Controlled User-Interface for Amyotrophic Lateral Sclerosis Patients
Author List:
Grewal, Harkishan ; Graduate, Computer Engineering, California State University, Fullerton, Presenting Author
Matthews, Aaron ; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
Tea, Richard ; Undergraduate, Computer Engineering, California State University, Fullerton
George, Kiran; Computer Engineering, California State University, Fullerton

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a rapidly-progressing neurological disease that attacks the motor neurons responsible for controlling muscle movement. Patients with ALS lose fine motor control in their limbs leaving them immobile and unable to use a conventional joystick-controlled electric wheelchair. Sip-and-puff controlled wheelchairs enable individuals with ALS to regain some mobility and autonomy. However, sip-and-puff controlled wheelchairs are cumbersome to use and lead to fatigue and discomfort after prolonged use. An autonomous wheelchair with a sip-and-puff controlled user-interface is proposed to alleviate the strain associated with a conventional wheelchair. A new user interface (UI) is developed to work with a sip-and-puff device, allowing a user to easily control the UI. The UI interfaces with an autonomous wheelchair navigation system (developed in our previous work) to navigate to a destination. In the proposed design, the user need only select the destination once using the sip-and-puff device. Once a destination is selected, the autonomous navigation system (ANS) takes control to perform the navigation and obstacle avoidance. The ANS uses the Robot Operating System (ROS) to communicate with sensors and microcontrollers to navigate. ROS takes input from the rotary encoders to track the positon of the wheelchair and a LiDAR to detect obstacles. The ANS shifts the burden of path-planning and obstacle avoidance away from the user, greatly reducing the strain on the user. Ten trials were conducted to test the autonomous navigation capabilities of the proposed system. The wheelchair successfully navigated a path of 10.26 meters in 100% of the trials with no collisions. The average time to navigate to the destination was 28.6 seconds. Additional ten trials were conducted to test the sip-and-puff device and the efficacy of the user interface. The average time to select a destination on the UI using the sip-and-puff device was 6.3 seconds. The average time for the user to cancel navigation and select a new destination was 4.86 seconds. These results show promise of greatly improving usability compared to conventional sip-and-puff controlled wheelchairs. The destination selection time and rerouting times are shorter due to the simple design of user interface and the overall navigation times are shorter due to the autonomous navigation system.

Poster #: 118
Campus: CSU Long Beach
Poster Category: Other
Keywords: animal model, meal patterns, gut peptides
Project Title: Preference for high energy diet in rats modified by alternate day fasting and satiety peptides
Author List:
Frankot, Michelle; Graduate, Psychology, California State University, Long Beach, Presenting Author
Carrillo, Audrey; Graduate, Psychology, California State University, Long Beach, Presenting Author
Treesukosol, Yada; Psychology, California State University, Long Beach

Abstract: Alternate day fasting (ADF), a paradigm involving free access to food for 24-h followed by no access to food for 24-h, leads to weight loss for both humans and rats. When rats receive free access to both a palatable high-energy (HE; i.e., high in fat and sugar) food and standard chow they tend to overeat HE food and gain weight. In order to examine how fasting alters diet preference and food intake, meal pattern analysis was conducted across daily 23-h sessions in male and female Sprague Dawley rats. Rats were assigned to one of three diet conditions: AD LIB rats were given access to both chow (3.43 kcal/g) and HE (4.73 kcal/g) food every day, INT rats were given access to chow every day with the addition of HE food every other day, and ADF rats were given access to both chow and HE food every other day; no food was administered on alternate days. Rats were also injected with satiety peptides CCK and exendin-4 to determine how gut peptide signaling may be involved in diet preference. Given the 24-h food restriction periods, it was anticipated that ADF rats would show the highest preference for HE food versus chow, yet the ADF group displayed decreased preference for HE compared to INT and AD LIB rats (p < .001). This effect was more pronounced in male rats than in female rats. Although HE preference decreased over time for all groups, HE preference increased when rats were injected with a combination of CCK and exendin-4 (p < .05), and this effect was most pronounced in ADF rats. Consistent with findings in the literature, access to the high caloric HE food in a fed state (INT rats) resulted in hyperphagia driven by increased meal size of the HE food (p = 0.008). This was not observed in ADF rats that were presented HE food and chow following 24-h food restriction. The decreased preference for HE food in ADF rats appears to be driven by changes in meal pattern parameters. Specifically compared to the INT group, ADF rats initiated more meals (p < 0.001) and displayed larger meal size (p = .035) of chow. The direct controls of meal size can be categorized as positive (e.g., oral) and negative (e.g., postoral inhibitory) signals; thus here, the ADF schedule appears to increase orosensory stimulation and/or decrease sensitivity to inhibitory cues towards chow. This shift in diet preference may contribute to the effectiveness of using ADF as a dietary strategy.

Poster #: 119
Campus: CSU Fullerton
Poster Category: Other
Keywords: Deep Learning , real-time recognition, Meltdown in Autistic Children
Project Title: Deep Learning Based Recognition of Meltdown in Autistic Children
Author List:
George, Feba; Graduate, Computer Engineering, California State University, Fullerton, Presenting Author
Patnam, Sindhoor; Graduate, Computer Engineering, California State University, Fullerton, Presenting Author
George, Kiran; Computer Engineering, California State University, Fullerton

Abstract: Dealing with children who are suffering from severe autistic disorders could be a tough challenge often. It gets worse especially when they experience sudden nervous breakdowns or meltdowns. Fortunately, studies have shown that children exhibit certain soothing gestures prior to the meltdowns. The overarching goal of the project is to recognize such gestures using an intelligent gesture recognition system and warn caretakers/parents about their child’s meltdown that may involve physical harm to themselves or others. In this project, deep learning neural networks, one of the efficient approachs for image recognition, is used to train the system. The data (pictures representing common signs of a meltdown) to train the system is gathered from MIT scene and other verified internet sources to create a suitable deep learning neural network using the DIGITs training platform from Nvidia©. After training, the trained model is deployed in a real-time image recognition program. With the help of multiple cameras, gestures exhibited by children with autism is captured in real time and compared against the trained model using the software. In the training phase, the model resulted in an accuracy of ~93%, which then reduced to ~92% in the deployment stage. After the functional testing was done on five healthy subjects, who performed all the gestures that are chosen for the training purpose, the system still resulted in an efficient accuracy percentage of ~92. The preliminary results reveal that the system has the potential to recognize gestures in real-time and warn caretakers/parents about a potential meltdown.

Poster #: 120
Campus: CSU Los Angeles
Poster Category: Other
Keywords: Boron heterocycles, Acinetobacter baumannii, antimicrobial susceptibility
Project Title: Antimicrobial Susceptibility and Cytotoxicity of Boron Heterocycles
Author List:
Rahman, Shakila; Graduate, Department of Biological Sciences, California State University, Los Angeles
Nunez-Flores, Rogelio; Graduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Luna, Breanna; Department of Biological Sciences, California State University, Los Angeles
Mora, Hugo; Graduate, Department of Biological Sciences, California State University, Los Angeles, Presenting Author
Groziak, Michael; Department of Chemistry & Biochemistry, California State University, East Bay
Xu, Howard; Department of Biological Sciences, California State University, Los Angeles

Abstract: Antibiotic drug resistance is increasing at an alarming rate during the recent decades which renders most currently available antibiotics ineffective. Coupled with the scarce antibiotic drug pipeline in the pharmaceutical industry, there is an urgent unmet need for the discovery of novel antibiotics. Previously, we reported synthesis and antibacterial property of 33 boron heterocycles. Some of these boron compounds were found to be active against Escherichia coli and enoyl ACP reductase was subsequently determined to be a potential target of these inhibitors. In this study, we report the antimicrobial susceptibility of select boron heterocycles against a few clinical isolates of Acinetobacter baumannii, one of the most problematic bacterial pathogens that are multidrug resistant. In addition, cytotoxicity of six boron heterocycles was also determined. Specifically, minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 26 boron heterocycles were determined against four clinical isolates of A. baumannii using established broth microdilution procedure according to Clinical Laboratory Standard Institute and the human embryonic kidney cell line HEK 293 was used to determine the cytotoxicity of six boron compounds using MTS as a cell viability indicator. Our results indicated that eight boron compounds exhibited solid potency against A. baumannii isolates, producing MICs values of 4-16 µg/mL and most of them exert cidal activities with MBC values the same as or close to their MIC values. Cytotoxicity results showed that IC50 values ranging between 18 µg/mL and 24 µg/mL, indicative of some cytotoxicity to the HEK 293 cells. We acknowledge grant support from CSUPERB’s Entrepreneurial Joint Venture Matching Grant program and the National Science Foundation (under Grant # HRD-1602210; for CSU LSAMP-BD Cohort XIII).

Poster #: 121
Campus: CSU Fullerton
Poster Category: Other
Keywords: Brain-computer interface, assistive technology,
Project Title: A Vibrotactile Approach Based on P300 for Brain Computer Interface Paradigm Benchmarked Against Auditory Steady State Response
Author List:
Anil, Divya; Graduate, Computer Engineering, California State University, Fullerton, Presenting Author
Mistry,, Krupal; Graduate, Computer Engineering, California State University, Fullerton
Pelayo, Pablo; Undergraduate, Computer Engineering, California State University, Fullerton, Presenting Author
George, Kiran; Computer Engineering, California State University, Fullerton

Abstract: Brain computer interface (BCI) is a popular assistive technology which serves as a communication channel between a person’s brain and an external system to assist the disabled individuals, especially those affected with neurodegenerative disorder, and enable them to lead an independent life. The most widely used BCIs are based on P300 event related potential, which is a positive deflection in electroencephalograph (EEG) signals that occurs after 300 milliseconds once the target stimulus is identified. This study aims in developing a P300 response based on the vibrotactile haptic approach by providing concentration to tactile stimulus delivered to the fingertips of the subject. This stimulus is obtained from motor vibrations that are attached to the fingers which run at different revolutions per minute. In this study, two approaches are used to measure the P300 response which is obtained by concentrating on specific motors. The first approach involves one motor attached to each of the five fingers on one hand, while the second approach made use of both hands with three motors on each hand.
Four subjects participated in the testing and the P300 response was obtained by placing electrodes on the scalp according to the 10 – 20 International standards, which describes the relevant brain positions for EEG experiments. The second approach provided better accuracy and faster P300 response since the users found it easier to distinguish the vibration when the motors were placed on separate hands. Also, this approach was more efficient with less number of motors, hence less options presented to the user. Moreover, the performance of this approach is compared with an audio steady state response (ASSR), by generating two tones with beat frequencies at 37 and 43 Hz, modulated with carrier frequencies at 2500 and 1000 Hz, respectively using MATLAB. For the testing stage, all four subjects were asked to perform five trials. In the setup, LED associated with a motor turns on once the relevant P300 response is detected. In the haptic approach, an average accuracy of 83.33% and a response time of 2.5 seconds were obtained, compared to 47.5% and 18.72 seconds, respectively, using the ASSR approach. The haptic approach proves to be less stressful and a better option compared to ASSR. From the preliminary results, the proposed vibrotactile system can be potentially utilized to operate an electric wheelchairs and electronic communication devices.

Poster #: 122
Campus: CSU Sacramento
Poster Category: Other
Keywords: Drosophila, gut microbiome, autism
Project Title: The autism-associated chromatin modifier, Chromodomain Helicase DNA Binding Protein 8, affects axon guidance and behavioral phenotypes in Drosophila
Author List:
Lew, Amy; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Nguyen, Darren ; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Hu, Alain; Undergraduate, Biological Sciences, California State University, Sacramento
Murphy, Lillian; Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Undergraduate, Biological Sciences, California State University, Sacramento
Crawford, Robert; Biological Sciences, California State University, Sacramento

Abstract: Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder that afflicts 1 in 68 children in the United States. Recent genomic studies have identified recurrent mutations within specific genes, providing insight into the complex genetic etiologies of this disorder. Mutations in Chromodomain Helicase DNA Binding Protein 8 (CHD8) are 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. While CHD8 is thought to play a role in ASD, the cellular phenotypes affected by CHD8 mutations remain unclear. Our research is aimed at examining both neural and GI phenotypes in Drosophila to elucidate how mutations in CHD8 impair development. Studies using germ-free mice have begun elucidating mechanisms by which species-specific microbiota metabolites modulate behaviors associated with ASD. Thus, the assays described here will ultimately be used to study connections between gut microbiota and neural phenotypes in fruit flies. METHODS AND RESULTS: Similar to other model organisms, the Drosophila homolog of CHD8, kismet (kis), is expressed during embryonic development and complete loss of kis is embryonic lethal, so we examined heterozygous kis mutants. We used immunohistochemistry and confocal microscopy to show that heterozygous loss of kis causes severe axon guidance defects in adult brains. Disrupted axon guidance is one of the cellular phenotypes common to ASD. We also used the courtship assay—a quantitative measure of an innate behavior—to show that heterozygous kis mutants exhibit significant deficits in courtship behaviors. This result reiterates the important role kis plays during neural development, and it will also allow us to quantitatively assess how variations in gut microbiota affect the severity of kis phenotypes. FUTURE DIRECTIONS: We are currently examining relative phylogeny and abundance of metabolic gene clusters for gut microbiomes of wild-type versus kis Drosophila and will examine how changes to gut microbiota influence neural phenotypes. We are also investigating cellular phenotypes within the gut epithelium in an effort to understand how loss of kis may influence changes in the gut microbiome and cause GI problems associated with the CHD8 subtype of ASD. FUNDING: CSUPERB New Investigator Award.

Poster #: 123
Campus: San José State University
Poster Category: Other
Keywords: muscle spindle afferent, proprioception, electrophysiology
Project Title: Effect of glutamate released from synaptic-like vesicles on muscle spindle afferent receptor ending sensitivity to muscle stretch in adult mice
Author List:
Than, Kimberly; Undergraduate, San José State University, Presenting Author
Chu, Sarah; Undergraduate, San José State University, Presenting Author
Klier, Nikola; Undergraduate, San José State University
Villegas, Natanya; Undergraduate, San José State University
Fuentez, Alyssa; Undergraduate
Wilkinson, Katherine; San José State University

Abstract: Proprioception is the sense of relative body position in space and is necessary for completing complex motor tasks. The most significant proprioceptors are thought to be the sensory neurons that innervate the muscle spindle, the Group Ia and Group II muscle spindle afferents (MSAs). MSAs are mechanosensors that increase firing in response to muscle stretch and movement. Synaptic-like vesicles (SLVs) are found within MSA nerve endings and release glutamate following muscle stretch. Prior studies suggest that exogenous glutamate increases the sensory response to stretch. However, the response of individual MSAs to glutamate was not measured and some of the increased firing could have been caused by activation of silent non-MSA afferents, like nociceptors. We tested the hypothesis that glutamate can act locally on MSA receptor endings and alter responsiveness to stretch. We used an in-vitro mouse muscle-nerve preparation to record MSA firing activity. Briefly, the extensor digitorum longus (EDL) muscle and innervating deep peroneal branch of the sciatic nerve were dissected and perfused in an oxygenated synthetic interstitial fluid tissue bath. An extracellular recording electrode was placed on the nerve and individual MSAs identified by waveform. A series of 4s ramp-and-hold stretches were applied to the EDL and instantaneous firing frequency before and during stretch was measured. Upon exposure to glutamate (1mM), there was an increase in static stretch response (n=12, 18.79 ± 24.15%). To confirm that SLV release of glutamate contributes to the alteration in stretch response, vesicular glutamate transporter (VGLUT) inhibitors were used to eliminate the packing of glutamate into SLVs. There was a decrease in individual afferent firing rates upon the addition of a potent VGLUT inhibitor, xanthurenic acid (3mM; n=17; 35.21 ± 64.79%), with 4 of 17 MSAs showing complete elimination of firing. These results support the hypothesis that glutamate can act locally in the absence of central nervous system circuits to increase MSA stretch sensitivity. Future studies will block glutamate reuptake of SLV-released glutamate to cause more physiological and localized increases in glutamate concentrations to probe the importance of this mechanism in the regulation of MSA sensitivity.

Poster #: 124
Campus: CSU Long Beach
Poster Category: Other
Keywords: animal model, binge eating, taste
Project Title: Sucralose profile as a marker for binge eating behavior
Author List:
Gould, Alexa; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Frankot, Michelle; Graduate, Psychology, California State University, Long Beach, Presenting Author
Treesukosol, Yada; Psychology, California State University, Long Beach

Abstract: Like humans, rats avidly prefer sweet substances. When presented a bottle of water and a bottle of sweet solution, rats will increase in preference for the sweet solution in a concentration-dependent manner. Sucralose is an artificial sweetener currently marketed as the primary sweetening component of the commercially available “Splenda”. When rats are presented water and sucralose, as sucralose concentration increases a subset of the rats drink more sucralose. We can categorize these animals as sucralose preferers (SP). In contrast, some rats drink less sucralose (sucralose avoiders (SA)). Here, male and female Sprague-Dawley rats were classified as either SP or SA via a two-bottle preference test. Rats were presented one bottle of water and one bottle containing a concentration of sucralose that ascended every two days. Previous findings have shown that the SP/SA profile is evident in female but not male rats. Yet here, SP and SA profiles were found in both male and female rats. Next, to assess whether SP/SA status would predict binge eating behavior later in life, a binge-access schedule was set into place for 6 weeks. Here, rats were given 30-min access to chow and sweetened fat-shortening, Crisco, after a 23-hour food deprivation twice a week. During these 30-min sessions, SP rats tended to eat more of the sweetened Crisco than SA rats, but this was not consistent across sessions. In contrast, while sweetened Crisco intake did not significantly differ by sex, male rats ate significantly more of the chow than female rats. Overall, these preliminary findings suggest sucralose acceptance correlates with intake of sweetened fat in 30-min sessions in a binge paradigm. These findings also show sex differences in diet preference.

Poster #: 125
Campus: CSU Los Angeles
Poster Category: Other
Keywords: Antifreeze Protein, Insulin Producing β Cell, Cold Preservation
Project Title: Hypothermic preservation of insulin producing rat INS-1 cells line using a recombinant antifreeze protein
Author List:
Quach, Kevin ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Gonzalez, Ignacio ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Perez, Rachel; City of Hope
Abdullah, Ismail ; City of Hope
Omori, Keiko ; City of Hope
Wen, Xin; Chemistry and Biochemistry, California State University, Los Angeles

Abstract: Organ preservation for transplant applications has saved life of many patients who were at stage of organ failure. Cold preservation of donor pancreas using University of Wisconsin (UW) solution has been a standard method for many years for whole pancreas transplantation or islet transplantation which requires isolating insulin producing islets from pancreas. Islet transplantation has been shown to be a safe and effective treatment for patients with type 1 diabetes (T1D), however, the success depends largely on the viability of the islets isolated after pancreas preservation. The current solution to preserve the pancreas is not optimum. Therefore, there is an urgent need to develop a better solution to preserve the organs prior to transplantation. Nature has adapted system that can achieve survive at extreme conditions. In particular, antifreeze proteins (AFPs) have been found in many species for survival during harsh winter at low temperature (-30ºC). AFPs prolong cell survival by binding to ice crystals inhibiting the crystallization inside the cells. They can create a gap between the melting point and freezing point of water, referred to as thermal hysteresis. In this study, we prepared recombinant AFP from a beetle, Tenibrio molitor (TmAFP) and investigated the efficacy to prolong in vitro rat insulin producing INS-1 cell line survival and viability during hypothermic preservation. INS-1 cells were used for preservation at 4 ºC for up to 7 days in UW solution, in the absence (Ctrl) and presence of TmAFP at concentrations range of (0-1000 µg/mL). The viability of the INS-1 cells was monitored on days 0, 3, 4, 5, 6 and 7 by trypan blue staining using a Cellometer Auto Cell Viability Counter. The addition of TmAFP (50 and 500 µg/mL) in UW maintained INS-1 viability for up to 6 days while viability of the control cells without TmAFP significantly decreased (>20% decrease as compared to pre preservation) on day 6. The results suggesting the potential of TmAFP for improving β cell viability during cold preservation.

Poster #: 126
Campus: CSU Sacramento
Poster Category: Other
Keywords: Drosophila, neurodevelopment, autism
Project Title: Exposure to the environmental neurotoxicant polychlorinated biphenyl-95 phenocopies a common autism risk gene in Drosophila melanogaster
Author List:
Murphy, Lillian; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Hindi, Zaed ; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Lopez, Ashlie; Undergraduate, Biological Sciences, California State University, Sacramento
Corona, Rafael; Undergraduate, Biological Sciences, California State University, Sacramento
Nguyen, Kimberly ; Undergraduate, Biological Sciences, California State University, Sacramento
Doan, To Hien
Tupikova, Angelina
Ghenta, Kristina

Abstract: Mounting evidence indicates that the interaction of environmental chemicals with specific genetic susceptibilities is linked to autism spectrum disorder (ASD). Yet, identification of specific environmental chemicals that interact with genes to cause ASD remains a critical gap in our understanding of ASD etiology. Given that there are over 80,000 chemicals in use with little to no toxicological data, the field urgently needs an efficient method for screening chemicals. This project involves the development of assays using the fruit fly, Drosophila melanogaster, for identification of chemicals that molecularly converge with fmr1 (fragile X mental retardation 1). Mutations in fmr1 are the most common single gene cause of ASD and the role fmr1 plays in neural development is conserved from flies to humans. Data from vertebrate model organisms suggest that gestational exposure to environmental chemicals, like polychlorinated biphenyl 95 (PCB-95), can increase the risk of ASD in humans with fmr1 mutations. Drosophila with fmr1 mutations have (1) decreased courtship index (CI; a quantitative measure of courtship behaviors), (2) impaired axon guidance in an adult neural structure called the mushroom body (MB), and (3) disrupted synapse formation in the larval neuromuscular junction; thus, we exposed embryonic and larval stage fruit flies to PCB-95 and examined these phenotypes. We used the courtship assay, an established paradigm for behavioral analysis of fruit flies, to determine that exposure to nanomolar concentrations of PCB-95 significantly decreases the CI in wild-type (wt) flies. We used immunohistochemistry and confocal microscopy to examine adult brains and found a dose-dependent increase in axon-pathfinding defects in the MB. We also used immunohistochemistry and confocal microscopy to analyze synapses within the larval body wall and found PCB-95 exposure disrupts synapse formation. Significantly, the phenotypes caused by PCB-95 in wt flies are the same phenotypes caused by loss of fmr1, supporting the observation that PCB-95 confers increased risk of ASD in individuals with this genetic risk factor—they affect the same neurodevelopmental processes. We are currently investigating the severity of these phenotypes in fmr1 mutant flies exposed to PCB-95. These results are consistent with findings in vertebrate model organisms and, thus, support the use of Drosophila for further chemical screening. Funding: CSUPERB New Investigator Award.

Poster #: 127
Campus: CSU San Marcos
Poster Category: Proteins (Include Proteomics)
Keywords: Amyloid, Protein aggregation, Fluorescence Quenching
Project Title: The C-terminus of CsgE Is Involved In Preventing Protein Aggregation
Author List:
Aranda, Isamar; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Binmahfooz, Ashwag; Graduate, Chemistry and Biochemistry, California State University San Marcos
Smidt, Gunnar; Undergraduate, Chemistry and Biochemistry, California State University San Marcos
Basham, Michael; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Jayasinghe, Sajith; Chemistry and Biochemistry, California State University San Marcos

Abstract: Introduction
Gram-negative bacteria, such as E.coli and Salmonella, contain proteinaceous, hair-like, cell surface organelles known as curli. Curli serve to facilitate cell-cell interactions and are essential for host cell colonization. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF act as chaperones to prevent the premature aggregation of CsgA and CsgB which are the main protein components of Curli. CsgE and CsgF help transport CsgA and CsgB proteins to the cell surface, where CsgA and CsgB begin to assemble to form Curli. We have observed that CsgE is able to inhibit the aggregation of CsgA, as well as human islet amyloid polypeptide (hIAPP), an amyloidogenic polypeptide that is unrelated to curli. We wished to characterize the interaction between CsgE and CsgA/hIAPP to determine mechanistic details of curli assembly.
Methods
The BL21 bacterial expression system was used to express four different mutants of CsgE (34C, 58C, 99C and 119C) where the wild type residue at the respective position was replaced by a cysteine. Cell pellets recovered from protein expression were lysed using a French Press and then were purified using Ni affinity chromatography. The cysteines were labeled using the environment sensitive probe IAEDANS, and purified again through Ni affinity and gel filtration chromatography. Fluorescence quenching was used to determine the ability of CsgE to interact with the non-aggregating rat Islet Amyloid Polypeptide (rIAPP).
Results and Discussion
rIAPP differs from hIAPP by only three amino acids, but does not aggregate to form amyloid and was used to reduce the complicating effects of hIAPP aggregation. We measured quenching of IAEDANS by Potassium Iodide (KI) in the absence and presence of rIAPP. In the absence of rIAPP IAEDANS labeled at positions 34, 58, 99 and 119 gave Stern-Volmer quenching constants of 9.6, 9.2, 12.1, and 7.6 respectively, while in the presence of rIAPP these values were 9.7, 8.7, 12.4, and 6.5. The significant reduction in the quenching constant for position 119 in the presence of rIAPP suggests that the fluorophore at this position becomes protected from quenching. The protection could either be due to a conformational change in CsgE driven by binding rIAPP, or could be due to residue 119 interacting directly with rIAPP, and suggests that the C-terminal region of CsgE is involved in its ability to prevent protein aggregation.

Poster #: 128
Campus: CSU Sacramento
Poster Category: Proteins (Include Proteomics)
Keywords: amyloid, protein folding, lipoprotein
Project Title: Stability of the amyloidogenic human apolipoprotein A-I variant G26R
Author List:
Tran, Thao; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Roberts, Linda; Chemistry, California State University, Sacramento, Presenting Author

Abstract: Apolipoprotein A-I (ApoA-I) is the main component of high density lipoprotein (HDL), which removes cholesterol from peripheral tissues and has anti-inflammatory and anti-oxidant cardioprotective properties. However, certain mutations increase the propensity for misfolding into amyloid structure, which leads to the formation of insoluble fibrils that deposit in tissues, counteracting its anti-atherogenic properties. Development of therapeutics to inhibit or reverse amyloid formation requires a thorough understanding of the mechanisms underlying amyloid formation. A key factor in amyloid formation in many amyloid forming proteins is decrease in stability. To determine whether stability is a factor in amyloid formation in the amyloidogenic apoA-I variant G26R, we analyzed the stability of the protein incubated for a period of 21 days at pH 7.2 using equilibrium solvent denaturation compared to wild-type (WT) protein. The unfolding of the protein in urea was monitored using intrinsic tryptophan florescence. Data were fitted to obtain ∆G° (Gibbs free energy of protein unfolding), m (cooperativity of unfolding), and D1/2 (denaturant concentration at the midpoint of denaturation). In the first week of incubation at 37 degrees C (measurements taken Day 1 and 7 after protein purification), the ∆G° of G26R was lower than that of WT (an average of 4.14 kcal/mol compared to 5.39 kcal/mol) and was accompanied by a reduction in D1/2 from 2.82 M for WT to an average of 2.66 kcal/mol for G26R. The cooperativity also decreased. At two and three weeks of incubation, the ∆G°, D1/2 and cooperativity all increased. The decrease in the first week reflects reduced stability of the G26R protein. The increase from two weeks onward likely reflects the increased stability of fibrils. Amyloid formation in vivo likely arises from multiple factors including pH. Future work (to be presented in part at the symposium) aimed at understanding the synergistic effect of multiple variables on amyloid formation in vivo will explore the stability of WT and G26R at acidic pH.

Poster #: 129
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: HdeB chaperone protein, NMR spectroscopy, structure/function studies
Project Title: NMR characterization of chemical shifts and backbone flexibility of chaperone HdeB at pH 6
Author List:
Abasi, Lannah; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge

Abstract: HdeB is a conditionally disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria, including E.coli. It, along with a homolog protein called HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from denaturing and aggregating in the highly acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria, and resulting proliferation of dysentery. At physiological pH, HdeB is found as an inactive folded homodimer. Interestingly, it remains a fully folded dimer in its active state (at pH 4-5), suggesting that the mechanism of chaperone activation is a result of the structural flexibility of the protein.
The long-term goal of this study is to gain insight into HdeB’s unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics (internal motions). In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to probe HdeB’s flexibility and structure at pH 6.0. I recombinantly expressed 13C and 15N isotopically labeled HdeB, then purified and concentrated the protein. Numerous two- and three-dimensional NMR experiments were then conducted on this sample to assign each peak, or chemical shift, to its respective residue in the sequence. This is a necessary step to commencing an intensive protein NMR study, since it enables us to know where changes are localized in the sequence.
Thus far, I have assigned 71% chemical shifts of backbone amide groups at pH 6.0. Furthermore, preliminary backbone dynamics experiments have revealed intermediate time scale dynamics (µs to ms) in the loop between the 2nd and 3rd helix (the binding region of the two monomers), especially in the region between residues 35 and 41. In addition, many of the residues in the loop region were not observable in the spectra, further suggesting intermediate time scale dynamics at those positions. Continuing research will focus on completing chemical shift assignments and investigating the dynamics at different pHs.
We gratefully acknowledge the support of the NIH for research funds (SC3-GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).

Poster #: 130
Campus: San Francisco State University
Poster Category: Proteins (Include Proteomics)
Keywords: aminoglycoside antibiotics, acetyltransferase,
Project Title: Constructing Acinetobacter aminoglycoside acetyltransferase AAC(6’)-Ig mutants to investigate their effects on catalysis and substrate specificity
Author List:
Macias, Jennifer; Graduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Pham, Olivia ; Graduate, Biology, San Francisco State University, Presenting Author
Vaca, Alexis; Undergraduate, Chemistry and Biochemistry, San Francisco State University
Pennings, Pleuni; Biology, San Francisco State University
Kuhn, Misty ; Chemistry and Biochemistry, San Francisco State University

Abstract: Aminoglycoside N-acetyltransferases (AACs) are one of three types of aminoglycoside modifying enzymes bacteria use to reduce the effectiveness of these antibiotics. Modification of the drugs renders them unable (or less able) to bind to the ribosome and prevent protein synthesis. While many AACs have been studied for decades, a subclass called AAC(6’)-Ig has only recently been investigated in detail. The relatively small amount of information about this subclass has come from the structural and functional studies of this enzyme from Acinetobacter. We previously found this enzyme was quite promiscuous in its ability to modify a variety of aminoglycosides, but showed significantly less activity toward these antibiotics compared to other known AACs. Additionally, we noticed the enzyme exhibited substrate inhibition only in the presence of some antibiotics. Since less is known about how this enzyme functions and the reasons why certain antibiotics cause substrate inhibition, we have designed a combinatorial computational and biochemical approach to investigate the effects of various point mutations on its activity toward a panel of antibiotics compared to other AAC enzymes. While we are in the beginning stages of this project, we have already made significant progress toward the design and construction of our 75 member mutant library and high-throughput approach to characterize these mutants. The knowledge we gain from studying the AAC(6’)-Ig constructs will provide a greater understanding of which amino acid residues in the protein may allow the bacterium to acquire greater resistance toward these antibiotics in the future. The ultimate goal is to be able to have clinicians sequence patient isolates of this bacterium to determine which mutations have been acquired in this gene as a mechanism to offer appropriate selection and dosing of antibiotics.
This project has been funded by the Ken Fong Translational Research Fund (to MLK and PP) and San Francisco State University Startup Funds (to MLK).

Poster #: 131
Campus: San Diego State University
Poster Category: Proteins (Include Proteomics)
Keywords: Protein Design, Novel Protein/Protein Interaction, Metal-mediated Interface Design
Project Title: Designed Metal-Mediated Protein Dimerization
Author List:
Maniaci, Brian; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Love, John; Chemistry and Biochemistry, San Diego State University

Abstract: The main goal of this Protein Design project is to engineer novel dimer interactions that are driven by interfacial metal binding sites. The design of protein/protein interactions is of high interest to the pharmaceutical and biotechnological industries. Incorporation of metal-binding sites at the target protein interface is one approach to improve the specificity and affinity of designed protein-protein interactions. We recently used this approach to create high-affinity, zinc-mediated protein homodimers.
Using a metal-templated approach we engineered interfacial metal binding sites to generate a high affinity homodimer using the normally monomeric β1 domain of Streptoccocal Protein G (Gβ1). Metal coordination enables focused interfacial redesign, which allows for the preservation of naturally occurring amino acids and enhanced affinity and binding specificity. Protein complex formation is driven by metal coordination with designed histidine side-chains that function to coordinate the metal atoms.
Complex formation and assembly of the metal-mediated homodimers was evaluated with size exclusion chromatography and x-ray crystallography. To measure the molecular weight of the designed complexes we used size-exclusion chromatography combined with multi-angle light scattering (SEC-MALS). This analytical technique separates proteins based on size, and the molecular weight is determined from scattered laser light. The Gβ1 monomer and metal-mediated dimers have molecular weights of 6.2 and ~12.8 kDa, respectively. In addition, the structures of the homodimers were solved via X-ray crystallography and revealed that the target structure was achieved and that the complexes form through the designed zinc metal coordination.
We are also engineering a library of rationally designed proteins that contain mutations in proximity to the metal binding sites. The goal is to explore key thermodynamic parameters that are essential for protein complex formation. We are currently measuring the binding affinity of the metal-mediated protein-protein interactions using fluorescence polarization. Preliminary results indicate that the measured affinities are in the range of 100 nM, which is similar to naturally protein complexes and provides additional support for successful design of high-affinity metal-mediated protein-protein interactions.
Funding Agencies: Department of Defense, California Metabolic Research Foundation, CSU Annual Biotechnology Symposium (CSUPERB)

Poster #: 132
Campus: CSU Fresno
Poster Category: Proteins (Include Proteomics)
Keywords: NMR, protein, IDP
Project Title: Protein Expression and Structural Characterization of Lunasin: An Anti-Cancer Peptide
Author List:
Singh, Jaideep; Graduate, Chemistry, California State University, Fresno, Presenting Author
Brooks, Cory; Chemistry, California State University, Fresno
Krishnan, Krish; Chemistry, California State University, Fresno

Abstract: Lunasin is a unique 43-amino acid peptide naturally derived from seed grains such as amaranth and soybean. Lunasin has been shown to exhibit novel therapeutic properties such as cancer prevention, immunity support, heart health, and the disruption of abnormal inflammatory response. Evidence suggests Lunasin participates in histone modification and gene regulation, but the lack of information on the three-dimensional structure and dynamics of the protein. Our research objective is to characterize the secondary structure of Lunasin by circular dichroism (CD) and solve the tertiary structure of the protein via solution-state nuclear magnetic resonance (NMR) spectroscopy.
We have developed a robust E.Coli (SHuffle) expression system, optimized for the production of isotopically enriched (15N) Lunasin. This approach enables expression of 15N-labeled Lunasin in mg quantities for three-dimensional structural determination. The secondary structure of Lunasin was characterized using CD spectroscopy. NMR and structural characterization of recombinant Lunasin show both peptides are redox sensitive and contain transient secondary structure elements. These results are further supported by molecular dynamics simulations of Lunasin performed in explicit water. The combination of these results suggests that Lunasin could be classified and functions as an intrinsically disordered protein (IDP), contrary to the belief that it forms a well-ordered structure.

Poster #: 133
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Chlorination, Drug Discovery, C-H Funcationalization
Project Title: The Catalyst-Controlled Regiodivergent Chlorination of Phenols Towards Drug Discovery Efforts
Author List:
Maddox, Sean; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Dinh, Andrew; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author

Abstract: Lewis base catalyzed chlorination often results in highly regioselective mixtures of isomers (>32:1), with the isomeric ratio closely correlating to the innate reactivity of the substrate. However, in the case of highly regioselective electrophilic aromatic substitutions, obtaining the non-favored constitutional isomer can become a synthetic challenge. Furthermore, the innate reactivity of substrates with multiple electron donating groups can lead to complex mixtures of isomers, rendering the catalysis inefficient. To overcome these challenges, we hypothesized the addition of catalyst functionality that can interact with the substrate could potentially circumvent the innate reactivity of the molecule, resulting in control of the regiochemical outcome of chlorination. Monitoring by nuclear magnetic resonance spectroscopy, we experimented with various Lewis and Brönsted acidic functionalities appended to Lewis basic catalysts to led us to Nagasawa’s bis-thiourea catalyst, which sufficiently overcame the regioselectivity of phenol chlorination. Moreover, further modification of Lewis base catalyst (BINAP-derived phosphine sulfide) was found to increase the innate selectivity of phenols. This breakthrough illustrates that the appropriate choice of catalyst can be used to control the site of chlorination in electrophilic aromatic substitutions. We experimentally determined the difference in Gibbs Free Energy between the regioselectivity of both catalysts to approach 2.77 kcal/mol, which corresponds to a 99:1 enantiomeric ratio in the realm of enantioselective catalysis. Future work on the Lewis base chlorination will focus on atroposelective chlorination towards the dynamic kinetic resolution of “non-traditional” atropisomeric scaffolds ubiquitous in drug discovery.

Poster #: 134
Campus: CSU Bakersfield
Poster Category: Synthetic Chemistry
Keywords: BAPN, LOX, inhibitor
Project Title: Synthesis of Small Molecule Inhibitors of Lysyl Oxidase
Author List:
Redden, Jordan; Undergraduate, Chemistry & Biochemistry, California State University, Bakersfield, Presenting Author
Cervantes, Francisco; Undergraduate, Chemistry & Biochemistry, California State University, Bakersfield, Presenting Author
Wan, Alexander; Undergraduate, Chemistry & Biochemistry, California State University, Bakersfield
Solano, Danielle; Chemistry & Biochemistry, California State University, Bakersfield

Abstract: Lysyl oxidase (LOX) is an enzyme involved in the maturation of the connective tissues collagen and elastin that is also involved in cancer metastasis from hypoxic tumor cells. Tumors that induce LOX are more likely to spread to other parts of the body. Research has shown that β-aminopropionitrile (BAPN) can inhibit the LOX enzyme and prevent the tumor from metastasizing. BAPN is toxic, however, so it cannot be directly introduced to the body without damaging connective and epidermal cells. The goal of this project is to synthesize small carrier molecules that can selectively deliver BAPN to hypoxic tumor cells. New aromatic amides and new benzylamines were prepared that incorporate the BAPN structure. Benzylamines were prepared by reductive amination with 2-picoline borane (pic-borane). Amides required method development and were eventually prepared via a coupling reaction with benzoic acid derivatives and BAPN mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). Our optimal reaction conditions as well as yields will be reported. These products were then purified via column chromatography and tested via gas chromatography – mass spectrometry (GCMS) and nuclear magnetic resonance (NMR) spectroscopy. In total, three new amides and three new benzylamines have been prepared with several more in progress. The NMR and GCMS indicated the target molecules were synthesized and purified. Once purity has been established, these molecules will be submitted to our collaborators for screening against lysyl oxidase.

Poster #: 135
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Atropisomer, Synthesis, Asymmetric Catalysis
Project Title: The Cinchona-Alkaloid Catalyzed Nucleophilic Dynamic Kinetic Resolution of Aryl-Naphthoquinones
Author List:
Maddox, Sean; Graduate, Chemistry and Biochemistry, San Diego State University
Dawson, Gregory; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author

Abstract: Atropisomerism is a form of chirality that arises from the hindered rotation about a bond, resulting in rotational enantiomers. Various natural products and bioactive small molecules exhibit atropisomeric biaryl motifs, yet no general strategies exist to produce these types of scaffolds with diverse functionality proximal to the chiral axis. A general enantioselective synthesis toward diverse enantioenriched biaryl-atropisomeric moieties would provide access to products with unique geometries, structural properties, and functionality. Providing diverse functionality proximal to the chiral axis allows for structure activity relationship (SAR) studies towards the design of efficacious bioactive small molecules. We have developed a dynamic kinetic resolution of biaryl naphthoquinone atropisomers via the nucleophilic addition of thiophenol proximal to the chiral axis. Various biaryl naphthoquinone atropisomers were amenable to this strategy, often yielding enantiomeric ratios of greater than 90:10. The resulting enantioenriched 1,4-dimethoxynaphthalene atropisomers can be further modified to contain desirable functional groups proximal to the chiral axis with high enantioretention. These results represent a general strategy towards the synthesis of biaryl atropisomers with diverse, useful functionality proximal to the atropisomeric axis.

Poster #: 136
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: Natural Product , Medicinal Chemistry, anti-cancer agent
Project Title: The Optimized Syntheses and AntiProliferative Activity of a Simplified Dactylolide Mimic
Author List:
Chen, Guanglin; Chemistry, California State University, Fresno
Vue, Bao; Chemistry, California State University, Fresno, Presenting Author
Wang, Rubing; Postdoc, Chemistry, California State University, Fresno
Chen, Qiao-Hong; Chemistry, California State University, Fresno

Abstract: Dactylolide was isolated from the marine sponge Dactylospongia sp. in 2001. Its core structure is the same as the macrocyclic core of marine macrolide (-)-zampanolide that has been identified as a promising anti-cancer lead compound due to its unique covalent-binding with tubulin and superior cytotoxic potency compared to paclitaxel against multi-drug resistant cancers. We envision that zampanolide possesses poor pharmacokinetic profiles as a drug candidate partially due to its chemically fragile side chain. The overarching goal of this project is to identify simplified (-)-zampanolide mimics with stabilized side chains as well as manageable synthetic methods. With this goal, this study aimed to develop a practical and manageable synthetic approaches to the simplified core mimic (i.e. simplified dactylolide mimic) that lacks the tetrahydropyran ring in dactylolide. To this end, two optimized methods have been successfully developed by us using allylic iodide or allylic tosylate as the starting material for the critical Williamson ether synthesis. Through these two methods, gram scale of this dactylolide mimic has been successfully synthesized in our laboratory. Its antiproliferative activity has been evaluated in three prostate cancer cell models using WST-1 cell proliferation assay. This presentation will report the full details of our optimized synthetic methods and the antiproliferative activities of the simplified dactylolide mimic.
This project is supported by NIH/NIGMS under Award Number SC2GM121185.

Poster #: 137
Campus: CSU Channel Islands
Poster Category: Synthetic Chemistry
Keywords: Ketonucleosides, Diastereoselective reduction, CBS catalyst
Project Title: Diastereoselective synthesis of xylo- and arabinofuranosides by catalytic hydrogenation of corresponding keto-nucleosides
Author List:
Maldonado, Jessica; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Mendoza, Jesica; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Chemistry, California State University Channel Islands

Abstract: Several nucleoside analogs have been approved by the FDA for cytotoxic therapy and for treatment of hepatitis, HIV, and HSV. 3′-azidothymidine (AZT) was the first antiretroviral treatment for HIV and is also used to treat several human cancers. Cytarabine has been used to treat various forms of leukemia and Hodgkin`s lymphoma as a chemotherapy drug. A challenging step in the synthesis of nucleoside analogs is to convert the 2′ and or the 3′-hydroxyl groups on the ribose moiety into desired modifications with a retention in the configuration. In view of this, we report an efficient, two-step approach to achieve desired diastereomeric configuration of the 2′,3′-hydroxyl moieties using an enantioselective reducing chiral catalyst. Oxidation to keto-nucleosides followed by enantioselective reduction using CBS catalyst provided an efficient route toward the synthesis of target molecules. This method produced the desired xylo- and arabino- configurations among both purines and pyrimidines, including adenosine, cytidine, and uridine derivatives, with up to 98% diastereomeric excess (de). The resulting nucleoside xylofuranoside derivatives were further modified by nucleophilic addition of N3 to elicit ribonucleoside guanidine (RNG) precursors. This approach may increase efficiency in the generation of nucleoside precursors of interest along with a more efficient production of xylo- and arabinofuranoside prodrugs such as cytarabine and vidarabine.

Poster #: 138
Campus: CSU Northridge
Poster Category: Synthetic Chemistry
Keywords: catalyst, enantioselective, pharmaceutical
Project Title: Induced enantioselectivity in inner-sphere transfer hydrogenation catalysts
Author List:
Weeks, Cal; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge

Abstract: Selective catalysis still has much promise for reducing cost and improving purity of vital pharmaceuticals. In particular, transfer hydrogenation catalysts have attracted much attention for the ability to produce chiral centers from the relatively safe and inexpensive transfer of hydrogen from alcohol solvents to ketone or alkene groups in drug precursors. Despite the initial promise of Noyori-type catalysts, their selectivity could not be generalized due to their dependence on weak (and unpredictable) non-covalent interactions between the catalyst and substrate. The Kelson group has developed a new catalyst system that uses electronically coupled ruthenium dimers to achieve transfer hydrogenation through metal-bound (inner-sphere) substrates. Since the position of bound substrate is known, the environment around the binding site could be deliberately designed to restrict binding to specific substrates in the desired orientations. We report here the test of possible design-for-selectivity in the Kelson ruthenium dimer catalyst system by incorporating large chiral phosphine ligands and surveying reaction yields for enantioselectivity. The precatalyst was prepared by first reacting (Tpy)RuCl3 (where Tpy=2,2’:6’,2”-terpyridine) with TpyTpy (bis-4’,4’-(2,2’:6’,2”-terpyridine)) to form [(Tpy)Ru(TpyTpy)]2+ and subsequent reaction of this complex with RuCl2(DMSO)4 (DMSO=dimethylsulfoxide) to form [(Tpy)Ru(TpyTpy)RuCl2(DMSO)]2+ (Complex 1). Typical reactions entailed reacting Complex 1 with commercially available chiral phosphines to form the catalyst in-situ followed by addition of substrate and supporting base at reflux. The reactions were sampled periodically, and the yields and enantioselectivities were measured by gas chromatography. While the chloride salt of Complex 1 was slow likely due to competitive binding of chloride, the perchlorate salt of Complex 1 exhibited good catalytic rates. Reactions incorporating chiral mono- and diphosphines resulted in significant enantiomeric excesses from the transfer hydrogenation of acetophenone [such as 18% from (1R,1’R,2S,2’S)-DuanPhos]. Smaller enantiomeric excesses were also measured for 3,3-dimethyl-2-butanone. These results confirm that this catalytic system has potential for design-for-selectivity. We thank CSUPERB (Research Development Grant), the NIH (SCORE S06 GM48680), ACS Petroleum Research Fund, and CSUN Office of Sponsored Projects for support of this work.

Poster #: 139
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: Organocatalysis, Bisphosphorylimides, Friedel–Crafts
Project Title: Chiral Bisphosphorylimides as Organocatalysts for Friedel–Crafts Reactions
Author List:
Apolinar, Omar; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Adamson, Natalie; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Ahlberg, Lina; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Thompson, Cristofer; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Diaz, Gloria; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Dean, Melisa; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Klasic, Brittney; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Maynard, Jessica; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Ghasb, Elie; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Iafe, Robert; Chemistry & Biochemistry, California State University San Marcos

Abstract: As an ever-increasing number of chiral bioactive molecules flood the pharmaceutical industry, the importance of efficient enantioselective synthesis and purification of these compounds also increases. Enantioselective organocatalysis has emerged as a powerful synthetic tool complementary to traditional metal-containing catalytic transformations. The use of organocatalysts are attractive because reactions tend to be operationally simple due to their stability to air and water, ready availability, and low toxicity. Taking this into account, we have developed the asymmetric Brønsted-acid catalysis of the Friedel–Crafts reaction of sesamols, a naturally occurring component of sesame oil that shares the chemical structure of many pharmaceutical compounds, with benzylidenecarbamates using chiral bisphosphorylimides as an organocatalyst. Despite their notable potential for catalysis, bisphosphorylimides are currently underutilized in this role. The chemical scaffold of bisphosphorylimides is highly structured and ordered, mimicking the active site of an enzyme. The long-term objective is the enantioselective synthesis of bioactive natural products, unnatural amino acids, and other various building blocks useful in the synthesis of chiral bioactive molecules.
Results: Progress towards the synthesis of two new bisphosphorylimide catalyst derivatives and characterization will be reported (R = SiPh3 and C6H4CF3). These bulky catalysts are currently being used to determine the substrate scope of the reaction. An improved synthetic route of the model bisphosphorylimide organocatalyst (45% yield over 2 steps) from enantiopure BINOL using liquid ammonia has been developed in our laboratory. Catalyst activity of the model bisphosphorylimide was optimized using sesamol with a benzylidenecarbamate electrophile (Ar = tolyl) and triethyl amine as an additive at reduced temperature to afford the target amine in quantitative yield in 1.5 hours. Catalyst loading is effective as low as 5 mol %, and nucleophilic weak bases hinder the rate of reaction. The bisphosphorylimide also shows catalytic ability with two other benzylidenecarbamate electrophiles: Ar = phenyl and anisyl.
Acknowledgements: We thank CSUPERB (New Investigator and Presidents’ Commission Program) for support of this work

Poster #: 140
Campus: CSU Sacramento
Poster Category: Synthetic Chemistry
Keywords: Glycodendrimers, Synthesis,
Project Title: The Synthesis of an Octavalent Dendrimer for Glycodendrimer Synthesis
Author List:
Cerney, James; Graduate, Chemistry, California State University, Sacramento, Presenting Author
McReynolds, Katherine; Chemistry, California State University, Sacramento

Abstract: Dendrimers have been found to be useful tools among a variety of biomedical applications, including as transport molecules, scaffolds, and inhibitory agents. These globular molecules consist of a central core which is then branched using linker molecules. The number of functional ends increases with each branching step. Our research is founded on the synthesis of glycodendrimers containing oxime linkages. By having a larger number of functional ends, the molecule is able to bind to an increased number of targets. The size of these molecules also plays an important role in their effectiveness through mimicking the proper size of the cell receptors normally targeted by these infectious agents. Many commercially available dendrimers exhibit high toxicity. Our research focuses on synthesizing molecules that have binding affinity to the desired biological targets with low toxicity.
Currently, work is being done to synthesize an aminooxy terminated octavalent core. The linker was first synthesized through a two-step process. First, one of the hydroxyl groups of ethylene glycol was attached to acrylonitrile in a 57% yield. The primary alcohol of the linker was then tosylated in a 74% yield. The tetravalent core was next synthesized through an SN2 reaction involving the central core molecule, 2,2’-oxybis(ethylamine), with the tosylated linker resulting in a 56% yield. The tetra-nitrile core then underwent a two-step, one pot reaction in which the nitrile termini were reduced and Boc protected resulting in a 47% yield. This then underwent deprotection to reveal amine terminated ends in a 92% yield. The nitrile ends then underwent further branching using acrylonitrile to yield the octa-nitrile product. The tetra-amine core then underwent a reduction protection followed by a deprotection to produce the octa-amine.
The octa-amine core will next be reacted with a short linker, synthesizing the aminooxy terminated core. The final dendrimer will then undergo glycosylations utilizing various sugars, such as maltotriose and maltose to give the desired glycodendrimers.

Poster #: 141
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Chirality, Medicinal Chemistry, Organic
Project Title: The Atroposelective Dynamic Kinetic Resolution of Diaryl Ethers: A New Route to Chiral Pharmacophores.
Author List:
Dinh, Andrew; Graduate, Chemistry and biochemistry, San Diego State University, Presenting Author
jackson, Amy; Undergraduate, Chemistry, San Diego State University, Presenting Author

Abstract: Atropisomerism is a mode of chirality that arises due to sterically hindered rotation about an axis. The axis of chirality may rapidly interconvert from one conformer to another depending on the amount steric bulk proximal to the axis. One overlooked class of atropisomers are diaryl ethers, which are ubiquitous in drugs and natural products, such as the antibacterial vancomycin and mammalian hormone thyroxine, Seminal work has shown that the different atropisomer conformations of diaryl ethers possess different pharmacological properties. However, in depth medicinal chemistry studies on diaryl ethers have been hindered by a lack of asymmetric routes to homochiral diaryl ethers.

To address this issue we have begun development of a chemical methodology that utilizes a dynamic kinetic resolution on a hindered, yet freely rotating, diaryl ether naphthoquinone. Preliminary results have shown that a urea-based quinine derived quaternary ammonium catalyst can effect the enantioselective alkylation of these quinones using nitroalkanes as the alkyl source with enantiomer ratios up to 85:15. This poster will discuss our recent efforts to further optimize this chemistry as well as apply it to the synthesis of atropisomeric analogs of diaryl ether based kinase inhibitors.

Poster #: 142
Campus: Humboldt State University
Poster Category: Synthetic Chemistry
Keywords: peptoid, biomimetic, lead
Project Title: Synthesis and Evaluation of a Lead Binding Peptoid
Author List:
Alizadeh, Tara; Undergraduate, Chemistry, Humboldt State University, Presenting Author

Abstract: Heavy metal contamination of soils and waterways, caused by industrial processes such as mining, continues to be an environmental problem. Lead is a particularly problematic environmental contaminant. Each year in the United States approximately 310,000 children between the ages of 1 and 5 are tested with elevated levels of lead, which can accumulate over months or years and lead to serious health related issues. The aim of this project is to prepare a peptoid with binding affinity for lead. Peptoids are synthetic oligomers similar to naturally occurring peptides which make up proteins. In a peptoid side chain groups are attached to nitrogen rather than the alpha carbon of a peptide. This results in heteropolymers capable of higher order folding with biological activity. Peptoids are simple to prepare, easy to purify, and are stable under biological conditions making them excellent biomimetics for natural peptides. Previous research has shown that the peptide sequence GGGTNTLSNNGGG has an affinity for binding lead particles. Utilizing solid phase reaction chemistry the peptoid analog was synthesized. The resin bound peptoid has been evaluated for lead binding affinity using Flame Atomic Absorption Spectrophotometry. Initial results show a 27:1 lead to peptoid binding with an average 14% lead decrease in the presence of 1.31× 10-5 moles of the peptoid. Treatment of peptoid bound lead with hydrochloric acid resulted in release of lead indicating the recyclability of this peptoid modified resin for lead binding. The decrease in lead concentration in solution coupled with the reusability of the peptoid resin are promising results for peptoid use in lead contaminated water supplies.

Poster #: 143
Campus: Sonoma State University
Poster Category: Synthetic Chemistry
Keywords: antimicrobial peptide, membrane peptide, peptide synthesis
Project Title: Organic Synthesis of the Putative Target Peptide for anti-Listerial Molecules
Author List:
Berhane, Faven; Undergraduate, Chemistry, Sonoma State University, Presenting Author
Baros, Mark; Undergraduate, Chemistry, Sonoma State University, Presenting Author
Whiles Lillig, Jennifer; Chemistry, Sonoma State University

Abstract: Listeria monocytogenes are pathogenic bacteria that are in unpasteurized dairy products and deli meats. There are antibiotics available to treat Listeriosis, but a possible alternative are antimicrobial peptides called bacteriocins. Class IIa bacteriocins, like Piscicocin V1B, target Listeria using its mannose phosphotransferase system, and domain C of this system (MPTC) is required for bacteriocin activity to induce cell sensitivity. Little else is known about the nature of the interaction between bacteriocins and Listeria cells. Current literature acknowledges the importance of the MPTC loop for bacteriocin induced cell sensitivity, but lacks data indicating that the bacteriocin and the loop make contact to induce cell sensitivity. The goal of this experiment was to synthesize the peptide loop of Listeria’s MPTC to later perform competition assays with. The method used to synthesize the peptide loop was solidphase peptide synthesis. Solidphase peptide synthesis involved anchoring the last amino acid of the peptide to a resin support, deprotecting the amino acid, adding the next amino acid for coupling (peptide bond formation), and repeating this process until the sequence was completed.
The synthesis was successful as indicated by UVvis absorbances and ninhydrin tests. The protecting group of the amino acids absorbs light at 280 nm. UVvis absorbances of the waste at 280 nm were taken after each rinse to confirm deprotection of the amino acids before coupling began. The UVvis absorbances of the waste per wash decreased, indicating removal of the protecting group. The ninhydrin test was used to confirm deprotection occurred, and that peptide bond formation was complete. Deprotection was confirmed to be successful for every amino acid except proline. However, this was common in the literature and the ninhydrin test supported successful deprotection of the proline. This issue was noted in literature as being common, and did not indicate that the amino acid was not successfully deprotected. The ninhydrin test also confirmed formation of peptide bonds for each amino acid, based on color change from blue to yellow. Mass spectrometry and high performance liquid chromatography was used to finalize peptide purity. Future work will be to study the interaction between bacteriocin and extracellular MPTC loop of Listeria.
This work was funded by a Sonoma State SOURCE award.

Poster #: 144
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: Broiler Chickens, Probiotics, Gut Colonization
Project Title: Detecting a Probiotic Product Colonizing the Gut of Broiler Chickens
Author List:
Pisula, Anneka; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
VanderKelen, Jennifer; Biological Sciences, California Polytechnic State University, San Luis Obispo
Bennett, Darin; Animal Science , California Polytechnic State University, San Luis Obispo
Niles von Kraut, Konor ; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Cates, Taylor ; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Bruce, Ashley ; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Porta, Bethany ; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Wack, Carson; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo
Kitts, Christopher L. ; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: The purpose of this study was to detect a probiotic product composed of four bacterial strains (Pedioccoccus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bacillus subtilis) during a feeding trial with broiler chickens. The probiotic is marketed to improve broiler chicken health, increase productivity, and aid public concerns about growing antibiotic pathogenic resistance. The working hypothesis was that birds given the probiotic would exhibit an improved growth performance and the administered bacteria would colonize some compartments of the gut. Ninety six broiler chickens were equally divided into 3 experimental and 3 control pens. The experiment was conducted over 25 days and birds were fed two diets during the experiment, a starter diet (days 0-11) and a finisher diet (days 12-25). Experimental birds were given the probiotic product, administered via the drinking water at a concentration of 10^4 CFU/ml. Feces were collected daily and on day 22 of the experiment, 6 birds from each group were sacrificed for gut sampling. Lumen and mucosa samples collected from each bird’s duodenum, jejunum, ileum, and ceca. P. acidilactici was detected in both feeds, confounding probiotic detection efforts. B. thuringensis was detected in feces within one hour of probiotic administration, and was predominantly detected in experimental birds only. Both P. acidilactici and L. plantarum were consistently detected in both control and experimental birds, making the source of these bacteria in the gut uncertain. B. thuringensis was seen only in experimental bird gut samples, although it was rarely detectable. P. pentosaceus was observed more often in experimental gut samples, but rarely seen in mucosa samples. P. acidilactici and L. plantarum were present in all gut samples and were common in the cecal mucosa, indicating probable gut colonization. Although P. acidilactici and L. plantarum showed the best potential for gut colonization, it was unclear if the administered probiotics were colonizing experimental birds. Even though the four bacterial strains were detectable in the gut and feces, probiotic supplementation did not result in significant differences in body weight, rate of weight gain, feed intake, or feed conversion ratio. However, birds growing in a more stressful environment than the carefully controlled experimental set up used here may have exhibited probiotic-related effects.

Poster #: 145
Campus: CSU Northridge
Poster Category: Agriculture/Biofuels/Environment
Keywords: plant biotechnology, ethylene, flood tolerance
Project Title: Development of flood tolerant Arabidopsis thaliana by over-expression of the gene ACO (aminocyclopropane-1-carboxylic acid oxidase)
Author List:
Ramadoss, Niveditha; Graduate, Biology, California State University, Northridge, Presenting Author
Gupta, Dinesh; Graduate
Vaidya, Brajesh; Staff
Joshee, Nirmal; Faculty
Basu, Chhandak; Biology, California State University, Northridge

Abstract: Floods are a big threat to human life and global food security. The number of severe floods has increased worldwide over the past three decades and it is one of the most anticipated consequences of global warming. To address the crop loss issue due to floods, we focused on developing plants that are tolerant to flood. Ethylene, a growth hormone is known for helping plants during flooding conditions. However, plants that are susceptible to floods are not capable of converting the ethylene precursor ACC (aminocyclopropane-1-carboxylic acid) to ethylene due to lack of oxygen in the submerged water. ACC to ethylene conversion is catalyzed by the enzyme ACC oxidase. Based on this, we hypothesize that overexpression of ACC oxidase enzyme may sustain higher ethylene production in plants thereby making them flood tolerant. The ACC oxidase gene was PCR amplified from Arabidopsis thaliana plants and cloned into a plant expression vector pBINmgfp5-er. The gene was placed under the control of a strong constitutive Cauliflower Mosaic Virus promoter. Transgenic plants were created through the floral dip of three-week-old T0 (transgenic zero or wild-type) plants. The plants were grown until T3 generation. Presence of the introduced gene was confirmed by regular polymerase chain reaction (PCR) and expression was confirmed by qPCR (quantitative PCR). The flood tolerance test showed that transgenic plants were more tolerant to flooding stress than the wild type. After 20 days, the wild-type plants died whereas the transgenic plants survived the flood stress. Physiological analysis exhibited faster growth rate of transgenic plants accompanied by tall stems and reduced number of trichomes when compared with controls. These traits are identified as adaptive traits to escape the damages caused by the flood. Research is underway in our lab to develop economically important flood-tolerant crops.

Poster #: 146
Campus: CSU Fresno
Poster Category: Agriculture/Biofuels/Environment
Keywords: Synergistic Effect, Nematodes, C.elegans
Project Title: Synergistic effect of Organic Chalcones on C. elegans and M. incognita
Author List:
Shinde, Shantanu; Graduate, Biology, California State University, Fresno, Presenting Author, Eden Award Nominee
Kendoyan, Sosse; Graduate, Biology, California State University, Fresno, Presenting Author, Eden Award Nominee

Abstract: Plant Parasitic Nematodes (PPNs), like Meloidogyne incognita, are responsible for large monetary losses in USA agriculture and worldwide. Conventional nematicidal agents, such as methyl bromide, have been banned because of its toxic effects on humans and the environment. To find alternative strategies for a safe and effective nematicide, a series of organic chalcones were tested and our previous research findings have established chalcones (17, 25 and 30) to be 100% effective against the model nematode C. elegans at concentrations of 10-4 M; chalcones belong to the flavonoid family of organic compounds. However, whether these two chalcones could work simultaneously to control nematodes with lower concentrations of chalcones has not been tested. The purpose of this research, is to study the nature of interactions (if any) between Chalcone 17, 25 and 30 on C. elegans using nematode based bioassays. Thus, work with C. elegans serves as a methodological and biological reference for work with M. incognita. Initially, different concentrations of chalcone 17, 25 and 30 were tested individually, and based on this single chalcone test results, we selected ranges of concentrations (1×10-5, 0.8×10-5, and 0.6×10-5, 0.4×10-5, 0.2×10-5, 10-6M) of each 17, 25 and 30 that caused a mortality rate of approximately 30% (lethal dose 30 or LD30). To examine the nature of interaction between them, we performed assays by mixing the chalcones at their corresponding LD30. For example, Chalcone 17 (LD30[A]) was mixed with chalcone 25 (LD30[B]) to attain the required concentration of the mixture. If the chalcones [A]+[B] have a simple additive effect, a mortality rate of around 60% would be observed. However, if the chalcone combination is synergistic the mortality rate should be higher than 60%. Our results support a synergistic effect since the combination of chalcone 17, 25, and 30 (pair wise and the triple combination) when used in lower concentrations (as low as 10-6 M), resulted in a mortality rate of 90%-100% in C. elegans within the 3-day exposure.
The results suggest that, although the cellular or physiological targets of these chalcones in the nematode are yet unknown, a combination of these chalcones could interact and destroy cellular or physiological functions more effectively.

Poster #: 147
Campus: Cal Poly San Luis Obispo
Poster Category: Agriculture/Biofuels/Environment
Keywords: Olive , Biorefinery, Methane
Project Title: Biochemical Methane Potential of Enzymatically Pre-treated and Destoned Olive Cake
Author List:
Tai, Patrick; Graduate, Food Science and Nutrition , California Polytechnic State University, San Luis Obispo, Presenting Author
Spierling , Ruth; Civil and Environmental Engineering , California Polytechnic State University, San Luis Obispo
Jung, Stephanie; Food Science and Nutrition , California Polytechnic State University, San Luis Obispo

Abstract: Olive cake is the solid residue which results from the production of virgin olive oil. While the cake is a major by-product that the olive industry must dispose of, it is also considered a low-value by-product. Our study attempts to establish a potential application of olive cake in the context of an olive oil mill waste biorefinery. We are focusing on converting olive cake into a value-added product, a substrate for methane production. Our central hypothesis is that an enzymatic pretreatment could be applied to olive cake, maximizing its conversion into soluble carbohydrates. Furthermore, removal of the stones could be beneficial for the previously mentioned conversion. 1 kg of olive pomace was enzymatically pre-treated with a cocktail of cellulase, xylanase, hemicellulase, and pectinase (BIO-CAT) at a final concentration of 1.6% (w/w, db) for 24 h. Destoning was achieved with a Leeson Horizontal Screw Press (Model: C145T17FB60D). The pre-treated cakes were anaerobically digested in batch digesters at a substrate to inoculum ratio (S:I) of 1:2 and 2:3 by volatile solids (VS) for 30 days at mesophilic conditions. The methane concentration of the biogas was determined using a gas chromatograph (SRI 8610) with a thermal conductivity detector and a six-foot concentric column (Alltech CTR I). The enzymatic pre-treatment increased the reducing sugar content of the olive cake by roughly 178% while the destoning removed 91% of the stones. After 30 days of digestion and at a S:I of 1:2, the control produced 287 ± 10.1 mL methane / g VS and the destoned produced 358 ± 9.4 mL methane / g VS, an increase of 25% of methane produced. The enzymatic pretreatment alone decreased methane production by 33%. Our data suggests enzymatic pre-treatment introduces inhibition to the digester while illustrating the destoning of olive cake can increase its methane potential so that it is comparable substrate to glucose (370 mL methane / g VS), signifying further conversion of olive cake could divert it into landfill, which will address a significant environment concern associated with this by-product for the growing olive oil industry.

Poster #: 148
Campus: Humboldt State University
Poster Category: Agriculture/Biofuels/Environment
Keywords: persistent organic polutants, HPLC, herbicides 2,4-D Triclopyr
Project Title: Analysis of Herbicides on Culturally Significant Plants Throughout Yurok Ancestral Territory
Author List:
Martinez, Amanda; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Cappuccio, Jenny; Chemistry, Humboldt State University

Abstract: Use of herbicides by public and private entities throughout the Yurok ancestral territory, has raised concern for the health of Native peoples exposed to these organic residues on plant materials. Plants play a significant role within the Yurok culture and are utilized in basket weaving, as medicinals, and for ceremonial purposes. The objective of this study is to determine if there are detectable levels of herbicide on culturally significant plants within the Yurok ancestral territory. Using organic extraction followed by High Performance Liquid Chromatography (HPLC) analysis, the herbicides 2,4-D and Triclopyr were identified on plant material collected from the Ah-Pah location. versus control samples from Fern Canyon. The HPLC results will be verified by using Gas Chromatography Mass Spectrometry (GCMS) and/or Liquid Chromatography Mass Spectrometry (LCMS) to further verify organic pollutants within culturally significant plant samples.

Poster #: 149
Campus: CSU Fullerton
Poster Category: Agriculture/Biofuels/Environment
Keywords: secondary organic aerosol, visibility, cloud formation
Project Title: Atmospheric Reactions of Glyoxal Aerosol: Geographic Effects on Visibility and Cloud Formation
Author List:
Kaak, Marissa L.; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
McCoy, Katey; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Saad, Ali; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Chavez, Damaris; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Hudson, Paula K.; Chemistry and Biochemistry, California State University, Fullerton

Abstract: In the past thirty years, there has been a significant increase in the frequency of wildfires in California. These wildfires directly release carbon containing compounds as gases (e.g., carbon monoxide, carbon dioxide, and methane) and particles (e.g., soot). The primary emissions from wildfires have an impact on air quality and human health in the immediate vicinity of the fire. Additionally, as gases and particles are transported away from the wildfire source, emitted biogenic compounds (e.g., terpenes and glyoxal) can react to form secondary organic aerosol (SOA) that affect visibility and can serve as cloud nuclei (seeds for cloud formation) affecting rainfall and climate. The resulting effects of SOA are governed by aerosol composition which is controlled by where aerosol particles are geographically transported. To better understand and predict visibility effects and cloud formation properties of SOA originating from wildfires, aerosol are generated from laboratory reactions of glyoxal (GX) with three atmospherically relevant compounds: glycine (GLY, the most abundant amino acid present in the atmosphere), ammonium sulfate (AS, prevalent in agricultural areas), and sodium chloride (NaCl, prevalent in coastal regions). Visibility effects are examined by measuring the absorbance of reaction products in the visible wavelength range. Cloud formation properties are conducted using a tandem differential mobility analyzer that measures the change in aerosol particle size as a function of increasing relative humidity (RH). Although aerosol composition controls absorbance and water uptake properties, results indicate complicated reaction mechanisms are occurring. For example, reactions of GX with GLY and AS are brown in color, likely from the formation of imidazole compounds, affecting visibility. However, while water uptake of GX:GLY mixtures is independent of relative reactant concentrations, allowing for easier prediction of cloud formation, the particle diameter of GX:AS mixtures is highly variable. Particle diameters increase from 11% to 33% at 85% RH as the relative concentration of AS is increased from 2 to 50%. Even though aerosol particles may have similar absorptive properties, this does not ensure other physicochemical properties, like water uptake, are conserved. Improved understanding of these processes allows climate modelers to reduce uncertainties in predictions for visibility and rainfall resulting from aerosol emitted from wildfires.

Poster #: 150
Campus: CSU Northridge
Poster Category: Astrobiology
Keywords: metagenomic, permafrost, exobiology
Project Title: Metabolic and survival strategies of microbial communities in ancient permafrost
Author List:
Thongsarn, Anthony; Undergraduate, Biology, California State University, Northridge, Presenting Author
Srinivas, Archana; Postdoc, Biology, California State University, Northridge
Mackelprang, Rachel; Biology, California State University, Northridge

Abstract: In polar regions, much of the landscape is underlain permafrost–dirt, rock, and sediment that is permanently frozen. Permafrost is an important research topic because (1) permafrost thaw contributes to climate change and (2) it serves as a model for exobiology. Permafrost is a reserve for organic undecomposed carbon, which is typically stored in a frozen state. However, climate change is thawing the permafrost exposing the carbon to microbial decomposition. Microbes degrade the newly-available carbon and respire it in the form of carbon dioxide or methane. Permafrost is a model for exobiology because many extraterrestrial bodies in the solar system including planets, comets, and moons are cryogenic. If we learn about survival strategies in permanently frozen soil on Earth, we may gain understanding of how life (if it exists) survives on cryogenic extraterrestrial bodies. However, studying life in permafrost is difficult because the majority (~99%) of microbes living in permafrost cannot be cultured in a laboratory setting. To circumvent this hurdle, we apply metagenomic techniques where DNA is isolated directly from the permafrost and subjected large-scale next generation sequencing. To understand how life survives and metabolizes organic material, we sequenced metagenomic DNA from Pleistocene permafrost from three different time periods (19kyr, 27kyr, and 33kyr), assembled the sequence into contigs, and binned the contigs in groups representing draft genomes. We binned thirty-six draft genomes representing many novel organisms including methanogens from the Methanobacteriaceae family, nitrogen oxidizers from the Nitrospiraceae family, and ammonia oxidizing archaea from the Thaumarchaeota phylum. Analysis of the draft genomes shows communities capable of diverse metabolic processes such as fermentation, sulfur and iron metabolism, nitrate oxidation, and ammonia oxidation. Currently, we are comparing the metabolic capabilities between permafrost age categories to determine how communities adapt metabolically to life in increasingly ancient cryoenvironments.
Funding provided by the NASA exobiology program.

Poster #: 151
Campus: CSU San Bernardino
Poster Category: Astrobiology
Keywords: microfluidics, cultivation, cell sorting
Project Title: Testing and validation of a microfluidic device and laser tweezers for use in single-cell isolation for cultivation
Author List:
Mosier, Damon; Graduate, Biology, California State University, San Bernardino, Presenting Author
Dodsworth, Jeremy; Biology, California State University, San Bernardino

Abstract: Of the many species of bacteria and archaea that are known, few have been successfully grown in pure culture. This is due to a variety of reasons, including the inability of some microbes to grow on solid media or their slow growth rates and low abundance in natural samples. Optofluidic cell sorting, a method for viewing and separating live microbial cells via microscopy, is one technique that can be used to isolate cells for cultivation, but is not often implemented due to the level of difficulty and associated costs. Based on a previous design used for single-cell genomics, a microscope was adapted for single-cell isolation using optical trapping (laser tweezers) and a microfluidic device made from polydimethylsiloxane (PDMS). The goal of this study was to address several concerns associated with this technique: maintaining sterility, preventing cells from sticking to the inner surfaces of the microfluidic device, validating single-cell isolation, and determining cell survival rates. It was found that the microfluidic devices remained fully functional after multiple autoclaving cycles when the control and flow channels were filled with water to prevent collapsing. This allows for the microfluidic devices to be sterilized and reused, eliminating the high cost and lengthy production time of obtaining new ones. To prevent individual cells from sticking to the inner walls of the microfluidic chip during sorting, Tween 20 and F127 pluronic were used in low concentrations of 0.02% and 0.04%, respectively. To validate that single, viable cells could reliably be obtained, control sorting experiments were performed using E. coli. Putative single-cell suspensions harvested from the device were split between five different aliquots of LB growth medium and incubated for 2 days. In all cases, only one of the five aliquots yielded growth, and between 40-87% of sorted cells yielded growth in three separate experiments. These tests validate that the technique can be reliably applied for isolation of viable microbes, and will be used in future studies for isolation of uncultivated thermophiles, including members of the candidate phyla Aigarchaeota, Fervidibacteria, and Calescamantes that are currently maintained in mixed enrichment cultures in the laboratory. Obtaining pure cultures or defined co-cultures of members of these candidate phyla will allow for a better understanding of their metabolic capabilities.
This work was supported by NSF grant DEB-1557042.

Poster #: 152
Campus: CSU Northridge
Poster Category: Astrobiology
Keywords: permafrost, exobiology, methane
Project Title: Isolation of Novel Psychrotolerant Organisms from Ancient Permafrost
Author List:
Romero, David; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Srinivas, Archana; Postdoc, Biology, California State University, Northridge
Mackelprang, Rachel; Biology, California State University, Northridge

Abstract: Approximately one quarter of the Earth’s surface is underlain by permafrost, which is permanently frozen soil. Life exists in this frozen and static environment, eking out an existence or even thriving under these harsh conditions. Despite the seemingly unassuming existence, these microorganisms are relevant to the fields of exobiology and climatology. In the search for life beyond Earth, permafrost can be used an analogue to Martian permafrost or other cryogenic bodies. If we can understand the adaptations that enable life to survive in Earth’s permafrost, we can gain insights into how life (if it exists) survives in other frozen environments in the solar system. Microbial communities in permafrost also impact our planet’s climate. Permafrost preserves undecomposed plant material and as the earth warms, massive reserves of organic matter become available for degradation. Microbes consume this organic matter and release greenhouse gasses, including methane, which is 20 times more potent than carbon dioxide. To determine how permafrost thaw contributes to climate change, we must understand the processes by which microbial communities degrade carbon and release greenhouse gasses.
To study the diverse organisms living in permafrost, we are isolating microbes from a chronosequence of Pleistocene permafrost frozen for 19kry, 27kyr, and 33kyr. To maximize diversity we used multiple media types and culture conditions. Anaerobes were grown in tubes of MSO6, pressurized with a gas mix of H2CO2. Agar plates of ISP4, RDM, and Modified PYG were used to grow anaerobic, microaerophilic, and aerobic organisms. Physiology and taxonomic identification experiments are ongoing and thus far we have cultured nine aerobic isolates and have characterized two, one from the genus Promicromonospora (phylum Actinobacteria) and another organism from the genus Bacillus (phylum Firmicutes). Anaerobic cultivation is ongoing and much of our effort is focused on methanogens because prior studies suggest they are present and active in our samples. Through characterizing the living microbes in this environment, we can gain insights into how individual species degrade permafrost organic matter and understand the survival strategies enabling life in permafrost through geologic time.
Funding is from the NASA exobiology program.

Poster #: 153
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: thirdhand smoke, ozone, cytotoxicity
Project Title: Impacts of Atmospheric Oxidation on the Cellular Toxicity of Cigarette Smoke
Author List:
Melton, Tanner; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Yang, Divine ; Graduate, Chemistry, California State University, Fresno, Presenting Author
Verhalen, Robyn; Undergraduate, Biology, California State University, Fresno
Hasson, Alam ; Chemistry, California State University, Fresno
Bush, Jason; Biology, California State University, Fresno

Abstract: Residual tobacco smoke that lingers after active smoking, termed Environmental Tobacco Smoke (ETS) or Third-Hand Smoke (THS), is believed to undergo reactions with atmospheric oxidants and have an increase in cytotoxicity. The scientific literature has demonstrated that compounds present in THS have the ability to be adsorbed on indoor surfaces and remain stationary. This stationary period can lead to either a re-emission of the compounds long after active smoking or heterogeneous reactions with atmospheric oxidants. This study analyzed the change in composition of tobacco smoke after being treated with ozone, nitrous acid, or hydroxyl radicals. The tobacco smoke was collected by smoking two standard Marlboro Red cigarettes in a custom smoking machine that pumped the smoke into a Teflon bag. The mixture was analyzed for Volatile Organic Compounds (VOCs) by Proton Transfer Reaction-Mass Spectrometer (PTRMS) to establish base concentrations of VOCs and the smoke was then treated with one of the atmospheric oxidants or UV light and assessed. There was a statistically significant change (p<0.05) in concentration for 19 of the 24 analyzed. Next, the objective was to evaluate potential cytotoxic effects of smoke residue conditions using these filtered extracts on biologically relevant cell lines, A549 (human lung epithelial carcinoma) and CCL-93 (Chinese hamster lung fibroblast), using a microplate-based colorimetric WST-1 cytotoxicity assay which measures cellular proliferation, viability, and cytotoxicity by the level of mitochondrial dehydrogenase activity. The untreated and ozone-treated smoke extract samples were applied at varying concentrations (2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.01%) and subsequently measured for colorimetric absorbance. This revealed mean IC50 values of 0.23 ± 0.03 mg/mL for CCL-93 cells and 0.08 ± 0.05 mg/mL for the A549 cells when ozone-treated smoke was applied. The IC50 values for untreated tobacco smoke were 0.33 ± 0.06 mg/mL and 0.12 ± 0.02 mg/mL for CCL-93 and A549 cells, respectively. Reactions with ozone therefore changed the chemical composition of tobacco smoke present in THS and resulted in an increase in cytotoxicity of THS. Taken together, these results suggest that the cellular impact of the smoke residue is deleterious, particularly when exposed to ozone.

Poster #: 154
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Drosophila melanogaster, neurodegenerative disease, NMDA
Project Title: Impact of N-Methyl-D-aspartate receptor antagonist MK-801 on the neurotoxic effects of beta-methylamino-L-alanine in Drosophila melanogaster
Author List:
Mueller, Catherine J.; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Goto, Joy J.; Chemistry, California State University, Fresno

Abstract: Beta-methylamino-L-alanine (BMAA) is a non-protein amino acid that may be a causative agent for amyotrophic lateral sclerosis-Parkinson dementia complex (ALS-PDC), a neurogenerative disease characterized by a loss of motor function, tremors, and dementia. Carbamylated BMAA has a similar structure to glutamate, and it therefore may act as an agonist to the glutamate receptor N-Methyl-D-aspartate (NMDA). BMAA can become biomagnified in the food chain from its production by cyanobacteria which are symbionts to the root nodules in cycad plants; the cycad seeds in turn are a source of food for flying foxes (P. mariannus) which are eaten by the indigenous Chamarro people in Guam. This biomagnification of BMAA in the Guam ecosystem may have contributed to the high prevalence of ALS-PDC among Guam inhabitants in the 1950s.
Drosophila melanogaster (fruit fly) is used as the model organism in this project as it is effective in studying human neurodegenerative diseases and modeling basic mechanisms of neurotransmission. MK-801 (dizocilpine), a non-competitive antagonist to the NMDA receptor, may mitigate the neurotoxic effects of the agonist BMAA by preventing its binding to NMDA. Canton S fruit flies were fed control food, 0.1 mg/mL MK-801, 25 mM BMAA, and a mixture of 0.1 mg/mL and 25 mM BMAA, consecutively for four days per each food type, for a total of 16 days. The flies’ locomotor ability was tested using a tap-down method in which the flies are tapped down inside a vial and the number of flies above a 5.5 cm mark after 10 seconds was recorded for each day of feeding.
It was found that on average 90.41%, 55.66%, and 13.18% of MK-801-fed, BMAA-fed, and co-fed (BMAA and MK-801) flies surpassed the 5.5 cm mark, respectively. These results demonstrate the expected progressive decrease in locomotor ability and viability from BMAA ingestion. However, the co-feeding of both MK-801 and BMAA resulted in an even further decrease in locomotor ability and viability as compared to BMAA alone, despite the expected mitigation that MK-801 was thought to have. Further studies will be conducted to observe whether MK-801 pre-feeding prior to BMAA administration may have any therapeutic effect.
Acknowledgements: CSU Fresno Undergraduate Research Grant Award (2017-2018); CSU Fresno Department of Chemistry.

Poster #: 155
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: Metal-organic frameworks, Enzyme encapsulation, Glucose oxidase
Project Title: Synthesis of Metal Organic Frameworks for Enzyme Encapsulation
Author List:
Nelms, Katherine; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Sosa, Joshua; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles
Liu, Yangyang; Chemistry and Biochemistry , California State University, Los Angeles

Abstract: Glucose oxidase (GOx) is an enzyme that catalyzes the oxidation of glucose to gluconic acid and is largely used to detect glucose activity in body fluids, among other biotechnological applications. However, GOx is only stable at low temperatures and select solvents with pH at or near 7.0. The enzyme cannot degrades outside of these conditions. This study presents the encapsulation of GOx in iron-based metal-organic frameworks (MOFs) a proposed method of stabilizing the enzyme in a greater range of conditions. MOFs are high surface area crystalline materials with permanent porosity that are composed of metal ion clusters and organic linkers. The MOF Zr PCN-222 (Fe), which is composed of zirconium clusters and iron-porphyrin ligands, was selected for encapsulation due to its large pore size (3.2 nm) and high thermal and chemical stability. GOx was successfully encapsulated by preparing identical samples of Zr PCN-222 (Fe) suspended in aqueous GOx solution of concentration equal to 0.936mg/mL. This procedure was repeated using a range of concentrations of GOx solution to ensure maximum loading. It was found that enzyme encapsulation is proportional to the concentration of the solution used. Ultraviolet-visible spectroscopy data was taken for each sample after a designated time frame, and the absorption of each sample was used to determine encapsulation percentage and optimum loading time. The resulting spectra showed the maximum loading capacity of GOx within Zr PCN-222 (Fe) to be 3.48% by weight. The time frame needed to achieve maximum GOx encapsulation was determined to be one hour, as the absorption of GOx within Zr PCN-222 (Fe) remains constant beyond a one hour increment. Future research will examine GOx encapsulation using MOFs with pore sizes larger than 3.2nm and measuring the thermodynamic and chemical stability of encapsulated GOx using quantitative measures.

Poster #: 156
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: nanobody, antibody, X-ray crystallography
Project Title: Role of a Non-canonical Interloop Disulfide Bond in the Stability and High-Affinity Binding of Camel VHH to Listeria monocytogenes
Author List:
Mendoza, Matthew; Undergraduate, Chemistry, California State University, Fresno, Presenting Author, Nagel Award Finalist
Toride, Moeko; Graduate, Chemistry, California State University, Fresno
Brooks, Cory; Chemistry, California State University, Fresno

Abstract: Listeria monocytogenes (L. monocytogenes) is a food-borne bacterial pathogen that causes the fatal disease listeriosis. Nearly 1,600 people contract listeriosis annually, with over 260 fatalities. Host cell invasion by L. monocytogenes is mediated by a virulence factor called Internalin B (InlB). Disruption of the interaction of InlB with host cell receptors represents a potential site for therapeutic intervention. A single domain antibody (VHH) called R303 is derived from the unusual heavy-chain antibodies of camels R303, and binds InlB with high affinity. The VHH also neutralizes L. monocytogenes and represents a potential Listeria therapeutic. R303 possesses three complementarity-determining regions (CDRs) that function in antigen binding. The CDR3 of R303 is characteristically longer than the other CDRs and contains a non-canonical interloop disulfide bond between CDR1 and CDR3 which may function to stabilize the binding interaction with InlB. To examine the role of this disulfide bond in InlB binding, the interloop disulfide bond was removed from R303 via site-directed mutagenesis. Indirect ELISA was performed to analyze the binding affinity of cys-mutant R303 for InlB. The binding affinities of wild-type and cys-mutant R303 were determined to be nearly identical. Furthermore, fluorescence microscopy demonstrated that cys-mutant R303 neutralizes L. monocytogenes invasion in vitro. To assess the role of the interloop disulfide bond on thermal stability of R303, circular dichroism spectroscopy was performed. The melting temperatures of cys-mutant R303 and wild-type R303 were determined to be 65oC and 75oC, respectively. X-ray crystallography was performed to analyze the structural intricacies of CDR1 and CDR3 in cys-mutant R303. The crystal structure and flexibility parameter analysis of cys-mutant R303 reveals that CDR3 is rigidified despite the absence of the interloop disulfide bond. This study provides evidence that the interloop disulfide bond is not critical to high-affinity binding of R303 for InlB, however, the interloop disulfide bond is significant to the thermal stability of R303. The project is funded by the National Institutes of Health (grant #: 1SC3GM112532-03).

Poster #: 157
Campus: Humboldt State University
Poster Category: Biochemistry
Keywords: Rickettisa, folate, HPLC-UV
Project Title: HPLC-UV quantitation of folate synthesized by Rickettsia endosymbiont Ixodes pacificus
Author List:
Chen, Junyan; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Topete, Joshua; Graduate, Chemistry, San José State University
Watanabe, Ichi; Graduate, Chemistry, San José State University
Pesek, Joseph; Chemistry, San José State University
Matyska-Pesek, Maria; Chemistry, San José State University
Zhong, Jianmin; Biological Sciences, Humboldt State University, Presenting Author

Abstract: Ixodes pacificus, also known as the western black-legged tick, is a vector of Lyme borreliosis and anaplasmosis. The tick is widely inhibited on the west coast and is also known as a host for Rickettsia species. Previously our lab has demonstrated the existence of all folate genes in the genome of Rickettsia endosymbiont Ixodes pacificus (REIP), a bacterial isolated from partially engorged I. pacificus. However, the nature of the symbiotic relationship between REIP and I. pacificus is unknown. In this study, we measured the folate concentration in REIP-infected ISE6, which is a tick cell line. By HPLC-UV, we successfully separated folic acid, 5-formyltetrahydrofolayte (5-THF) and 5-methyltetrahydrofolate (5-MTHF) standards by aqueous normal phase using Diamond HydrideTM liquid chromatography column. We also measured the levels of folate in culture medium, uninfected ISE6 cells, and REIP-infected ISE6 cells. We found that the level of the folate concentration is higher in REIP-infected ISE6 cells than that in uninfected ISE6 cells. In addition, we found that the level of folate concentration is higher when bacteria were starved for two hours in folate-free medium, compared with the non-starved control. The findings of this study give insights on the nutritional interactions between REIP and its tick hosts.

Poster #: 158
Campus: CSU Bakersfield
Poster Category: Biochemistry
Keywords: Coffee, Cancer, Phenolic acids
Project Title: The Impact of Roasting Level on the Phenolic Acid Profile of Coffee
Author List:
Biju, Denny; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author
Rios, Diana; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Mojica, Benigno; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Vela, Klarisse; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Davis, Isabel; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield
Forester, Sarah; Chemistry and Biochemistry, California State University, Bakersfield

Abstract: According to the American Cancer Society, more than 59,000 men and women are diagnosed with oral cancer, and part of the treatment includes consuming a healthy diet rich in phenolic compounds. Phenolic acids are a class of phenolic compounds that are produced by plants in response to ultraviolet light, pests and disease. Chlorogenic, caffeic, and gallic acids are particularly abundant in coffee, a widely consumed beverage. Consumption of these coffee-derived components could have many beneficial health effects including prevention of oral cancer. The concentration of these phenolic compounds are dependent on roast levels of coffee. In this study, the anticancer activities of various freeze dried coffee beverages were compared, differing only in their roast treatments. The different roasting stages selected for this study were “Green”, “Cinnamon”/Blonde, “City”/Medium, “Full City”/Medium-Dark, and “Full City Plus”/Dark. The freeze-dried coffee samples were re-suspended in water and methanol and levels of phenolic acids were determined by high-performance liquid chromatography (HPLC). The total phenolic content and total antioxidant activity of each extract was also determined by the Folin-Ciocalteu and ORAC (oxygen radical absorbance capacity) assays, respectively. Data collected from the Folin-Ciocalteu method showed that the “Cinnamon”/Blonde roasted coffee contains the greatest concentration of phenolic compounds. Observed data, through HPLC, displayed high peak areas at 280 nanometers (mAUs) for caffeic acid and gallic acid in the “Cinnamon”/Blonde roasted coffee and high peaks of chlorogenic acid were observed in the “Green” roasted coffee. The concentrations of caffeic acid and gallic acid in the “Cinnamon”/Blonde roasted coffee were calculated to be 734.3 µmol/g of extract and 2.5 µmol/g of extract. A concentration of chlorogenic acid, 8,285.7 µmol/g of extract, was calculated from the high peaks observed in the “Green” roasted coffee. The “Cinnamon”/Blonde also showed the most amount of total antioxidant activity compared to the other extracts according to the data collected from the ORAC assay. In conclusion, the consumption of lighter roasted coffee (“Cinnamon”/Blonde), along with a healthy and balanced diet, may contribute to the prevention of oral cancer.

Poster #: 159
Campus: San José State University
Poster Category: Biochemistry
Keywords: chemoenzymatic, light-driven process, directed evolution
Project Title: LIght-driven Chemoenzymatic oxytrifluoromethylation towards the production of valuable synthons
Author List:
Leong, Kasey; Undergraduate, Chemistry, San José State University, Presenting Author
Toledo, Alejandra; Undergraduate, Chemistry, San José State University, Presenting Author
Sulca, Carolina; Undergraduate, Chemistry, San José State University
Melkie, Marya; Graduate, Chemistry, San José State University
Foley, Bridget; Undergraduate, Chemistry, San José State University
Kato, Mallory; Chemistry, San José State University
Cheruzel, Lionel; Chemistry, San José State University

Abstract: Chemoenzymatic approaches offers the advantage of combining the potential of chemocatalysis with the selectivity and evolvability of biocatalysis. Photoredox catalysis has emerged as a new and innovative approach for the activation of small molecules capitalizing on the photochemical properties of the Ru(II)-diimine photosensitizer to initiate radical processes upon visible light excitation. Our laboratory has developed hybrid P450 enzymes where a covalently attached Ru(II)-diimine photosensitizer is able to deliver the electrons required to harness the enzyme synthetic potential upon visible light excitation.
Recently we have been interested in merging the synthetic potential of photoredox catalysis with the light-driven enzymatic approach to yield, in two light-driven steps, valuable synthons. Both rational design and directed evolution approaches were used to enhance the photocatalytic properties of the light-driven P450 enzymes. Using a recently established colorimetric assay compatible with our light-driven system as well as HPLC methodology, a set of hybrid enzymes suitable for functionalizing small aromatic compounds were identified. A series of substituted arenes were then trifluoromethylated and subsequently submitted to the hybrid enzyme reaction conditions to generate selectively oxytrifluoromethylated compounds. The products were thoroughly characterized by a wide range of techniques including HPLC and GC/MS as well as 1H, 13C, and 19F NMR. We have thus developed a chemoenzymatic approach to perform the selective oxyfunctionalization of substituted arenes upon visible light excitation.

Poster #: 160
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Methylobacterium extorquens, tetrahydromethanopterin biosynthesis, crystallography
Project Title: Purification and Pre-crystallization of a Dihydromethanopterin Reductase Vital in Tetrahydromethanopterin Biosynthesis in Methylobacterium extorquens
Author List:
Ortega, Randall ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Nepomuceno, Ahide; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Rasche, Madeline; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Methane is a potent greenhouse gas in the Earth’s atmosphere. It is produced by archaeal microorganisms (methanogens), commonly found in the digestive tracks of both humans and animals. In contrast, methylotrophic bacteria utilize one-carbon compounds like methane or methanol as the primary energy source. The one-carbon metabolism of methane-producing archaea and methylotrophic bacteria requires the cofactor tetrahydromethanopterin (H4MPT), an uncommon tetrahydrofolate analog. In archaea, the final step in H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT by dihydromethanopterin reductase X (DmrX). However, no DmrX homolog exists in the genome of the bacterium Methylobacterium extorquens AM1. Instead, the final reaction is catalyzed by dihydromethanopterin reducatase A (DmrA), a homolog of bacterial dihydrofolate reductase (DHFR). This finding has led to the hypothesis that DmrA evolved from duplication and mutation of an ancestral dhfr gene. To gain insight into evolutionary changes that may have altered the substrate specificity from dihydrofolate to H2MPT, computational modeling and crystallography studies were initiated. Using DHFR as a template, a Molsoft modeling program predicted the overall protein fold of DmrA and the NADPH binding site, but was unsuccessful in modeling a H2MPT binding site. In preparation for crystallography, a strategy was developed to increase the solubility and decrease the aggregation of recombinant DmrA. Shortening the N-terminal histidine tag from six to four histidines and storing the cells at pH 8 instead of pH 6.8 allowed purification of a single quaternary structure. After nickel affinity chromatography and desalting, a protein concentration of 4.1 µg/µL was obtained. SDS-PAGE showed greater than 98% purity with a main band at 17 kDa. A single band at ~400 kDa was obtained by native PAGE, consistent with a quaternary structure of 24 subunits. A pre-crystallization trial resulted in successful light precipitation using a Tris hydrochloride and ammonium sulfate buffer at a 2:1 ratio of sample:buffer. However, a crystallization screen with 288 conditions resulted in no precipitate or crystals, indicating that a higher protein concentration might be required for crystallography. Future research will focus on identifying protein concentration, salt, and buffer conditions suitable for DmrA crystallization and structure determination. (Funding provided by National Science Foundation and CSUPERB.)

Poster #: 161
Campus: Cal Poly San Luis Obispo
Poster Category: Biochemistry
Keywords: Kinase, Expanded genetic code, Structure-function relationships
Project Title: Integration of Biotechnologies for the Study of Phosphorylated Human Protein MEK1
Author List:
Ravatt, Leandre; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author, Nagel Award Nominee
Lee, Rebecca; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Runco, Caroline; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Gallant, Allison; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Oza, Javin; Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo

Abstract: The MAPK phosphorylation cascade is one of the most common cell proliferation signaling pathways. 90% of cancerous melanomas contain mutations in this pathway. The interactions between MEK1 and its substrate ERK2 causes a bottleneck in the cascade, through which all signals must pass, making MEK1 an ideal drug target. However, MEK1 is activated upon phosphorylation in the cell, a process which is difficult to replicate using traditional protein expression methods, limiting access to homogenous, active MEK1 samples for biochemical characterization. This technical limitation creates an obstacle in our ability to study the active form of MEK1 and to establish kinetic parameters for it. We aim to overcome this limitation by utilizing an expanded genetic code to site-specifically install phosphoserine into MEK1 for biochemical characterization. This technology leverages an orthogonal translation system to install phosphoserine in response to the Amber codon (UAG). We have combined this biological machinery with traditional in vivo protein synthesis to produce active, phosphorylated MEK1. Here we report our progress. To elucidate the structure-function relationships of MEK1, we are pursuing a hypothesis-driven, structure-guided approach based on conserved kinase motifs. Specifically, we have identified 16 residues in the catalytic and P+1 loops of MEK1 which we hypothesize are involved in ERK2 binding and phosphorylation. To date, we have successfully completed the mutation of 10 resides to alanine using site-specific mutagenesis. The activity of these mutants will further the understanding of MEK1’s structure-function relationships. We have also expressed, purified, and concentrated stocks of unphosphorylated, inactive MEK1 and its substrate ERK2. Using these stocks, we have established a high-throughput 96-well dot blot assay for the evaluation of MEK1’s kinetic parameters. Our data suggests that this platform is viable for the quantification of phosphorylated ERK2 in a given sample, which provides information on MEK1 activity. Going forward, our work will be the first to establish kinetic parameters for the phosphorylated, active form of human MEK1 using this validated immunoassay. We will subsequently characterize the kinetics of MEK1 mutants to identify residues vital to ERK2 binding and phosphorylation. Our research was funded by the Center for Applications in Biotechnology and the Bill and Linda Frost Fund.

Poster #: 162
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: Metal-organic frameworks, drug delivery, pH-responsive
Project Title: PEGylation of Metal-Organic Frameworks as Drug Carriers
Author List:
Liu, Brandon; 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 a class of hybrid organic-inorganic materials with high stability and internal surface area. MOFs are synthesized by the self-assembly of metal ion clusters and organic bridging ligands in solvothermal conditions. The abundant surface area facilitates chemical adsorption by intermolecular forces, and this has demonstrated applications in drug delivery, chemical catalysis, and compact gas storage, among other fields.
MOFs have demonstrated potential as drug delivery vehicles, overcoming challenges in controlled drug release, loading capacity, and toxicity due to functionalizable surfaces and tunable syntheses. Research on the surface modifications of MOFs for controlled drug release is underdeveloped. Thus, we aim to study the effects of surface adhesion of the acid-sensitive polymer poly(ethylene glycol) (PEG), known as PEGylation, in pH-responsive drug release. PEG introduces enhanced therapeutic properties to drug nanocarriers including reduced immune system recognition and controlled drug release. Due to the acidic nature of cancer cells, pH-responsive drug carriers promote controlled cancer drug release in intravenous administration.
We report the procedures and drug encapsulation properties of the modified drug carrier MOFs, correlating the effects of MOF structure and polymer length to drug encapsulation and pH-responsive drug release. We have synthesized and characterized one drug carrier MOF of interest, the channel MOF NU-1000, which harnesses large pores for larger drug loading. The organic ligand synthesis was monitored by nuclear magnetic resonance spectroscopy and product MOF crystallinity was confirmed by powder X-ray diffraction. Additionally, a partition of our NU-1000 sample has been surface-modified by solvent-assisted ligand incorporation of 4-(azidomethyl)benzoic acid, which contains an azide reactive in “click chemistry” with alkynyl-functionalized PEG derivatives. Future work encompasses the synthesis and PEGylation of alkynyl-functionalized PEG derivatives and the investigation of drug encapsulation and pH-responsive drug release kinetics of various effective cancer drugs in vitro. This research will be extended to similar drug delivery MOFs to compare the effects of PEGylation on MOF pore size and structure.
Overall, these studies aim to improve drug carrier efficiency in the delivery of therapeutic drugs and ultimately improve medicine through the advancement of drug carrier development.

Poster #: 163
Campus: CSU San Marcos
Poster Category: Biochemistry
Keywords: Chemical Biology, Fluorescence, Protein Labeling
Project Title: Ligation Independent Cloning and Purification of Peptide Deformylase for Single Molecule Fluorescence Resonance Energy Transfer Experiments
Author List:
Hite, Nicholas; Undergraduate, Chemistry and Biochemistry, California State University San Marcos, Presenting Author
Hamadani, Kambiz; Chemistry and Biochemistry, California State University San Marcos

Abstract: Recently, our lab demonstrated proof-of-principle for the ability to monitor nascent protein structure on the ribosome by single molecule Fluorescence Resonance Energy Transfer (smFRET). In order to report on nascent chains conformation this method requires the attachment of two different dyes, an acceptor and a donor, at two sites in the nascent chain. To achieve dual-labeling, we previously used a residue-specific tag-and-modify approach, in which homopropargyl glycine (HPG)–an alkyne-bearing analogue of methionine—replaced methionine and was charged onto methionyl-tRNA and introduced using a purified and reconstituted in-vitro translation system at pairs of sites in a nascent protein chain. After stalling the nascent protein chain, we then installed donor and acceptor azido-dyes at these sites via copper-catalyzed azide-alkyne cycloaddition (CuAAC). However, this AUG-codon reassignment approach limits the scope of smFRET measurements to constructs with one dye always positioned at the N-terminus of the nascent chain. In our present work, in order to enable more flexible labeling at pairs of internal methionine residues, we use the proteins peptide deformylase (PDF) and methionine aminopeptidase (MAP) to remove the N-terminal formyl-HPG from the nascent chain. Cloning of PDF into a His-tagged expression vector was accomplished using a Ligation Independent Cloning strategy. PDF was also purified using Ni-NTA technology with good yield. MAP is currently being cloned and expressed for future N-terminal nascent chains processing and smFRET analyses.

Poster #: 164
Campus: San José State University
Poster Category: Biochemistry
Keywords: intracellular pH, Drosophila, cancer
Project Title: Roles for pHi dynamics in cell cycle regulation
Author List:
Chire, Ismahan; Graduate, Biological Sciences, San José State University, Presenting Author
Virk, Harnoor; Undergraduate, Biological Sciences, San José State University, Presenting Author
Grillo-Hill, Bree; Biological Sciences, San José State University

Abstract: Intracellular pH (pHi) is tightly regulated by cells, and emerging evidence suggests that regulated pHi dynamics modulate distinct cell behaviors, including regulated proliferation, metabolic pathway activity and cell migration. In pathological conditions with dysregulated pHi, these normal cell behaviors are aberrantly regulated. For example, increased pHi is recognized as a conserved characteristic of cancers, and is thought to facilitate increased proliferation, metabolic adaptation, and metastatic invasion. We previously developed methods to increase pHi in developing Drosophila tissues by overexpression of the sodium-proton exchanger DNhe2. Using these methods, we show that increased pHi increases proliferation and causes tissue overgrowth in both eye and wing imaginal discs in Drosophila. To better understand how increased pHi promotes proliferation, we have performed preliminary and unpublished genetic screens to identify candidate pH-regulated proteins. We identified several pathways and genes with known roles in growth control: the oncogene Myc; cyclins A, B and D; EGF pathway components; and Wnt/Beta-catenin pathway components. Our immediate plans will focus on interactions with direct cell cycle regulators (cyclins and their regulatory kinases). Identifying proteins that directly respond to pHi dynamics and regulate proliferation will lend insight to both normal cell cycle progression and cell cycle dysregulation in cancer.

Poster #: 165
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Drosophila melanogaster, amino acids, neurodegenerative disease
Project Title: How Nutrition Affects the Locomotor Ability of a Fruit Fly Model of Lou Gehrig’s, Parkinson’s and Alzheimer’s Disease
Author List:
Singh, Harmala; Undergraduate, Biology, California State University, Fresno, Presenting Author
Goto, Joy J.; Chemistry, California State University, Fresno

Abstract: The neurotoxin β-N-oxalyl-amino-L-alanine (BOAA) is linked with the neurodegenerative disease, neurolathyrism, which leads to paralysis in the lower limbs. This project investigated how a nutrient-deprived diet alters the locomotor ability of an organism. Drosophila melanogaster, a model organism for studying human neurodegenerative disease, was used to measure these debilitating locomotor effects.
The specialized diet was administered to the flies using standard food pellets. Four different parameters were used to observe any locomotor alterations: a control diet, a control diet with BOAA, a drop-out diet, and a drop-out diet with BOAA. The control media includes yeast, which contains all of the 21 amino acids. On the contrary, the “drop-out media” for the fruit flies’ diets excluded the thiol-containing amino acids cysteine and methionine. BOAA lacks sulfur containing amino acids, so we wanted to see if removing these amino acids imposes a more drastic result in the flies’ locomotor abilities, which is analogous to the deleterious effects of BOAA.
We used the negative geotaxis assay, the tap-down test, which encompasses the natural tendency of flies to move against gravity when tapped to the bottom of a vial. Observations were recorded for three consecutive days, approximately 24 hours from the first food pellet administration. Within a given time period, a success rate of the flies climbing up to the 5.5 centimeter mark was noted to assess the locomotor abilities. On average, approximately 72% of control fed flies, 84% of BOAA-fed flies, 70% of drop-out media fed flies, and 80% of drop-out + BOAA fed flies had the capacity to climb to the top of the chamber, after three successive days. Our results demonstrate that a diet deficient in thiol amino acids with BOAA did not significantly decrease the locomotor activity in comparison to control-fed flies. These results demonstrate the importance of amino acids in the viability and locomotor ability of fruit flies. Prior studies, showed that a cysteine-amino acid deficient diet enhanced the BOAA-related locomotor deficiencies. We are investigating the role of how exactly BOAA and these thiols containing amino acids interact to produce the loss of motor ability, which may have implications for proteinogenic diet and viability.
Acknowledgements: This study was funded through the Fresno State Chemistry Department and Associated Students Inc. Undergraduate Research Grant.

Poster #: 166
Campus: Cal Poly Pomona
Poster Category: Biochemistry
Keywords: bioinorganic, spectroscopy, computations
Project Title: Spectroscopic and computational studies of metal nitrosyl complexes to elucidate the mechanism of nitric oxide reductase
Author List:
Phu, Phan N.; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author, Nagel Award Finalist
Stieber, S. Chantal E.; Chemistry & Biochemistry, California State Polytechnic University, Pomona

Abstract: Nitrous oxide (N2O) is the third most potent greenhouse gas with a global warming potential 310 times higher than carbon dioxide. Reducing the concentration of N2O is chemically challenging, however biological systems can reduce N2O through chemical reactions at copper centers within enzymes. Understanding the mechanism of N2O reduction would benefit pollutant control and have possible environmental significance. Different binding modes of NO at metal centers that are proposed as intermediates in the conversion of N2O to NO were investigated through X-ray emission spectroscopy (XES) and calculations. XES probes transitions from filled valence orbitals and can be used to inform ligand identity, metal ligand bonding, and metal spin state. While XES is a sensitive probe for small-molecule activation and coordination modes, the method is still being developed. We have used XES in combination with computational chemistry to understand binding modes between NO and nickel. Our results demonstrate that various binding modes of nitrosyl at nickel can be experimentally distinguished by XES. This highlights the sensitivity of XES for determining small molecule coordination to metal centers. To gain further insight into the electronic structures of the nickel nitrosyl complexes, density functional theory (DFT) calculations were carried out. The DFT-calculated XES spectra have good agreement with experiment and reveal metal-ligand interactions that contribute to resulting spectral features. Notably, the calculated XES spectra suggest that XES is sensitive to Ni-N-O bond angle changes of 10 degrees. The good correlation between experiment and theory suggests that XES can characterize small molecule intermediates in biological systems. Applying our XES calculations to proposed intermediates in the copper-containing nitrous oxide reductase (N2OR) supports that XES could experimentally distinguish key intermediates in proposed mechanisms that are currently highly controversial. The XES development in this work offers new experimental and computational techniques for characterizing complex systems, understanding mechanisms of N2O reduction, and detecting NO in biological settings.

Poster #: 167
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: Cholinesterases, Enzyme inhibition, Alzheimer’s disease
Project Title: Examining Fmoc-amino acid derivatives as selective inhibitors of butyrylcholinesterase
Author List:
Ramirez, Jennifer; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Finalist
Gonzalez, Jeannette; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Schwans, Jason; Chemistry and Biochemistry, California State University, Long Beach

Abstract: The design of enzyme inhibitors to act as drugs or as substrates for biocatalysts remains a challenge in biochemistry, pharmacology, and biotechnology. To mitigate the effects of Alzheimer’s disease (AD), a neurodegenerative disease characterized by progressive memory loss and the sixth leading cause of death in the United States, numerous enzyme inhibitors have been developed. Activity of the enzyme butyrylcholinesterase (BChE) increases in Alzheimer’s patients, and this increase is suggested to lead to a depletion of the neurotransmitter, acetylcholine. To counter this increase in activity, BChE-specific inhibitors have been developed to manage the progression of AD. We hypothesized 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could provide a new class of inhibitors, as they contain features found in BChE inhibitors currently available as pharmaceuticals. The readily available chemistry makes amino acids an attractive scaffold for identifying BChE inhibitors. We found Fmoc-Leu-O- as an effective and selective BChE inhibitor with an inhibition constant (KI) value of 115 µM. A series of Fmoc-Leu-O- derivatives with side chain modifications were then evaluated. The Fmoc-amino acids were purchased or synthesized from the corresponding amino acid. The KI value for each compound was determined using kinetics assays and UV-absorbance spectroscopy. For the four Fmoc-Leu-O- derivatives tested, Fmoc-neopentylglycine-O- showed the greatest potency with an inhibition constant of 36 µM, ~three-fold lower than the original compounds. A series of Fmoc-di-, tri-, tetra-, and pentapeptides were synthesized by solid phase peptide synthesis. The products were purified by HPLC and characterized by NMR and mass spectrometry. Our initial results identified the dipeptide (Fmoc-Leu-Leu-O-) as an effective inhibitor with KI value of 28 µM. We are currently investigating if longer Fmoc-Leu-O- peptides and other Fmoc-peptides are potent and selective BChE inhibitors. The results explore a new class of potent and selective BChE inhibitors and may identify features important for binding in the enzyme active site.
This project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) under award numbers T34GM008074, UL1GM118979, and RL5GM118978, and the National Science Foundation (NSF; MRI CHE-1337559). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.

Poster #: 168
Campus: San Francisco State University
Poster Category: Biochemistry
Keywords: Cardiogenesis, Nitric Oxide, Chick Embryo
Project Title: Nitric Oxide Focally Elevated in Endoderm at the Heart Forming Regions (HFRs) Signals Cardiogenesis in Chicken Embryos
Author List:
Shah, Devan; Graduate, Cell and Molecular Biology, San Francisco State University, Presenting Author
Denetclaw, Wilfred; Cell and Molecular Biology, San Francisco State University

Abstract: Nitric oxide synthases (eNOS, nNOS, iNOS isoforms) in chick produce the nitric oxide (NO) messenger gas molecule. NO signals primarily through a canonical NO/sGC/cGMP pathway in the regulation of myogenesis, neurogenesis, gastrulation, and of other embryo development events. The heart is the first organ to form in higher vertebrates and NO signals in cardiogenesis. However NO signaling has not been well-studied in the cardiogenesis event in early chicken embryo development where the Heart Forming Regions (HFRs), populations of mesodermal cardiac progenitors in which cardiogenic specification and determination occur, migrate during gastrulation to form the heart tube. To investigate NO signaling for spatial-temporal dynamic changes, gastrula stage chick embryos (HH3 – H5) were isolated to albumin-agar plates, labeled by 5 M DAF-2DA fluorescent NO indicator, and embedded in agarose for imaging by confocal microscopy (n=19). In HH3 embryos, NO signaling was found to be elevated in the endoderm and cardiogenic mesoderm of the anterior primitive streak, over lapping with the HFRs (n=4). In HH4 and HH4+ embryos, the HFRs migrate laterally and anteriorly to form bilateral fields, where endoderm NO paracrine signaling to subjacent cardiac mesoderm was shown to be focal at left and right HFRs over a diameter of 200 ± 20 µm (n=6). However, an elevated NO presence was not observed in the ectoderm layer dorsal to the HFRs. By HH5, cardiac progenitors migrate cranial-ward and fuse at the embryo midline, in the cardiac crescent, to begin heart formation. Endoderm NO signal was elevated throughout the HFRs and included cardiac mesoderm along the length of the cardiac crescent where a strong NO fluorescence signal was shown in cardiac cells at the heart fusion zone at the embryo midline. (n=3) On HH 4-5 embryo treatments with nitric oxide synthase inhibitor, 10 mM L-NAME, embryos developed to heart tube formation (HH 9 -10) stages and had 20 ± 5% smaller heart tubes than comparable early hearts from control embryos (n=4). Furthermore, the L-NAME treated embryos showed noticeably reduced myosin (n=4). qPCR analysis of HH9-10 whole embryo RNA isolate is now in progress to investigate NO signaling to regulate cardiac transcription factors functional in cardiogenesis. We conclude that the endoderm signals to the cardiogenic mesoderm through paracrine NO signaling in the HFRs, suggesting a role for NO in coordinating cardiogenesis in heart tube formation. NSF-STC-CCC-1548297

Poster #: 169
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Yeast, Peroxisome, Rab GTPase
Project Title: A membrane-anchored Rab GAP (GTPase activating protein) localizes to peroxisomes in S. cerevisiae
Author List:
Gutierrez, Miguel; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Quinn, Monique; Graduate, Biology, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Biology, California State University, San Bernardino

Abstract: Rab GTPases (Rabs) are lipid-anchored, small G-proteins that cycle between signaling active and inactive states based upon their association with GTP (active) or GDP (inactive). Rabs use effector proteins such as tethers to control docking and fusion of vesicles and membrane-bound organelles. Most Rabs are quite slow in hydrolyzing bound GTP to form GDP, and therefore Rabs rely upon GAPs (GTPase accelerating proteins) in order to control the time and place where Rab signals terminate. Rab GAPs across most eukaryotes tend to be soluble, cytosolic proteins that localize transiently to their membranes of activity, but a small number are known to possess transmembrane anchors. Of the nine known Rab GAPs in the yeast S. cerevisiae, all are soluble proteins except Gyp8, whose cellular function remains poorly understood. Computational analysis of Gyp8 peptide sequence predicts a single-pass transmembrane domain near the carboxy terminus of Gyp8. Observed by fluorescence microscopy, GFP-tagged Gyp8 co-localizes with markers of peroxisomes, which are organelles that function in breaking down long chain fatty acids. The carboxy terminus, including the transmembrane domain, is both necessary and sufficient for peroxisomal localization. To our knowledge, this is the first report of a Rab GAP associated with peroxisomes in any eukarotic system. In mutant yeast cells lacking peroxisomes, GFP-tagged Gyp8 no longer localizes to cortical punctae, instead localizing throughout the endoplasmic reticulum (ER). Ongoing studies are investigating whether Gyp8 regulates peroxisome function, dynamics or transport, such as vesicular transport from the ER to peroxisomes, peroxisomal biogenesis at the ER, and/or pexophagy.

Poster #: 170
Campus: CSU Los Angeles
Poster Category: Biochemistry
Keywords: metal-organic frameworks, enzyme encapsulation, glucose
Project Title: The Application of Iron(Fe) Based MOFs in Enzyme Encapsulation
Author List:
Papazyan, Edgar; 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) consist of organic ligands and metal clusters that are used to create highly porous three-dimensional structures. In this project, MOFs are used to encapsulate an enzyme, glucose oxidase (GOx). By being encapsulated into a MOF, the enzyme can stabilize in conditions outside of its strict requirements (when it is not encapsulated). We found that Fe based MOFs were the best for the encapsulation of GOx due to their exceptional stability and their activities that mimic peroxidase. PCN-222(Fe) was used to encapsulate GOx, using PCN-222(Free Base) as a control. The following method of encapsulation were used: placing the enzyme in solution with MOF and adding light agitation to the system in order to get the MOF to encapsulate. Since we cannot measure the concentration of the GOx enzyme inside the MOF directly, the concentration of GOx in the supernatant was measured using a UV-vis scan and the difference in the concentration was measured to calculate how much of the GOx enzyme was encapsulated into the MOF. These values were also tested for samples with varied agitation time, and we found approximately 1 hour was the best time period for maximum encapsulation. Some other values we calculated (for a certain time t) was peak wavelength, peak absorption, concentration of GOx, mg GOx encapsulated per mg MOF, weight percent of GOx encapsulated, and mol GOx encapsulated per g of MOF. Our results showed that PCN-222(Fe) was not practically able to encapsulate our target amount of GOx, so the future of this project lies in performing this experiment with a different iron based MOF. We will choose a MOF with bigger pore size than PCN-222(Fe)(3.7nm). The MOF that will be used next for encapsulation is PCN-333(Fe)(5.5nm) and isostructural PCN-333(Al) will be used as a control. The Al based MOF is used as a control because only Fe based MOFs exhibit peroxidase activity, which is necessary to induce a color change in the glucose detection. Through this experiment, we demonstrated that it is possible to use MOFs to encapsulate enzymes in order to help it survive in conditions it would otherwise be deactivated.

Poster #: 171
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Ambient Particulate Matter, Alveolar Macrophages, Reactive Oxygen Species
Project Title: PM2.5 Exposure and ROS production in NR8383 Rat Alveolar Macrophages
Author List:
Olivas, Micah; Undergraduate, Biology, California State University, Fresno, Presenting Author
Waterston, Anthony; Graduate, Biology, California State University, Fresno
Castillo, Joel; Undergraduate, Chemistry, California State University, Fresno
Hasson, Alam; Chemistry, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno

Abstract: Previous studies have established a clear link between adverse health effects and exposure to aerosols consisting of particulate matter 2.5 micrometers in diameter or less (PM2.5). The small size of these particles allows them to reach the distal ends of the respiratory tract, where they induce an inflammatory response mediated by a high production of reactive oxygen species (ROS) in alveolar macrophages (AM). Redox-active components of urban PM2.5, such as copper and quinones, are key suspects in PM2.5 toxicity, because these chemicals can generate ROS on the particles and inside a cellular context. However, the relative contribution of the above components to the AM global ROS response remains to be determined. Our group has collected a number of ambient PM2.5 samples during the winter months of 2013 and summer months of 2012 in Fresno and Claremont, which have historically coincided with peak periods of air pollution in each respective city. These samples were tested for their potential to induce a ROS response in the rat NR8383 AM cell line using the intracellular ROS-sensitive fluorescent probe 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA). 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 depending on the sample origin; strongly suggesting that the chemical composition of PM2.5 was a major determinant of their effect on AM ROS metabolism. These same PM2.5 samples were also characterized for their chemical content using chromatography and mass spectrometry based techniques. Two types of quinones (1,2-Naphtaquinone, and 1,4-Naphtaquinone) and one transition metal (Cu2+) were associated to samples with high capacity to induce ROS production in NR8383 AM cells. We are currently estimating the potential of each of these above components to induce a cell ROS response separately using mock samples containing concentration ranges similar to that found in the different PM2.5 samples.

Poster #: 172
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Cancer, Signal transduction, β-catenin
Project Title: Structure-based Discovery of Novel Small Molecule Wnt/β-catenin Signaling Inhibitors
Author List:
Guzman, Stacy; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Nagel Award Finalist
Nguyen, Romie; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Adkins, Lauren; Graduate, Biological Science, California State University, Fullerton
Patel, Nilay; Biological Science, California State University, Fullerton
de Lijser, Peter; Chemistry and Biochemistry, California State University, Fullerton

Abstract: The canonical Wnt signaling pathway is an essential signal transduction pathway which leads to the regulation of cellular processes such as differentiation and migration. Deregulation of components involved in Wnt/β-catenin signaling has been implicated in a wide spectrum of diseases including several cancers and degenerative diseases. Recent data suggests pre-cancerous cells overexpress the protein β-catenin when they become cancerous cells by Wnt stabilization and nuclear localization, ultimately activating the expression of β-catenin mediated genes downstream. However, in the absence of Wnt ligand stimulation, several proteins participate in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis, resulting in the inhibition of downstream gene expression. To inhibit β-catenin accumulation, we designed and synthesized a library of novel small molecules that can inhibit Wnt signaling by blocking protein–protein interactions or the activity of specific enzymes. We hypothesize that small molecules that reduced β-catenin levels can attenuate cell proliferation in cervical cancer cells (HeLa). To test this hypothesis, various small molecules with modified structures were synthesized and purified. To assess and quantify cell proliferation, the fluorescence-based CyQUANT assay was used to measure DNA content. HeLa cell lines were maintained for 24 hours containing 10 uM of each respective drug. Total β-catenin levels that are associated with raised nuclear and cytosolic pools were determined by immunocytochemistry (ICC). Our SAR studies indicated that: (1) inhibition of Wnt signaling by our small molecules appeared unique among structurally-related anthelmintic agents used commercially; (2) Wnt/β-catenin functional response was dependent on small changes in the chemical structure of each compound; and (3) long alkyl chain substituents with a polar end have consistently shown better results than those with a smaller alkyl chain. Our findings suggest that this series of small molecules presents a novel approach for treatment of Wnt-dependent cancers. Acknowledgements: HHMI, McNair, LSAMP, CSUF Chemistry & Biochemistry Undergraduate Research Award.

Poster #: 173
Campus: CSU Fresno
Poster Category: Biochemistry
Keywords: Caenorhabditis briggsae, Mitochondria, Speciation
Project Title: Altered Mitochondrial Function in Dysfunctional Hybrid Nematodes
Author List:
Johnson, Emma; Graduate, Biology, California State University, Fresno, Presenting Author
Rawson, Marissa; Undergraduate, Biology, California State University, Fresno, Presenting Author
Ross, Joseph; Biology, California State University, Fresno
Dejean, Laurent; Chemistry, California State University, Fresno

Abstract: Previous data suggest that Caenorhabditis briggsae experimental inter-population hybrid lines, in which the nuclear genome from one population is combined with the mitochondrial genome from the other, exhibit reduced organismal fitness. Phenotypes include reduced fecundity, increased reactive oxygen species (ROS) production, and increased lipid content. All of these changes could be related to mitochondrial-nuclear genetic incompatibilies that produce hybrid mitochondrial dysfunction. Mitochondria utilize the electron transport chain (ETC) to convert nutrients into fuel the body can use. The complexes of the ETC, with the exception of complex II, are encoded by both the mitochondrial and nuclear genomes. No connection has yet been made between the genetic basis of this dysfunction and the hybrid biochemical and organismal phenotypes. We hypothesize that reduced hybrid fitness is due to a reduction in ETC efficiency. To test this hypothesis, two mitochondrial enzyme assays were conducted to compare ETC function in four dysfunctional hybrid lines relative to two control lines. First, the enzyme catalase helps to protect cells from oxidative damage by ROS. A significant increase in catalase activity in replicate hybrid lines relative to the control line was observed. We interpret this difference as evidence of compensation for an increase in hybrid ROS levels. Second, a significant difference between the same lines in citrate synthase activity was also observed. Citrate synthase activity is correlated with the amount of mitochondrial mass present, suggesting that there are more, or larger, mitochondria in hybrids. In conclusion, biochemical evidence suggests that activity of at least two enzymes related to mitochondrial function are altered in the experimental inter-population hybrid lines. This is the first step in understanding the potential relationship between the genetic, biochemical, and organismal basis of mitochondrial dysfunction, and possibly the process of speciation, in inter-population hybrids.

Poster #: 174
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: site-specific recombination, plasmids, osmotic pressure
Project Title: Comparative analysis of Klebsiella pneumoniae Xer site-specific recombination sites
Author List:
Mercado, Elizabeth; Undergraduate, Biology, California State University, Fullerton, Presenting Author
Babayan, Melin; Undergraduate, Chemistry, California State University, Northridge
Guzman, Jimmy; Undergraduate, Chemistry, California State University, Northridge
Sherratt, David; University of Oxford
Tolmasky, Marcelo; Biology, California State University, Fullerton

Abstract: Xer site-specific recombination (XSSR) target sites are present in numerous plasmids. For some of them like the case of the cer site in ColE1, it has been established that XSSR confers stability by resolving multimers formed by homologous recombination. In other plasmids, the role of the target site is not so clear. For example, mwr, present in the Klebsiella pneumoniae plasmid pJHCMW1, seems to be too inefficient to confer stability. However, the efficiency as target for XSSR increases when the osmotic pressure of the environment is lowered, and as a consequence, the plasmids increases the negative supercoiling density. To understand more about the characteristics and role(s) of the XSSR sites we studied other sites present in plasmids isolated from clinical K. pneumoniae strains. Comparative analysis of the nucleotide sequences of the sites showed that a subset of sites share a mutation at the binding site of ArgR, one of two architectural proteins needed for formation of a synaptic complex previous to the Xer-mediated strands exchange. In vivo dimer resolution was assessed transforming E. coli DS941 with dimers followed by incubation overnight at 37 °C, plasmid isolation and analysis in agarose gel electrophoresis. Plasmid stability was determined culturing cells without selection for a known number of generations, followed by plating in the presence and absence of selection. The results of the experiments showed that Xer site-specific recombination sites that include the mutation at the ArgR site (Xer* sites) are abundant in plasmids of Gram-negatives. The in vivo resolution assays of recombinant clones including three representatives of Xer* sites showed that resolution was poor in L broth, but it was efficient in the same medium lacking NaCl. Concomitantly, plasmid stability was poorer in medium containing NaCl. Our results suggest that the mutation at the ArgR binding regions in Xer* sites is not exceptional, but it is present in a large number of sites in numerous plasmids of different bacterial genera. However, these sites may not be sufficiently active in various environments to ensure plasmid stability. These results could indicate that there are others functions played by XSSR sites in selected plasmids. Some of these functions could include promoting vertical and horizontal dissemination of genes participating in DNA exchanges as well as during the conjugation process.
Funding: 2R15AI047115-04 (NIH) and MHIRT 2T37MD001368 (NIH)

Poster #: 175
Campus: CSU San Bernardino
Poster Category: Biochemistry
Keywords: Lipid droplet, Cell signaling, Rab GTPase
Project Title: Constitutive signaling by an endosomal Rab5 isoform causes lipid metabolism dysfunction and ‘obesity’ in S. cerevisiae
Author List:
Lombere, Mindy; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Sayed, Sajida; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Nickerson, Daniel; Biology, California State University, San Bernardino

Abstract: Yeast cells build lipid droplets (LDs) in anticipation of starvation. LDs consist of amphipathic lipid monolayers surrounding a neutral lipid interior. Surface proteins allow lipid droplets to dock with other cellular compartments, but the unusual structure of LDs prevents compartments from fusing together. Instead, LDs are thought to dock with other organelles in order to enable lipid exchange between organelles. Identifying the proteins that mediate interactions between LDs and other organelles is a priority for understanding lipid metabolism and transport. The Rab GTPase Vps21 (yeast ortholog of human Rab5) controls endosomal tethering and vesicle fusion. In the signaling-active, GTP-bound state, Vps21 interacts with a set of effector proteins that function as endosome membrane tethers. The Vps21 mutant Vps21Q66L cannot hydrolyze GTP and is therefore ‘locked’ in the active, GTP-bound state and (it is thought) locked in interactions with effector protein tethers. We analyzed yeast cells expressing signaling-active Vps21Q66L using quantifiable enzyme-coupled endosomal cargo transport assays (e.g. CPY-invertase and Sna3-FLuciferase) and were surprised to find little to no misregulation of endosomal cargo transport. However, we observed vps21Q66L mutants display a pronounced cellular growth defect compared to wild type VPS21 cells. Because the vps21Q66L growth defect first as yeast exit logarithmic phase and should begin adaptation to starvation conditions, we investigated whether or not vps21Q66L mutants correctly build LDs. Using the LD marker Erg6-GFP, we observed by fluorescence microscopy that vps21Q66L mutants display higher LD signal than wild type cells, and vps21Q66L LDs possess an aberrant, distended LD morphology. Biochemical analysis (gas chromatography-mass spectrometry) of lipid content from vps21Q66L mutants indicates an ‘obese’ phenotype in which cells accumulate an overabundance of neutral lipids. These phenotypic data suggest a new link between Rab signaling at endosomes and cellular lipid metabolism. Ongoing studies are investigating whether the obese phenotype is a result of increased synthesis of neutral lipids or slower consumption. Also, we are pursuing both genetic and biochemical approaches to identify proteins that interact with GTP-bound Vps21 to trigger aberrant lipid metabolism and LD morphology.

Poster #: 176
Campus: San José State University
Poster Category: Biochemistry
Keywords: Beta-catenin, cancer, Drosophila
Project Title: Probing the molecular mechanism of the cancer-associated Beta-catenin H42R mutation
Author List:
Josipovic, Andin; Graduate, Biological Sciences, San José State University, Presenting Author
Grillo-Hill, Bree; Biological Sciences, San José State University

Abstract: Beta-catenin (Beta-cat) is a dual-function protein with roles in both adherens junctions to facilitate cell-cell adhesion, and as a signaling molecule in the Wnt pathway. Beta-cat protein levels are tightly regulated by cells, as inappropriate activation of Wnt signaling is associated with pathological tumor growth. We recently discovered that Beta-cat protein is degraded in a pH-sensitive manner at high intracellular pH, and identified a Beta-cat Histidine to Arginine (H>R) mutation that abolishes this pH-sensitive regulation. This Beta-cat^H>R mutation was identified in multiple human tumor samples in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, suggesting a possible role in tumorigenesis. Currently, the molecular mechanism by which this mutation contributes to tumorigenesis is unknown. We found that overexpression of Beta-cat^H>R in the Drosophila eye induces dysplasia and pronounced tumors which are more severe than phenotypes observed with previously characterized oncogenic Beta-cat alleles. In this poster, we will present data that describes the specific effects of this mutation. Our preliminary data suggests that Beta-cat^H>R shows increased transcription of Wnt target genes, using in vivo transcription reporters in whole mount, dissected Drosophila tissues. We see both an increase in the number of cells that express Wnt target genes, and increased intensity of expression levels of these reporters. We are currently working to quantify these effects. We will also look at effects on endogenous Wnt target genes to verify the biological significance of our findings. Understanding how this recurrent cancer-associated Beta-cat^H>R mutation affects Wnt signaling will increase our knowledge of this pathway, and may contribute to development of therapeutic approaches for human tumors that result from increased Wnt signaling.

Poster #: 177
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: Alternative Splicing, RNA binding proteins, post-translational modifications
Project Title: Understanding the Role of Post-Translational Modifications on the Splicing Activity of two Related RNA Binding Proteins
Author List:
Reynaga, Janice; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Nagel Award Nominee
Pina, Jeffrey; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Keppetipolla, Niroshika; Chemistry and Biochemistry, California State University, Fullerton

Abstract: The Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that controls the alternative splicing of many gene transcripts. PTBP1 belongs to a gene family with paralogs expressed tissue specifically. PTPB1 is expressed in most cell types but is absent in differentiating neurons and muscle. Paralog PTBP2 (also known as neuronal PTB) is highly expressed in differentiating neurons. The amino acid sequence of PTBP1 is 74% identical to PTBP2 and the two proteins have a similar domain organization with 4 RNA binding domains (RBDs) connected by 3 linker regions and an N-terminal region. The two proteins have over-lapping and non-overlapping target exons; PTBP1 functions as a splicing repressor of many neuronal exons while PTBP2 does not. PTBP1 is expressed in neuronal progenitor cells, but is down-regulated during neuronal differentiation while the level of PTBP2 is up-regulated. The switch in the levels of the two proteins effects the splicing of many exons that are sensitive to PTBP1, thus causing a change in the neuronal splicing program, that is critical for the development and maturation of neurons. How the two proteins exert different splicing outcomes is not well understood. Recent studies in the lab, using PTBP1-PTBP2 chimeric proteins, where portions of PTBP1 replaced the corresponding domain of PTBP2, highlighted regions of PTBP1 that change the splicing activity of PTBP2. This study also demonstrated that the two proteins have different protein-protein interactions, highlighting that additional factors such as post-translational modifications (PTMs) may dictate PTBP splicing activities. We have previously shown that splicing reaction mixtures containing bacterial expressed recombinant PTBP1 and PTBP2, in HeLa nuclear extract, exert different splicing effects on certain exons such as the N1 exon of the c-Src pre-mRNA. To capture splicing relevant PTM’s, we conducted mass spectrometry studies on purified, bacterial expressed, recombinant PTB proteins that were incubated in HeLa nuclear extract. Results of these studies highlight many PTMs in PTBP1 and 2. We also observe significant differences in PTM’s between PTBP1 and PTBP2 in the degree of phosphorylation and ubiquitiylation, in regions that changed the splicing activity of PTBP2, per chimera study. Currently, we are conducting a mutational analysis of those modified residues that are different between PTBP1 and PTBP2, to determine the role of PTM’s in PTBP splicing activity.

Poster #: 178
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: RNA binding proteins, nuclear cytoplasmic shuttling, Immunocytochemistry
Project Title: The Role of Ubiquitin on Polypyrimidine Tract Binding Protein Nuclear-Cytoplasmic Shuttling
Author List:
Barrios, Eduardo; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Al Bassri, Sara; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Keppetipola, Niroshika; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Polypyrimidine Tract Binding Protein 1 (PTBP1), is a heterogeneous ribonucleoprotein protein (hnRNP I) that plays an important role in many cellular functions including alternative splicing regulation, cap-independent translation initiation and mRNA localization. PTBP1 contains 4 RNA bindings domains (RBDs) connected by 3 linker regions and an N-terminal region, that each play a role in the diverse functions of the protein. Upon translation, PTBP1 is shuttled into the nucleus via its nuclear localization sequence (NLS) located in the N-terminal region. Previous studies have identified that translocation from nucleus to cytoplasm is dictated by Protein Kinase A mediated phosphorylation of Serine 16 in the N-terminal region and RBD2. Proteomic studies conducted in the lab have identified many residues in RBD2 post translationally modified, by the addition of acetate and ubiquitin groups. In this study, we aimed to understand the role of RBD2 ubiquitination, on PTBP1 nuclear cytoplasmic shuttling. To test this, we conducted an alanine scan of the modified lysine residues; Lys206, Lys212, Lys218, Lys238, Lys259 and Lys266. The mutants were created via two-step PCR and cloned into the mammalian expression vector pcDNA3.1(+). Upon sequence confirmation, the mutants were transfected into mouse neuro2A cells and assayed for localization in vivo using immunocytochemistry. Our results highlight that mutant K212A and K266A are localized in the nucleus, in contrast to wild-type PTBP1 which is localized in both the nucleus and the cytoplasm. These results imply that Lys212 and Lys266 are important for PTBP1 nuclear export. We are currently assaying RBD2 with anti-ubiquitin antibodies to determine the type of ubiquitin linkage, to design experiments to probe the role of RBD2 ubiquitination on PTBP1 nuclear export.

Poster #: 179
Campus: Stanislaus State University
Poster Category: Biochemistry
Keywords: cigarette, DNA damage, radical
Project Title: Aged Catechol: A Model System for Studying Chemistry and DNA Damaging Effects of the Cigarette Tar Radical
Author List:
Lopez, Christopher; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Mendez, Suzanne; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Aleman, Elvin; Chemistry, California State University, Stanislaus
Stone, Koni ; Chemistry, California State University, Stanislaus

Abstract: Catechol is present in large (milligram) amounts in tobacco cigarettes. Previous work demonstrated that oxidized solutions of catechol mimic the DNA binding and nicking activity of mainstream cigarette smoke extracts. An aqueous solution of catechol was allowed to stand for three weeks in order to compare the resulting polymer of catechol to second and third hand cigarette tar extracts. The aqueous solutions of catechol were purified by Sephadex chromatography to remove non-polymerized catechol and 48 fractions were collected. The fractions were analyzed by UV spectroscopy, and 12 of the samples had absorbance at 275 nm, with spectra similar to the cigarette tar fractions. Gel electrophoresis was used to analyze the nicking of plasmid (pUC18) DNA. Aqueous extracts of tar extracts from second and third hand cigarette smoke nick the pUC18 DNA, causing the DNA to unwind after a single nick. This creates two separate bands of DNA in the agarose gel; nicked and intact DNA. Therefore, this same method was used to analyze the 12 aged catechol fractions. The DNA samples that were incubated with buffer (no tar) exhibited 0-3% nicking. The catechol polymer when incubated with the DNA nicked 5-9%. An oxygraph instrument was used in order to analyze the production of hydrogen peroxide by the aged catechol. When the aged catechol fraction was added, the amount of available oxygen decreased by a rate of 12 nmol/mL/second. This rate is similar to the rate obtained (11 nmol/mL/second) when second hand smoke extracts were analyzed. Methylene blue becomes colorless upon reaction with hydroxyl radicals, thus it was use to qualitatively detect the presence of hydroxyl radicals in solution. Filter Paper was cut into strips, and then dipped into a 1.0mM solution of Methylene Blue dye. After drying for 24 hours, a 50 μL aliquot of the catechol fraction was added and the dye became colorless. Thus, the aged catechol solutions have hydroxyl radicals. Nuclear Magnetic Resonance spectra of the fractionated catechol polymer solutions were obtained. Fresh catechol has proton peaks at 6.7 and 6.8 ppm, the fractionated catechol has peaks at 1.2, 1.8, 5.0, and 8.3 ppm. This indicates a significant structural change of the aged catechol sample. Aged catechol solutions allow us to determine the chemical properties of the cigarette tar radical with only one chemical species in the polymeric mixture.

Poster #: 180
Campus: CSU Long Beach
Poster Category: Biochemistry
Keywords: siRNA delivery, hybrid peptide, drug delivery
Project Title: Collagen Hybrid Peptides as Carriers for Nucleic Acid Therapeutics
Author List:
Gamboa, Alicia; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Slowinska, Katarzyna; Chemistry and Biochemistry, California State University, Long Beach

Abstract: Short interfering RNA (siRNA) causes sequence specific gene silencing of mRNA, and has been shown to be a very promising therapeutic agent for a wide range of diseases. The delivery of siRNA faces distinct challenges: degradation by nucleases, efficiency of uptake, and release into the cell to silence the targeted gene. To address these issues we have developed hybrid collagen/cell penetrating peptides (CHP) that allow internalization of the peptide with cargo into cells. The CHP-siRNA complex forms via self-assembly that do not require chemical modification. The binding interactions between CHP and siRNA were analyzed using isothermal titration calorimetry (ITC) and results were analyzed using a single site independent model to determine the reaction stoichiometry. Analysis of the ITC thermogram indicates that the CHPs form stoichiometric complexes with siRNA at molar ratios depending on the number of arginines in the sequence. To further characterize interaction between the CHP and siRNA, the CHP-siRNA complex was prepared and visualized using polarized light microscopy, revealing the formation of highly crystalline nanoparticles. ImageJ software was used to measure the diameter of the crystals and statistical analysis indicated an average size of 290 nm with a standard deviation of 85 nm. These results indicate that CHPs are able to form structurally stable complexes with siRNA. The uptake efficiency of siRNA into cells was measured using flow cytometry and compared to Lipofectamine. Our results showed that CHPs were able to deliver siRNA with higher efficiency (98%) than Lipofectamine (78%). Confocal microscopy was used to perform co-localization studies of CHP-siRNA complexes in 3T3 Swiss mouse fibroblast cells. The results suggest that the number of arginines in a CHP sequence affects the release of siRNA into the cytoplasm. Flow cytometry analysis was used to examine gene knockdown efficiency of the CHP-siRNA complex in comparison with Lipofectamine. 3T3 Swiss mouse fibroblast cells stably expressing green fluorescent protein (GFP) were treated with siRNA targeting GFP utilizing either CHP or Lipofectamine as the delivery vehicle. The results showed that delivery of siRNA using CHP had a better gene knockdown efficiency of about 50% with comparison to Lipofectamine (about 10%). The data suggests that our strategy to employ CHP to deliver nucleic acid therapeutics is very effective.

Poster #: 181
Campus: CSU Northridge
Poster Category: Biochemistry
Keywords: Three-point attachment model, Stereoselectivity, Conformational Flexibility
Project Title: Chiral Recognition Framework to Describe Stereoselectivity (or lack thereof) in Protein-Ligand Interactions
Author List:
Mikhael, Simoun; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Chemistry and Biochemistry, California State University, Northridge

Abstract: Understanding stereoselectivity in biological systems is crucial for drug design as the number of chiral drugs is rising and pharma companies can afford to test only one or two stereoisomers of a chiral drug in the clinic. The stereocenter recognition (SR) model has been proposed that reduces ligands with multiple stereocenters and complex topologies into Chiral Graphs, whose interaction maps with proteins provide an atomistic-level framework to understand protein-ligand interactions in terms of the minimum number of interactions required for stereoselectivity.
The protein data bank (PDB) contains structures of about 24,000 ligands bound to different proteins. After a detailed mining, filtering, and analysis of these ligands, we have so far identified more than 5,900 unique stereoisomeric ligands, which bind to the same or similar protein (Sequence Identity ≥ 90%). This is a surprisingly large number of stereoisomeric ligand pairs that do not show strong stereoselectivity as they are found bound to the same protein. For all these ligands, their stereochemical characteristics and noncovalent interactions with proteins have been analyzed. As a first application of the SR model, we have compared several such ligand stereoisomeric pairs in terms of their interactions maps (with the same protein) for ligand topologies covering up to 7 stereocenters, to understand the lack of stereoselectivity in terms of the underlying noncovalent interactions between ligands and one or more protein residues.
In one of the cases, where the human Angiotensin converting enzyme (AnCE) was found binding to a phosphinic tripeptide FI or its stereoisomeric tripeptide FII, both stereoisomers bound to the enzyme equally well using the same number of interaction locations, consistent with very similar binding affinities measured for these tripeptide ligands for the AnCE enzyme. That was also the minimum number of interaction locations prescribed by the SR model necessary for stereoselective recognition. On the other hand, in the Drosophila AnCE, the FI tripeptide binds to the enzyme using five interaction locations, whereas the FII peptide binds utilizing only four of those locations. This is consistent with the observed binding affinity of FI being stronger than that of the FII for the Drosophila enzyme. Several such cases will be presented to explain the lack of stereoselectivity in protein-ligand interactions, which stems from protein’s and/or ligand’s conformational flexibility.

Poster #: 182
Campus: San Diego State University
Poster Category: Biochemistry
Keywords: pancreatic cancer, biomarkers, label-free
Project Title: Novel Detection of Biomarkers of Pancreatic Cancer Using Microfluidics and Nonlinear Multi-Photon Laser Wave-Mixing Detector
Author List:
Liang, Jie; Graduate, Chemistry and Biochemistryq, San Diego State University, Presenting Author
Venturini, Filippo; Graduate, Chemistry nd Biochemistry, San Diego State University
Tong, William; Chemistry and Biochemistry, San Diego State University, Presenting Author

Abstract: We present our preliminary results for sensitive detection of biomarkers including carbohydrate antigen 19-9 (CA 19-9) and carbohydrate antigen 242 (CA 242). Current detection methods require complicated and time-consuming labeling steps. Our patented nonlinear laser wave-mixing methods offer significant advantages including label-free native detection, excellent sensitivity, small sample requirements, short optical path length, high spatial resolution and portable detector designs. The wave-mixing signal is generated when the two input beams are mixed inside the analyte and it can be collected with virtually 100% efficiency and maximum signal-to-noise ratio. The signal has a quadratic dependence on analyte concentration, and hence, small changes can be monitored more effectively. Since wave-mixing probe volume is small (nanoliter to picoliter), it is inherently suitable for microfluidics or capillary-based electrophoresis systems (e.g., 75 μm i.d. fused silica capillary). Different biomarkers can be immobilized on a custom 3D printed slide. Since wave mixing is an absorption-based method, we could use both fluorophore and chromophore labels, if desired. We obtained excellent sensitivity levels for CA 19-9 using Chromeo P503 and a 473 nm solid-state excitation laser. Preliminary detection limits of 1×10-17 mole and 90 U/mL were obtained for methyl red and CA 19-9, respectively. One can run a standard protein ladder to estimate capillary electrophoresis retention time for CA 19-9. We 3D printed our own glass slides and microarrays used in these detection methods for early diagnosis of pancreatic cancer.
Acknowledgment: We acknowledge partial support of this work by the U.S. Dept. of Homeland Security Science and Technology Directorate, U.S. Dept. of Defense, Army Research Office, NSF, NIH (R01), and NIH IMSD.

Poster #: 183
Campus: CSU Fullerton
Poster Category: Biochemistry
Keywords: alpha-2-macroglobulin, copper binding, pigs, ,
Project Title: Purification and copper binding of alpha-2-macroglobulin from plasma of humans and pigs
Author List:
Garcia, Alejandra; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Linder, Maria C.; Chemistry and Biochemistry , California State University, Fullerton

Abstract: Alpha-2-macroglobulin (a2M) is a major component of the blood plasma of most mammals, and is not only a trap for circulating proteases, but transports immune signaling agents as well as zinc. Some time ago, the Linder laboratory showed that a2M, and its homolog in rodents (alpha-1-inhibitor 3) are also important in the transport of copper (Weiss & Linder, Am J Physiol, 249, E77, 1985) and directly deliver copper to cells (Kidane et al., Biometals 25: 697, 2012). In humans, a2M is a tetramer of 180 kDa subunits, but the details of its structure have still not been worked out. Preliminary titration studies determined that it probably contains 2 Cu atoms per molecule. The goal of the studies presented here was to establish how Cu is bound to this molecule, and whether pig a2M has a similar structure.
Blood plasma from human volunteers (obtained with IRB approval) and from pigs (Bioreclamation IVT) was used for purification of a2Ms using the procedure established by the laboratory of Salvatore Pizzo (Duke University), which involves fractionation with PEG 8000 (4-16%), and Zn(II)-charged metal affinity chromatography (IMAC) on Chelating Sepharose. Further separation from contaminants and fragments was by size exclusion chromatography (SEC) on Sephacryl S300. Protein concentration was determined by the Bradford assay with BioRad reagents and bovine serum albumin as the standard. Copper was determined by graphite furnace atomic absorption.
Human a2M was almost pure after the IMAC, as shown by SDS- and native PAGE. The remaining contaminants were removed by SEC, and a2M eluted with an Mr of about 800 kDa. The Cu:protein ratio was ~4 atoms/molecule. Pig plasma a2M was half the size of human a2M, but the Cu:protein ratio was also ~4/molecule. Cu-EPR spectra (Peter Faller, University of Strasbourg France) for human and pig a2M were the same, but there was evidence of two kinds of Cu-binding, which is being further explored.
We conclude that pig a2M differs from that of the human in being present in the blood plasma as a dimer rather than a tetramer, but that similar ligands are binding the copper. Differences in total copper per subunit may reflect variations in loosely bound copper acquired from buffers and equipment during purification.

Poster #: 184
Campus: San José State University
Poster Category: Bioengineering
Keywords: , ,
Project Title: Impedance Sensing of Blood Clots
Author List:
Mireles, Salvador; Graduate, Biomedical, Chemical, and Materials Engineering, San José State University, Presenting Author
Kim, Max; Graduate, Biomedical, Chemical, and Materials Engineering, San José State University
Ramasubramanian, Anand; Biomedical, Chemical, and Materials Engineering, San José State University
Lee, John; Mechanical Engineering, San José State University

Abstract: An injury to a blood vessel exposes the extracellular matrix proteins at the wound site. Circulating platelets get activated at this site, and together with plasma coagulation factors, generate thrombin, which converts fibrinogen to an interconnected network of fibrin polymer with enmeshed cells or the clot. The strength and stability of the clots are critical for homeostasis since hypo-function may lead to hemorrhage, while hyper-function may lead to thrombotic complications. The current laboratory assays for monitoring coagulation and clot properties are not optimal as they do not provide comprehensive assessment, continuous measurements, may require additional expertise or physician(s) and large sample volumes, and not suitable for point of care measurements. To alleviate these issues, we propose to utilize impedance sensing as an alternative. Using standard photolithography and lift-off techniques, we fabricated interdigitated electrodes on aluminum coated microscope slides. Various electrode geometries ranging from 10 – 100 µm digit spacing were selectively chemically etched, and the electrodes were integrated into a 1 mL/min continuous flow microfluidic device. Impedance measurements were made at 20 Hz – 1 MHz to record 100 data points over 16 averaged cycles at 50 mV excitations using a spectrometer. After calibrating the electrode assembly using standard electrolytes, we measured the impedance and phase angle of uncrosslinked fibrin clots polymerized using with 0.001-1 U/ml thrombin. We observed a maximum change in impedance magnitude at 100 kHz due to fibrin polymerization. We are currently measuring the effects of crosslinking of fibrin, retraction due to platelets, and cellular composition on these properties. Our results demonstrate that impedance sensing is a viable alternative to mechanical measurements of clot properties; and successful completion of the objectives may provide a desirable diagnostic tool for coagulation disorders.

Poster #: 185
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: Muscle , Collagen, Engineered Tissues
Project Title: Engineered Constructs with Aligned Topography for Muscle Growth
Author List:
Anderson-Fears, Keenan; Undergraduate, Biochemistry, California State University, Long Beach, Presenting Author
Guerrero, Daniel; Undergraduate, Electrical Engineering, California State University, Long Beach
Nguyen, Jeffrey; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Kane, Madison; Undergraduate, Biomedical Engineering, California State University, Long Beach
Suarez, Jessica; Undergraduate, Biomedical Engineering, California State University, Long Beach
Mata, Daniel; Undergraduate, Biomedical Engineering, California State University, Long Beach
Elder, August; Undergraduate, Bioemdical 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: Injuries that result in volumetric muscle loss (VML) lead to fibrosis, scarring, and loss of tissue function. Skeletal muscle possesses great capacity for regeneration in response to minor damage, but severe defects are not effectively repaired. In this work, we lay the groundwork for engineering advanced biomaterials with defined physical and biochemical signals that support myoblast growth in vitro and skeletal tissue formation in vivo. Collagen type 1 was extracted from porcine dermal tissue (obtained from an ISO/FDA certified provider) using a simple acid solubilization method. After purification, the collagen was formed into thin films (h = 15 µm) and also as soft hydrogels (h = 200 µm). Compression tests were performed on the hydrogels to determine stiffness. Tensile testing will be used to analyze the films. The hydrogels and thin films were cast in a poly-dimethyl siloxane (PDMS) mold with micro channels. The model to fabricate the mold was designed in SolidWorks and fabricated using a Project 6000 3D Printer. To assess cell proliferation and organization, C2C12 myoblasts and 3T3 fibroblasts were cultured in these substrates for 1-2 weeks. Collagen films and collagen type 1 gels were fabricated with aligned microtopography as a physical signal for myoblast development. Compression tests of the collagen hydrogels showed a Young’s modulus value of 3.2 kPa. The engineered hydrogel constructs supported fibroblast and myoblast proliferation, and directed growth within the channels. Myoblasts cultured on thin films in the micro channeled molds proliferated and demonstrated desired myoblast phenotype. These designed collagen bioengineered constructs demonstrate potential for applications in muscle tissue engineering. Acknowledgements: Jeremy Bonifacio facilitated printing of 3D Models.
Funding: CSULB ORSP, CSULB COE, CSUPERB.

Poster #: 186
Campus: CSU Northridge
Poster Category: Bioengineering
Keywords: Cooling of Biomedical Devices, Jet Impingement, Porous Filled Channel
Project Title: Numerical Investigation of Thermal Transport through Porous Filled Channels Consisting of Multiple Injection Jets for Cooling of Biomedical Devices
Author List:
Zing, Carlos; Graduate, Mechanical Engineering, California State University, Northridge, Presenting Author
Mahjoob, Shadi; Mechanical Engineering, California State University, Northridge, Presenting Author

Abstract: Development of advanced cooling techniques and thermal management are key issues to design advanced biomedical devices, such as imaging equipments, PCR (polymerase chain reaction) machines, lasers, ultrasound equipments, radiography machines and surgical instruments. Due to high usage of electronics and micro processors in advanced biomedical devices, high heat fluxes will be produced that threatens the safety of these devices. As such, the temperature needs to be kept below a maximum operating temperature. In this work, an advanced porous filled heat exchanger is numerically investigated and developed based on the technology of jet impingement, indirect cooling and utilization of highly conductive metal foams. High conductive porous materials provide a large surface area for a given volume that is an essential parameter in heat transfer augmentation and thermal control. The heat exchanger consists of multiple impinging jets, through diverse geometrical configurations. In order to cool the biomedical device, the base of the heat exchanger will be in contact with the device part to be cooled and therefore the base will be subjected to high heat flux leaving the devices. The coolant will enter the porous filed channel through multiple jet injection channels. The study has been performed by varying certain parameters such as the number of the jet inlet channels, their orientations, the size of the central and peripheral jet inlet channels, the inclined walls of the porous filled channel, the channel wall structure, the channel maximum and minimum thickness, and the coolant velocities and flow rates. This study is performed for two and three dimensional models and physical characteristics and temperature distribution on the base and within the porous field channel are investigated in depth to develop optimum designs. The study indicates that by placing the peripheral jets more closely to the central one and by properly arranging the jet inlets, the cooling effectiveness can be improved substantially while providing a more uniform temperature distribution.

Poster #: 187
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: heart, flow, bioengineering
Project Title: The Effect of Inflow Cannula Angle on the Intraventricular Flow Field of the LVAD-Assisted Heart
Author List:
Marquez, Nikolas; Undergraduate, Mechanical Engineering, San Diego State University, Presenting Author
Montes, Ricardo; Graduate, Mechanical Engineering, San Diego State University
Salim, Saniya; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
May-Newman, Karen; Mechanical Engineering, San Diego State University

Abstract: Positioning of the LVAD inflow cannula has proven to be important for left ventricular (LV) flow. Previous studies have identified cannula malposition as a significant risk for pump thrombosis. Thrombus development is a consequence of altered flow dynamics, which can produce areas of flow stasis or high shear that promote coagulation. The goal of this study was to examine the effect of LVAD inflow cannula angle on the LV flow field for a range of cannula lengths and support conditions. Experimental studies were performed using a mock circulatory loop with a customized silicone LV and the EVAHEART LVAD (EVI, Inc.) to simulate the biomechanics of the LVAD-assisted heart. Two LVAD speeds were studied, one which produced aortic valve opening (1800 rpm), and a higher speed in which the aortic valve was closed (2300 rpm). A transparent inflow cannula was tested in two orientations, the standard orientation in which the inflow cannula is oriented parallel to the septal wall and an angled orientation in which the cannula is rotated 15° toward the septum. Vortex structures were analyzed by calculating the vorticity from the measured velocity, and using the Q criterion to identify the vortex boundaries. Circulation and kinetic energy (KE) were computed for the clockwise (CW) and counter-clockwise (CCW) vortices, as well as vortex size, shape and position during the cardiac cycle. Localized stasis was evaluated in small regions of interest (ROI) surrounding the inflow cannula, and combined with a map of normalized pulsatile velocity (NPV). The velocity fields measured for each cannula orientation during the cardiac cycle were similar during diastolic filling for both LVAD speeds, but revealed more apical stasis and lower cannula velocities for the angled orientation. The CW vortex became more elongated with LVAD speed for the standard cannula, but this effect was blunted when the cannula was angled toward the septum. Regional velocity was greater within the standard inflow cannula than for the angled orientation, and NPV revealed low pulsatility within as well as adjacent to the inflow cannula. Overall, the effect of angulating the LVAD inflow cannula toward the septum results in reduced velocity and pulsatility of blood flow into the inflow cannula from the LV. These findings support the practice of positioning the LVAD inflow cannula parallel to the septum, and discourage angulation that increases flow stasis and the risk of thrombus formation.

Poster #: 188
Campus: Cal Poly San Luis Obispo
Poster Category: Bioengineering
Keywords: keratinocytes, RT-qPCR,
Project Title: Gene Expression in Sphingomyelin-Treated Keratinocytes post UVB Irradiation
Author List:
Bingham, Trevor; Undergraduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Switalski, Stephanie; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Laiho, Lily; Biomedical Engineering, California Polytechnic State University, San Luis Obispo

Abstract: Non-melanoma type skin cancer accounts for a large proportion of skin cancer cases. This laboratory uses the addition of sphingomyelin to keratinocytes to observe its impact on reducing UV photodamage. Past research done by the lab has demonstrated that the addition of sphingomyelin (SM) to keratinocytes in cell culture led to a reduction in p21 protein expression 24 hr post UVB (30mJ/cm2) exposure, an indication of less DNA damage. The goal of this study was to use quantitative reverse transcription PCR (RT-qPCR) to provide additional quantification of the reduction of p21 expression and to gain an understanding of how the addition of SM to keratinocytes effects their RNA expression levels. Comparisons of RNA expression were made between two groups: keratinocytes exposed to UVB irradiation and keratinocytes cultured with SM and exposed to UVB irradiation. RNA was extracted from cell cultures using TRIzol in tandem with silica spin columns (TRIzol® Plus RNA Purification Kit). The RNA was then analyzed for quantification via spectroscopy and integrity via RNA gel (SYBR Gold). Contaminating gDNA were quantified using a designed 18S primer on samples. Two step RT-qPCR was completed using reverse transcription, and quantification of RNA was completed using SYBR green. Standards and controls were applied to ensure designed primers matched standards. Final analysis let to the observation of a 22.59 % decrease in p21 fold expression in keratinocytes cultured with SM, further demonstrating the protective effects of sphingomyelin against UVB irradiation.

Poster #: 189
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: heart, flow, bioengineering
Project Title: Patient-specific Left Ventricle Models for Studying Flow Dynamics in the LVAD-Assisted Heart
Author List:
Vu, Vi; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Montes, Ricardo; Graduate, Mechanical Engineering, San Diego State University
May-Newman, Karen; Mechanical Engineering, San Diego State University

Abstract: Left ventricular assist devices (LVADs) are pumps developed to resolve heart failure symptoms through increased circulatory support. The LVAD is surgically connected to the left ventricle (LV) and aorta. Upon LVAD implantation, many sudden cardiac alterations are experienced by the cardiovascular system, including a reduction in LV volume. During periods of high LVAD support, the aortic valve (AoV) remains closed throughout the entire cardiac cycle (CC) and all blood flow occurs through the LVAD. This intraventricular flow alteration creates a stasis region along the LV outflow tract (LVOT), which is associated with a risk of thromboembolism. The purpose of this study was to measure patient flow dynamics and develop a model of patient specific LV geometry and function to evaluate vortex behavior and flow transport during LVAD support.
Clinical study: Following a method described in our previous study, ultrasound imaging of a patient before and 8 days post – LVAD implantation were obtained and used to calculate vortex circulation, kinetic energy (KE), and residence time (RT). EchoPAC software was used to determine the change in LV volume throughout the CC. Before LVAD implantation, the patient’s ejection fraction was calculated as the ratio between stroke volume (SV) and end diastolic volume (EDV), and the cardiac output (CO) was calculated by multiplying SV with average heart rate. Post-LVAD implantation, CO was estimated by the LVAD flow output.
Experimental study: The SDSU Cardiac Simulator (CS) has the capability to match patient-specific flow conditions, was setup using different silicon LV models with volume variation. For pre-LVAD case, the system was setup to match the patient’s EDV, and the CS program was chosen to reproduce similar EF and CO of the patient. For post-LVAD case, the patient’s EDV, LVAD speed and total CO was matched. Data were collected using Particle Image Velocimetry technique and further analyze using MATLAB to obtain vortex parameters and RT.
The result shows the average results for both clinical and experimental studies. The CW and CCW circulation obtained from CS were within 18% and 35% of Pre-LVAD values. For the post-LVAD condition, CW and CCW circulation from the CS had less agreement with patient data, as they were within 69% and 25% of patient data respectively. Further modification in setup and program will be performed to obtain better agreement between post-LVAD CS and patient-specific parameters.

Poster #: 190
Campus: CSU Long Beach
Poster Category: Bioengineering
Keywords: microfluidics, lab on a chip, open-source
Project Title: An Open-source Centrifugal Microfluidic Platform for Chemical and Biological Applications
Author List:
Venkatraman, Rahul; Graduate, Chemical Engineering, California State University, Long Beach
Mith, Shafi; Chemical Engineering, California State University, Long Beach
Le, Tiffany; Graduate, Chemical Engineering, California State University, Long Beach, Presenting Author
Esqueda, Genesis; Undergraduate, Chemical Engineering, California State University, Long Beach
Tang, Christine; Undergraduate, Biomedical Engineering, California State University, Long Beach, Presenting Author
Lo, Roger; Chemical Engineering/Biomedical Engineering, California State University, Long Beach

Abstract: Microfluidics is the study of fluid behaviors, manipulations, and design of devices that can efficiently perform these tasks at the micrometer scale. Highly sophisticated microfluidic chips or lab-on-a-chip (LOC) devices could potentially revolutionize healthcare, biology, chemistry, and all related disciplines, but the wide adoption of this technology is still a challenge due to the lack of an established, standardized manufacturing process. Most devices in the lab are primarily based on lateral flow and fabricated with tedious photolithographic techniques of master preparation, followed by casting and bonding to glass substrates. These devices typically feature microchannels in the 1-100 µm range on a 75 x 25 mm glass slide. Although the microfluidic device itself has a small form factor, the actual apparatus for fluid manipulation and data collection/analysis is much larger due to the need for pumps, microscopes, and computers. For point-of-care applications, an instrument must be portable, simple to operate, economical in consumables, and inexpensive for production.
In this work, we seek to design and construct such an instrument that meets these requirements through the combination of centrifugal microfluidics, 3D printing, and open-source electronics. Fluid manipulations based on centrifugal motion are a function of spin speed and do not require large external pumps. The microfluidic discs are fabricated with micromachined polycarbonate sheets and double-sided adhesive films. The instrument for fluid manipulation and data collection/analysis was built with a Raspberry Pi 3 board, Arduino Uno board, on-board stroboscopic imaging camera, and 3D-printed gear train capable of high speeds from 1,000-15,000 rpm using a stepper motor. The whole setup is the size of a conventional shoebox. The prototype has been built and is being tested for a model process of synthesizing microbeads from alginate solutions.

Poster #: 191
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: microfluidics , immunoassay, Stem Cells
Project Title: Cancer Stem Cell Cluster Capturing Microfluidics Platform
Author List:
Rodriguez, Ygnacio; Graduate, Mechanical Engineering , San Diego State University, Presenting Author

Abstract: There is a consensus among researchers that cancer stem cells are the cause of metastasis in cancer patients. Isolation and capture of these rare cells continues to be a daunting task that is still looking for an innovative and efficient method. While a variety of approaches have been suggested over the past several years, immunocapturing in a microfluidics platform carries a substantial promise as shown by recent published works. In this work, we present a microfluidic device to capture cancer stem cells. Using a polydimethylsiloxane (PDMS) mold, we have created a series of 32 bifurcating microfluidic channels that utilizes a herringbone design for the slowing and chaotic mixing of the patients’ blood. This creates more cell-to-wall interaction time increasing the chances of capturing these rare cells. Through the help of our clinical collaborators, we are able to coat the channel walls with anti-bodies to target and capture specific cancer stem cells. Patients’ blood is then passed through the microfluidic cancer stem cell capture chip and released where it can then be sequenced and analyzed. The goal is to be able to test different drugs to identify the most optimal therapy for each individual patient. We also plan on adding a triangular prism ‘obstacle’ design which will work as a trap to capture more malignant cells, specifically cancer stem cell clusters, as well as increase the surface area of the walls.

Poster #: 192
Campus: San Diego State University
Poster Category: Bioengineering
Keywords: heart, valve, flow
Project Title: The Effect of Mitral Valve Prosthesis Design and Orientation on Intraventricular Flow Studied in a Mock Circulatory Loop
Author List:
Montes, Ricardo; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Vu, Vi; Graduate, Mechanical Engineering, San Diego State University, Presenting Author
Marquez, Nikolas; Undergraduate, Mechanical Engineering, San Diego State University
May-Newman, Karen; Mechanical Engineering, San Diego State University

Abstract: Thrombus formation associated with implanted cardiovascular devices increases the risk of stroke and pulmonary embolism. Abnormal blood flow patterns are linked with thromboembolic events (TE), especially in the presence of medical devices such as heart valve prostheses (HVP). The goal of this study was to characterize flow patterns, vortex formation, pulsatility and predict TE potential for a variety of mitral valve prosthesis designs.
Particle image velocimetry was used to measure 2-D velocity on the midplane of a silicone left ventricle (LV) in a mock circulatory loop. The velocity map was measured for three different HVP in the mitral position: a porcine bioprosthesis (BP), a tilting disk (TD) valve in two orientations: towards the LV free wall (TD-F) or the septum (TD-S), and a bileaflet valve in three orientations: anti-anatomical (BL-AA), anatomical (BL-A), and at a 45° angle (BL-45). The baseline hemodynamics simulated a heart failure patient with an aortic flow rate of 3.5 ± 0.5 L/min at a heart rate of 70 bpm, at a mean aortic pressure of 65 ± 5 mmHg. Lagrangian analysis of blood transport and LV washout was assessed, where particle positions through several cardiac cycles were calculated, and the number of particles remaining at the end of the first, second and third cardiac cycle tabulated and normalized by the initial particle number to obtain the flow fraction. The reduction of flow fraction at the end of the first cardiac cycle constitutes the direct flow (DF), subsequent reduction during the second cardiac cycle is the delayed ejection fraction (DEF), and the remainder is the Retained Flow (RF).
The BP valve exhibited a DF of 94% and DEF of 6%, resulting in minimal RF. Transport through the bileaflet HVP was slower, with a DF of 75% for the BL-AA, and approximately 50% for the other two orientations. DEF and RF were lower for BL-AA than for BL-A or BL-45. The tilting disk HBP demonstrated the worst flow transport, with low DF (48% for TD-F and 38% for TD-S) and high DEF (22% for TD-F, 24% for TD-S) and RF (17% for TD-F, 28% for TD-S).
The BP valve, with a single central orifice, provides the best flow transport with the highest DF and lowest RF values. The TD valve exhibited the worst performance, especially in the septal orientation (TD-S), which resulted in low DF and high RF. Despite demonstrating reduced LV pulsatility, the bileaflet valves performed well, especially in the anti-anatomical orientation which is clinically preferred.

Poster #: 193
Campus: San José State University
Poster Category: Bioengineering
Keywords: , ,
Project Title: Microstructure and Micromechanics of Blood Clots
Author List:
Kim, Max; Graduate, Biomedical, Chemical, and Materials Engineering, San José State University, Presenting Author
Mireles, Salvador; Graduate, Biomedical, Chemical, and Materials Engineering, San José State University
Saha, Amit; Postdoc, Biomedical, Chemical, and Materials Engineering, San José State University
Ramasubramanian, Anand; Biomedical, Chemical, and Materials Engineering, San José State University
Lee, John; Mechanical Engineering, San José State University

Abstract: A blood clot is a biocomposite material composed of a fibrin network with the inclusion of platelets and red cells. The strength and stability of clots demarcate hemostasis from bleeding and thrombosis as weak clots give in to hemorrhage while thrombotic occlusions compromise the patency of blood vessels. Hence, understanding clot biomechanics is critical for the treatment of cardiovascular diseases. The objective of this work is to investigate the relationship between clot micromechanics and microstructure under physiologically relevant loading conditions. Fresh frozen plasma and fibrinogen was polymerized with 0.1 U thrombin and immersed with 2 µm diameter fluorescent microspheres. These gels were loaded onto a custom developed extensometer platform to apply a constant strain at a prescribed displacement rate of 60 µm/s. The microspheres were individually tracked using fluorescence microscopy coupled with an ImageJ bead-tracking plugin. Under similar loads, AF- 594 conjugated fibrinogen was stretched beyond the limits of structural integrity to visualize the deformation of fibrin networks. The displacement and the deviation in rate of displacement suggests shows that in the absence of crosslinking in fibrin gels, the individual fibers may stretch uniformly while enhanced crosslinking in fibrin networks may present a combination of stretching of individual fibers and the network itself resulting in non-uniform local deformation. To expand this work, we will visualize simultaneously, fibrin networks, platelet microdomains and microspheres. Our results demonstrate that, under constant strain, clot deformation at the microscale depends on local clot microstructure. This suggests that clot microenvironment may dictate the clot mechanical properties and ultimate rupture.

Poster #: 194
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: addiction, treatment, developmental
Project Title: Administration of Zolmitriptan, a 5-HT1B receptor agonist, attenuates the expression of nicotine reward in female, but not male adolescent rats
Author List:
Franco, Daniela; Graduate, Psychology, California State University, Long Beach, Presenting Author
Cabrera, Ryan; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Hernandez, Kimberly ; Undergraduate, Psychology, California State University, Long Beach
Iñiguez, Sergio; Psychology, University of Texas, El Paso
Zavala, Arturo; Psychology, California State University, Long Beach

Abstract: Preclinical research demonstrates an involvement of 5-HT1B receptors in the rewarding effects of cocaine and alcohol. Recently, we have shown that stimulation of 5-HT1B receptors with Zolmitriptan decreases the rewarding effects of nicotine in male and female adolescent rats. In the present study, we examined if giving Zolmitriptan also decreases the expression of nicotine-induced conditioned place preference (CPP). Beginning on postnatal day (PD) 27, male and female adolescent rats underwent an 11-day nicotine CPP procedure. On days 1 and 11, rats were tested for their pre-conditioning and post-conditioning place preferences, respectively, during 15-min sessions. On days 3-10, rats were conditioned for 15-min with saline or nicotine (0, 0.022, 0.067, or 0.2 mg/kg) on alternating days. To examine the role of 5-HT1B receptors in the expression of nicotine-induced CPP, Zolmitriptan (0 or 10 mg/kg) was administered 15 min before the start of the post-conditioning test session. Results indicate that activation of 5-HT1B receptors decreased the expression of nicotine-induced CPP in female, but not male rats in a dose-dependent manner. Altogether, these results indicate that 5-HT1B receptors play a critical role in the rewarding properties of nicotine and further suggest that 5-HT1B receptors may be a novel target for nicotine dependence.

Poster #: 195
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: reward, developmental, addiction
Project Title: Pattern of drug dosing during conditioning affects the expression of alcohol-induced conditioned place preference in adolescent male and female rats
Author List:
Blevins, Kennedy; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Franco, Daniela; Graduate, Psychology, California State University, Long Beach
Brown, Yohanna; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Iñiguez, Sergio; Psychology, University of Texas, El Paso, California State University, Long Beach
Zavala, Arturo; Psychology, California State University, Long Beach

Abstract: Preclinical research examining the rewarding effects of alcohol during adolescence is limited, despite alcohol being the most commonly abused recreational substance during adolescence. Using the conditioned place preference (CPP) paradigm, a validated animal model of drug reward, prior research has shown that the pattern of drug dosing during conditioning is important, with more robust cocaine-CPP being produced using ascending doses of cocaine compared to fixed doses. Similarly, the present study examined whether ascending doses of alcohol during conditioning produced more robust alcohol-induced CPP compared to a fixed dosing regimen during conditioning. Beginning on postnatal day (PD) 31, male and female adolescent rats underwent a 10-day alcohol CPP procedure. On days 1 and 10, rats were tested for their pre-conditioning and post-conditioning place preferences, respectively, during 15-min sessions. On days 2-9, rats were conditioned for 15-min with saline or ethanol on alternating days. During ethanol conditioning days, rats were randomly assigned to receive either ascending alcohol doses (0.0063-2.0 g/kg, intraperitoneally) or fixed alcohol doses (0.5, 1.0, or 2.0 g/kg, intraperitoneally). The results indicated that males exhibited CPP preference using the ascending dose pattern of ethanol compared to fixed dosing pattern, whereas females only exhibited CPP using a fixed high doses (2.0 g/kg) of ethanol. Overall, these results suggest that the pattern of doses used during conditioning sessions may play an important role in elucidating the rewarding effects of alcohol in preclinical research.

Poster #: 196
Campus: CSU Long Beach
Poster Category: Clinical
Keywords: addiction, opioid, development
Project Title: Sex differences in oxycodone-induced conditioned place preference in early adolescent male and female rats
Author List:
Manoogian, Adam; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Brown, Yohanna; Undergraduate, Psychology, California State University, Long Beach
Geraghty, Cassandra; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Iñiguez, Sergio; Psychology, University of Texas, El Paso
Zavala, Arturo; Psychology, California State University, Long Beach

Abstract: Oxycodone abuse among adolescents has increased in recent years. Surprisingly, little preclinical research has been done to establish the effects of oxycodone during this critical period of development. We examined the rewarding effects of oxycodone in male and female adolescent rats using the established conditioned place preference (CPP) paradigm, a model used to assess vulnerability to reward. Male and female rats were assessed for oxycodone-induced CPP using a 10-day CPP procedure beginning on postnatal day (PD) 27. During pre-conditioning (PD 27) and post-conditioning (PD 36) sessions, rats were tested for their baseline and final place preference, respectively, in 15-min sessions. During conditioning (PD 28-35), rats underwent daily 30-min sessions, during which they received alternating oxycodone (0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, or 9.0 mg/kg) and saline injections in distinct compartments. Results indicated that rats showed a significant shift towards the oxycodone-paired compartment at the higher doses, with males requiring a lower dose than females to show a significant shift for the oxycodone-paired side. This data suggests that sex differences in oxycodone reward are evident early in adolescence and that there is a need to understand the neurobiology of oxycodone abuse in adolescence.

Poster #: 197
Campus: CSU San Marcos
Poster Category: Clinical
Keywords: , ,
Project Title: Intrathecal (intraspinal) catheters: a possible adjunct for spinal infections?
Author List:
Shweikeh, Faris; Biological Sciences and Biotechnology, California State University San Marcos, Presenting Author

Abstract: Infections afflicting the Central Nervous System are relatively rare, though recent reports are indicative of a rise in their incidence over the more recent years. Spinal infections in particular are troublesome to treat and result in severe morbidity and mortality. They include spinal epidural abscess, osteomyelitis, and spondylodiscitis. While they may occur de novo (primary), secondary infections following surgery or procedures are more common. The conventional treatment is with antibiotics combined with surgery, if deemed necessary. Intravenous antibiotic therapy can be prolonged for several weeks in some patients with resistant microbes. On the same token, invasive surgery may be necessary to eradicate the infection and could be performed during the immediate hospital admission or even many days or weeks after discharge in other cases. It is proposed that intrathecal (intraspinal) catheter or needle administration of antibiotics as an adjunct treatment may refine the management of this grim condition. A prototype of such a device is introduced as a proof-of-concept in this research. The design of this instrument includes an insertion point, a vessel for delivery of the drug (antimicrobial) therapy, and a storage capsule (reservoir) where the drug is stored for future release. Insertion of the apparatus is via a fine needle using the standard Seldinger technique. Following puncture, insertion, and appropriate positioning of the needle/catheter system, it is fastened robustly in place. The unit is then connected to a reservoir that contains the medication. It allows for timely infusion of the drug so that appropriate dosing is achieved. Infusion of the drug can be performed either manually (by the patient or care giver) or, if to be administered according to a schedule, programmably and automatically. This three component system of inserting securely, a vessel for safe and accurate drug delivery, and a storage reservoir allows for a safe and accurate release of antibiotics or other chemotherapeutics to the spine and surrounding structures, including other areas of the Central Nervous System. Finally, its modifiability and applicability (perhaps to other diseases) garners freedom to both the clinician and the patient. It is hoped that such a treatment device would result in quicker infection clearance, a less invasive option for patient and clinician, and an overall better outcome.

Poster #: 198
Campus: CSU Northridge
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: GPCR DataBase, Computational Biophysics, Structural Bioinformatics
Project Title: An Integrated G Protein Coupled Receptor KnowledgeBase (GPCRKB)
Author List:
Zhao, Benjamin; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Abrol, Ravinder; Chemistry and Biochemistry, California State University, Northridge, Presenting Author

Abstract: G-protein-coupled receptors (GPCRs) are a large family of integral transmembrane proteins that play important roles in a large number of physiological processes. Their profound significance is highlighted by the fact that about half of the clinical drugs target these receptors. Significant advances in uncovering their signaling and atomistic structures is generating a wealth of knowledge about these receptors, which is available in separate databases. There has been a need to create a knowledgebase for these GPCRs that brings together information about their sequences, structures, mutations, signaling, physiology, and disease associations in a single easy to use interface, which can be used by GPCR researchers as well as educators. Currently, other GPCR databases like IUPHAR and GPCRDB contain only subsets of above mentioned knowledge.
We have developed a new integrative knowledgebase on GPCRs called GPCRKB (to be located at www.gpcrkb.org) that brings together their sequences, structures, mutations, signaling, physiology, and disease associations in an easy to navigate as well searchable user friendly interface. GPCRKB was developed using MySQL relational database framework. It brings together sequence information from the UniProt database, structure information from the RCSB protein data bank (PDB), mutation information from NCBI’s dbSNP database, signaling information from IUPHAR Pharmacology database, physiological information from Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and disease information from multiple sources like UniProt, dbSNP, and IUPHAR databases. Each human GPCR has a dedicated GPCRKB page with sections on the protein’s sequence, structure, various mutations along with their frequencies, and signaling pathways with physiological processes. The structure viewer was developed using NGLviewer, which can highlight transmembrane domains, ligand binding sites, GPCR hot-spot residues, and mutations on top of the GPCR structures. GPCRKB provides a useful platform for researchers to generate hypothesis about GPCRs of interest to probe their functional biology.
As future developments, a number of structural bioinformatics and structural biophysics tools are being added to facilitate standard analysis of proteins at the sequence and structure levels as well as to predict structures of GPCRs for which there are no experimental structures available.

Poster #: 199
Campus: San Diego State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: viral metagenomes, motifs, signature genes
Project Title: The use of signature genes and motifs to improve annotations of viruses
Author List:
Turner, M. Ben; Graduate, San Diego State University
Cavalcanti, Giselle ; Postdoc
Papudeshi, Bhavya ; Graduate
Dinsdale, Elizabeth; Faculty

Abstract: Viruses are important in the biogeochemical cycling of the oceans and drive bacterial evolution; however, the majority of viruses cannot be cultivated. Metagenomics, a culture independent method helps in the studying of these undescribed viral communities. The taxonomic and functional characterization of viruses and phages still remains a challenge because a significant proportion of viral metagenomes have low similarity with known viruses. In this study I investigate viral ‘dark matter’ within environmental metagenomes. I aim to improve current viral annotation methods through identification of virus-specific genetic motifs. Studies have identified certain conserved sequences called signature genes in some taxa which could be used to identify the presence of those taxa in unknown sequences. I first utilized these viral taxonomy-inferring signature genes and their corresponding motifs to identify virus families using homology searches by BLAST and MAST. For the reads that did not have homology with the known viral sequences in the databases, I identified de novo motifs using the MEME tool. By implementing these two distinct approaches, I increased the number of annotated viral reads. I was able to decrease the percentage of unknown sequences from 90 % to 70 %. The next aim of this study is to reconstruct near-complete viral genomes from the metagenomic data, through extracting, assembling and binning all the sequences. The motifs that I identified for the specific viral family or genus will be searched in the binned sequences to help with the reconstructions of novel viruses. Overall, my study improves upon current bioinformatics methods in the identification of viral sequences.

Poster #: 200
Campus: San Francisco State University
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: alternative splicing, plants, bioinformatics
Project Title: A Bioinformatics Approach to Uncovering the Role of Alternative Splicing in Plants
Author List:
Nyung, Jeanice; Graduate, Cell and Molecular Biology, San Francisco State University, Presenting Author
Everson, Cameron
Roy, Scott; Cell and Molecular Biology, San Francisco State University

Abstract: Alternative splicing (AS) is a cellular process by which multiple messenger RNA (mRNA) species arise from the selective splicing of introns within a gene. RNA-sequencing (RNA-seq) data analysis of various places has yielded estimates of 61% of intron-containing genes undergoing AS. Despite this prevalence, little is known about AS’s role in transcriptome complexity and genetic regulation in plants. We leveraged the availability of RNA-seq data from diverse plant species and life stages to compare profiles of AS across the plant kingdom. Using an iterative multi-step transcriptomics pipeline, we have performed an exhaustive genome-wide survey to systematically identify AS events in various plants and shed light on the role of AS in plants. This work revealed substantial variation in rate and form of AS across plant lineages, and insights into the functional pathways affected by AS in various lineages.

Poster #: 201
Campus: CSU Fullerton
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: heat-shock proteins, natural variation, molecular evolution
Project Title: Assessing the extend of natural variation of the major chaperone sub-network in humans
Author List:
Chavez, Christina; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Nikolaidis, Nikolas; Biological Science, California State University, Fullerton

Abstract: Elucidating how genetic variation contributes to human evolution, adaptation, and disease predisposition is an overarching goal in modern human genetics. Molecular chaperones as key orchestrators of cellular homeostasis and adaptation are critical for cell health and disease in humans. Therefore, it is of paramount importance to determine how natural mutations alter the function of molecular chaperones and how these changes affect cell survival. However, the impact of the evolutionary process on their function and diversification remains largely unknown. In this study, we determined the natural variation and identified the modes of evolution of an important component of the human chaperone network, composed by Hsp70s, Hsp40s, and BAGs. Specifically, we collected single nucleotide polymorphisms (SNPs) from the 1000Genome and Exome Aggregation Database projects and analyzed those mutations using descriptive statistics and sequence evolution tools. The results can be summarized as follows: (I) the vast majority of the SNPs are rare having a frequency below 5% within humans. (II) In 90% of the genes the number of non-synonymous SNPs (nsSNPs) is significantly higher than the number of synonymous SNPs (sSNPs). (III) Fifty percent of the genes had a significantly higher SNP density than the surrounding genes. (IV) Eighty percent of the genes had significantly higher SNP density within their exonic regions as compared to both intronic and un-translated regions. (V) The majority of genes contained higher proportion of sSNPs than nsSNPs within known functional regions (domains), while the number of sSNPs is similar between domains and non-domain protein regions. (VI) On average only 15% of the genes contained nsSNPs on an amino acid position of known function. (VII) Only 5% of the nsSNPs were predicted to be deleterious and have a functional outcome. (VIII) Calculations of synonymous and non-synonymous distances revealed the action of strong purifying selection, the intensity of which varied dramatically both between and within the gene families with some exceptions in which the analyses supported positive selection. Collectively, these results reveal that these heat-shock genes are very well conserved and suggest that strong purifying selection due to functional constraints shaped their evolution in humans.

Poster #: 202
Campus: CSU Long Beach
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Posture, Center of Pressure, Hurst’s Exponent
Project Title: The Effect of Light Touch on Stability During Quiet Standing
Author List:
Moore, Aisha; Graduate, Computer Science and Computer Engineering, Biomedical Engineering, California State University, Long Beach
Asgari, Shadnaz; Computer Science and Computer Engineering, Biomedical Engineering, California State University, Long Beach
Krishnan, Vennila; Physical Therapy, California State University, Long Beach, Presenting Author

Abstract: Human beings employ multiple strategies to maintain body balance in standing position. One such strategy is light touch, which can be used as a proprioceptive means of maintaining balance. The main objective of this study is to use the Hurst’s exponent (a measure of long-term memory of time series) to quantify the subtle changes that occur with light touch compared to without light touch. To achieve this goal, ground reaction forces from a force plate (AMTI) were collected from 15 healthy adults during quiet standing under the following conditions: eyes open while lightly touching a force sensor (EOLT), eyes closed while lightly touching a force sensor (ECLT), eyes open without light touch (EO), eyes closed without light touch (EC). Three 30-second trials were collected from each condition, and light touch was defined as any force less than 1 Newton. Ground reaction force data was used to calculate the center of pressure (COP) and its Hurst’s exponent (HE). ANOVA results revealed that HE was significantly different between conditions. Post-hoc analysis showed that the HE for ECLT was significantly closer to 1 than the HE for EC (p=0.0006) and EO (p<0.000001). We conclude that compared to traditional analysis, HE analysis would discriminate the subtle postural changes associated with the displacement of COP in healthy adults. This parameter could potentially be employed to discriminate the postural changes associated with aging process and with neurological disorders.

Poster #: 203
Campus: CSU Monterey Bay
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: Cancer, Systems Biology, Cellular Metabolism
Project Title: Classification of Cancer Samples through Inferred Metabolic States using Flux Balance Analysis
Author List:
Hansen, Blake; Undergraduate, Mathematics and Statistics, California State University, Monterey Bay, Presenting Author
Jue, Nathaniel; Biology, California State University, Monterey Bay

Abstract: Cancer is characterized by uncontrolled cell growth as the result DNA damage that compromises the cell cycle. Additionally, cancer is heterogeneous and phenotypic differences are common across cancer types. The classification of different kinds of cancer into separate groups can be used to quickly inform on effective treatment regimes and disease progressions associated with of individual incidences of cancer. Many previous studies use genomic characteristics to classify cancers, both at the DNA and RNA level. However, these methods cannot classify all cancer types and may obscure a true cancer “phenotype” given the complex patterns of transcriptional noise and DNA mutations that underlying shifts in transcriptional profiles. Additionally, many phenotypic changes are not necessarily reflected in gene expression, meaning that many genetically dissimilar cancers may express this phenotype. We propose to use of cell metabolism as a phenotype for improved classification of cancer type and prediction of cellular functions. Previous studies have developed the use of flux balance analysis to predict possible metabolic states. Using CORDA, an algorithm for identifying which of the 7,785 metabolic reactions in general human metabolic reconstruction model, Recon2.2, define cellular metabolic potential, and gene expression data from 50 cancer tumor samples on the Cancer Genome Atlas, we define and classify cancer samples based on model composition and metabolic predictions using Bray-Curtis clustering. Preliminary results indicate that phenotypes based on the composition of metabolic models create more distinct clusters than gene expression data alone. Results of flux balance analysis on the metabolic models may be used to improve the classification of cancer types through further examination of metabolic differences. In the future, we plan to apply these methodologies to single cell RNA-seq data to identify metabolic heterogeneity within tumors and during tumor development.

Poster #: 204
Campus: CSU Fresno
Poster Category: Computational (Bio, Chem, Math, Eng, etc.)
Keywords: neuroscience, behavior,
Project Title: Modeling visual perception, learning, and memory of wood ants navigating in naturalistic environments
Author List:
Lent, David; Biology, California State University, Fresno, Presenting Author
Mendoza, Austin; Undergraduate, Biology, California State University, Fresno

Abstract: Experiments have revealed how visual features are used to guide foraging routes in wood ants. Using these data, we have developed algorithms to extract visual features that ants use for guidance from panoramic scenes. Through simulation we have characterized how visual cues that ants use are extracted, prioritized and stored during navigation. A foraging model was created in MATLAB to simulate navigation in a procedurally generated environment where the visual cues could be precisely characterized. In these environments, our algorithms extracted and stored the visual cues that were available during a single Levy walk foraging event. Following a random foraging event, the success on subsequent foraging bouts using the stored information was examined. When we examined subsequent foraging walks we found the success of the simulated ant in finding the goal location using only a particular cue or a combination of cues depended on two factors – the length of the route and decay rate of information in a memory network. To further explore this we simulated the Levy walk foraging event over various sampling points (100-10000) and implemented linear or exponential decay in the networks storing the information. Our data suggests that the optimal strategy is to sample and store between 1000 and 2000 points along the foraging route, independent of scale, with a network subjected to exponential decay. These parameters resulted in a stored representation that allowed the simulated ant to best find the goal on subsequent foraging bouts. We then produced several novel random foraging walks with the same goal location. The subsequent walks for these foraging events had similar success demonstrating sufficient information was stored and resulted idiosyncratic foraging routes due to the varied information encountered during the random walk. Additionally, we explored how multiple subsequent walks updated and modified memory to produce more robust walks over time. Lastly, we compared the success of subsequent of the model when foraging in sparse and cluttered environments. The results of these simulations have provided insight into the mechanisms involved in prioritization and perception of visual information, it supports that ants need only processes relevant cues intermittently and they do not continually process visual information. Additionally, it has let us investigate how learning and storage of spatial information can be optimized in simple networks and nervous systems.

Poster #: 205
Campus: Stanislaus State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Corrole, Photochemistry, Spectroelectrochemistry
Project Title: Photophysical, Electrochemical, and Spectroelectrochemical Characterization and Solvent Effect on the Tautomerism of Free-Base Corrole
Author List:
Reed, Christopher; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
León, Aliz; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Calvillo, Guadalupe; Undergraduate, Chemistry, California State University, Stanislaus
Kohl, Forrest; Undergraduate, Chemistry, California State University, Stanislaus
Mendez, Suzanne; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Alemán, Elvin A.; Chemistry, California State University, Stanislaus

Abstract: Corroles are aromatic compounds consisting of four pyrrole-like subunits connected in a ring via three methane bridges and one direct bond between the alpha carbons of two of the subunits. They have a structure and photophysical properties similar to that of porphyrins, which are currently being researched in blocking tumor progression and metastasis. In photosynthetic bacteria, tetrapyrrolic molecules are used in light harvesting complexes to channel solar energy towards the reaction complex where photosynthesis occurs. Similar to porphyrins, corroles demonstrate exceptional photophysical properties in regards to their absorbance of light and fluorescence. Corroles exist in two separate tautomeric states causing them to exhibit noticeably different photophysical and electrochemical behaviors in different solvents. A proper understanding of corroles interaction with solvents and how they affect corroles light absorbing and emitting capabilities are necessary to develop potential uses of corrole in artificial photosynthetic devices, petroleum explorations, cancer diagnosis, and tumor treatment.
We investigated the photophysical, electrochemical, and spectroelectrochemical properties of free-base triphenyl corrole (H3TPCor). We have characterized corroles ability to absorb light in various solvents and in a multitude of concentrations. We have also characterized the excited state emission of H3TPCor and the role of solvent in the lifetime of the same excited state. UV-Visible data shows a trend indicating that the transition from one tautomer to another is dominated by the H-bonding donating ability of the solvent molecules with the core of H3TPCor. One tautomer shows four oxidation potentials in the electrochemistry experiments, while the other shows three oxidation potentials. Both tautomers have only one reduction potential. UV-Visible spectra of reduced H3TPCor species all followed the same trend: The results indicate that the reduced species prefers this form irrespective of the solvent used. When oxidized, H3TPCor’s spectrum resembles the spectrum of the most stable tautomer. We have also characterized the free-base corrole with electron withdrawing and donating groups. Preliminary results have shown that many of the same properties that we found for H3TPCor and the functionalized corrole have many similar trends in regards to the solvent effect in the tautomerization equilibrium.

Poster #: 206
Campus: CSU East Bay
Poster Category: Diagnostics/Imaging/Analytical
Keywords: Imaging, Oligonucleotides,
Project Title: Development of a Glutathione Detection Tool to Assess Cellular Oxidative Stress
Author List:
Sin, Davina; Graduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Liu, Dorothy; Undergraduate, Chemistry and Biochemistry, California State University, East Bay
Halim, Marlin; Chemistry and Biochemistry, California State University, East Bay

Abstract: The project goal is to develop a tool for live-cell monitoring of the levels of glutathione, which plays an important role as a cellular antioxidant. Oxidative stress, which is caused by an imbalance in harmful free radical production and cellular anti-oxidative capability, has been implicated in tumor cell proliferation and neurodegeneration. Glutathione helps prevent the damage caused by these free radical species and monitoring its level may provide valuable insights into the cellular health status.
Currently, the most widely used methods for glutathione detection involve cell lysis. We aim to develop a tool that can be used to monitor changes in real time in living cells. Our design uses oligonucleotide strands (also known as aptamers) that can recognize the oxidized and reduced forms of glutathione, and translate their cellular concentrations into optical (fluorescence) signals.
In order to isolate aptamers that bind specifically to glutathione, multiple rounds of a technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was employed. In the first round, starting from a large diverse combinatorial library of aptamers, several steps ensue. These are: 1) incubation of the aptamer library with either the reduced or oxidized glutathione, 2) partitioning of aptamers that bind to the molecule from the rest of the pool, 3) amplification via PCR (Polymerase Chain Reaction) the binding aptamers, and 4) separation of the PCR amplicons into single-stranded aptamers. Using this newly generated product, we perform steps 1 through 4 again. As many rounds as necessary will be repeated until we isolate aptamers with a high binding affinity.
We have completed a total of 45 SELEX rounds for reduced and oxidized glutathione. In each selection round, the binding of aptamers to glutathione was checked by electrophoresis and imaged using ethidium bromide. The amplification yield was quantified by UV absorbance. In all the rounds, we obtained between 35-65 ng/μL of oligonucleotides. Presently, we have isolated aptamers that indicate binding to 5 mM of glutathione.
In conclusion, our data has demonstrated the success of finding aptamers that has an affinity to glutathione in the mid-mM concentration. We will perform additional SELEX rounds to obtain those that can bind to 1 mM glutathione. These aptamers will subsequently be adapted into a tool that can translate the presence of glutathione into light emission.

Poster #: 207
Campus: CSU Los Angeles
Poster Category: Diagnostics/Imaging/Analytical
Keywords: scanning ion conductance microscopy, resistive pulse sensing, atomic force microscopy
Project Title: A hybrid sensing and imaging platform for single cell analysis in biomedical applications
Author List:
Chieng, Andy; Undergraduate, California State University, Los Angeles
Wong Su, Stephanie ; Undergraduate, California State University, Los Angeles
Chang, Megan; Undergraduate, California State University, Los Angeles
Parres-Gold, Jacob; Undergraduate, California State University, Los Angeles
Jehlar, Tristan; Undergraduate, California State University, Los Angeles
Wang, Yixian ; Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author

Abstract: A novel hybrid sensing and imaging platform combining scanning ion conductance microscopy (SICM), atomic force microscopy (AFM) and resistive-pulse sensing has been developed for real-time determination of biological processes at single cell or sub-cellular level in various biomedical applications. SICM and AFM both provide high spatial and temporal resolution in topographic imaging. Moreover, the pipette probe in SICM can be used for localized delivery in a resistive-pulse sensing mode, while AFM can be operated in PinPoint mode to monitor the mechanical change of the cell membrane. We have successfully applied the system for several biomedical applications. The first one is to investigate how alpha-synuclein (α-Syn) and its aggregates affect the neuronal cells for understanding the pathological mechanism of Parkinson’s disease (PD). As the first step, we have successfully detected dramatic disruption of cell membranes caused by α-Syn aggregates in real time. The next step is to further improve the imaging spatial resolution for monitoring the possible pore formation on the membrane. If the pore-forming hypothesis involving a certain form of α-Syn (e.g. oligomers) is demonstrated, our data can help guide the development of drugs that inhibit or reverse the α-Syn aggregation and thus prevent the progression of the disease. The second project is to use the system to demonstrate the fusion of antibacterial liposomes and bacteria and acute bacterial damage. We have observed the morphological change at bacteria caused by added liposomes, and are planning to set up detection for real-time visualizing the fusion process. Elucidating the underlying mechanism would lead to an improved understanding of how multicellular organisms control bacteria. On the other hand, the delivery mode has been demonstrated using 10nm-gold nanoparticles as the model system. We are currently working on adding a surface plasmon resonance detection feature onto the system, which will provide kinetic information of reactions occurring at individual cells, such as small molecules binding to the cell membranes. This project is supported by CSULA Startup Fund and CSUPERB New Investigator Grant.

Poster #: 208
Campus: San Diego State University
Poster Category: Diagnostics/Imaging/Analytical
Keywords: CEA, biomarkers, label-free
Project Title: Ultrasensitive Detection of Cancer Biomarker CEA Using Multi-Photon Nonlinear Laser Wave-Mixing Spectroscopy
Author List:
Suprapto, James; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Pradel, Jean-Sebastien; Graduate, Chemistry and Biochemistry, San Diego State University
Tong, William ; Chemistry and Biochemistry, San Diego State University, Presenting Author

Abstract: Nonlinear laser wave-mixing spectroscopy interfaced to capillary electrophoresis is demonstrated as an ultrasensitive, label-free, antibody-free native detection method for cancer biomarker carcinoembryonic antigen (CEA). Wave mixing offers inherent advantages over conventional methods including zeptomole-level detection and high spatial resolution suitable for single-cell analysis. A single laser was used to generate two input beams that were then focused and mixed inside the sample cell. The input beams created interference gratings, which in turn diffracted incoming photons to produce two coherent laser-like signal beams. The stronger signal beam was collected by a photodetector with high optical collection efficiency and excellent S/N. Since CEA absorbs in the UV wavelength range, a 20 mW 266 nm UV solid-state laser was used to excite the analyte in its native form without the use of labels. The wave-mixing signal has a cubic dependence on laser power and a quadratic dependence on analyte concentration, and hence, small changes in the analyte can be monitored efficiently. A preliminary CEA detection limit of 6.6 fg/mL was determined and it is comparable or better than those of ELISA or fluorescence-based techniques. Wave-mixing detection is applicable to both fluorescing (labeled) and non-fluorescing (label-free) samples and it can be easily configured as battery-powered portable detectors that are suitable for field use. Potential applications include detection of various biomarkers, cancer cells, viruses, single cells and reliable and early detection of diseases.

Poster #: 209
Campus: Cal Poly Pomona
Poster Category: Diagnostics/Imaging/Analytical
Keywords: aptamer, shiga toxin, food safety
Project Title: Selection of DNA aptamers targeting shiga toxin type 1
Author List:
Chen, Andrew; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Garrett, Michael; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Angeloni, Shawnee; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona
Liu, Junjun; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Shiga toxins (Stx) are virulence factors produced by Shiga toxin-producing E. coli (STEC) that are sometimes found in food. Infection with STEC causes life threatening hemolytic uremic syndrome. Stx is produced by O157:H7 STEC and many other non-O157 STEC serotypes. Current contamination management focuses on detecting each individual serotype of STEC, which is very inefficient. A more effective way to ensure food safety would be detecting the toxins rather than the serotypes of the bacteria. There are two main types of Stx, Stx1 and Stx2, and the latter is more virulent. The effective detection of Stx in foods usually includes rapid on-site screening followed by laboratory confirmation. The rapid methods include instrument-based approaches and antibody based immunoassays. The instrument-based methods require expensive equipment, trained personnel as well as costly and time-consuming pretreatments. Antibody based immunoassays are widely used due to their rapidity and simplicity. However, these methods heavily rely on antibodies, which may encounter some drawbacks due to the cost and difficulty associated with production, limited stability, and the complexity of modification of suitable antibodies.
Recently, aptamers have emerged as a promising alternative to antibodies. DNA aptamers are short, synthetic, single stranded DNA with unique 3-dimensional conformations, which bind to a target molecule with high specificity and affinity. Aptamers are cheaper to produce, have higher specificity, are easier to label for different applications, have much longer shelf lives, and thus, are far more cost effective and are considered ideal alternatives to antibodies in food safety test. By using a method called systematic evolution of ligands by exponential enrichment (SELEX), we have successfully selected a DNA aptamer that specifically binds to Stx1. In a direct ELISA, the aptamer was able to detect as little as 100 ng of recombinant Stx1, and the sensitivity could be improved further if the sample is cultured in enrichment media before the test. The selection of aptamers targeting Stx2 is underway. In conclusion, we have selected a DNA aptamer that could be used in food safety test for rapid, specific and sensitive detection of Stx1. The project was supported by a grant from California Sate University Agriculture Research Institute (ARI).

Poster #: 210
Campus: CSU San Bernardino
Poster Category: Disease (Pathogens)
Keywords: Influenza virus , siRNA, export
Project Title: Utilizing siRNA to Evaluate the Importance of Host Factors in Influenza Replication to Identify Novel Therapeutic Targets
Author List:
Thompson, Michael; Graduate, Biology, California State University, San Bernardino, Presenting Author
Ortiz-Simon, Alicia; Undergraduate, Biology, California State University, San Bernardino
Medina, Pedro; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Heredia Valdovinos, Yolanda; Undergraduate, Biology, California State University, San Bernardino
Newcomb, Laura ; Biology, California State University, San Bernardino

Abstract: Influenza virus is the causative agent of seasonal flu with significant health and economic impacts worldwide. Current treatments include annual vaccination, which has variable efficacy, and antivirals, which become ineffective with use due to selection of resistance. Host proteins are integral for viral replication; therefore, a novel approach to treat viral infection centers on targeting host proteins that play an essential role in viral replication but a redundant role for the host cell. Here we use siRNA-mediated knockdown to determine the importance of host factors on influenza viral RNA expression to establish if they may be targets for development of novel antiviral therapies. Tissue culture cells were transfected with targeted siRNA SMARTpools (Dharmacon) or non-target siRNA control for 48 hours. Cells were then infected at high multiplicity of infection with influenza A virus (Udorn H3N2) or mock treated for 4 hours and collected for RNA isolation. RNA concentration was determined by optical density and integrity by observation of rRNA resolved by gel electrophoresis. Equal concentration of RNA was reverse transcribed with oligo dT and qPCR performed with specific primers to assess relative abundance of target gene and influenza mRNAs. Our results in A549 cells corroborate the importance of host nuclear mRNA export factor Nxf1 as essential for viral replication, and reveal novel results for the role of host microRNA nuclear export factor Xpo5. Targeted knockdown of Nxf1 results in a severe decrease in viral RNA expression while targeted knockdown of Xpo5 results in a massive increase in viral RNA expression. We also performed targeted knockdown of UAP56 and URH49, paralog cellular DEAD box RNA helicases implicated in efficient influenza replication. Our results in A549 cells reveal URH49 siRNA targets the DDX family, resulting in inhibition of both UAP56 and URH49, and inhibiting viral RNA as expected. UAP56 siRNA treatment resulted not only in UAP56 knockdown and a decrease in viral RNA, but interestingly, increased URH49 RNA expression. To understand if our results are related to host cellular responses to viral infection such as the innate antiviral IFN pathway, we have repeated these experiments in Vero cells which lack IFN. RNA analysis is currently underway.
This research is funded by CSUPERB Research Development Grant to LLN.

Poster #: 211
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Trypanosoma brucei, mechanosensitive channels, endogenous tagging
Project Title: Endogenous localization of a mechanosensitive channel in the human protozoan parasite Trypanosoma brucei.
Author List:
Hernandez, Monica; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Nguyen, Kristy; Undergraduate, Biological Science, California State University, Fullerton
Dave, Noopur; Graduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Biological Science, California State University, Fullerton

Abstract: Trypanosoma brucei (T. brucei) causes devastating disease in humans and livestock across many regions of Africa. It is a protozoan parasite transmitted by the tsetse fly and the causative agent of African Sleeping Sickness in humans. Once the parasite has established infection in the host it can pass the blood brain barrier causing important psychological and neurological symptoms. At this stage, death is inevitable. Current treatment of the disease requires intravenous administration and is associated with severe side effects, including seizures. Only one medication is FDA approved increasing the need for new therapeutic options. In bacteria, mechanosensitive channels (MSCs) have been shown to contribute to virulence by regulating cell response during times of stress such as changes in osmotic pressure. They also play a role in other mechanosensory mechanisms such as social motility and quorum sensing, important for regulating the size of the parasite population. Screening of T. brucei’s genome revealed a putative mechanosensitive channel, TbMcS. TbMcS shares 64% identity with a characterized MscS-like in Trypanosoma cruzi and 31% identity with the E. coli small conductance MSC (MscS). In E. coli, this channel regulates cell response during osmotic stress. TbMcS may therefore play a similar role in T. brucei and participate in other mechanosensory mechanisms contributing to its virulence. We propose that TbMcS is a mechanosensitive channel important for regulating physiological responses like osmotic compensation, quorum sensing and social motility. To localize the channel and establish its physiological role we have incorporated a C-terminal 3xmyc tag by homologous recombination in the endogenous locus of the gene, rendering expression controlled by its native promoter. Our findings indicate that the channel is localized in the membrane of the procyclic forms of the parasites suggesting the channel plays a role in detection of mechanical cues. Ongoing RNAi experiments will provide insight about the role of TbMcS in signaling cascades during mechanical and physiological stress. There are currently no identified MscS-like channels in humans, making TcMcS a promising drug target against an economic and medical burdensome disease.
Funding: CSUPERB New Investigator grant.

Poster #: 212
Campus: CSU Fresno
Poster Category: Disease (Pathogens)
Keywords: klebsiella, biofilm,
Project Title: The role of spheroplast formation in antibiotic tolerance of Klebsiella pneumoniae biofilms
Author List:
Cunningham, Christian; Undergraduate, Biology, California State University, Fresno, Presenting Author
Okonkwo, Justin; Undergraduate, Biology, California State University, Fresno, Presenting Author
Van Laar, Tricia; Biology, California State University, Fresno

Abstract: Klebsiella pneumoniae is a multidrug resistant bacterium commonly found in nosocomial infections. Previous research in our lab has demonstrated that K. pneumoniae cells within biofilms form spheroplasts under carbapenem antibiotic stress. Carbapenems are last-resort beta-lactams used to inhibit cell wall synthesis. Normally, the lack of a cell wall will lead to death from osmotic pressure, but some cells have shown tolerance and recovery when found within the biofilm. We performed RNA sequencing of carbapenem-treated biofilms and identified many differentially expressed genes. A potentially interesting gene that is upregulated during this stress response is spy. This gene is predicted to encode a homolog of a periplasmic, ATP-independent chaperone protein that is upregulated during spheroplast formation. Our hypothesis is that a spy mutant will be defective for spheroplast formation and therefore reduce K. pneumoniae antibiotic tolerance within a biofilm. We have obtained a transposon mutant in spy and used a crystal violet assay to determine that the mutant forms an equivalent biofilm to the wild-type strain. We identified that the minimum inhibitory concentration (MIC) of the carbapenem antibiotic meropenem was similar for both the spy mutant and wild-type strains. We then determined whether the defect in spy had an effect on biofilm formation and found no difference in biomass production between wild-type and mutant strains. We then used meropenem at increasing concentrations and determined whether there was disruption of preformed biofilms. We found no difference in biomass between the wild-type and spy mutant biofilms when treated with meropenem suggesting that a lack of spy itself does not affect antibiotic tolerance within the biofilm. We are now in the process of determining whether the spy mutant is defective for spheroplast formation using electron microscopy. We will continue to analyze spy and its potential importance for antibiotic tolerance as well as examine other genes that could be involved in stabilizing spheroplasts and promoting antibiotic tolerance within the biofilm.

Poster #: 213
Campus: CSU Long Beach
Poster Category: Disease (Pathogens)
Keywords: Toxoplasma gondii, Calcium, Parasitism
Project Title: Characterization of putative calcium-binding proteins in the human parasite Toxoplasma gondii
Author List:
Meepe, Imara; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Sandoval-Olmos, Dalia; Undergraduate, Biological Sciences, California State University, Long Beach
Chetsawang, Jason; Undergraduate, Biological Sciences, California State University, Long Beach
Pace, Douglas; Biological Sciences, California State University, Long Beach

Abstract: Toxoplasma gondii is an Apicomplexan obligate intracellular parasite which produces the disease toxoplasmosis. The reliance of T. gondii on calcium (Ca2+) for the initiation of invasion-linked traits is well established, yet many of the molecular mechanisms by which T. gondii regulates Ca2+ remain unknown. This study seeks to explore the roles of putative Calcium Binding Proteins (CBP1 and CBP2) as mechanisms for regulating Ca2+ flux during the tachyzoite lytic cycle. CBP1, which contains 2 EF hand domains and 4 transmembrane domains, exhibited stage-dependent changes in cellular localization. During the intracellular stage it localized in the apical cytoplasm of the parasite. In extracellular stages, CBP1 localized to the limiting membrane of the Plant-like Vacuole (PLV), an important site of protein maturation and Ca2+ storage. Intracellular Ca2+ regulation (measured using the ratiometric probe FURA2-AM) in CBP1 over-expressing (OE) mutants revealed an elevated cytosolic Ca2+ concentration when compared with parental RH parasites. Furthermore, induction of cytosolic Ca2+ influx, either through thapsigargin treatment (which inhibits the SERCA) or activation of extracellular Ca2+ entry pathways, was diminished in OE mutants. This response is presumably due to the extra buffering capacity resulting from the over-expression of CBP1. CBP2, a soluble protein containing 2 EF hand domains, localized in the apical cytoplasm of the parasite during the extracellular stage. GIEMSA invasion efficiency experiments, which determine the ratio of parasitophorous vacuoles formed to host cell nuclei, revealed divergent responses in the two OE mutants. Compared with parental strains, CBP1-OE exhibited significant (Student’s t-test, p<0.01) reduction in invasion efficiency, whereas CBP2-OE mutants displayed significant (Student’s t-test, p<0.05) enhancement in invasion efficiency. Overall, our results suggest that these CBPs may have Ca2+ buffering capacities and likely play distinct roles during the lytic cycle based on their localization. Currently, we are endogenously tagging CBP1 and CBP2, as well as generating knockout mutants utilizing the CRISPR/Cas9 system. Full characterization of these CBPs will elucidate their biological importance during the lytic cycle and their potential as targets for therapeutic strategies in combatting infection.

Poster #: 214
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Microbiome shift, Long chain fatty acids, Acne
Project Title: Microbiome-Free Fatty Acid Cross-Talk in the Human Pilosebaceous Gland and Consequences for Colonization Resistance
Author List:
Valdez, Nico; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Fuentes, Jaime; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: The outgrowth of Cutibacterium acnes in sebaceous glands is a hallmark of inflammatory skin disorders including acne, yet the underlying mechanisms remain largely uncharacterized. Of particular importance during shifts to dysbiosis are unknown pathways of host-microbiome crosstalk and epigenetic regulation. To that end, we hypothesize reciprocal interactions in which microbial-induced free fatty acid (FFA) production by sebocytes reduces colonization resistance to infection as resident species of the skin microbiota secrete an abnormal array of metabolites into the microenvironment. Previous work in our lab established a novel co-culture assay in which primary sebaceous cells obtained from human donors at the UC Davis Medical Center produced significantly more of the lipogenesis regulatory factors mTOR and AKT following exposure to live C. acnes relative to uninoculated controls, heat killed bacteria, or filtered bacteria supernatants. Here we used silencing RNA inhibition and confocal laser scanning electron microscopy to elucidate Toll-like receptor 2 as the host ligand mediating bacterial-induced lipid synthesis. A corresponding increase in inflammatory cytokines and decrease in antimicrobial peptide production was determined by quantitative real-time PCR using RNA extracted from sebocytes exposed to live cultures of C. acnes. To elucidate the identities of bacterial-induced FFA fractions, we performed gas chromatography-mass spectroscopy analysis. Our results show that the predominant lipids purified from co-culture supernatants are long chain derivatives C:12, C:14, C:16, and C18. Interestingly, a similar profile was elucidated following analysis by the same methods of tissues from acne patients. When applied to commensal or pathogenic Staphylococcus species, long chain FFAs differentially increased or decreased growth rates and minimal inhibitory concentrations, suggesting species-specific mechanisms of selection that alter colonization resistance. Interestingly, RNA sequencing analysis and metabolomics profiling showed that bacteria exposed to FFAs produced by sebocytes have an altered transcriptome and metabolite profile compared to organisms grown in isolated broth cultures. Collectively, our results are beginning to elucidate portions of a novel feed-forward mechanism. A better understanding of the interplay at the host-microbiome interface promises to elucidate novel therapeutic approaches for treating inflammatory skin disorders.

Poster #: 215
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: aerosol, liposomes, amphotericin B
Project Title: Aerosolization of Liposomal Amphotericin B (AmBisome) Maintains Antifungal Drug Concentrations in Lungs for up to Five Days Post-Treatment
Author List:
Dave, Janam; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author, Nagel Award Finalist
Olson, Jon; Biological Sciences, California State Polytechnic University, Pomona
Adler-Moore, Jill; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Background: In immunocompromised patients, Aspergillus causes severe pneumonia with 50-90% mortality in untreated patients and 50% mortality even with treatment. The high patient mortality indicates that treatment for this disease needs to be improved. Although AmBisome (AmBi) a potent antifungal drug,is used intravenously (IV) to treat this infection, aerosol AmBi treatment has the potential to deliver the drug to the lungs at greater concentrations and be used alone or in combination with IV AmBi for treatment. We developed an aerosol delivery system for AmBi and evaluated its pharmacokinetics in mice to determine baseline drug levels for future treatment of pulmonary aspergillosis.
Methods: AmBi (1.33 mg/mL) was nebulized with a Schuco S5000 nebulizer and delivered to mice in a compartmentalized, isolation chamber. Swiss-Webster female mice received one, two or three daily, twenty-minute aerosol treatments (12 mice/treatment group). Mice (n=4/treatment group/timepoint) were sacrificed at 24,72 or 120 hours after their last aerosol treatment. The lungs, livers, kidneys and spleens were collected, homogenized, and amphotericin B extracted from the tissues via methanol, and analyzed for drug concentration via bioassay using AmBi standards and Candida albicans as the indicator organism.
Results: The aerosol system was successful in delivering at least the MIC level (2-4 μg/g) for Aspergillus spp. in the lungs of all treatment groups at all timepoints. The mean lung drug concentration after 24h was 17.7, 5.34, and 3.64 μg/g for three, two or one treatments, respectively, and the concentration decreased over time to 11.13, 3.04, and 3.69 μg/g, respectively, after 120h. None of the kidneys and livers from any treatment group contained detectable levels of amphotericin B. Only the spleen had detectable drug levels, but only at 120h after one, two or three treatments (1.53, 1.64 and 3.33 μg/g, respectively).
Conclusions: Aerosol delivery after three treatments with AmBi achieved lung drug concentrations well above the MIC for many Aspergillus spp. (17.7 vs 2-4 μg/g). This aerosol regimen will be used to investigate its potential to treat pulmonary aspergillosis. The low concentrations of amphotericin B in the kidneys and livers is advantageous as AmBi can be associated with some limited nephrotoxicity and hepatotoxicity.

Poster #: 216
Campus: San José State University
Poster Category: Disease (Pathogens)
Keywords: Breast cancer, Walnut Extract, Doxorubicin and Paclitaxel
Project Title: Determining synergistic effects between doxorubicin or paclitaxel with a walnut extract in human breast cancer cells
Author List:
Khalsa, Gurbhej; Undergraduate, Biological Sciences, San José State University, Presenting Author
White, Brandon; Biological Sciences, San José State University

Abstract: Background: The most common treatment for human breast cancer involves the use of chemotherapeutics. However, these treatments are also accompanied by various side effects as well decreased efficacy sometimes resulting in reoccurrence of the cancer. Previous research has shown that combinations of chemotherapeutics show improved efficacy at lower doses. Consumption of fruits, vegetables, and nuts has been shown to reduce cancer incidence as well as exert anti-tumorigenic activities both in vivo and in vitro. Our lab has been studying how an extract created from walnuts can target breast cancer. We have shown that a walnut extract (WE) can target breast cancer cells to induce apoptosis. However, the extracts only work at large concentrations that might not be viable as a potential therapy alone. The goal of this study is to determine if there are any synergistic effects between a WE and a commonly used chemotherapeutic agents for treating breast cancer, Doxorubicin (Dox) or Paclitaxel (Pac).
Methods: MDA-MB-231 cells were grown in Dulbecco’s modified Eagle medium with 10% Fetal Bovine Serum. The cells were seeded into 96-well plates at 25,000 cells/well and incubated for 24 hours and then treated in triplicate with WE, Dox, Pac or various combinations. The cells were then incubated for 72 hours and cell viability assays were performed using Cell Titer Glo following the recommended protocol. Percent cell death was normalized to DMSO treated cells. The Combination Index (CI)-Isobologram developed by Chao and Talalay was used to analyze the combination cell viability assays.
Results: Consistent with previous published work, treatment of MDA-MB-231 cells with Pac (100µM-0.0001µM), Dox (100µM-0.001µM), and WE (1-0.0325 mg/ml) resulted in cell death in a dose dependent manner. Combinations of WE, Dox and Pac were tested for synergistic effect. Starting combinations of WE and Pac at 1:100, 1:50, and 1:25 (mg/ml:µM) and two-fold serially diluted displayed antagonistic effects (CI> 1) or antagonistic effect (CI > 1). Combinations of WE and Dox at ratios at 1:100, 1:50, and 1:25 (mg/ml:µM) and two-fold serially diluted displayed synergistic effects (CI < 1.0).
Conclusion: Together these data suggest that combinations of WE and Pac do not display synergistic effect or sufficient additive effect for the treatment of human breast carcinomas. However, results from combinations of WE and Dox in MDA-MB-231 cell lines suggest that there is a synergistic effect.

Poster #: 217
Campus: CSU Northridge
Poster Category: Disease (Pathogens)
Keywords: chemotherapy, terpyridine, intercalator
Project Title: Anti-tumor activity of ruthenium terpyridine DNA intercalators
Author List:
Hernandez, Sergio; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Melgar, Estelli; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Kelson, Eric; Chemistry and Biochemistry, California State University, Northridge

Abstract: Tpys (2,2’:6’,2”-Terpyridines) have been reported to be promising anti-tumor agents that intercalate DNA and block replication and transcription. Tpys also exhibit a promising selectivity for tumors that likely exploit an active means of transport into cancer cells. The Kelson group has prepared Tpys modified with intercalating groups and ruthenium complexes bearing them. These products have been shown to strongly intercalate DNA with the cationic ruthenium complexes being the most effective. We report here a toxicity study of three promising ruthenium complexes as anti-tumor agents: (AnthTpy)Ru(Bpy)Cl+ (Complex 1) [where AnthTpy=4’-(9-phenanthryl)-2,2’:6’,2”-terpyridine and Bpy=2,2’-bipyridine], (AnthTpy)RuCl2(DMSO) (Complex 2) (where DMSO=dimethylsulfoxide), and (Tpy)Ru(Dppz)Cl(+) (Complex 3) (where Dppz= dipyrido[3,2-a:2′,3′-c]phenazine). All three complexes are strong DNA intercalators designed to enter cells by the same transport mechanisms as Tpy. In addition to this, Complex 2 could diffuse into cells as a neutral compound and become cationic by chloride dissociation within the cell. In a typical experiment, the cell lines were cultured in well plates, dosed with 100 µM to 5.0 µM of Complex 1, 2, or 3 in OptiMEM, incubated for 48 hours, and tested for cell viability by a standard colorimetric assay (MTT). In initial experiments, these complexes exhibited relatively low toxicity (requiring 75 µM or more to kill 50% of a culture). In analogy to Tpy, the toxicity of all three complexes improved when cytochrome P450 detoxification was inhibited with 1.0 mM cimetidine. The addition of 25 µM chloroquine amplified the effect resulting in toxicity at 20 µM, 40 µM and 20 µM for Complexes 1, 2, and 3, respectively. The results suggest that Complexes 1, 2 and 3 enter HepG2 cells by endocytosis and are released into the cytoplasm when endosome pH is raised by chloroquine. Overall, these results confirm Complexes 1, 2, and 3 have anti-tumor activity and are likely entering HepG2 tumor cells by an active process (endocytosis). We hope this work will eventually lead to selective anti-tumor agents without the traditional side effects. We thank CSUPERB (Research Development Grant), the NIH (SCORE S06 GM48680), ACS Petroleum Research Fund, and CSUN Office of Sponsored Projects for support of this work.

Poster #: 218
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: biofilm inhibition, sulfated polysaccharides, P. aeruginosa and S. aureus
Project Title: The Effects of Sulfated Carbohydrates on Bacterial Biofilm Formation
Author List:
Suasin, RJ; Undergraduate, Chemistry, California State University, Sacramento, Presenting Author
Varela, Adriel; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Crawford, Robert; Biological Sciences, California State University, Sacramento
McReynolds, Katherine; Chemistry, California State University, Sacramento

Abstract: Antimicrobial resistance is a growing concern for global public health. As a result of resistance to antibiotics, treatments for infections become ineffective and can prolong infections. In a chronic wound, the body can’t heal properly due to the presence of biofilm embedded skin pathogens. The biofilm is a protective extracellular matrix that allows the bacteria to resist antimicrobials as well as actions by the host immune system. An alternative approach to treating infections with antibiotics is to inhibit the biofilm production of these bacteria. By targeting the pathogenesis of biofilm formation, bacterial infections become less severe and can heal properly.
Using microtiter biofilm assays and kinetic growth curves we screened various carbohydrates such as dextran sulfate, heparin sulfate, colominic acid, and sulfated colominic acid for their ability to interfere with biofilm formation. We tested 5, 50, and 500 µg/ml concentrations of each polysaccharide on Staphylococcus aureus and Pseudomonas aeruginosa which represent Gram positive and negative skin pathogens, respectively. In the kinetic growth curves, we do not see a significant increase or decrease in growth of S. aureus or P. aeruginosa when treated with dextran sulfate, heparin sulfate, colominic acid, and sulfated colominic acid. This would suggest that these sugars are not bactericidal, or being used as a carbon source. This is ideal for non-selective pressure, which may reduce the ability of resistance.
We have also found that dextran sulfate and heparin sulfate increase the ability of S. aureus and P. aeruginosa to form biofilms in a concentration dependent manner. Heparin sulfate is more effective at increasing biofilm formation than dextran sulfate. We also were able to demonstrate that sulfated colominic acid reduces biofilm activity, while colominic acid does not have an effect on biofilm activity. Sulfated colominic acid is able to reduce S. aureus biofilm formation by up to 20%, and was able to greatly diminish P. aeruginosa biofilms by up to 70%. This was confirmed qualitatively by growing these bacteria on collagen coated chamber slides, probing them with fluorescent labels, and analyzing them using confocal microscopy. Resulting confocal images showed a reduction in biofilm surface area correlated with increasing concentrations of sulfated colominic acid. This suggests that sulfated colominic acid is a promising inhibitor of bacterial biofilm formation.

Poster #: 219
Campus: CSU Sacramento
Poster Category: Disease (Pathogens)
Keywords: Microbiome, Next Generation Sequencing, Fluorescence In-Situ Hybridization
Project Title: Characterizing Shifts in Microbiome Biogeography Mediated by Inflammatory Skin Disorders
Author List:
Dominic, Natalie; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Truong, Catalina; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author

Abstract: Interest in studying shifting roles for microbial diversity and function in health and dysbiosis has emerged thanks in part to novel technologies facilitating the elucidation of transcriptomic and phylogenetic signatures of the human microbiome. One clinical characterization remaining largely unknown is the composition of microbes and microbial-generated metabolites in sebaceous glands associated with inflammatory skin disorders such as acne and rosacea, which are commonly treated by topical administration of antibiotics and steroids. Here we use next generation high throughput metagenomic sequencing (NGS) and fluorescence in-situ hybridization (FISH) to identify species diversity and functional gene clusters in follicular casts obtained from human subjects at the UC Davis Medical Center. Briefly, affected and control tissues were obtained from patients with acne or rosacea using strips that extract 6 – 8 deep skin follicles per sampling event. The total microbial genomic DNA was extracted from follicular pools for each grouping of inflamed tissue and analyzed for relative phylogenetic abundance and distribution of bacterial operons mediating critical metabolic functions. Our results show an overrepresentation of Cutibacterium acnes, Corynebacterium granulosum, Staphylococcus aureus, Propionibacterium humerusii, and Propionibacterium avidum at inflamed versus control sampling sites. Among the intriguing genes implicated by our findings toward colonizing this lipid-rich niche include those mediating ammonia oxidation and long chain fatty acid synthesis. Additionally, we developed and extensively optimized DNA probes to visualize microbiome biogeography by confocal scanning laser microscopy within fixed follicle tissues using FISH analysis. Our results confirm the microbial diversity observed by genomic sequencing methodologies and present one of the first clinical depictions of shifting microbiota in deep follicle tissues affected by inflammatory skin disorders. Follow-up studies in our lab include model development and testing of the pre- and pro-biotic potential for key metabolites and microbiota species as mechanisms to restore health-associated phylogeny and metabolic landscapes to affected areas. A better understanding of microbial biogeography using long-term longitudinal studies promises to elucidate novel non-antibiotic and non-steroid treatment strategies for combatting microbiome shifts in acne, rosacea, and related dysbioses.

Poster #: 220
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Parasites, calcium, CRISPR/Cas9
Project Title: Role of a mechanosensitive channel in Trypanosoma cruzi calcium homeostasis
Author List:
Tiwari, Megna; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Dave, Noopur; Graduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Biological Science, California State University, Fullerton

Abstract: In order to complete its life cycle, Trypanosoma cruzi-the protozoan parasite that causes Chagas disease- must adapt to drastic environmental changes as it moves from an insect vector to a mammalian host. However, little is understood about the mechanisms utilized by T. cruzi and other protozoan parasites to sense these changes.
We have identified and characterized a bacterial-like mechanosensitive channel in T. cruzi (TcMcS). TcMcS, has shown to participate in sensing environmental changes in the parasite by mediating the flow of ions and osmolytes across membranes. Previous studies in several human-disease related protozoans, including T. cruzi, show that calcium is a major signaling molecule that regulates multiple physiological processes in the parasites including motility, replication, invasion and differentiation.
We have developed epimastigote cell lines in which TcMcS has been knocked down or knocked out by CRISPR/Cas 9 gene targeting. To investigate the intracellular levels of calcium by fluorometry, we have loaded the parasites with Fluo4-AM and analyzed the intracellular calcium concentration under normal conditions or in response to different external stimuli. Our results show a decrease in intracellular levels of calcium in knockout strains when compared to wild type parasites, as well as a difference in the dynamic of the calcium responses. These findings support the hypothesis that TcMcS plays an important role in intracellular calcium homeostasis in T. cruzi, and that its activation could be linked to calcium signaling pathways.
Small conductance mechanosensitive channels such as TcMcS are not found in mammalian host cells, making them a suitable target for development of new therapeutics that could minimize detrimental side effects observed with the currently available drugs to treat Chagas disease.
Funding: CSUPERB New Investigator grant, AHA 16GRNT30280014 and NIH R15 AI122153.

Poster #: 221
Campus: CSU Fullerton
Poster Category: Disease (Pathogens)
Keywords: Trypanosoma cruzi, CRISPR/Cas9, Mechanosensation
Project Title: CRISPR/Cas9 mediated endogenous tagging of Trypanosoma cruzi mechanosensitive channel TcMcS
Author List:
Ghebrial, Michael; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Hernandez, Monica ; Graduate, Biological Science, California State University, Fullerton
Jimenez, Veronica; Biological Science, California State University, Fullerton

Abstract: Trypanosoma cruzi is the parasite that causes Chagas disease, or American Trypanosomiasis, in humans. This parasite is found in insects from the Triatominae family, also known as kissing bugs. T. cruzi is transmitted to mammalian hosts during the kissing bug’s blood-feeding process. Insect vector to mammalian host transfer involves drastic changes in pH and osmotic conditions. The parasite’s ability to survive these environmental stressors is attributed, in part, to the presence of a bacterial-like mechanosensitive channels (TcMcS). In order to establish the localization of the channel in the three life stages of the parasite, we have tagged the protein using CRISPR/Cas9 methods. Because trypanosomes do not possess non-homologous end joining DNA repair systems, the double-strand DNA cut caused by the Cas9 nuclease activity is used to force repair by homologous recombination. We have designed a DNA template containing a 3xHA tag incorporated at the position of the stop codon, followed by a hygromycin resistance cassette. A CRISPR/Cas9/sgRNA all-in-one plasmid and the DNA repair template were transfected using electroporation and the parasites were selected with neomycin and hygromycin. We used immunostaining and fluorescence microscopy to observe the localization of these channels in the parasites. Our next step is to use antibodies against the 3xHA tag for immunoprecipitation assays followed by mass spectrometry, in order to identify proteins interacting with TcMcS. These studies will allow us to establish the molecular mechanism and signaling pathways by which TcMsS mediates compensatory responses against environmental stressors.
Funding: AHA 16GRNT30280014 and NIH R15 AI122153

Poster #: 222
Campus: CSU Long Beach
Poster Category: Disease (Pathogens)
Keywords: Toxoplasma gondii, protein synthesis, parasitism
Project Title: Determining the rates and importance of protein synthesis during the lytic cycle of the intracellular human parasite Toxoplasma gondii
Author List:
Monahan, Colleen; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Salladay, Ivan; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Pace, Douglas; Biological Sciences, California State University, Long Beach

Abstract: Toxoplasma gondii is an obligate intracellular parasite member of the Apicomplexan phylum. During its lytic cycle, T. gondii invades host cells as extracellular tachyzoites and subsequently replicates inside host cells as intracellular tachyzoites. Various critical proteins guide the invasion process (i.e., invasion-linked proteins). Although the functions of many of these proteins have been determined, their quantitative expression and timing of synthesis is unknown. This study aims to 1) determine whether proteins regulating parasite invasion of host cells are synthesized in the intracellular or extracellular stages of the lytic cycle and 2) measure absolute rates of protein synthesis during these stages. To investigate objective 1, Giemsa counterstaining invasion assays were performed in parasites exposed to the protein synthesis inhibitor emetine. Extracellular parasites treated with emetine (100 μM) showed no significant inhibition of invasion (N=3, One-way ANOVA, p>0.50), while intracellular parasites treated with a much lower concentration of emetine (10μM) exhibited diminished invasion (N=3, Student’s T-test, P<0.05). While preliminary, these results imply that protein synthesis is more sensitive to pharmacological agents in the intracellular stage. The observed decrease in invasion also suggests that invasion-linked proteins may be synthesized in this stage, prior to host cell egress and invasion. To connect the overall inhibition of protein synthesis to invasion-linked proteins, the expression levels of invasion-specific and non-specific proteins will be quantified. Additionally, a strong dependence of parasite replication on host cell protein synthesis was observed. When host cells exposed to emetine (100µM) were challenged with parasites, parasite invasion was found, however no parasite replication was observed over the course of 4 days (N=3, One-way ANOVA, p<0.001). Measurements of absolute rates of protein synthesis (objective 2), as well as metabolic cost, are currently being performed. Understanding parasite protein metabolism will provide a more comprehensive understanding of invasion proteins, their dependence on host cell protein synthesis, and their sensitivity to pharmacological agents. Ultimately, this may be useful in the treatment of T. gondii infection.

Poster #: 223
Campus: Cal Poly Pomona
Poster Category: Disease (Pathogens)
Keywords: Garlic, Macrophages, Candida albicans
Project Title: Garlic’s (Allium sativum) effects on lipopolysaccharide or Candida albicans stimulated tumor necrosis factor-α secretion from varying macrophage types
Author List:
Soto, Benjamin; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author, Nagel Award Nominee
Buckley, Nancy; Biological Sciences, California State Polytechnic University, Pomona

Abstract: For centuries many societies have hailed garlic (Allium sativum) as a remedy for various types of ailments. One proposed target of garlic’s biological actions is the immune system which consists of cells such as macrophages. Macrophages destroy pathogens and release signaling molecules known as cytokines. Cytokines are proteins used by immune cells to direct an immune response. One important cytokine used by macrophages is tumor necrosis factor-alpha (TNF-α). It was recently found in our lab that in J774A.1 (J7) murine macrophages, garlic and allicin, the major component of crushed garlic, stimulate lipopolysaccharide (LPS) induced TNF-α production. In our laboratory, we have also found that garlic decreases Candida albicans (C. albicans)-induced TNF-α production from J7 cells. LPS is an outer cell membrane component of gram negative bacteria and Candida albicans (C. albicans) is an important opportunistic fungal pathogen, and they both stimulate macrophages via different receptors. Contrary to our findings in J7s, other labs have reported that garlic reduces LPS-induced TNF-α production from RAW 264.7 (RAW) murine macrophages. However, no one has reported on the effects of garlic on C. albicans-induced TNF-α production in RAW or other cell types. Thus, we set out to do a comparative study of the effects of garlic using three different cell types. We used J7, RAW and thioglycollate-induced macrophages derived from male and female mice. The cells were treated with an aqueous garlic preparation in the presence or absence of LPS or C. albicans. Twenty four hours after treatment, cell supernatants were collected and analyzed by Enzyme Linked Immunosorbent Assay (ELISA) to quantify TNF-α secretion. In garlic treated cells, LPS stimulated J7 cells and male and female thioglycollate-derived macrophages, TNF-α secretion levels were increased, but no significant change was observed in RAW cells under these conditions. In garlic treated, C. albicans stimulated J7, RAW and male and female thioglycollate-derived macrophages TNF-α secretion levels were decreased. These results suggest that the immunomodulatory effects of garlic depend on the cell type and on the type of stimuli exerted on the cell.

Poster #: 224
Campus: CSU San Marcos
Poster Category: Disease (Pathogens)
Keywords: obesity, T cells, Immunology
Project Title: Restoration of T cells in the epithelial layer of the small intestine in obese mice by diet-induced weight loss
Author List:
Miranda, Nadia; Undergraduate, California State University San Marcos, Presenting Author
Cruz, Michelle; Graduate, California State University San Marcos, Presenting Author
Jameson, Julie; California State University San Marcos

Abstract: According to the CDC, more than one third of the U.S population is obese. Preliminary data in our laboratory has shown that obesity reduces the number of T cells in the intestines of mice. This reduction of T cells leads to impaired tissue repair in DSS colitis models and compromises the protective barrier against pathogens and exposure from the outside environment. Thus, our goal was to identify how to restore the number of T cells to improve intestinal homeostasis. We investigated whether T cell numbers in the intestine can be fully restored through diet-induced weight loss. Briefly, 6 week old male mice were placed in one out of three cohorts: One cohort was placed on a normal chow diet for 14 weeks, the second cohort was placed on a high fat diet for 14 weeks, and the third cohort was placed a high fat diet for 7 weeks and switched to a normal chow diet for the remaining 7 weeks. While mice placed on a high fat diet exhibited a significant reduction in T cell number, mice that underwent diet-induced weight loss exhibited a full restoration of T cell number in the small intestine. In addition, the dieted mice also showed improved survival when challenged with DSS-induced colitis. These studies are now being followed up with RNAseq experiments to investigate how gene expression is changing within the intestine and better understand the mechanisms of T cell loss in obesity.

Poster #: 225
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zebrafish, morpholino, skin development
Project Title: Investigating the function of a transcription factor, Znfx1, during zebrafish early development
Author List:
Corona, Leobardo; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Sandoval, Roberto; Undergraduate, Biology, California State University, Dominguez Hills
De La Torre, Maricruz; Graduate, Biology, California State University, Dominguez Hills, Presenting Author
Wang, Fang; Biology, California State University, Dominguez Hills

Abstract: Although the skin is the largest organ in the body its early development and the interactions between skin cells and touch-sensing neurons have not been well studied. Our research aims at improving our understanding on the molecular basis of skin development and general skin cell biology thus paving the way for better skin disease diagnosis and treatment. Using zebrafish as our model organism, we have begun our investigation by studying the gene expression profiles of the skin. Early in development, the zebrafish epidermis consists of two layers; the top layer (periderm) and the bottom layer (basal). Our RNA-Seq study indicates Zinc finger NFX1-type containing 1 (znfx1), a little known transcription factor, is highly expressed in the basal cell layer at 52 and 72 hours post fertilization: the expression in basal cell is 20 times higher than non-skin cells (p < 1×10–5), and 4 times higher than its expression in periderm cells (p < 1×10–4). We have used microinjection of morpholinos to further investigate this gene. These antisense oligonucleotides can bind to target mRNA molecules and inhibit gene expression. We used two morpholinos to inhibit the normal RNA splicing of znfx1, one targets the exon-intron boundary of Exon 2 and the other targets the intron-exon boundary of Exon 3. After microinjection of the morpholinos into zebrafish embryos at the one cell stage, we isolated RNA at 2 dpf and our RT-PCR results show a significant reduction of the wild type PCR product and two additional shorter fragments. Subsequent TA cloning and sequencing confirm that znfx1 gene in the morpholino injected embryos mainly produced altered mRNA molecules: the most common form is a frameshift mutation with a 158-base pair deletion. Thus, the morpholino injection resulted in a partial knockdown of znfx1. Preliminary observations detected a wide range of phenotypes after morpholino injection, from wild type, mild skin deformity, to severe developmental defects (embryos lack of normal head and tail). We are currently quantifying and confirming the phenotypes caused by morpholinos as well as conducting in situ hybridization to obtain better spatial and temporal expression of znfx1. In the future, we will 1) use various transgenic fish lines to detect the specific type of cells or components that are affected by the inhibition of znfx1, and 2) create genomic mutants using CRISPR/Cas9 to verify phenotype.
Norris Grant, CSUDH RSCA, and CSUDH Faculty Scholar Program, LS-AMP

Poster #: 226
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Synaptic Partner Recognition, C. elegans, synapse
Project Title: Elucidation of the molecular mechanisms that link synaptic partner recognition and synaptogenesis
Author List:
Le, Nghi; , Biological Sciences, San José State University, Presenting Author
Clippard, Erik; , Biological Sciences, San José State University, Presenting Author
Varshney, Aruna; , Biological Sciences, San José State University
Watters, Katie; , Biological Sciences, San José State University
Marigal, Angela; , Biological Sciences, San José State University
Shankar, Raakhee; , Biological Sciences, San José State University
Tatarakis, David ; , Biological Sciences, San José State University
Magallanes, Khristina; , Biological Sciences, San José State University
Pollock, Sierra; , Biological Sciences, San José State University
VanHoven, Miri; Biological Sciences, San José State University

Abstract: Our nervous system is a network of neural circuits that mediate perception, behavior and thought. To form circuits, neurons must identify their correct synaptic partners among the many neurites in a target region, a process called synaptic partner recognition (SPR). The recruitment of synaptic components must be linked to SPR to ensure the fidelity of neural circuit formation. However little is known about how these processes are linked. Elucidating these molecular mechanisms may aid in understanding neurological disorders, such as autism. To study neural circuit formation, we utilize a trans-synaptic marker called Neuroligin 1-mediated GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP), which fluorescently labels specific synapses in live animals. Our group previously found that the UNC-6/Netrin ligand and its receptor, UNC-40/Deleted in Colorectal Cancer (DCC), regulate SPR between PHB sensory neurons and AVA interneurons in C. elegans. We discovered that in animals with mutations in unc-69, the homolog of the human Small Coiled-Coil protein (SCOCO), unc-76, the homolog of the human FEZ1, and mig-2, the homolog of the small GTPase Rho, synapses between PHB and AVA neurons are severely reduced. To determine if these molecules acts in the unc-40/DCC synaptic pathway, we used classical genetic techniques. We generated and analyzed the synaptic phenotypes of double mutants between unc-69/SCOCO and unc-40/DCC, unc-76/FEZ1 and unc-40/DCC, and mig-2/Rho and unc-40/DCC. Our results indicate that all three molecules act in the unc-40/DCC synaptic pathway. By analyzing the synaptic phenotype of unc-69/SCOCO animals over-expressing unc-6/Netrin, we also found that UNC-69/SCOCO likely functions downstream of UNC-6/Netrin. unc-69/SCOCO and unc-76/FEZ1 are thought to act as kinesin cargo adaptors for synaptic components, and mig-2/Rho is thought to regulate actin, another critical component of pre- and postsynaptic sites. Thus, our results indicate the first link in our system between synaptic recognition and targeting of synaptic components. We are currently conducting experiments aimed at understanding which synaptic components are transported by unc-69/SCOCO and unc-76/FEZ1. Additionally, we will determine if the transportation of pre- and postsynaptic components in these mutants are affected. Our ultimate goal is to elucidate the entire pathway from recognition of synaptic partners to synaptic assembly and maintenance.

Poster #: 227
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: ADCY5, cAMP,
Project Title: Examining the role of the ADCY5 point mutation p.R418W on cellular cAMP and cAMP effects on neuronal differentiation of murine stem cells
Author List:
Zepeda, Eliabeth ; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Robertson, Kiana ; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Zuniga, Nicholas; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Sprowles, Amy; Biological Sciences, Humboldt State University

Abstract: ADCY5-related Dyskinesia is a rare movement disorder, with early onset in childhood and adolescence. Previous studies have linked this disease to various point mutations in the ADCY5 gene; one study has demonstrated two of these mutations cause an increase in cyclic adenosine monophosphate (cAMP). However, the molecular basis of this disease phenotype has yet to be fully understood. Our study seeks to characterize the effects of one specific point mutation, p.R418W, on cellular cAMP levels using HEK293T cells, and to determine if increased cAMP levels affect neuronal differentiation using mouse embryonic stem cells (mESCs). Our initial experiments demonstrated successful differentiation of mESCs into a neuronal lineage, with 1-2% dopaminergic neuron production, detected by Immunocytochemistry (ICC). This preliminary data showed that treatment with 1400 uM cAMP did affect neuronal differentiation. The most striking finding was the presence of contracting neuronal clusters in the treated neurons, versus non-treated. Current experiments include creating a CRISPR model: a HEK293T cell line containing the p.R418W mutation. We have designed the appropriate guide RNA, confirmed a high transfection efficiency of the CRISPR components using fluorescence microscopy, and are currently screening colonies by sequence analysis. Once the mutant cell line is verified, it will be treated with pharmacological agonists to stimulate cAMP production in mutated cells as well as control cells. The amount of cAMP produced in mutated cells versus wild-type will be quantified using enzyme-linked immunosorbent assay (ELISA). Once we characterize the amount of cAMP produced in the ACDY5 p.R418W 293T cells, we will see if this level of cAMP affects differentiation of mESCs to dopaminergic neurons using our optimized mESC differentiation protocol. This data will not only help us better understand the role of ADCY5 in neuronal development, it will be beneficial to our collaborators, in choosing appropriate drugs for screening of induced pluripotent stem cells (iPSCs), collected from ADCY5 patients.

Poster #: 228
Campus: San José State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Neuron, Streptomyces, G protein coupled receptor
Project Title: The C. elegans G Protein Coupled Receptor srb-6 is Required for Detection and Avoidance of Streptomyces
Author List:
Tang, Angelina; Undergraduate, Biological Sciences, San José State University, Presenting Author
Ali, Nebat; Undergraduate, Biological Sciences, San José State University, Presenting Author
Tran, Alan ; Undergraduate, Biological Sciences, San José State University
O’Loughlin, Colleen; Postdoc, Department of Bioengineering and Therapeutic Sciences
Farah, Fatima; Undergraduate, Biological Sciences, San José State University
Balistreri, Anthony; Graduate, Department of Chemistry, San José State University
Chang, Eric; Undergraduate, Biological Sciences, San José State University
Coto Villa, Doris; Graduate, Biological Sciences, San José State University
Varshney, Aruna; Graduate, Biological Sciences, San José State University
VanHoven, Miri; Biological Sciences, San José State University

Abstract: Gram positive Streptomyces bacteria secrete nematicidal and antinematodal compounds, some of which are used to treat illnesses that stem from nematodal infections in humans. However, little is known about the interaction between nematodes and these bacteria. Our group determined that the nematode C. elegans utilizes chemosensory neurons in the head and tail to effectively detect and avoid Streptomyces secretions. We hypothesized that a chemosensory receptor was responsible for this avoidance behavior. To test our hypothesis, we conducted an RNAi screen of possible receptors expressed in the two chemosensory neurons in the tail, and at least one sensory neuron in the head. Of the 38 different receptors we knocked down, the most severe defect was observed for the srb-6 G Protein Coupled Receptor. A presumptive null mutation in srb-6 resulted in a similar defect in both the head and tail response, confirming the requirement for this receptor. Previous work from the Bargmann lab showed that srb-6 is expressed in PHA and PHB chemosensory neurons in the head, and ASH, ADL and ADF chemosensory neurons in the head. To confirm that srb-6 functions in these neurons, we cloned the srb-6 cDNA and expressed it in these neurons using the ocr-2 promoter, and expression of srb-6 in these neurons restored the avoidance behavioral. To confirm that the srb-6 receptor was required to sense Streptomyces, we imaged calcium dynamics in ASH neurons in live wild-type and srb-6 mutants. We observed a robust influx of calcium into ASH in response to Streptomyces extract that was significantly reduced in srb-6 mutants, consistent with a requirement for this chemoreceptor. We conclude that the srb-6 G protein coupled receptor is responsible for sensing Streptomyces and its secretion product. Future work will focus on identifying the remaining members of this chemosensory pathway. This work is funded by the National Institutes of Health (NIH) (NS087544 to MV and NL, GM089595 to MV and NS087544 to NL, 5SC3GM118199 to LMC), the National Science Foundation (NSF) (1355202 to MV), and CSU-LSAMP (HRD-1302873 CSU-LSAMP fellowships to A. Tran and S. Matthews).

Poster #: 229
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Apoptosis, Gene Regulation, Paclitaxel
Project Title: Effects of Paclitaxel on Glucocorticoid-induced apoptosis of Human Leukemic CEM Cells
Author List:
Aranas, Ivymarie; Undergraduate, Biology, California State University, Northridge, Presenting Author
Macias, Alejandro; Undergraduate, Biology, California State University, Northridge, Presenting Author
Medh, Rheem; Biology, California State University, Northridge

Abstract: Glucocorticoids (GCs) are steroid hormones widely used in anti-leukemia therapy as they can induce apoptosis through certain gene-regulatory pathways that affect pro- and anti-apoptotic genes. In cell culture models of human acute lymphoblastic leukemia (ALL), the synthetic GC, Dexamethasone (Dex), has been shown to induce expression of pro-apoptotic genes including E4BP4 (a transcription factor that upregulates pro-apoptotic BIM), and regulators of apoptosis such as FOXO3 and BIRC3, in correlation with apoptosis. Paclitaxel (Ptx), a taxol derived anti-proliferative alkaloid, induces apoptosis via a distinct but overlapping transcriptional response, and is often used in combination with Dex as an anti-leukemic agent. In this study we are investigating the effect of Ptx alone, and in combination with Dex, on apoptosis and regulation of key pro- and anti-apoptotic genes, in an effort to understand the molecular implications of combined treatment with Dex and Ptx. The CCRF-CEM cell lines CEM-C7-14 and CEM-C1-15 were previously found to be sensitive and resistant, respectively, to Dex. Sensitivity to Dex was restored in CEM-C1-15 cells upon ectopic expression of mouse E4BP4 (line CEM-C1-15mE#3). We are testing the hypothesis that E4BP4 and BIM play a central role in regulating apoptosis triggered by Ptx as well, and that combined treatment synergistically triggers apoptosis via a greater magnitude of upregulation, in addition to activating a broader spectrum of pro-apoptotic pathways. All three CEM cell lines were treated with 1uM Dex and 1uM Ptx either alone or in combination for 24-96h. All three cell lines were sensitive to Ptx, but CEM-C1-15mE#3 was least susceptible. Apoptosis was confirmed by staining cells with Annexin V-FITC followed by epifluorescence microscopy. Reverse transcription and end-point PCR followed by ImageJ analysis was employed to evaluate relative gene expression in response to each treatment. Our data suggest that Ptx upregulated FOXO3, BIRC3, and BIM, but down-regulated E4BP4 in the CEM-C7-14 cells, suggesting that Dex and Ptx have distinct effects on E4BP4 regulation but may share common downstream pathways for apoptosis via BIM upregulation.

Poster #: 230
Campus: CSU Monterey Bay
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: zinc finger protein, transposable element, primate genome
Project Title: Evidence for Coevolution of KRAB Zinc Finger Protein ZNF649 and L1PA Transposable Elements
Author List:
Thisner, Tiffany; Undergraduate, Biology, California State University, Monterey Bay, Presenting Author
Fernandes, Jason; University of California, Santa Cruz
Haeussler, Max; University of California, Santa Cruz
Salama, Sofie; University of California, Santa Cruz
Jue, Nathaniel; Biology, California State University, Monterey Bay
Haussler, David; University of California, Santa Cruz

Abstract: Genomes are continuously invaded by transposable elements (TE), sequences of DNA that insert and replicate themselves through various mobilization mechanisms. TE insertions, which can be both constructive and destructive, have given rise to mutations, disease, and genetic diversity. One type of TE, long interspersed nuclear element-1 retrotransposons (L1), have amplified themselves in primate genomes for the past 150 million years and now constitute ~17% of the human genome. L1 elements display a pattern of evolution in which new lineages develop under pressure from repressive factors that constitute the host genome’s defense. One line of defense is a family of transcription factors known as KRAB Zinc Finger proteins (KZNF), which silence TEs through recruitment of the KAP1 protein, inducing heterochromatin. This cycle of repression by KZNFs and escape by L1 elements, represents an evolutionary “arms race”. In this study, one KZNF-TE interaction is investigated; between ZNF649 and its binding of the L1PA family. ChIP-SEQ data from KZNFs were mapped to L1PA elements and ZNF649 was found to overlap a prominent KAP1 peak, suggesting ZNF649 represses expression of L1PA elements. L1PA consensus sequences were taken from the Repeat Browser (a repeat analysis tool based on the Human Genome Browser) and aligned, revealing that ZNF649 does not bind L1PA7, but binds lineages that evolved after L1PA7. Our current work investigates whether nucleotide changes occurred within or after the L1PA7 lineage’s transition to L1PA6 that contributed to ZNF649’s ability to bind the L1PA family. This analysis will deepen our understanding of the impact of KZNF-TE interactions on the human genome.

Poster #: 231
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: (p)ppGpp, polyhydroxybutyrate, starvation
Project Title: Regulation of a dynamically-localizing polyester by three Caulobacter crescentus transcription factors
Author List:
Rocha, Alexa; Undergraduate, Biology, California State University, Northridge, Presenting Author
Pajulas, Abigail; Undergraduate, Kineseology, California State University, Northridge, Presenting Author
Schreiner, Shannon; USC
Stott, Kristina; JPL
Vu, Son; Olive View Hospital
Bueno, Diana; Pasadena City College
Arvizu, Ignacio; Olive View Hospital
Murray, Sean; Biology, California State University, Northridge

Abstract: This investigation studies how the model organism Caulobacter crescentus responds to fatty acid starvation. To study this response, we created a FabH-depletion strain which induces fatty acid starvation. Starvation triggers the production of the intracellular signaling molecule (p)ppGpp in various bacterial species, including Caulobacter crescentus. (p)ppGpp helps RNA polymerase turn on genes whose products promote survival under starvation conditions. We discovered that one set of (p)ppGpp-dependent genes leads to the production of the carbon storage molecule (and biodegradable plastic) polyhydroxybutyrate (PHB). Immunoblot, transcriptional reporters, fluorescence microscopy, and mutant strains were used to create a model for PHB production during fatty acid starvation. (p)ppGpp is responsible for the expression of the transcription factor CtrA, which activates the expression of the transcription factor PhaR, necessary for PHB accumulation. PHB biosynthesis results from the efforts of three unique enzymes: PhaA, PhaB, and PhaC. We propose a model in which (p)ppGpp modulates phaC expression and (p)ppGpp, CtrA and PhaR stimulate phaA and phaB expression. Our model illustrates how these three transcription factors work together to regulate expression of the PHB biosynthetic genes in response to fatty acid starvation.
This work was supported by NIH grant SC2 GM084860 and a CSUPERB Development grant to SM, NIH R25 GM063787 to MariaElena Zavala in support of AR, NIH T34 GM008395 to MariaElena Zavala in support of AP, NIH R25 GM063787 to MariaElena Zavala in support of IA, and CSUN Interdisciplinary Research Institute in the Sciences Fellowships to KS and SS.

Poster #: 232
Campus: CSU Dominguez Hills
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Escherichia coli, experimental evolution, bacterial genetics
Project Title: Characterization of Genes Necessary for Escherichia coli survival in Long-Term Stationary Phase
Author List:
Salgado, Katia; Undergraduate, Biology, California State University, Dominguez Hills, Presenting Author
Kram, Karin; Biology, California State University, Dominguez Hills

Abstract: Bacterial evolution can lead to antibiotic resistance, new forms of pathogenesis, and more efficient production of industrial products. Experimentally evolving bacteria in the laboratory helps us better understand these processes, as well as basic evolutionary principles. Escherichia coli has the ability to adapt and survive during long-term stationary phase (LTSP), which follows lag, exponential, stationary, and death phases in the bacterial life cycle. During this period 99% or more of the initial cells in the culture have died and only a few cells possess the necessary mutations that allow the cell to survive and grow. Although some genes important for this survival have already been identified, there are likely many more genes important for this phenotype. The main objective of this study is to identify genes that play an important role during LTSP survival in E. coli. First, we identified genes that were only expressed during LTSP using RNA-seq data. We chose five of these genes to characterize: entC, feaR, glcE, prpB, and ytfS. We deleted the genes of interest through transduction using the KEIO collection of knock-out strains of E. coli and competed the mutant strain with a wild type strain to determine if the missing gene was important for LTSP survival. Each mutant and wild type strain were inoculated at a 1:1000 dilution in 5-ml of LB for competition, and incubated for 10 days – through LTSP. To differentiate the strains, mutant strains were marked with a kanamycin-resistance cassette, while wild type strains were marked with a chloramphenicol-resistance cassette, both in the lacZ gene. Through the course of ten days, serial dilutions were performed and plated on LB agar with each antibiotic. Our results indicated that the loss of glcE and prpB provided no significant changes during LTSP, while deletions in ytfS, feaR and entC showed some possible changes. feaR mutants showed the most significant results: wild type cells significantly outcompete the mutant strain, indicating a possible importance of the feaR gene during survival of E. coli during LTSP. The feaR gene is activated when the cell is experiencing catabolic repression and high levels of succinate are present, and FeaR controls a pathway for removing potential toxic aromatic compounds, which could be an essential mechanism during LTSP survival. Combining RNA-seq and genetic data can lead to more accurate identification of LTSP-specific genes than using either type of data alone.

Poster #: 233
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Developmental Biology, MicroRNA, In Situ Hybridization
Project Title: Analyzing the Function of Muscle Specific MicroRNAs in Xenopus laevis
Author List:
Bugay, John Paul; Undergraduate, Biology, San Francisco State University, Presenting Author
Ramirez, Julio; Postdoc, Biology, San Francisco State University
Domingo, Carmen; Biology, San Francisco State University

Abstract: laevis
MicroRNAs (miRNAs) are small non-coding sequences of RNA that regulate genes post-transcriptionally by binding to complementary mRNA and inhibiting translation. miRNAs are highly conserved between diverse species and play key roles in developmental pathways. Our laboratory is interested in understanding how miRNAs control muscle differentiation during vertebrae development. Previous studies have shown that two muscle specific miRNAs, miR-1a and miR-206, are expressed in muscle progenitor cells and are thought to play a role in muscle formation and maintenance. However, most studies have been conducted in cell culture and thus, the role of these microRNAs during embryonic development is limited. Results from our lab show that morpholino knockdown of miR-206 disrupts the formation of muscle fibers during X. laevis embryogenesis. Examination of the resultant phenotype suggests that miR-206 may regulate changes in cell adhesion and cell movement that are essential to muscle cell formation. Although miR-206 and miR-1a are expressed in a similar pattern, knockdown of miR-206 has a more severe effect on muscle formation than knockdown of miR-1a. In addition, ectopic expression of miR-1 appears to partially rescue the phenotype of a miR-206 knockdown, demonstrating some redundancy between miR-1 and miR-206. Together, these microRNAs appear to play an important role in regulating the cell behaviors necessary for proper vertebrate muscle morphogenesis. This work was funded by NIH MBRS SC3 1SC3GM111118-01 and MARC T34-GM008574.

Poster #: 234
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: morphogenesis, development, Drosophila
Project Title: Genetic Screen to Analyze Interactions between the Toll-like Receptor, 18-wheeler, and genes on the X-chromosome
Author List:
Villalba, Jacquelyn; Undergraduate, Biology, California State University, Long Beach, Presenting Author
Molina, Anel; Undergraduate, Biology, California State University, Long Beach
Eldon, Elizabeth; Biology, California State University, Long Beach

Abstract: Cleft lip and palate involves abnormalities of epithelial cells during development and occurs 1 in every 700 birth, yet very little is known about the genetic defects that might be involved. The 18-wheeler gene found in Drosophila melanogaster directs epithelial cell migration during development. Previous work has demonstrated that homozygous 18-wheeler mutant embryos have abnormal salivary glands. Drosophila salivary glands are an excellent model for mammalian organ development. To identify genes that interact with 18-wheeler during epithelial organ development we take advantage of the observation that an embryo heterozygous both for 18-wheeler and for a gene that interacts with18-wheeler will produce defective salivary glands. We are systematically searching the X-chromosome for interacting genes using a collection of 93 X-chromosome-linked deficiencies (Df(1)). Together these deficiencies delete 2,288 of the 2,331 euchromatic genes on the X-chromosome (98.1%). To obtain embryos that are heterozygous for both 18-wheeler and an X-linked deficiency, males carrying an 18-wheeler mutation and a green fluorescent protein (GFP) reporter expressed in salivary glands (stock 84-1) are mated with females heterozygous for an X-linked deficiency. Their other X-chromosome is a GFP-expressing “balancer”. Control embryos are obtained by using males that are wild type at the 18-wheeler locus, but still carry the GFP salivary gland reporter (stock 15-1). Embryos are collected, fixed, and subjected to immunocytochemistry to detect GFP which is expressed in the salivary glands of the 18-wheeler; Df(1) embryos. If the mutations interact, salivary gland morphogenesis will be abnormal. Defects include, but are not limited to, glands lengthening, shortening, or migrating asymmetrically. Df(1)BSC719 (stock 26571) show no gene interaction since wild type glands are observed in both the 15-1 and 84-1 heterozygous embryos. Df(1)BSC644 (stock 25734) shows a genetic interaction but not in the manner predicted. When crossed to the 18-wheeler mutant wild type glands are observed but when crossed to wild type 18-wheeler the glands are shorter. This suggests that the deficiency causes a defect in gland morphogenesis that is rescued by reducing the dosage of the 18-wheeler protein. Further work will examine the genes within Df(1)BSC644 in more detail by ordering flies with smaller deficiencies within that deficiency to narrow down the location of gene responsible for the defect in morphogenesis.

Poster #: 235
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: ceruloplasmin, kidney, estrogen, ,
Project Title: Potential contribution of the kidney to the increased levels of blood plasma ceruloplasmin induced by estrogen treatment in rats
Author List:
Goodrum, Mark; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Hernandez, Brisamar; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Linder, Maria C.; Chemistry and Biochemistry, California State University, Fullerton

Abstract: Ceruloplasmin (Cp) is the main copper binding protein in the blood plasma and has several 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 gestation and in women taking estrogen-containing oral contraceptives. It has generally been accepted that most Cp in plasma is produced by hepatocytes in the liver, where copper is incorporated in the trans Golgi network after being pumped from its cytosolic carrier (ATOX1) by ATP7B. After exocytosis into the blood, Cp interacts directly with the cell surface of cells, which results in uptake of its copper, and formation of apoCp without entry into cells (Ramos et al., PLoS ONE 11(3): e0149516, 2016). The kidney also expresses Cp mRNA, but its potential role in producing Cp for the blood has not previously been explored, which is the purpose of these studies.
Adult, female Fisher rats were injected subcutaneously daily for two weeks, with 9.5% ethanol-0.9% NaCl without and with estradiol (10 mg), before euthanization and collection of kidney, liver and blood plasma. Relative levels of plasma Cp were determined by rocket immuno-electrophoresis. Relative amounts of apo and holoCp were determined by Western blotting after native PAGE. Tissue expression of mRNA for Cp, Atp7b and Atp7a were determined by qPCR after extraction of RNA and reverse transcription into cDNA, using primers and probes from Dharmacon.
Estrogen treatment doubled the levels of Cp protein in the plasma and increased the proportion of holo to apoCp. In liver, estrogen treatment slightly increased expression of Cp mRNA, and also for Atp7b and its homolog, Atp7a – which is not normally thought to provide Cu to Cp. In contrast, levels of these three mRNAs more than doubled in the case of the kidney. Interestingly, relative to 18S RNA, expression of Cp and Atp7b/a mRNAs in kidney were in the same range as for liver.
These results suggest that formation of Cp by the kidney may rival that of the liver, at least in rats, and that in response to estrogen it may be providing more of the additional Cp found in the blood.

Poster #: 236
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Breast Cancer, Gene Regulation, apoptosis
Project Title: Gene regulatory pathways that modulate response to Dexamethasone and Daunorubicin in breast cancer cell lines
Author List:
Macias, Alejandro; Undergraduate, Biology, California State University, Northridge, Presenting Author
Aranas, Ivymarie; Undergraduate, Biology, California State University, Northridge, Presenting Author
Medh, Rheem; Biology, California State University, Northridge

Abstract: Glucocorticoids (GCs) such as Dexamethasone (Dex) are anti-cancer agents that induce apoptosis (programmed cell death) and counter side effects of chemotherapy through regulation of pro- and anti-apoptotic genes such as E4BP4, FOXO3A, BIM and BIRC3. Anthracyclines, such as Daunorubicin (Dauno) are effective anti-cancer agents for leukemias, breast cancer and ovarian cancer. Several regimens combine both classes of chemotherapy agents, although Dex is known to promote rather than retard cell proliferation in some breast cancer cell lines. E4BP4, a transcriptional regulator, can either induce apoptosis or promote cell survival in a cell type and stimulus-specific context. The phosphorylation state of E4BP4 and its crosstalk with the FOXO3A and AKT pathways modulate its pro-survival or pro-apoptosis functions. E4BP4 is upregulated by GCs and mediates Dex-evoked apoptosis in leukemia cells, but its role in breast cancer has not been explored. We hypothesize that E4BP4 regulation and its crosstalk with other pathways modulate either pro-survival or pro-apoptosis responses to chemotherapeutic agents in breast cancer. We are investigating the response of breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (triple negative) to Dex and Dauno (single agent and in combination) with respect to cell viability, apoptosis and regulation of pro- and anti-apoptotic genes including E4BP4, FOXO3A, BIM and BIRC3. MTT cell viability assays determined that MDA-MB-468 and MCF-7 cells are resistant to 1 uM Dex, with viable cells being about 92% and 140% compared to untreated controls, respectively. MDA-MB-468 cells were sensitive to 50 nM Dauno with viability at 62% compared to control. Cells are stained with Annexin V-FITC followed by epifluorescence microscopy to detect apoptotic cells. Evaluation of Dex and Dauno-mediated regulation of gene expression was done via reverse-transcriptase and end-point PCR analysis, followed by ImageJ quantitation, or by RT-qPCR analysis. Our data suggest that Dauno upregulates while Dex downregulates E4BP4 in MDA-MB-468 cells. Combined treatment significantly enhances FOXO3 upregulation compared to either single agent, in correlation with increased cell death. Our data suggest that Dex and Dauno may cooperatively promote apoptosis in MDA-MB-468 cells, and that E4BP4 expression correlates with cell death.

Poster #: 237
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Alternative splicing, Neural development, Tau
Project Title: Alternative Splicing of Tau Exon 10 in the Developing Mouse Cortex and Hippocampus
Author List:
Milla, Allyson; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author
Franklin, Michael; Graduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Department of Biological Sciences, California State University, Long Beach

Abstract: Formation of neurofibrillary tangles (NFTs) is one of the defining neuropathological hallmarks of Alzheimer’s disease (AD). The NFTs result from aberrant tau proteins, which are encoded by the gene of microtubule associated protein, tau (Mapt). We have previously found that in the developing mouse cortex/hippocampus, mRNA levels of splicing factor, suppressor of white-apricot homolog (Sfswap) decreased with age. Its encoding protein, SFSWAP, is an alternative splicing factor that inhibits the inclusion of exon 10 in Mapt mRNA. Alternative splicing of Mapt exon 10 generates two isoforms containing three (3R, without exon 10) or four (4R, with exon 10) microtubule-binding repeats. Thus, we hypothesized that there would be an age-dependent increase in the inclusion of Mapt exon 10 occurring in the developing mouse cortex/hippocampus due to down-regulation of Sfswap. To test our hypothesis, we used reverse transcription with polymerase chain reaction (RT-PCR) and quantitative polymerase chain reaction (RT-qPCR) to characterize and measure the expression of total, 3R, and 4R Mapt mRNA in the cortex/hippocampus of male and female C57BL/6J mice collected on postnatal day (PN) 0, 7, 14, and 21 (N=8 per sex per age). We first observed that regardless of sex, PN0 mice expressed only 3R mRNA and PN7 animals generated equal amounts of 3R and 4R mRNA, while only 4R mRNA was produced at PN14 and PN21. Next, we used RT-qPCR to quantify relative expression of the total and 4R Mapt mRNA. Total Mapt mRNA levels in the developing mouse cortex/hippocampus showed an age-dependent decrease (p<0.001) and female-biased expression (p=0.008) whereas ratios of 4R vs. total Mapt increased with age (p<0.05). Overall, our data support our hypothesis that during early development, the inclusion of Mapt exon 10 in the mouse cortex/hippocampus increases, possibly due to down-regulation of Sfswap. We are currently investigating the effects of sex and age on mRNA levels of three other splicing factors, CUGBP Elav-like family member 3 (Celf3), CUGBP Elav-like family member 4 (Celf4), and RNA binding motif protein, X-Linked (Rbmxl1), which may also involve regulating the splicing of Mapt exon 10. Our new data will help us better understand the regulation of Mapt exon 10 splicing and elucidate its functional importance in normal neural development and differentiation as well as disease.

Poster #: 238
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: NFkB, RCAN1, apoptosis
Project Title: Role of NFkB pathway in RCAN1-dependent apoptosis of human leukemia cells
Author List:
Papelian, Teni; Undergraduate, Biology, California State University, Northridge, Presenting Author
Shrestha, Alsha; Undergraduate, Biology, California State University, Northridge, Presenting Author
Hurtado, Joanna; Undergraduate, Biology, California State University, Northridge
Medh, Rheem; Biology, California State University, Northridge

Abstract: Our laboratory is studying the molecular basis of glucocorticoid (GC) induced apoptosis and GC resistance in human leukemic CCRF-CEM sister cell lines. Upregulation of RCAN1 correlates with sensitivity to Dexamethasone, a synthetic GC used in the clinic as an anti-leukemic agent. Recent studies suggest that RCAN1 interacts with the NFkB pathway to modulate its activity. NFkB promotes cell survival and proliferation, and is known to antagonize apoptosis. To understand the role of RCAN1 in lymphoid cell apoptosis, and its effect on NFkB, we are employing RCAN1 knockdown/knockout leukemia cell lines along with their wild-type counterparts. CRISPR-Cas9-mediated genome editing of GC-sensitive CEM C7-14 cells yielded heterozygous knockout in two distinct clones. An RCAN1 knockout derived from the haploid cell line HAP1 was obtained from Horizon Discovery. Western blotting using specific antibodies for intermediates of the NFkB pathway including NFkB1, IkB and NIK evaluated correlation of their expression to that of RCAN1 in wild type and knockout lines. Our data suggest that RCAN1 knockdown correlates with decreased abundance of IkB, suggesting that RCAN1 may mediate IkB abundance via promoting its phosphorylation and subsequent degradation. Interestingly, NFkB1 (p50) abundance is also reduced in knockout cells, suggesting additional levels of RCAN1-mediated regulation of the NFkB pathway. Different dimeric combinations of p50 with other NFkB family proteins modulate transcriptional activation or repression; hence further studies are underway to understand the precise molecular crosstalk between the two pathways. To evaluate the effect of NFkB activity on RCAN1 expression, HEK293FT cells are being transiently transfected with a construct harboring 1400bp of the RCAN1 promoter upstream of the Gussia Luciferase (GLuc) reporter, with Secreted Alkaline Phosphatase (SEAP) serving as an internal reference. Two inhibitors of the NFkB pathway, BV6 and Cardamonin (Card), and an activator of NFkB, Betulinic acid are being used to modulate the NFkB pathway and study its effect on GC-dependent and independent RCAN1 promoter activity. Our data suggest that RCAN1 and NFkB pathways crosstalk to modulate each other’s’ activity and that this interaction may affect GC-evoked apoptosis of leukemia cells.

Poster #: 239
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: EMAST, aggressive colorectal cancer, mouse models
Project Title: The first demonstration of EMAST, a biomarker for aggressive human colorectal cancer, in a mouse model of disease
Author List:
Luu, Jennifer; Undergraduate, Chemistry and Biochemistry , San Diego State University, Presenting Author, Nagel Award Nominee
Bhaskaran, Nitya; Graduate, Biology, San Diego State University
Mamindla, Priyadarshini; Graduate, Bioinformatics and Medical Informatics Program, San Diego State University
McGuire, Kathleen; Biology, San Diego State University

Abstract: Colorectal Cancer (CRC) is the second leading cause of cancer-related deaths in the United States. In CRC there is a significant racial disparity, with African Americans having higher incidence and mortality than Caucasians. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a biomarker for aggressive CRC because it is associated with inflammatory responses that promote tumor growth and invasion. It has been found to be more common in African American rectal cancer. EMAST is found through insertions/deletions of tetranucleotide repeats in the DNA and it is caused by a malfunction of DNA mismatch repair due to inflammation. Studies on EMAST have been limited because of the low availability of patient samples and the previous lack of an appropriate animal model. No animal model of cancer has ever been shown to display EMAST. Our aim was to look at specific tetranucleotide repeats in mouse tumors to see if EMAST can be found in mice. The goal of the project was to provide unique and essential tools to cancer researchers for the study of the EMAST biomarker of aggressive cancer in mouse models.
Previously in our lab, an algorithm was developed to identify mouse tetranucleotide repeats that contain characteristics of human EMAST sequences. For this project, seven of the identified potential EMAST repeats were studied further. Our mouse model of cancer is a ‘Cre/lox’ model in which colon-specific expression of the Cre recombinase results in a defective Apc gene, leading to colon polyps and CRC. PCR primers were created for the selected repeat sequences and the products generated were tested by DNA sequencing. The repeats were analyzed in both tumor-adjacent, i.e. normal, colon tissue and tumors from seven mice that displayed either precancerous or cancerous colon lesions. One repeat was also analyzed in seven normal (non-tumor bearing) parental mouse colon tissues.
Five out of seven sequences tested showed EMAST in tumors, while a sixth displayed instability in individual mice, and five/seven large and invasive tumors tested showed EMAST in our mouse model of CRC. Parental mouse colon tissues showed no instability in the marker tested, showing that a tumor-microenvironment was required for the observed instability. Therefore, for the first time ever, an approach to study EMAST in mouse models of cancer has been developed and a mouse model of CRC has been shown to display EMAST.

Poster #: 240
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: transcription, cell cycle, supercoiling
Project Title: Gyrase-mediated Supercoiling Drives the Expression of the Caulobacter crescentus sciP Promoter
Author List:
Requena, Kiana; Undergraduate, Biology, California State University, Northridge, Presenting Author
Heasley, Keaton; Undergraduate, Biology, California State University, Northridge
Kumar, Priya; Undergraduate, Psychology, California State University, Northridge
Murray, Sean; Biology, California State University, Northridge

Abstract: The location of genes on the Caulobacter crescentus chromosome is thought to regulate gene expression based on the methylation states of the promoters. As the replisome copies the chromosome, it also leads to major changes in the supercoiling of DNA. We believe DNA supercoiling controls the transcription of a subset of cell-cycle regulated genes. We treated C. crescentus cultures with the antibiotic Coumermycin A1, a DNA gyrase inhibitor. As reported for other bacteria, high levels of the antibiotic inhibited growth, as expected, whereas low levels improved growth. SDS-PAGE analysis of cell lysates suggested a notable change in the presence of a few C. crescentus proteins under Coumermycin dosing, one of which is the master regulator SciP. There appeared to be no significant change in accumulations of the other cell cycle master regulators: DnaA, GcrA, CtrA, and CcrM, suggesting that supercoiling only regulates a subset of genes rather than all of them. Miller Assays and immunoblots confirmed that a decrease in sciP transcription leads to less SciP accumulation when gyrase activity is inhibited. We observed similar results in cells treated with another antibiotic and gyrase-inhibitor, novobiocin. Morever, we identified that plasmid pJC326C can serve as a reporter for changes in DNA supercoiling in C. crescentus cells. When DNA supercoiling was inhibited, the plasmid relaxed and migrated more slowly through an agarose gel. From this data, we conclude that DNA supercoiling is important in the transcription of at least one cell cycle master regulator in C. crescentus.

This work was supported by NIH R25 GM063787 to MariaElena Zavala in support of KH and by NIH 8TL4GM118977-02 to Crist Khachikian in support of PK and KR.

Poster #: 241
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Drosophila, genetics, morphogenesis
Project Title: Genetic Screen to Analyze Interactions between the Toll-like Receptor, 18-wheeler, and Genes on the X-Chromosome
Author List:
Villalba, Jackie; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Molina, Anel; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Eldon, Elizabeth ; Biological Sciences, California State University, Long Beach

Abstract: Cleft lip and palate involves abnormalities of epithelial cells during development and occurs 1 in every 700 birth, yet very little is known about the genetic defects that might be involved. The 18-wheeler gene found in Drosophila melanogaster directs epithelial cell migration during development. Previous work has demonstrated that homozygous 18-wheeler mutant embryos have abnormal salivary glands. Drosophila salivary glands are an excellent model for mammalian organ development. To identify genes that interact with 18-wheeler during epithelial organ development we take advantage of the observation that an embryo heterozygous both for 18-wheeler and for a gene that interacts with18-wheeler will produce defective salivary glands. We are systematically searching the X-chromosome for interacting genes using a collection of 93 X-chromosome-linked deficiencies (Df(1)). Together these deficiencies delete 2,288 of the 2,331 euchromatic genes on the X-chromosome (98.1%). To obtain embryos that are heterozygous for both 18-wheeler and an X-linked deficiency, males carrying an 18-wheeler mutation and a green fluorescent protein (GFP) reporter expressed in salivary glands (stock 84-1) are mated with females heterozygous for an X-linked deficiency. Their other X-chromosome is a GFP-expressing “balancer”. Control embryos are obtained by using males that are wild type at the 18-wheeler locus, but still carry the GFP salivary gland reporter (stock 15-1). Embryos are collected, fixed, and subjected to immunocytochemistry to detect GFP, which is expressed in the salivary glands of the 18-wheeler; Df(1) embryos. If the mutations interact, salivary gland morphogenesis will be abnormal. Defects include, but are not limited to, glands lengthening, shortening, or migrating asymmetrically. Df(1)BSC719 (stock 26571) show no gene interaction, since wild type glands are observed in both the 15-1 and 84-1 heterozygous embryos. Df(1)BSC644 (stock 25734) shows a genetic interaction, but not in the manner predicted. When crossed to the 18-wheeler mutant, wild type glands are observed, but when crossed to wild type 18-wheeler, the glands are shorter. This suggests that the deficiency causes a defect in gland morphogenesis that is rescued by reducing the dosage of the 18-Wheeler protein. Further work will examine the genes within Df(1)BSC644 in more detail to narrow down the location of gene responsible for the defect in morphogenesis.

Poster #: 242
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR, NPC1, mCherry
Project Title: Construction of Niemann Pick Disease Type C1 HEK 293 Cell Model utilizing CRISPR Gene editing
Author List:
Valencia , Stephanie ; Undergraduate, Humboldt , Humboldt State University, Presenting Author
Kraff, Austin ; Undergraduate, Biology, Humboldt State University
Nisson, Haley; Undergraduate, Biology, Humboldt State University
Steele , John; Biology , Humboldt State University

Abstract: Niemann Pick Disease Type C1 (NPC1) is a rare lysosomal storage disorder that affects 1:150,000 people. The disease is characterized by cholesterol accumulation within lysosomes, as well as clinical cognitive decline and neurodegeneration. These symptoms can be attributed to a mutation in the NPC1 gene that leads to the interruption of the intracellular cholesterol transport. Using CRISPR Cas9 technology, the ability to make a cellular model with inducible CRISPR gene regulation can give researchers considerable insight into the cellular pathology of Niemann Pick Disease Type C1 as well as the ability to apply targeted drug therapy and potentially lead to drug discovery. We hypothesize that by using bioinformatics, a CRISPR cas9 system, and genome editing we will make a functioning HEK293 NPC1 cell model with inducible CRISPR gene regulation. To build this system, we 1) used online bioinformatic tools to build primers, 2) Hifi assembly of mCherry coding sequence with pUC19 plasmid, 3) utilized gene editing technology with the purpose of inserting the mCherry (a fluorescent protein) coding DNA sequence into the NPC1 gene and 4) inserted the built sequence into HEK 293 cells for use in inducible CRISPRi/a. Preliminary results show a successful build of the DNA sequence insert containing the mCherry coding sequence which has been confirmed by restriction digest and gel electrophoresis. Once completed the model will be tested in the hopes that it will follow the cellular mechanisms that lead to the NPC1 cholesterol transporter dysfunction. With the cellular model successfully made, and functionality proven, future work will include testing the cells for targeted drug therapy.

Poster #: 243
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Peroxisome Proliferator-Activated Receptors, ATP-Binding Cassette A1, Lipoprotein Lipase
Project Title: Peroxisome Proliferator-Activated Receptor Gamma Regulates Transcription of Lipoprotein Lipase and ATP-Binding Cassette A1 in THP-1 Macrophages
Author List:
Christian, Bryan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Robinson, Cache; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge

Abstract: Background: Peroxisome proliferator activated receptors (PPAR) are members of the nuclear-receptor gene super family of transcription factors. Upon activation, PPARs bind to specific DNA sequences called peroxisome proliferator regulatory elements (PPRE) and regulate the transcription of genes involved in various physiological functions. Lipoprotein lipase (LPL) is a secreted enzyme that hydrolyzes lipoprotein triglycerides for cellular uptake. LPL has a PPRE in its promoter region suggesting a role for PPAR in regulating its transcription. ATP-Binding Cassette A1 (ABCA1) facilitates cholesterol efflux from cells toward high-density lipoprotein (HDL) formation. ABCA1 is also regulated by PPAR-γ, via another nuclear receptor transcription factor, Liver X Receptor alpha (LXR-α). In previous studies, we demonstrated an upregulation of ABCA1 in LPL-knock-down (KD) THP-1-derived macrophages.
Objective: To examine a role for PPAR-γ in LPL-dependent ABCA1 regulation, and to investigate how these proteins regulate lipid metabolism in THP-1 macrophages.
Methods: Gene silencing was accomplished using RNA interference technology. Lentivirus particles expressing gene-specific short hairpin (sh) RNA were used to silence the LPL gene, and small interfering (si) oligonucleotides were used to repress PPAR-γ1 expression in THP-1 macrophages. End-point and quantitative real-time PCR analysis were used to determine transcription of various genes from wild-type (WT), LPLKD, and PPAR-γKD macrophages. Cholesterol efflux assays were performed in 3H-cholesterol loaded cells to investigate the functional ability of ABCA1 to transport lipids from macrophages to HDL.
Results: RT-PCR analysis showed that the level of PPAR-γ transcripts in LPLKD cells was 93% of the level in WT cells. The qPCR showed specific repression of PPAR-γ resulted in a remarkable down-regulation of LPL transcript levels by 82% and ABCA1 by 99.9%, and this was confirmed with end-point RT-PCR. ABCA1-mediated cholesterol efflux in PPAR-γKD cells is 42% of the level seen in WT cells. Ciglitazone, a PPAR-γ agonist, increases cholesterol efflux in WT cells, but to a much lower level in PPAR-γ1KD cells.
Conclusions: LPL transcription is directly correlated to PPAR-γ transcript levels. PPAR-γ expression level also regulates ABCA1 transcription and function, through the PPAR-γ/LXRα/ABCA1 pathway.
Acknowledgements: This work was supported by National Institutes of Health Awards R15HL083946 and SC3GM095413.

Poster #: 244
Campus: San Francisco State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Developmental Biology, Gene regulation, microRNA
Project Title: Determining Target Genes Regulated by miR-206 and miR-1 during Early Skeletal Muscle Development in Xenopus Laevis
Author List:
Garcia, Jason; Undergraduate, Biology, San Francisco State University, Presenting Author
Lopez-Pomino, Maria Pilar; Graduate, Biology, San Francisco State University
Ramirez, Julio; Postdoc, Biology, San Francisco State University
Domingo, Carmen; Biology, San Francisco State University

Abstract: MicroRNA’s (miRNAs) are small non-coding RNA’s that regulate genes post-transcriptionally to modulate various aspects of development including growth, differentiation, and tissue maintenance. In particular, we have demonstrated that miR-206, a muscle-specific miRNA, regulates muscle formation in Xenopus laevis. miR-206 and miR-1, a closely related muscle-specific miRNA, likely regulate signaling pathways controlling proliferation, protein trafficking, adhesion and maintenance of muscle cell fate. We identified approximately two hundred potential miR-206/miR-1 targets in a Xenopus cDNA library using the web server, TargetScan. We optimized a GFP/RFP reporter system to confirm a selected number of putative miR-206/miR-1 targets. In our assay, we co-inject a GFP RNA bearing putative miR-206/miR-1 binding sites, and a RFP RNA to normalize relative GFP/RFP levels, into fertilized embryos at the one-cell stage. We used the 3’UTR of pax7, a gene expressed in muscle satellite cells and a known target of miR-206 and miR-1, to optimize our assay. We demonstrate that miR-206 and miR-1 down regulate GFP levels as predicted. We show that a predicted target, cav2, a protein involved in the inner surface of the caveola that is associated with cellular growth and lipid transduction, is not down regulated in the presence of exogenous miR-206 or miR-1 and thus, is unlikely to be a miR-206 target. However, we do show that sdpr, a protein coding gene that is associated with the caveola pathway, is up regulated compared to control samples. Thus, sdpr is likely a direct miR-206/miR-1. These results indicate that our GFP reporter assay will be useful in identifying new miR 206/miR-1 targets, which will help us better understand their role in muscle development and disease.

Poster #: 245
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: p38, Wnt catenin, cancer
Project Title: Identification of a Novel Group of Cell Cycle Inhibitors’ Effects on β-catenin through the p38 MAPK Signaling Pathway
Author List:
Banuelos, Allison; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Spolsdoff, Devin; Undergraduate, Biological Sciences, California State University, Fullerton
Kasper, Logan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Patel, Nilay; Biological Science, California State University, Fullerton

Abstract: Mitogen-activated protein kinases (MAPKs) mediate a variety of cellular behaviors in response to extracellular stimuli. The p38 MAPK signaling pathway has been shown to mediate a wide range of biological processes, such as the modulation of cell cycle progression, proliferation, and apoptosis. The dysregulation and over-activation of p38 MAPK levels are often found in various cancers. It has been suggested that p38 MAPK also participates in cross-talk with the canonical Wnt pathway to regulate β-catenin. In the canonical Wnt pathway, a destruction complex composed of Axin, APC, and GSK3β, marks β-catenin for degradation, preventing it from interacting with the TCF/LEF family to transcribe downstream genes involved in cell proliferation. Mutations in this pathway that lead to increased levels of β-catenin are also associated with cancer. Thus, compounds that downregulate p38 MAPK activity to reduce β-catenin levels may serve as an anti-cancer therapeutic strategy. We screened a group of novel cell cycle inhibitors’ effects on HeLa cell proliferation and found that a group of compounds reduced cell proliferation by >40%. Immunocytochemistry (ICC) and Western blotting showed that certain compounds reduced β-catenin levels in HeLa cells compared to vehicle (0.1% DMSO) treated cells. Quantitative polymerase chain reaction (qPCR) suggested that these compounds are also downregulating mRNA levels of p38 MAPK and its downstream target genes BMP2, COX2, and SERPINE2 relative to no treatment. Our data suggests that our compounds may be reducing β-catenin and cell proliferation through a p38 MAPK/Wnt dependent manner. The results of our planned experiments will provide a more thorough understanding of the cross-talk between the p38 MAPK and Wnt/β-catenin pathway, and identify the anti-proliferative effects of these compounds.

Poster #: 246
Campus: CSU San Bernardino
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Influenza virus , RNA expression , RT-qPCR
Project Title: Establishing the role of NS1 – NP interaction and effect on viral RNA expression.
Author List:
Lopez, Larry; Graduate, Biology , California State University, San Bernardino, Presenting Author
Aquino, Edmar Jack; Undergraduate, Biology , California State University, San Bernardino
Gallardo, Jennifer ; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Gazca, Cinthia; Undergraduate, Biology, California State University, San Bernardino
Newcomb, Laura ; Biology, California State University, San Bernardino

Abstract: Influenza virus is of economic and global health concern. Influenza rapidly evolves to evade vaccines and gain resistance to antiviral therapies. While yearly vaccination is required to control seasonal influenza, pandemic prevention relies on effective antivirals. Here we aim to establish if the viral nonstructural NS1 protein enhances viral RNA expression through interaction with NP. If so, the NS1-NP protein interaction could represent a novel antiviral target. Influenza vRNP complexes are responsible for viral RNA synthesis and comprise a vRNA segment bound by the viral RNA dependent RNA polymerase complex (RdRP) and multiple copies of nucleoprotein (NP). To examine the effect of NS1, reconstituted viral ribonucleoproteins (vRNPs) were expressed in the presence or absence of NS1 in human tissue culture cells (293T). Cells were collected and total RNA was isolated. RNA concentration was determined by optical density and equal concentrations resolved on a 1% agarose bleach gel to confirm integrity. RNA was reverse transcribed with oligo dT and quantitative PCR performed with specific primers. Our results confirm addition of NS1 enhances RNA expression from reconstituted vRNPs. Although we hypothesized del20NP, an NP mutant with 20 nucleotides deleted from the N-terminus, would not respond to addition of NS1, our analysis reveal that while overall activity of del20NP vRNPs is diminished compared to WT-NP vRNPs, enhancement of RNA expression by addition of NS1 is still observed. These results suggest NS1 effect on viral RNA expression is not through the N-terminus of NP. To directly evaluate NP-NS1 interaction we have initiated sucrose gradient ultracentrifugation to examine NP complexes. Human tissue culture cells (293T) were transfected with plasmids to express WT-NP or del20NP in the presence of absence of NS1. Total protein extracts were isolated and examined for NP and NS1 expression by Western blot. Protein extracts were separated on 5 – 40% sucrose gradient with a 16-hour spin at 50K rpm at 4°C. Gradient fractions were collected in thirteen parts and each fraction will be analyzed by Western blot to detect NP and NS1. Based on the reconstituted vRNP assay results, we expect both WT-NP and del20NP to demonstrate NS1 interaction. Future experiments will examine effect of NS1 mutants on viral RNA expression and NP interaction. Research is funded by CSUPERB Research Development Grant to LLN. JVG is a MARC scholar via NIGMS training grant GM083883.

Poster #: 247
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: metabolism, tissue regeneration, stem cells
Project Title: Glutamine sustains Cripto expression, enabling MyosinII/RAB11A-dependent trafficking in stem cells to promote tissue regeneration
Author List:
Duell, Erika; Undergraduate, Biology, California State University, Northridge, Presenting Author, Nagel Award Nominee
Hoover, Malachia ; Graduate, California State University, Northridge
Booker, Evan; The Salk Institute
Williams, Blake; Undergraduate, California State University, Northridge
Arellano-Garcia, Caroline; Undergraduate, Biology, California State University, Northridge
Fisher, Wolfgang; The Salk Institute
Gray, Peter; The Salk Institute
Kelber, Jonathan; Biology, California State University, Northridge

Abstract: Stem cells are necessary for proper tissue homeostasis and regeneration while dysregulation of their activity leads to multiple pathologies. We hypothesized that the stem cell marker, Cripto (TDGF1), is a regulator of tissue regeneration. Using the zebrafish model of caudal fin wound healing, we demonstrate that Cripto is necessary and sufficient for stem cell-mediated regeneration in this in vivo model. We identified non-muscle Myosin IIs (MYH9/10) as novel Cripto binding proteins using proteomics and confirmed these interactions by Co-IP/Western blot and immunofluorescence (IF) in mesenchymal stem cells. Notably, dual pharmacological inhibition of Cripto and Myosin IIs did not reduce caudal fin regeneration beyond that of either inhibitor alone, suggesting that these proteins function in the same pathway during this process. Our previous work demonstrated that soluble (GPI anchor-cleaved) Cripto promotes tissue-specific stem cell functions via cell-surface binding to GRP78 (glucose-regulated protein 78). Here, we report that Myosin II inhibition reduces cell surface localization and production of soluble Cripto. Since GRP78 primarily functions as an ER chaperone that responds to cellular stress such as nutrient or oxygen depletion, we tested whether nutrient deprivation affects Cripto expression, sub-cellular localization or binding to Myosin IIs in mesenchymal stem cells. Interestingly, we found that glutamine is necessary, but not sufficient, to sustain Cripto expression and its Myosin II-associated functions. Using the BioGrid database, we discovered Rab11A as a co-binder of GRP78 and Myosin IIs. Given the endosomal/exosomal trafficking functions of Rab11A, we hypothesized that Cripto may in turn help traffic GRP78 or other cell surface proteins into these compartments in a Myosin II-dependent manner. Inhibition of Myosin IIs in mesenchymal stem cells transfected with wild-type, constitutively active or dominant negative GFP-tagged Rab11A revealed that Myosin IIs act upstream of and in concert with Rab11A regulate Cripto localization to Myosin II-rich membrane protrusions. Based upon these data, we propose a new model of Cripto function whereby Myosin II/Rab11A signaling promotes membrane localization of Cripto to facilitate unique endosomal/exosomal transport processes. Furthermore, reduced glutamine metabolism limits cellular Cripto levels, restricting protein sorting and autocrine/paracrine signaling during stem cell-mediated tissue regeneration.

Poster #: 248
Campus: CSU Long Beach
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: inositol, diabetes, Drosophila melanogaster
Project Title: Inositol Reduces Hyperglycemia and Obesity in Drosophila melanogaster larvae
Author List:
Flores, Angelina M.; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Rivera, Maria J. ; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Eldon, Elizabeth D. ; Biological Sciences, California State University, Long Beach
Klig, Lisa S. ; Biological Sciences, California State University, Long Beach

Abstract: Gestational diabetes predisposes pregnant women to type 2 diabetes. Myo-inositol supplementation of pregnant women’s diets has been shown to reduce the incidence of gestational diabetes, and as such type 2 diabetes, by decreasing insulin resistance. Myo-inositol is a six-carbon sugar alcohol that is a precursor for phosphatidylinositol (PI), a cell membrane phospholipid. PI is involved in the phosphoinositide-signaling pathway, which is essential for the regulation of cellular functions. Myo-inositol has also been implicated in diseases and complications such as polycystic ovary syndrome and cancer, as well as metabolic diseases. In this study hyperglycemia and obesity induced by a high sucrose diet are examined in Drosophila melanogaster third instar larvae. The hypothesis is that inositol, both dietary and endogenously synthesized, will have a positive effect relieving hyperglycemic and obese conditions. Wild-type like larvae grown on semi-defined food with high sugar had an obese-like phenotype that was demonstrated in a float buoyancy assay. Inositol supplementation of this semi-defined food reduces the number of obese-like larvae. Similar to the high sucrose diet, deletion of the inositol synthetic gene (MIPS) also caused an obese-like phenotype, which was dramatically improved with inositol supplementation. This study also addresses hyperglycemia by measuring the glucose content within the hemolymph of the larvae. High sucrose food increases the levels of glucose in the hemolymph of wild-type like larvae more than 25%, while inositol supplementation reduces hemolymph glucose levels by more than 10% in all conditions. Similar to high sucrose food, the lack of the MIPS gene results in elevated hemolymph glucose, which is also reduced by the addition of inositol. These studies were further expanded to include an examination of the expression levels of the MIPS gene via qPCR for the wild-type like larvae fed either a diet to induce obesity and hyperglycemia, or the same diet supplemented with inositol. These studies contribute to a better understanding of inositol’s role in obesity, hyperglycemia, and diabetes.
This project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638 and CSULB RS114.

Poster #: 249
Campus: Cal Poly Pomona
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Human Mesenchymal Stem Cells, Adipogenesis, SUV39H1
Project Title: Epigenetic Regulator SUV39H1 Is a Negative Regulator of Human Adipogenesis
Author List:
Tan, Lun; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Ibili, Esra; Undergraduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Zhao, Yuanxiang; Biological Sciences, California State Polytechnic University, Pomona

Abstract: Human adipogenesis is the process through which uncommitted human mesenchymal stem cells (hMSCs) differentiate into adipocytes (fat cells). Understanding its molecular and cellular regulation may provide a way to prevent and/or treat obesity and obesity related diseases. HMSCs normally reside in the bone marrow and adipose tissue. They can be easily isolated, expanded in vitro and upon receiving the appropriate external stimuli, differentiate into specific mature cell types including adipocytes. Based on a high throughput screen using siRNAs targeting 5000 genes in the human genome, a list of genes whose expression knock-down by its siRNAs led to enhanced adipogenic differentiation of hMSCs were uncovered. This study has focused on one of the uncovered genes, SUV39H1, which encodes a histone lysine methyltransferase and its role in human stem cell fate commitment has never been studied. Here we report that SUV39H1 is down-regulated during adipogenic differentiation of hMSCs. Knocking down the expression of SUV39H1 by around 65% using siSUV39H1 consistently promoted adipogenesis, resulting in near two-fold of oil droplets compared to siControl, even though total cell number was reduced by about 10%. Comparison between siControl and siSUV39H1 treatment groups after 14, 30, and 60 days of adipogenic differentiation demonstrated that transient transfection with siSUV39H1 during differentiation initiation not only accelerated the process of adipogenesis by allowing cells to accumulate fat faster, but also significantly increased the percentage of cells becoming mature adipocytes, likely by potentiating cells that typically do not respond to adipogenic inducing media to commit to adipogenic lineage. In addition, transient transfection with siSUV39H1 during the differentiation initiation stage promoted cells to commit to adipogenic lineage even in the presence of osteogenic inducing media, which normally commit the cells to osteogenic lineage in the absence of siSUV39H1. Finally, we demonstrated that the expression of CEBPα, a master regulator of adipogenesis, was markedly increased by around five-fold in siSUV39H1 cells as compared to siControl cells. Taken together, our study indicates that SUV39H1 is a negative regulator of human adipogenesis, whose expression knock-down significantly promotes adipogenic differentiation efficiency by both accelerated speed and increased adipogenic fate commitment, at least partly through promoting the expression of CEBPα.

Poster #: 250
Campus: Stanislaus State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Recombineering, CRISPR, Mutagenesis, ,
Project Title: CRISPR Induced High Frequency of Site Specific Mutagenesis in E. coli
Author List:
Youngblom, Jim; Biology, California State University, Stanislaus
Gonzales, Allysa; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Collins, Heather; Undergraduate, Biology, California State University, Stanislaus, Presenting Author
Vergara, Carlos; Undergraduate, Biology, California State University, Stanislaus
Soriano, Elizabeth; Undergraduate, Biology, California State University, Stanislaus

Abstract: CRISPR technology has revolutionized genome editing and has been subject to significant attention both in the science literature and in the mass media. The purpose of this study was to determine whether an E. coli site-specific mutagenesis system using CRISPR mediated cutting as a stimulus to promote recombineering (recombination-mediated genetic engineering) could be applied to predictably, with high frequency, mutate a particular gene in E.coli. The LacZ gene was chosen because a reliable selection technique is available. To initiate this project we constructed two plasmids. One plasmid contains the coding sequence for CAS9, a tracrRNA, and a tetracycline resistance gene. The other plasmid contains 30 nucleotides of the LacZ gene expressed as a guide RNA and a kanamycin resistance gene. Following electroporation into the recombineering strain XTL118 of E. coli and selection on standard bacterial growth media containing kanamycin, tetracycline, IPTG, and x-gal we were able to conclude that we had directed a single-stranded 59-nucleotide DNA fragment containing an in-frame stop codon to replace the homologous region in wild-type LacZ. Modified colonies were white; unmodified were blue. More than 50% of the post-transformation colonies were white, indicating an unusually high rate of mutagenesis. White colonies were subjected to colony PCR and sequencing to confirm the presence of the specific nucleotide change (G to C) in the 204th codon. Ongoing experiments are testing the correlation between the efficiency of site-specific mutagenesis and 1) strand selection (leading vs. lagging strand) for the hybridization of the guide RNA and 2) the number and position of mismatches between the single-stranded DNA fragment and the host chromosome. These experiments are relatively easy and cheap, allowing undergraduate students in our advanced genetics lab class to use recombineering in conjunction with CRISPR directed cleavage to successfully modify an E. coli. ribosomal protein gene. Students committed to pursuing further education and careers in molecular biology are benefiting from first-hand experience with CRISPR and a greater depth of understanding of a new and powerful technique. Students not pursuing careers in molecular biology are benefiting as CRISPR technology is forcing society to consider new bioethical scenarios and students exposed to the technology will be able to address such scenarios with a more scientifically informed perspective.

Poster #: 251
Campus: Cal Poly San Luis Obispo
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Yeast, Meiosis, Chromosome segregation
Project Title: The roles of the Ndj1 and Tid1 proteins in an alternative system that directs chromosome segregation during meiosis
Author List:
Shaw, Ethan; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Hillers, Ken; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: Meiosis is a specialized form of cell division in sexually reproducing eukaryotes. Crossovers are physical connections formed between homologous chromosomes during meiosis; these connections help ensure normal segregation of homologous chromosomes at meiosis I. However, the yeast Saccharomyces cerevisiae and other eukaryotes can still segregate homologs properly even in the absence of some crossovers. This is due to a backup mechanism known as distributive segregation. This mechanism in yeast properly segregates non-crossover chromosomes at a higher rate than if segregation were completely random. To study distributive segregation, we have generated diploid yeast with one chromosome pair that will cross over only infrequently, due to DNA sequence divergence. Appropriate segregation of this chromosome pair during meiosis will depend on distributive segregation; we can then assess the possible roles of candidate proteins in distributive segregation through determination of the effect of mutation on segregation of this chromosome pair. Our initial work has focused on the roles of two proteins, Ndj1 and Tid1. These two proteins affect meiosis in many ways, including the efficiency of crossover regulation and the overall timing of meiosis, but their roles during distributive segregation are not fully known.
After yeast undergo meiosis, they form a four-spore tetrad; each spore represents one of the four products of meiosis. These tetrads can be dissected using a micromanipulator, and the four spores set out onto growth medium and germinated. Failure to segregate a chromosome pair during meiosis I will result in nondisjunction, producing two haploid spores lacking one chromosome; these aneuploidy spores are inviable, and will not germinate. Thus, meiosis I nondisjunction produces an elevated incidence of 2-spore-viable tetrads after meiosis; we use this phenotype as a measure of the efficiency of distributive segregation.
Comparison of spore viability among WT, ndj1-, and tid1- strains reveals an elevated incidence of 2-spore-viable tetrads in ndj1-, but not tid1-; these results suggest that the Ndj1 protein, but not the Tid1 protein, plays some role in distributive segregation. However, the overall spore viability in these strains was low enough to complicate analyses; thus, we are currently preparing to redo the experiments in the SK1 strain, which is a better strain for meiotic analyses.
Funded by the Cal Poly College-Based Fee program.

Poster #: 252
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Lipoprotein Lipase, cholesterol efflux, scavenger receptors
Project Title: The down-regulation of ABCA1 in LPL-expressing THP-1 macrophages may be mediated by fatty acid products of lipolysis
Author List:
Kim, Aidan; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Moctar, Khadija; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge

Abstract: Introduction: Atherosclerosis is a condition in which the artery walls harden and get blocked due to a buildup of cholesterol-loaded macrophages. Cellular cholesterol accumulation is the net result of receptor-mediated uptake and transporter-mediated efflux. Scavenger receptors (CD36, SR-AI, SR-BI) mediate cellular cholesterol uptake whereas the ATP-binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. Lipoprotein lipase (LPL) is secreted by macrophages and catalyzes the hydrolysis of circulating triglycerides, facilitating cellular lipid uptake. We have recently demonstrated that the expression of ABCA1 is upregulated in LPL-knock-down THP-1 cells.
Hypothesis: We hypothesized that in addition to regulating cholesterol efflux, LPL may also regulate the expression of macrophage scavenger receptors responsible for cholesterol uptake. Further, we hypothesized that fatty acid products of LPL’s lipolytic activity may repress ABCA1 transcription or function in WT cells.
Methods: The LPL gene was silenced in THP-1 macrophages (LPL-KD) by siRNA interference technology. Transcript levels of scavenger receptors were compared in WT and LPL-KD macrophages by RT-PCR. The mass of total cholesterol in cell lysates was compared using a cholesterol quantification assay. To mimic the presence of lipolysis products, WT cells were incubated in the absence or presence of various unsaturated or saturated fatty acids, followed by measurement of ABCA1-mediated cholesterol efflux.
Results: LPL-KD cells showed an increase in ABCA1 expression (161% of WT). Cholesterol content in LPL-KD cells was 75% compared to WT macrophages. The expression of scavenger receptors SR-BI and CD36 was lower in LPL knock down THP-1 cells compared to WT macrophages (55% and 60%, respectively). Treatment of WT cells with Palmitic acid and Stearic acid increased cholesterol efflux by approximately 50%, whereas Oleic acid did not significantly alter cholesterol efflux.
Conclusion: LPL may modulate cellular cholesterol content by regulating the expression of scavenger receptors and the cholesterol transporter. Contrary to our hypothesis, certain fatty acid products of lipolysis increased cholesterol efflux from THP-1 macrophages. Experiments are underway to distinguish the effects of fatty acids on ABCA1 transcription, stability and function.
Acknowledgements: This work was supported by National Institutes of Health Awards R15HL083946 and SC3GM095413.

Poster #: 253
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: CRISPR-Cas9, extracellular matrix, proprotein convertase cleavage
Project Title: Investigating enzymatic cleavage of endogenous MAGP2 by gene disruption via CRISPR-Cas9
Author List:
Song, Ann; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Perez, Aldwin Apollo; Graduate, Biological Science, California State University, Fullerton
Miyamoto, Alison; Biological Science, California State University, Fullerton

Abstract: Microfibril-associated glycoprotein 2 (MAGP2) is a small secreted protein associated with elastic fibers in the extracellular matrix; it promotes elastic fiber formation, angiogenesis, anti-inflammatory effects, and regulates Notch and integrin signaling. Alterations of MAGP2 are correlated with an inherited vascular disorder and ovarian cancer. MAGP2 function may be regulated by proprotein convertases (PC) cleavage of its last 20 amino acids; this cleavage promoted the binding of exogenous MAGP2 to microfibrils in immunofluorescence studies (IF). To study MAGP2 at its endogenous levels, a cell culture model carrying a mutation in the PC cleavage site of MAGP2 was generated using CRISPR-Cas9. Specifically, one plasmid carrying a MAGP2 gRNA with Cas9 and a second carrying the donor PC site mutation were co-transfected into HEK293 cells to activate site-specific homology-directed repair (HDR). After neomycin selection, 15 drug-resistant clones were generated; PCR genotyping and sequencing analysis showed that three clones integrated the PC mutation into a single MAGP2 allele. These results revealed that mutant cell lines were heterozygous, which is consistent with reported CRISPR studies using HDR; homozygosity is much more difficult to obtain. Nevertheless, HEK293 lines carrying one mutant allele of MAGP2 were successfully created. Therefore, the CRISPR plasmids could be used to investigate the role of MAGP2 PC cleavage in multiple cell models, including ovarian cancer cells, endothelial cells, and dermal fibroblasts. Thus, the CRISPR plasmids were electroporated into immortal human dermal fibroblasts (GM5659T) to study the role of PC cleavage in MAGP2 microfibril binding; however only one clone with random integration of the mutation was achieved. This may be due to low transfection efficiency of GM5659T since mutant cell lines were able to be generated in HEK293, a high transfection efficiency cell line. Published studies also reported that low transfection efficiency reduces HDR-mediated mutagenic efficiency. Together, these results indicate a more efficient gene delivery system, such as lentivirus, is required for site-specific integration. Additionally, HDR efficiency could be increased using small molecule inhibitors to repress other competing repair pathways. In conclusion, these studies have generated heterozygous MAGP2 PC mutant cell lines and identified conditions for generating homozygous cell lines. Funding: CSUF Junior Faculty Intramural grant

Poster #: 254
Campus: CSU Fullerton
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: cancer, cell cycle, drug discovery
Project Title: A Novel Group of Anti-proliferative Compounds Mediate their Effects through p21 Related Cell Cycle Proteins
Author List:
Daoudi, Sarah; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Youn, Stephanie; Undergraduate, Biological Science, California State University, Fullerton
de Lijser, Peter; Chemistry and Biochemistry, California State University, Fullerton
Patel, Nilay; Biological Science, California State University, Fullerton

Abstract: We have screened a small library of compounds synthesized at California State University, Fullerton, for their anti-proliferative activity in three human cell lines: HeLa, HUTU80, and MG63. CyQUANT cell proliferation assay also showed some cell line specificity of ‘hit’ compounds. These select group of compounds have been shown to reduce b-catenin levels and cellular proliferation rates (unpublished). The current project focuses on identification of the effects of these compounds on p21, a cell cycle regulatory protein. The protein p21, is a major target of p53 activity and is linked to DNA damage and cell cycle arrest. p21 is a member of the CIP/KIP family which are CDK (cyclin-dependent kinase) inhibitors. Our preliminary results also show that these compounds increase the amount of p21 protein and could be affecting the cell cycle, specifically the interaction between p53 and p21. qPCR results suggest CDKN1A expression levels increase in the presence of the compounds and CDK1NA induction could possible attenuate cell cycle arrest. Expression of p53 target genes also increase after treatment with anti-cancer compounds, implying the activation of p53 activity. P21 inhibition is attributed to two activities, which include carboxyl-terminal PCNA-domain and an amino-terminal CDK-cyclin inhibition domains. The interaction between p21 and PCNA (proliferating cell nuclear antigen), a DNA polymerase accessory factor, results in direct inhibition of DNA synthesis in the S phase of the cell cycle. Whereas p21’s interactions with CDKs occur through binding at the N-terminal and interfering with the phosphorylation of CDK1. It was shown that p21 inhibits kinase activity of CDK1 and induces arrest in the G2 phase of the cycle. Treatment of HeLa cells with these hit compounds indicated a decrease of CDK1 gene expression levels but did not impact PCNA; implying the inhibition of CDK1 and potential cell cycle arrest. Our ongoing experiments focus on determining whether p53 is being activated. These analyses will help identify mechanism of action for the hit compounds in this library of compounds.

Poster #: 255
Campus: San Diego State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Genomics, Bioinformatics, Microbiology
Project Title: Genome Sequence of USA100 MRSA strain 209
Author List:
Chen, Yingfeng; Graduate, Biology, San Diego State University, Presenting Author
Crosby, Heidi ; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora,
Oosthuysen, Wilhelm; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora,
Diekema, Daniel; Department of Internal Medicine, University of Iowa, Iowa City
Kelley, Scott; Biology, San Diego State University
Horswill, Alexander; Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora

Abstract: USA100 strains are a significant contributor to the overall burden of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infections. Strain 209 (also called IA209) is a throat culture isolate that is spa type 002, SCCmec type II, PVL negative, and classified by pulse field gel electrophoresis as USA100. The strain is resistant to antibiotics methicillin, oxacillin, erythromycin, clindamycin, levofloxacin, and cefoxitin. Here, we used de novo genome assembly method to generate a nearly complete draft genome for this strain. Cells grown in pure culture were lysed with lysostaphin and DNA was purified using the Puregene Yeast/Bacteria Kit B (Qiagen). DNA libraries were generated using the KAPA preparation kit (Roche) and sequenced using the Illumina MiSeq platform. Sequencing generated a total of 5,320,190 paired end reads. The raw sequences were pre-processed using Cutadapt to trim out adaptors, and Prinseq was used to perform quality filtering. Initial genome assembly was performed with SPAdes genome assembler program, which produced 17 contigs (>500 bp) with a combined total of 2,777,773 bp (N50 value was 324,561 bp) and with a G+C content of 32.73%. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) predicted the genome contain a total of ~2400 proteins. Comparisons of the genome annotations to related strains will be used to determine novel mechanisms of antibiotic resistance in this strain.

Poster #: 256
Campus: CSU Northridge
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: Glycolysis, Lipoprotein Lipase, Gene Silencing
Project Title: Effects of Silencing the Lipoprotein Lipase Gene on Enzymes of Glycolysis in Skeletal Muscle Cells
Author List:
Mogul, Adam; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Ramos Correa, Michele; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Medh, Jheem; Chemistry and Biochemistry, California State University, Northridge

Abstract: Background: Lipoprotein lipase (LPL) is an enzyme required for the hydrolysis of triglycerides to free fatty acids and glycerol. In earlier reports, we demonstrated that muscle LPL levels are directly correlated with insulin resistance, and reducing LPL expression increased insulin-stimulated glucose uptake. Additionally, LPL-knock down (LPL-KD) L6 rat muscle cells showed increased glucose oxidation compared to wild type (WT) L6 cells. Glycolysis is the first pathway in glucose oxidation, and two enzymes regulating glycolysis are hexokinase (HK) and phosphofructokinase (PFKM).
Objective: To compare expression and activity of the glycolytic enzymes hexokinase and phosphofructokinase in LPL-KD and WT L6 muscle cells.
Methods: The LPL gene was silenced in rat skeletal muscle cells (L6 cells) by lentiviral-mediated RNA interference. Total RNA was isolated and specific primer pairs were used for end-point and real-time PCR amplification of LPL, Hexokinase 2, and PFKM. β-actin was used as a housekeeping gene during all PCR analyses. The threshold cycle (CT) values from quantitative PCR were used to calculate fold change in transcript levels using the 2^(-ΔΔCT) method. The amplicons from end-point PCR were resolved by 2% agarose gel and the DNA bands were quantified by ImageJ (NIH) analysis.
Results: Quantitative PCR has shown that LPL-KD cells have <3% of the LPL expression as their WT counterparts due to the shRNA silencing. Silencing of the LPL gene resulted in a dramatic repression of transcription of HK (12.5% of WT levels) and PFKM (3.8% of WT levels). These findings were confirmed via end-point PCR which showed significantly lower intensity for HK and PFKM amplicons (54% and 57% respectively) in LPL-KD samples compared to WT.
Conclusion: LPL-KD cells showed decreased transcription of glycolytic enzymes but an increase in glucose oxidation. Thus, enzyme activity assays will be carried out to provide more insight on this disparity. We hypothesize that in WT cells, lipolytic products may inhibit glycolysis, thereby triggering an upregulation of transcription of glycolysis regulatory enzymes including HK and PFKM.
Acknowledgements: This work was supported by NIH Award SC3GM095413.

Poster #: 257
Campus: Humboldt State University
Poster Category: Molecular Biology (Include Regulation and Genomics)
Keywords: pluripotent, cell fate, germ layer
Project Title: A regulatory role for cJun in overexpression of Oct4 leads to increased GATA4 expression and cardiomyocyte formation
Author List:
Brewer, Rachel; Graduate, Biological Sciences, Humboldt State University, Presenting Author
Pan, Meiling; Undergraduate, Biological Sciences, Humboldt State University, Presenting Author
Koepke, Lauren; Undergraduate, Biological Sciences, Humboldt State University
Sprowles, Amy; Biological Sciences, Humboldt State University

Abstract: As cells transition from the point of fertilization through the process of embryonic development many critical choices are made which affect cell fate. At the blastocyst stage, the earliest distinction, two separate populations arise. One will generate all of the extraembryonic tissues while the other will yield all of the embryonic tissues. These cells, which generate the organism, are termed pluripotent at this stage and found within the inner cell mass (ICM). Here, we examined the effect of cJun expression in regulating Oct4, a gene known to be a master regulator of pluripotency. Luciferase assays demonstrated that our wild type (WT) cJun plasmid increased the activity of the Oct4-luciferase reporter construct, which encompasses approximately a 3Kb sequence directly upstream of the transcription start site. Additionally, we corroborate previous reports that cJun can synergize with beta-catenin, a member of the Wnt pathway, to significantly increase the promoter activity of Oct4. To assess the developmental implications of this regulatory relationship, we carried out transfections with WT-cJun followed by differentiation to embryoid bodies. Western blot analysis showed an increase in Oct4A expression 36 hours post-transfection, when cells were treated with cJun compared to the vehicle alone. Embryoid bodies were harvested four and six days following initiation of differentiation and it was revealed that an increase in cJun correlated with an increase in GATA4 expression. Together, these suggested that an increase in Oct4A driven by cJun regulation could lead to selection toward the endodermal germ layer. In light of this, we carried out directed differentiation into cardiomyocytes, using a hanging drop intermediate, and found that an increase in cJun led to a significant increase in cardiomyocyte formation and beating. Overall, this suggests a new role for cJun in the regulation of potency and the formation of the endoderm during those early cell fate choices.

Poster #: 258
Campus: Sonoma State University
Poster Category:
Keywords: kinetics , Photo-CORMS, Fuel Cells
Project Title: Synthesis and Kinetic Experiments of Solvent Dependent Photochemical [Fe-Fe] Hydrogenase Model Complexes
Author List:
Jolly , Brandon ; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Ortiz , Lilia ; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Works, Carmen ; Chemistry , Sonoma State University

Abstract: The iron-iron hydrogenase enzyme, as well as some of its structural and functional models, have been shown to catalyze the reversible oxidation of molecular hydrogen. Their catalytic activity is inhibited by carbon monoxide but this inhibition is photochemically reversible. Photolysis of [Fe-Fe] hydrogenases model compounds release carbon monoxide ligands, and our data suggests that this photoproduct is an active catalyst for hydrogen utilization. In addition, carbon monoxide is known to have therapeutic benefits at low concentrations in mammilla tissues. This has inspired our group to study the CO releasing ability of iron-iron hydrogenas model compounds both for use as potential fuel cell technology and as possible CO releasing molecules (Photo-CORMS) for therapeutic applications. Kinetic studies were employed to obtain temperature dependent rate constants for the thermal reaction of the photoproduct of such model compounds with CO in various solvents. Our data suggests that this reaction is solvent dependent with rate constants varying from 0.9 M-1s-1 (in THF) to 8.0 x 108 M-1s-1 (in hexane) and Eyring plots, suggest a negative entropy of activation. This data is important to determine the utility of these molecules to biotech applications. In addition, we have made a variety of synthetic modifications to determine how electronic properties determine photochemical, thermal and solvent reactivity.

Poster #: 259
Campus: Stanislaus State University
Poster Category:
Keywords: DNA-Repair, FRET, Single-Molecule-Fluorescence
Project Title: Development of an Undergraduate-Friendly Single-Molecule Fluorescence System to Study DNA Base Flipping and Protein-DNA-Interaction in UV Damaged DNA
Author List:
Godínez, José; Undergraduate, Chemistry, California State University, Stanislaus, Presenting Author
Lo, Jonathan Y.; Undergraduate, Chemistry, California State University, Stanislaus
Brummel, Connor; Undergraduate, Computer Science, California State University, Stanislaus
Alemán, Elvin A. ; Chemistry, California State University, Stanislaus

Abstract: Cyclobutane pyrimide dimers (CPD) in DNA, which are formed when DNA is exposed to ultraviolet irradiation, block replication if the systems designed to repair the CPD sites fail. The repair enzyme T4 endonuclease V (T4 endo V) eliminates CPD damages. The crystal structure of T4 endo V shows that when the protein complexes with a double helix DNA containing a thymine dimer, the 5’ side complementary adenine flips out and makes the dimer accessible for the catalytic reaction without affecting the adenine base. The crystal structure also shows the DNA is bent ~60o. We have developed single molecule fluorescence (SMF) and fluorescence resonance energy transfer (FRET) spectroscopy approaches to study the repair mechanism and kinetics of T4 endo V in real time.
The SMF experiment is done using a Luca-Andor EM camera and a Nikon microscope. Micro-Manager, an open-source software that was modified to permit the monitoring of intensity in two regions of interest, in real time. We have modified the software to produce an accessible data file with which the recorded data can be analyzed proceeding the experiment. As a proof of principle, the optical setup built for the SMF experiments and the software were tested using quantum dots to prove the efficiency of the equipment in imaging and monitoring particles approaching the picometer scale and these results will be presented.
To study the bending mechanism of CPD damaged DNA we used FRET spectroscopy. The FRET spectroscopy was done by using a Cy3 donor fluorophore and a Cy5 acceptor fluorophore on both damaged and undamaged DNA. Compared to the crystal structure of the T4 endo V-DNA complex, Cy3 and Cy5 fluorophores were strategically localized to use FRET to study the bent conformation. We have characterized the FRET properties using SMF, steady-state measurements, and gel electrophoresis. The decrease of the Cy3 fluorophore and increase of the Cy5 from the emission intensities indicates that FRET is occurring between both fluorophores. With FRET occurring, the steady state and SMF experiments has helped us to understand the bending of DNA in CPD damaged DNA. This project is important because it will lead to a better understanding of how T4 endo V finds damaged sites; and how, when, and why the structure of the DNA is distorted during the repair process.

Poster #: 260
Campus: Cal Poly San Luis Obispo
Poster Category: Other
Keywords: , ,
Project Title: Myoblasts enhance collateral capillary arteriogenesis during chronic ischemia
Author List:
Hamzeinejad, Vahid; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Cardinal, Trevor R.; Biomedical Engineering, California Polytechnic State University, San Luis Obispo

Abstract: Peripheral arterial occlusive disease (PAOD) involves peripheral artery occlusion, which causes ischemia (i.e. insufficient blood flow) and is the leading cause of amputation. Natural bypass vessels known as collaterals can preserve downstream tissue viability by creating an alternative route for blood flow. Increased blood flow through preexisting collateral increases shear stress and triggers arteriogenesis, the outward remodeling of arterial vessels. The presence of collaterals improves prognosis in patients with ischemic disease, but many patients never develop identifiable collaterals to relive peripheral ischemia. Animal models that lack preexisting collaterals are capable of forming collaterals from capillaries that undergo arteriogenesis to become arterialized capillary collaterals (ACC). A treatment to induce the formation of ACCs, particularly in diseased or aged tissues that lack the propensity to form new collateral vessels, could be highly effective in giving protection against ischemia. Exercise is a great therapy for PAOD, since following exercise, resident muscle stem cells known as satellite cells (SCs) activate and regulate local angiogenesis. SCs and their progeny myoblasts, secrete factors known to be involved in arteriogenesis (many of these compounds involved in angiogenesis are also involved in arteriogenesis) and the recruitment of other cells involved in remodeling. Therefore, transplanting these putative arteriogenic cells is a potential therapy to recapitulate some of the beneficial effects of exercise therapy. In this study, the lateral feed artery in the spinotrapezius muscle of Balb/C mice was ligated to induce chronic ischemia; myoblasts were transplanted during surgery to enhance arteriogenesis of collateral capillaries. Implanted mice exhibited increased arteriogenesis, ACC diameter increased to 28.11 ± 0.85 μm vs 16.25 ± 0.62 μm in a no transplant control. As corollary, we evaluated the process for cell construct preparation, and discovered that cells cultured on scaffold overnight (20.86 ± 1.6 μm) before transplant did not stimulate arteriogenesis as significantly as those who were cultured for 6-hours only. Limited nutrient supply and gas exchange while incubating on scaffolds could induce stress on myoblast and cause limited reactivity/apoptosis post implant. In summary, myoblast transplantation increases ACC arteriogenesis; however, they need to be transplanted as early as possible post-harvest from culture flask.

Poster #: 261
Campus: CSU Fresno
Poster Category: Other
Keywords: Alzheimer’s disease, Apolipoprotein E, Small Organic Molecule Drugs
Project Title: Tertiary Sulfonamides and arylmethyl amines to modulate apoe, abca1/ldlr for Alzheimer’s Therapy
Author List:
Kumar, Prarthana; Undergraduate, Chemistry, California State University, Fresno, Presenting Author, Nagel Award Nominee
Bajwa, Bhvandip; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Kim, Byungwook; Department of Neuroscience, Mayo Clinic, Jacksonville, FL
Kim, Jungsu; Department of Neuroscience, Mayo Clinic, Jacksonville, FL
Maitra, Santanu; Chemistry, California State University, Fresno

Abstract: One of the more prominent traits of Alzheimer’s disease (AD) is the accumulation of insoluble Amyloid Beta (Aβ) plaque that are deposited in the brain. Aβ plaque is caused by accumulation and aggregation of soluble Aβ monomers created from the proteolysis of amyloid precursor protein (APP). Apolipoprotein E (apoe) is a transport lipoprotein that helps with lipid regulation in the body and brain. Some of the genetic variations of apoe have been studied to reveal their ineffectiveness at removing soluble Aβ monomers before the formation of the final insoluble plaque. Upregulation of ATP binding cassette transporter a1 protein (ABCA1) has also been shown to inhibit amyloid formation. Apoe functions as a ligand for Low Density Lipoprotein Receptor (LDLR) mediated endocytosis and inducing LDLR had a direct effect on Abeta plaque reduction in transgenic mice brains. Our collaborative research aimed at developing small organic molecules to inhibit apoe and induce abca1 or ldlr in order to ultimately reduce Ab plaque accumulation in the brain. We hypothesize that by reducing apoe and increasing abca1/ldlr, we might be able to reduce Amyloid plaque from the brains and provide protection or prevention. Preliminary studies successfully identified a triarylmethyl amine scaffold that inhibited apoe in human astrocytoma cell. Structure-Activity Relationship (SAR) studies subsequently helped develop tertiary aryl sulfonamides and tertiary arylmethyl amines as lead molecules. Biological assays showed several sulfonamides capable of inducing abca1 which regulates lipid regulation and metabolism while inhibiting apoe production. One of the five identified leads is a potent abca1 inducer (EC50 in nM range) and another lead is an apoe inhibitor (IC50 in the low uM range). The research project is currently focusing on in vivo studies, mechanistic investigation, and further refinement of the pharmacaphore.

Poster #: 262
Campus: Sonoma State University
Poster Category: Other
Keywords: Chrysomela aeneicollis, evolutionary ecology, SNP genotyping
Project Title: Environmental and genetic influences on larval development rate of montane leaf beetles in nature
Author List:
Michel, Nicolette; Undergraduate, Biology, Sonoma State University, Presenting Author
Regello, Richard; Undergraduate, Biology, Sonoma State University
Rank, Nathan; Biology, Sonoma State University

Abstract: Species at high elevations can be indicators of climate change based on their sensitivity to changes in temperature, snow cover and habitat. Willow leaf beetles, Chrysomela aeneicollis, live in the mountain regions of the Eastern Sierra Nevada range in California in diapause under snow cover most of the year, except in summer when they surface to reproduce. Beetle populations fluctuate significantly due to climate variability and tend to decline and retreat to higher elevations after dry winters. Several study populations that we had tracked since 1998 became extirpated after the most recent drought of 2012-2016. Prior studies have shown that allele frequencies at the nuclear metabolic enzyme locus phosphoglucose isomerase (PGI) vary greatly along a 40-kilometer latitudinal gradient. A single non-synonymous single nucleotide polymorphism (SNP) at PGI is responsible for this latitudinal variation and PGI variation has been shown to influence metabolic rate and the stress response. We hypothesize that environmental and genetic factors influence beetle performance and persistence in local habitats. To test this hypothesis, we surveyed 14 beetle populations throughout the summers of 2011, before the drought, and 2012, after the first unusually dry winter. Populations were located in different drainages along the latitudinal gradient. We also analyzed temperature and light availability data throughout this period. For the present study, we used an allelic discrimination assay on an ABI 7900HT real time PCR system to obtain PGI genotypes for that SNP. Results showed more rapid beetle development in 2012 even though there was less snow cover in 2012 than 2011. We also observed that larval development proceeded more rapidly in populations with a higher frequency of the northern PGI allele (PGI-G) than those with a higher frequency of the southern allele (PGI-A). This relationship occurred in both years, suggesting that the same pattern exists under both wet and dry conditions. Our results support the hypothesis that environmental and genetic factors influence survival and performance in nature. The National Science Foundation and the White Mountain Research Center provided financial support for this work.

Poster #: 263
Campus: CSU Fresno
Poster Category: Other
Keywords: neurodegeneration, behavior,
Project Title: Characterization of motor deficits from expression of mutant α-synuclein in Parkinson’s disease model Drosophila melanogaster
Author List:
Taha, Abu; Undergraduate, Biology, California State University, Fresno, Presenting Author
Diaz, Antonio; Undergraduate, Biology, California State University, Fresno, Presenting Author
Lent, David; Biology, California State University, Fresno

Abstract: Parkinson’s disease (PD) is one of the most common neurodegenerative diseases in the world. Research estimates that approximately one million people in the United States are affected by PD. A common factor in found in all the brains affected by PD is the overabundance of presence of a defunct protein named α -synuclein. The mutated version of α -synuclein protein in PD forms insoluble aggregates that are toxic to the function of dopamine producing neurons. These aggregates make up a large portion of Lewy bodies—structures that inhibit the overall function of a neuron. In the present study, two mutant versions of α-synuclein protein were expressed in the entire nervous system of Drosophila melanogaster using the Gal4-UAS genetic system. The point mutations were induced at loci A30P and A53T. This study looked to examine how the misfolded α-synuclein affected the motor control in the limbs of the fruit fly by employing an obstacle avoidance assay. Previous research showed that if α-synuclein was present in excess in the brain regions of the fruit fly, it reduced the ability of a fly to navigate through the obstacles successfully. The present study aimed to exacerbate behavioral decline associated with abnormal expression by expressing the protein in the entire nervous system. For each mutation type, a batch of 10 day old and 30 day old flies were tested in a novel behavioral assay. The behavioral assay consisted of a visually guided high-contrast obstacle, exploiting the negative geotaxis behavior of the fly. To characterize changes in behavior, the motion of the fly including orientations, distance travelled, time taken, and overall completion of the obstacle were quantified. It was observed that flies had significant impairment in their motions and were unable to successfully complete the course of the obstacle. A large fraction of the tested flies had completely failed to finish negotiating around the obstacle. These conditions were more pronounced in flies that were aged. The results also demonstrated that pan-neural expression of α-synuclein had more prominent effects than the previous studies which restricted the protein expression to brain specific regions of the fly.

Poster #: 264
Campus: CSU Northridge
Poster Category:
Keywords: HdeA bacterial chaperone, NMR spectroscopy, protein dynamics
Project Title: Use of NMR techniques to describe the changes in protein motions of chaperone protein HdeA as a function of pH
Author List:
Benson, Jonathon; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Chemistry and Biochemistry, California State University, Northridge

Abstract: HdeA is one of several proteins found in the periplasm of acid tolerant bacteria such as E. coli that confer acid resistance. Under physiological conditions HdeA is an inactive folded dimer. At low pH, HdeA unfolds and acts as a chaperone by complexing with acid sensitive proteins to prevent their aggregation. This activation of HdeA enables acid tolerant bacteria such as those found in the Shigella genus to survive the acidic environment of the human stomach and cause disease.
The goal of our group is to understand and characterize the mechanism of activation and unfolding of HdeA using modern nuclear magnetic resonance (NMR) techniques. Previous experiments have demonstrated that HdeA unfolds and is activated between pH 3.0 and 2.0. My project’s current goal is to describe the changes in protein motions that occur as HdeA is transitioned from physiological pH to its activation in low pH conditions. Our hypothesis is that these changes in molecular motions are responsible for the dissociation of the dimer and activation of HdeA. Protein backbone and side chain motions were examined at pH 6 and pH 5 using R2 and R1 relaxation NMR experiments. From pH 6.0 to pH 5.0 large decreases in the R2/R1 ratio of N14, E46 and A48 were observed. These amino acids are structurally adjacent and are part of the dimer interface. This decrease in R2/R1 ratio suggests reduced intermediate (µs-ms) timescale motions in this portion of the dimer interface, and provides evidence that this cluster ultimately initiates HdeA dimer dissociation at low pH. Further research will continue to explore these molecular motions at pH 4.0.
Thank you to the NIH for research support (SC3-GM116745) and the NSF for funding the purchase of our NMR spectrometer (CHE-1040134).

Poster #: 265
Campus: San Francisco State University
Poster Category: Other
Keywords: , ,
Project Title: Branching Out: Mechanisms of WNT1 Gradient Formation in the Chick Spinal Cord
Author List:
Goodfellow, Sam; Graduate, Biology, San Francisco State University, Presenting Author
Santana, Frederick R.; Graduate, Biology, San Francisco State University, Presenting Author
Galli, Lisa ; Biology, San Francisco State University
Burrus, Laura; Biology, San Francisco State University

Abstract: Abstract: Dysregulation of the Wnt signaling pathway is implicated in embryonic defects, neurodegenerative diseases and cancers. Using the chick model for vertebrate development, we focus on understanding the mechanisms underlying Wnt gradient formation. WNT1 is known to form a dorsal-ventral gradient in the spinal cord of developing vertebrate embryos. Previous studies have identified that Porcupine (PORCN) and Wntless (WLS) play critical roles in regulating Wnt secretion and gradient formation. Despite these advances, little is known about how Wnts are transported to target cells. Though believed that Wnts were primarily transported via diffusion, recent studies in Drosophila and zebrafish have shown that Wnts can be transported via actin-based signaling filopodia. Thus, we hypothesized that filopodia are involved in the transport of WNT1 in the developing spinal cord. To test this hypothesis, we first generated biologically active WNT1-GFP and WLS-mCherry fusion proteins as tools for live imaging studies. We then transfected expression constructs for WNT1-GFP and/or WLS-mCherry into the spinal cord and collected live images using confocal microscopy. In the absence of WLS-mCherry, WNT1-GFP was primarily localized to the ER and Golgi and no filopodia-like projections were visualized. Upon co-expression of WLS-mCherry with WNT1-GFP, much of the WNT1-GFP was redistributed to the cell surface. Strikingly, we observed large numbers of filopodia-like projections containing both WNT1-GFP and WLS-mCherry. Thus, our results show that co-expression of WLS with WNT1 1) induced the formation of new actin-based filopodia-like projections and 2) promoted the localization of WNT1 to these structures. To further understand the molecular mechanisms underlying the localization and transport of WNT1 in filopodia, we are further characterizing the molecular profile of these filopodia-like projections and other intracellular factors that may play a role in projection formation.
Acknowledgements: NSF IOC (1244602), NIH-MARC (T34-GM008574), NIH-SF BUILD (8ULIGM118985-03), NSF RUI (MCB-1244602), CSUPERB, and NIH-RIMI (P20MD000262).

Poster #: 266
Campus: Cal Poly San Luis Obispo
Poster Category: Other
Keywords: glucuronidase, steroid hormones, MUG assay
Project Title: B-glucuronidase activity of Escherichia coli from hormonal contraceptive users and non-users
Author List:
Gadberry, Emma; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
VanderKelen, Jennifer; Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo
Yep, Alejandra; Biological Sciences, California Polytechnic State University, San Luis Obispo

Abstract: The main causative agents of urinary tract infections (UTIs) in women are uropathogenic Escherichia coli which migrate from the nutrient-rich environment of the intestine to inhabit the nutrient-poor environment of the bladder. A major risk group for UTIs are sexually active young many of whom use hormonal contraceptives containing synthetic steroid hormones at concentrations much higher than natural hormone levels. In the body, insoluble sex steroid hormones circulate to their target tissues bound to a sex hormone binding globulin. Once they reach the liver, they are prepared for removal from the body by conjugation to sulfates or glucuronides. In this more soluble form, they can either be excreted with the bile to the small intestine, or they can reenter the circulation, and be filtered out by the kidneys for excretion in the urine. In the presence of glucuronidated molecules, E. coli upregulate their gus operon which encodes a glucuronide importer and a glucuronidase enzyme. Removal of the glucuronic acid provides a carbon source for the bacteria. Thus, E. coli with increased glucuronidase activity, whether in the gut or the bladder, would have a selective advantage over cells lacking that ability. We hypothesized that the presence of high levels of glucuronidated hormone in hormonal contraceptive users would select for E. coli with higher b-glucuronidase activity compared to E. coli from hormonal contraceptive non-users. To test this, the b-glucuronidase activity of fecal E. coli isolates from 16 hormonal contraceptive users and 11 hormonal contraceptive non-users was determined using a reporter molecule, 4-methylumbelliferyl-beta-D-glucuronide (MUG), that fluoresces upon removal of the glucuronide by the glucuronidase enzyme. Results showed a significantly higher average b-glucuronidase activity in E. coli isolates from hormonal contraceptive users compared to non-users (p<0.02). These results are consistent with the hypothesis that hormonal contraceptive use selects for fecal E. coli with higher β-glucuronidase activity. If these isolates get established in the bladder, the increased enzyme activity may give them a selective advantage in this low-nutrient environment. Further experiments will compare the growth of the E. coli isolates with high and low glucuronidase activity in minimal media or urine containing glucuronidated molecules to explore the possible effects of hormonal contraceptive use on urinary tract infections.

Poster #: 267
Campus: San Francisco State University
Poster Category: Other
Keywords: cytotoxic compounds, marine-derived bacteria, anticancer screening
Project Title: A New Cytotoxic Furaquinocin Isolated from the Marine-Derived Streptomyces sp. CP53-67
Author List:
Luu, Gordon; Undergraduate, Chemistry and Biochemistry, San Francisco State University, Presenting Author
Bray, Walter; UC Santa Cruz, Chemistry and Biochemistry
Lokey, R.; UC Santa Cruz
Valeriote, Frederick; Josephine Ford Cancer Center
Amagata, Taro; Chemistry and Biochemistry, San Francisco State University

Abstract: Our research group has applied an organic extract library of marine-derived actinomycetes to a unique in vitro cancer cell screening system designated CP-DDA (cytological profiling – disk diffusion assay), which enables rapid identification of the mode action for a cytotoxic compound and extract. One of the selected actinomycetes strains, Streptomyces sp. CP53-67, displayed significant cytotoxic effects against solid tumor cell lines as well as solid tumor selectivity against OVC-5, an ovarian cancer cell line. Thanks to data from CP, it is believed that extract from CP53-67’s cytotoxic effect is derived from protein synthesis inhibition. We have already isolated two known cytotoxic compounds, reveromycins A and B, whose molecular targets have been identified as isoleucyl tRNA synthetase. Interestingly, cytotoxic effects of DDA were not observed in the HPLC fractions containing reveromycins. Instead, more polar HPLC fractions displayed cytotoxic effects. Detailed analysis and purification of the polar cytotoxic fraction led to isolation of a new cytotoxic compound. This compound has been identified as a new furaquinocin derivative based on the comprehensive analysis of 1D and 2D NMR data, as well as high resolution MS data. This new compound showed significant solid tumor selective effect against the ovarian cancer cell line (OVC-5). We have also determined that the production of the new furaquinocin derivative depend on the media components used for the liquid culture of this strain. In fact, this strain produced the new furaquinocin in quantities increased 10-fold when cultured in media conditions containing mannitol and soybean meal as carbon and nitrogen sources, respectively, as opposed to our standard media conditions that utilize soluble starch, glucose, yeast extract, and meat extract as nutrient sources.

Poster #: 268
Campus: CSU Fresno
Poster Category: Other
Keywords: climate change, extinction, birds
Project Title: The phylogenetic distribution of climate change vulnerability in California’s birds
Author List:
Moshier, Shelby; Undergraduate, Biology, California State University, Fresno, Presenting Author, Nagel Award Nominee
Dhaliwal, Gurjap; Undergraduate, Biology, California State University, Fresno
Reece, Joshua; Biology, California State University, Fresno

Abstract: Anthropogenic climate change is a major issue for many threatened and endangered species. One way of assessing the impact of climate change is through vulnerability assessments, which quantitatively evaluate the susceptibility of species to climate change. While vulnerability assessments can identify which species are most at risk, it can be unclear as to how species’ vulnerability is distributed phylogenetically and what would be lost in terms of evolutionary diversity if certain lineages were extirpated. We examine the distribution of quantitative climate change vulnerability scores (CCVSs) of 299 California avian taxa on a phylogenetic tree to determine if vulnerability is distributed randomly or non-randomly. The 299 species have vulnerability scores ranging from 12 to 72 and are separated into six bins based on natural breaks in the distribution of vulnerability scores on a scatterplot. Beginning with the bin ranked High vulnerability, the bins are cumulatively analyzed in R to calculate Faith’s Index of Phylogenetic Diversity (PD) and disparity as assessed by the Net Relatedness Index (NRI). The analysis of phylogenetic diversity indicates that if the two highest vulnerability bins were lost, more phylogenetic diversity would be lost than if the same number of taxa were randomly removed from the tree. Culling the three highest vulnerability bins from the phylogeny also results in a non-random loss of PD. This means that climate change vulnerability is clustered in certain clades. Overall disparity (NRI) in the California bird tree of life is significantly different from random losses of taxa when the two highest vulnerability bins and the four highest vulnerability bins are removed. Thus, climate change threatens both overall evolutionary diversity and disparity in California’s birds

Poster #: 269
Campus: Cal Poly San Luis Obispo
Poster Category: Product-focused Innovation
Keywords: Cell-Free Protein Synthesis, Genetic code, Learning Module/Curriculum Development
Project Title: Utilization of Cell-Free Protein Synthesis as an Educational Platform for Learning Transcription and Translation
Author List:
Gregorio, Nicole; Undergraduate, Chemistry & Biochemistry , California Polytechnic State University, San Luis Obispo, Presenting Author, Nagel Award Finalist
Kao, Wesley; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Hight, Christopher; Undergraduate, Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo
Oza, Javin; Chemistry & Biochemistry, California Polytechnic State University, San Luis Obispo

Abstract: Currently, there is no learn-by-doing classroom module that allows early-career STEM students to interact directly with the cellular processes of transcription and translation due to the closed nature of the cell. However, cell-free protein synthesis (CFPS), an emerging biotechnology, has enabled scientists to harness the genetic code and cellular machinery of transcription and translation in vitro. The open nature of CFPS allows direct manipulation of the components of transcription and translation without the cellular barrier. Reconstitution of cellular machinery in vitro for CFPS has been transformative for biotechnology, but its execution can be more limiting to early-career students. We have successfully adapted the CFPS biotechnology to develop a learning module accessible to early-career STEM students focusing on learning objectives involving the genetic code. Here we report the reformulation of the CFPS reaction setup for simplicity and improved reagent shelf life in order to adapt CFPS for the classroom. First, our simplified platform requires the pipetting of only 3 pre-mixed reagents, an 80% reduction in setup steps, making it more accessible to an early-career student with little-to-no laboratory experience. Second, our simplified platform is shelf-stable at -20°C rather than -80°C, making it accessible for undergraduate and high school classrooms. Most importantly, our data show that the reformulated CFPS reaction performs just as well as the original laboratory CFPS setup. We believe that this educational technology will be transformative to the way the genetic code is taught in classrooms today. To this end, we have developed a CFPS “kit” which includes the necessary reagents and equipment for the module; only an incubator and average freezer must be supplied by the user. The kit will also include written materials for students to understand and execute the experiments. Materials for the instructor will include worksheets, instructions, and a troubleshooting guide. This effort provides the first opportunity for students to explore the processes of transcription and translation in a truly hands-on fashion within the classroom environment. Our goal is to disseminate this simplified form of CFPS as a learning module to high school and undergraduate science classrooms in our community. Our research was funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF-1708919.

Poster #: 270
Campus: Cal Poly Pomona
Poster Category: Proteins (Include Proteomics)
Keywords: Maillard reaction, Protein stabilization, Protein functionality
Project Title: Creating Maillard Conjugates by use of Pea Protein Isolate and Various Molecular Weight Dextrans
Author List:
Steiner, Benjamin; Graduate, Human Nutrition and Food Science, California State Polytechnic University, Pomona, Presenting Author
Davidov Pardo, Gabriel; Human Nutrition and Food Science, California State Polytechnic University, Pomona

Abstract: Improvements in the stability of proteins and protein-coated colloidal particles have been achieved by employing the Maillard reaction to form conjugates with high-molecular-mass polysaccharides. Pulse proteins have been of recent interest within the food science industry due to their sustainability and nutritional content. Pea protein is of particular interest as it is not an allergen though it contains essential amino acids. Therefore, the inclusion of pea protein isolate (PPI) in conjunction with saccharides poses good possibilities for Maillard conjugation. The first step in creating Maillard conjugates was purifying pea protein, achieved by precipitating the protein at its isoelectric point to separate it from the rest of the pea components. Samples containing PPI and sodium caseinate (control) were mixed with dextran of different molecular weights at 1:1 w/w ratio, freeze dried, and placed in a climactic chamber set at 60oC and 77.5% relative humidity for 72 hours. Samples were removed every 24 hours to determine an appropriate timeframe for conjugation to take place. O-phtaldehyde assays were conducted to determine conjugation efficiency through reduction in the unconjugated amino groups. Lowry assays were conducted to determine conjugation yield which relates to the stability of the protein at the isoelectric point (Ip). Results thus far have indicated that PPI with dextran (40 KDa) yield the greatest conjugation efficiency showing a 22% reduction of available amino groups after 72 hours of conjugation. Conjugation yield after 24 h was 81% which indicates that 81% of the protein remained in solution after adjusting to the Ip, though the yield was drastically reduced to 72% and 57%, for 48 and 72 h respectively. This is possibly due to a degradation of the covalent bond between the protein and the polysaccharide, as well as possible coagulation and even polymerization of the PPI due to the heat treatment. Preliminary testing with sodium caseinate and dextran (40 KDa), following the same procedures as with PPI, showed 97% conjugation yield and 9% conjugation efficiency at 24 h while 48 h resulted in 93% conjugation yield and 19% conjugation efficiency. Future research will be conducted to determine the best Maillard conjugates complexed with polyphenols to encapsulate the lutein-enriched emulsion and enhance the physical and chemical stability of lutein. This work was supported by SCIFTS educational grants and ARI project number #17-04-239.

Poster #: 271
Campus: CSU Fresno
Poster Category: Proteins (Include Proteomics)
Keywords: pancreatic cancer, Palladin, proteomic
Project Title: An evaluation of Palladin overexpression in pancreatic cancer cells
Author List:
Vander, Karamjot; Graduate, Biology, California State University, Fresno, Presenting Author
Chu-Bacher, Rowena; Graduate, Biology, California State University, Fresno
Bush, Jason; Biology, California State University, Fresno

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with an overall survival rate of less than 5%. It is one of the most aggressive malignancies due to its chemotherapy-resistant nature and poor prognosis. A hallmark for many cancers including PDACs is the epithelial to mesenchymal transition (EMT) resulting in increased migration and invasion capabilities of these cells. EMT is a normal pathway in early developmental stages; however, the mechanism underlying this re-programing towards metastasis is not fully understood. Evidence suggests that aberrant expression of genes involved in cytoskeletal organization play a crucial role. Abnormal expression of the protein Palladin (PALLD) has been seen in PDAC and could induce EMT. Normally PALLD functions to stabilize a cell by forming stress fibers and focal adhesions (FA) and the rate of cell migration is proportional to the turnover of FA component. Furthermore, elevated levels of integrin beta5 (ITGB5) is known to stimulate attachment to the extracellular matrix and regulate invasion and migration of PDAC cells. We hypothesize that PALLD overexpression is associated with enhanced motility in pancreatic cancer cells through interaction with ITGB5. Preliminary data using a proteomic screen to evaluate the secreted protein profile of pancreatic cancer cell lines demonstrated a strong PALLD expression in late stage pancreatic cancer cells. Additional results show that ITGB5 is also overexpressed. To better test the hypothesis, we have subcloned PALLD into a strong expression vector and created stable cell clones for biochemical and biological evaluation. The overexpressed PALLD profile for early stage PDAC cells is being compared to the baseline expression of PALLD in drug (Gemcitabine)-resistant cells. Evaluating the role of PALLD in EMT could provide an improved therapeutic strategy to suppress metastasis of pancreatic cancer cells.

Poster #: 272
Campus: CSU San Bernardino
Poster Category: Proteins (Include Proteomics)
Keywords: Influenza virus , protein interactions , gradient ultracentrifugation
Project Title: Using Sucrose Density Gradient to Investigate NP Interactions and Identify Novel Antiviral Targets in Influenza A Viruses
Author List:
Gallardo , Jennifer ; Undergraduate, Biology , California State University, San Bernardino, Presenting Author
Proakatok, Susan ; Undergraduate, Biology , California State University, San Bernardino
Gazca , Cinthia; Undergraduate, Biology, California State University, San Bernardino, Presenting Author
Newcomb, Laura ; Biology, California State University, San Bernardino

Abstract: Influenza A viruses cause respiratory infections that can be life-threatening and sometimes even fatal. The influenza viral ribonucleoprotein (vRNP) is responsible for influenza RNA synthesis and is comprised of highly conserved proteins among influenza subtypes. The vRNP consists of many nucleoproteins (NP), the RNA genome segment, and the RNA dependent RNA polymerase (RdRP), a trimer of PA, PB1, and PB2. NP is a structural component of the vRNP, but also interacts with both viral and host factors to regulate viral RNA expression, making NP interactions of interest as possible antiviral targets. In addition to direct protein interaction with both the PB1 and PB2 subunits of the viral RdRP, NP also interacts with influenza NS1, a multifunctional protein, and host UAP56, a DEAD box RNA helicase. The overall goal of this project is to investigate NP interactions to identify novel antiviral targets. We will examine interactions among WT-NP and mutant NP with PB2 individually and as part of the RdRP, NS1, and host UAP56. Human tissue culture cells (293T) were transfected with WT-NP or mutant NP in combination with either the RdRP, PB2 or NS1. Two different NP mutants were examined, NPbd3, speculated to disrupt interaction with PB2, and del20NP, reported to disrupt interaction with UAP56. Cells were collected 48 hours post transfection and total protein isolated. Western blot was performed to confirm NP, RdRP, and NS1 transfection and expression, and endogenous UAP56 expression. Protein extracts were separated on 5 – 40% sucrose gradient with a 16-hour spin at 50K rpm at 4ºC. Gradient fractions were collected in ten parts and each fraction will be analyzed by Western blot to detect NP, PB2, NS1, and UAP56. It is expected WT-NP will sediment with each of its interacting partners and therefore be found in the same fraction when co-expressed, but not when expressed alone. We also expect sedimentation will demonstrate mutant NP proteins do not maintain targeted interaction. Our data will provide evidence of NP interacting domains that may serve as viable antiviral targets.
This research is funded by CSUPERB Research Development Grant to LLN. JVG is supported as a MARC scholar through NIGMS training grant GM083883.

Poster #: 273
Campus: CSU Bakersfield
Poster Category: Proteins (Include Proteomics)
Keywords: Proteins, Cancer, Inhibition
Project Title: Inhibition of Lysyl Oxidase in Breast Cancer Cells by Small – Molecule Inhibitors
Author List:
Johnston, Kathryn; Undergraduate, Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author, Nagel Award Finalist
Lopez, Karlo; Chemistry and Biochemistry, California State University, Bakersfield, Presenting Author

Abstract: Lysyl oxidase is an extracellular matrix, copper – dependent, amine oxidase that catalyzes a key crosslinking step in collagen and elastin. This enzyme has also been shown to play a role in promoting metastasis. The correlation between high LOX activity and cancer metastasis is strong enough that upregulated LOX activity can be used as a diagnostic marker for the severity of cancer in patients. β – aminopropionitrile (β – APN) is a known potent inhibitor of lysyl oxidase; however, this inhibitor is not selective and, therefore, cannot be used as a therapeutic agent. In order to increase selectivity for lysyl oxidase in cancer cells over normal cells, β – APN has been derivatized using aromatic sidechains. It is postulated that the hypoxic environment of cancer cells will cleave off the aromatic side chains and release the active inhibitor in cancer cells. This will not happen in the aerobic environment of normal cells and, therefore, it is a potential way to target a drug to specific cell types. Derivatives of β – APN were synthesized by the Solano research group at CSU Bakersfield and structures were verified by NMR. Inhibitors were then incubated with cancer cells at concentrations varying from 100 µM to 5000 µM and cell viability was determined as a percent of control. The presence of lysyl oxidase was determined by western blot analysis, and effectiveness of the drug was determined using an amplex red fluorimetric assay. Of the inhibitors that have been synthesized to date, two inhibitors, LP – 1 – 2 and MW – 1 – 5 (shown below) have been shown to reduce breast cancer cell viability with a 100 mM dose and 72 – hour incubation period. The effect on cell viability increased with increasing amounts of inhibitor. The selective targeting of lysyl oxidase was verified using western blot analysis and lysyl oxidase activity assays. The activity assays showed that addition of increasing amounts of inhibitor proportionally decreased the activity of lysyl oxidase with LP – 1 – 2 being the most effecive. The highest level of inhibition detected was with lysyl oxidase isolated from cancer cells treated with 5000 μM of LP – 1 – 2 for three days, which decreased the activity three – fold as compared to lysyl oxidase isolated from untreated cancer cells.

Poster #: 274
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: lipid droplets, alkane production,
Project Title: Comparative transcriptomic insights into cyanobacterial lipid droplet production
Author List:
Arias, Daisy; Graduate, Biology, California State University, Northridge, Presenting Author
Pinto, Kevin; Undergraduate, Biology, California State University, Northridge, Presenting Author
Summers, Michael; Biology, California State University, Northridge

Abstract: Nostoc punctiforme is a filamentous cyanobacterium capable of producing cytoplasmic lipid droplets (LDs). LDs contain triacylglycerols, a feedstock for biodiesel production, and long-chain alkanes typically found in diesel or jet fuel. Synthesis of these combustible compounds by photosynthetic microorganisms could reduce the use of fossil fuel in the future if LD formation can be enhanced. Previous work from our lab has shown that overexpression of two genes involved in alkane biosynthesis, aar and adc (2-gene), resulted in an increase in heptadecane and increased the number of LDs in N. punctiforme. The addition of a putative lipase gene in conjunction with aar and adc (3-gene) further increased the amount of heptadecane and number LDs. Comparative transcriptomic analysis of an empty vector wild-type control (WTC) and the two LD/alkane overproducer (OE) strains was conducted via RNA-Seq to identify gene regulation associated with increased LD formation and alkane production. Genes that showed a statistically significant >1.5-fold increase in the 2 and 3-gene OE strains relative to the WTC were identified and hypothesized to enhance over-production of LDs or cope with increased alkane production. To see if overexpression of the up-regulated genes could further enhance biofuel production, sixteen genes showing upregulation in the overproduction strains were cloned into a multi-copy plasmid and transformed into the WT strain either alone, in an operon group, or as the fourth gene or operon group in conjunction with the 3-genes previously shown to enhance alkane/LD production. The strains were screened following fluorescent staining of neutral lipids by epifluorescence microscopy. Strains expressing several individual genes alone displayed altered neutral lipid production in large areas of the membrane or numerous small lipid droplets with abnormal cellular location. Alterations in alkane and lipid composition are under analysis using a GC-MS. These data will ultimately help us develop strains capable of producing LDs enriched in high value compounds that can be utilized in biotechnological applications.

Poster #: 275
Campus: CSU Northridge
Poster Category: Proteins (Include Proteomics)
Keywords: lipid droplets, ABC1 kinase, alkanes
Project Title: Characterization of a Putative Kinase Protein Associated with Lipid Droplets in Nostoc punctiforme
Author List:
Janami, Sara; Undergraduate, Biology, California State University, Northridge, Presenting Author
Korsakov, Kirill; Undergraduate, Biology, California State University, Northridge, Presenting Author
Wadhwani, Elana; Chaminade College Preparatory High School
Polin, Jenevieve; Biology, California State University, Northridge
Summers, Michael; Biology, California State University, Northridge

Abstract: Nostoc punctiforme is a filamentous cyanobacterium capable of producing cytoplasmic lipid droplets (LDs) that contain triacylglycerides, alpha-tocopherol (vitamin E), and alkanes. Alkanes are the primary components of biofuel and therefore are of interest as a potential renewable energy source. To enhance our knowledge of LDs, proteins that co-purified with isolated LDs were identified by mass analysis. One relatively abundant LD protein, Npun_F2818, belongs to the putative AarF/ABC1/UbiB-family of unusual protein kinases. This study aims to determine whether overexpression or deletion of this protein affects fatty acid, alkane, or LD production. The Npun_F2818 protein and its cotranscribed neighbor, Npun_F2819, were transformed into WT, either alone (2818-19_OE) or in conjunction with 3 genes previously shown to increase alkane and LD production on a multicopy plasmid (aar, adc, and a putative lipase – termed the 3-gene plasmid). LDs from each strain were stained with a fluorescent dye for neutral lipids and viewed using epifluorescence microscopy. A CFP-Npun_F2818 protein fusion strain showed increased expression following exposure to high light and oxidative stress. An insertional mutant of Npun-F2818 (2818-) was constructed and phenotypically characterized. The mutant grew more slowly than either WT or 2818-19_OE under both normal vegetative conditions and oxidative stress. Both the mutant and 2818-19_OE grew more slowly than WT under high light. Western blot with anti-GFP antibody confirmed the presence of the CFP-2818 fusion protein in LDs. Preliminary results of fatty acid methyl ester analysis by GC-MS indicate increased levels of double-bonded C18 fatty acids and decreased levels of C17 alkanes in the 2818-19_OE when cloned into the 3-gene plasmid compared with the strain bearing only the 3-gene plasmid. These findings further our understanding of proteins associated with LDs, facilitating future production of high-value compounds for biotechnological use.

Poster #: 276
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: supramolecular, materials, organic chemistry
Project Title: Solid-State Assembly of Pyrogallol[4]arene Hexameric Capsules
Author List:
Garcia, Cesar; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Journey, Sara; Undergraduate, Chemistry and Biochemistry, San Diego State University
Teppang, Kristine; Undergraduate, Chemistry and Biochemistry, San Diego State University
Brim, Shaylyn; Undergraduate, Chemistry and Biochemistry, San Diego State University
Onofrei, David; Graduate, Chemistry and Biochemistry, San Diego State University
Addison, Bennett; Chemistry and Biochemistry, San Diego State University
Holland, Gregory; Chemistry and Biochemistry, San Diego State University
Purse, Byron; Chemistry and Biochemistry, San Diego State University

Abstract: Pyrogallol[4]arene is a curved, hydrogen-bonding molecule that is capable of self-assembling in solution to form molecular capsules. It serves as a model compound for molecular self-assembly, which has important biotechnological applications in medical materials and drug delivery. Pyrogallol[4]arene capsules enclose approximately 1300 Å3 of space, enough to trap up to seven or eight small molecules and isolate them from solution. Previous research performed by the group revealed that decyl pyrogallol[4]arene is the most kinetically stable hydrogen-bonded molecular capsule, making it ideal for further testing and applications in the material sciences. As a part of our studies on these capsules, we investigated the ability of the decyl pyrogallol[4]arene capsules to self-assemble in the solid state, specifically by mechanically inducing the transformation by the use of ball milling. In this experiment we determined how effective ball milling is at inducing encapsulation of selected guests. The ball milled sampled were prepared by mixing a 1:1 stoichiometric ratio of host and guest and milling the samples for 50 minutes, dissolving, and analyzingin solution (CDCl3) via 1H NMR. It was determined that 50 minutes was the optimal milling duration because most of the guest reached peak encapsulation by that time. Further testing revealed that extending the milling time does not result in more encapsulation and the guest-host complex reaches a percent encapsulation plateau. With these experiments, we extended the scope of molecular encapsulation by discovering unique molecular guest-host assemblies encapsulating [2.2]paracyclophane, an assembly unattainable by any other means of encapsulation. Diffusion NMR spectroscopy experiments were performed and confirmed that the ball milled complexes were formed in solution. Furthermore we tested the kinetics of the decyl pyrogallol[4]arene and propyl pyrogallol[4]arene by milling for 90 minutes and periodically removing samples to be analyzed by 1H NMR. Our experiments found no relationship between the nature or size of guest and rate of encapsulation. However, it did reveal a 15-fold decrease in fluorene encapsulation in propyl pyrogallol[4]arene as opposed to decyl pyrogallol[4]arene. The experiments on the solid state assembly of pyrogallol[4]arene performed by our group extend the scope of molecular encapsulation as well as provide insightful information on the mechanism of the assembly.

Poster #: 277
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Organic Chemistry, Medicinal Chemistry, Antibiotics
Project Title: The design of new antibacterial drugs against GTP cyclohydrolase I (GCYH-I)
Author List:
Samaan, George; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Paranagama, Naduni; Graduate, Chemistry and Biochemistry, San Diego State University
Swairjo, Manal; Chemistry and Biochemistry, San Diego State University
Purse, Byron; Chemistry and Biochemistry, San Diego State University

Abstract: The emergence of resistant bacteria necessitates the development of new antibacterial drugs to combat these potentially fatal microorganisms. This project focuses on the design of novel inhibitors as potential drug leads that target highly resistant bacteria, especially N. gonorrhea and S. aureus. This project targets an interesting enzyme that is involved in the synthesis of folate and other essential compounds in the cells. Inhibition of this enzyme kills the bacteria by stopping the synthesis of folate. This enzyme, GCYH-I, is present in the bacterial cells and human cells as well. Depending on the differences in structure (using X-ray crystallography) and binding between the human enzyme (GCYH-IA) and the bacterial enzyme (GCYH-IB), we try to design a molecule that can bind to the bacterial enzyme, not the human one. After screening many compounds in silico based on mechanistic rationale, 8-oxo-GTP was identified as a substrate analogue inhibitor and showed potent inhibition of GCYH-I in fluorescence-based enzymatic assays, but with 30-fold more selective inhibition for the human enzyme. This project’s goal is to modify this compound and make different substitutions to improve the differential inhibition of the bacterial enzyme. To test our design hypotheses, we used organic synthesis to prepare three compounds for testing against the bacterial and human enzymes. The first compound, was named G1, containing a functional group at O6 of the guanosine derivative to test the binding to the enzyme at this site. Having this large group at this site will differentiate between the bacterial and the human enzymes because of the larger size of the bacterial enzyme at this binding site. G1 showed inhibition of the bacterial enzyme 2.5 times more than the human enzyme at sub-micromolar concentrations. The percent reduction of the activity at 50 µM inhibitor concentration are 69% and 28% for the bacterial and human enzymes, respectively, which means more than 70-fold reversal of the differential inhibition over 8-oxo-GTP. The other two compounds are still being tested to measure their activities against the enzyme. In conclusion, G1 shows differential inhibition against the bacterial enzyme over the human one, which supports our strategy of further optimization it to develop a novel antibacterial drug against our target enzyme.

Poster #: 278
Campus: CSU Channel Islands
Poster Category: Synthetic Chemistry
Keywords: Appel reaction, N. Meningitides, Nucleosides analogs
Project Title: Brominated Nucleosides for Aromatic Substitution: Synthesis and in Vitro Studies
Author List:
Jacobsen, Casey; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
McLaughlin, Cameron; Undergraduate, Chemistry, California State University Channel Islands, Presenting Author
Awad, Ahmed; Chemistry, California State University Channel Islands

Abstract: The ability to mimic biological functions is a key advantage in using modified nucleosides for chemotherapy and antibiotics. Brominated modified nucleosides are convenient intermediates for a wide array of nucleoside derivatives including aromatic substitutions. The chemotherapy pharmaceutical Imatinib uses aromatic structures to emulate adenosine triphosphate; by using brominated nucleosides, similar aromatic substructures could be added. We hypothesize that the resulting compound would be functionally similar to Imatinib while also expressing the benefits of being a modified nucleoside. Furthermore, modified nucleosides have previously been shown to have antibacterial functionality. Herein we report the synthesis of brominated and phenolated nucleoside intermediates along with antibacterial application. Brominated compounds were synthesized by the use a modified Appel reaction while phenolated compounds utilized a modified Gabrial-Mitsunobu reaction and Williamson ether reaction. The brominated modified nucleosides were taken forward to antibacterial testing due to their ability to react with a large variety of aromatic substructures. The bacteria chosen for testing included three gram positive and three gram negative bacteria, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Enterococcus faecalis, Proteus vulgaris, and Neisseria meningitides. 5’-bromo adenylyl, 5’-bromo uridyl, and 3’,5’-bromo uridyl compounds had percent inhibition of 7.5%, 12%, and 12% respectively against N. meningitides. These results show the promising effectiveness of brominated compounds as antibiotics while simultaneously serving as accessible intermediates for further reactions with aromatics including phenolation.

Poster #: 279
Campus: San José State University
Poster Category: Synthetic Chemistry
Keywords: infrared emission, quantum dot, surface chemistry
Project Title: PbSe Quantum Dot Photophysics after Ligand Exchange Chemistry
Author List:
Robinson, Ryan; Undergraduate, Chemistry, San José State University, Presenting Author
Leroy, Michael; Graduate, Chemistry, San José State University, Presenting Author
Kazemi-Daliri, Roksana; Undergraduate, Chemistry, San José State University
Nordlund, Dennis; Stanford SYnchrtron Radiation Lightsource, Chemistry, San José State University
Wolcott, Abraham; Chemistry, San José State University

Abstract: Lead selenide (PbSe) quantum dots (QDs) are an interesting platform for the study of quantum confinement and surface induced defect states. PbSe QDs exhibit strong quantum confinement and are an ideal model to probe quantum mechanical behaviour. PbSe QDs are ideal for infrared emission spectroscopy and photovoltaic applications because of their size tunable absorption and emission properties. PbSe QDs are synthesized via a metallo-organic hot injection technique and their size is controlled by reaction time. Upon reaction completion, the PbSe QDs are insulated within a shell of oleic acid (OA) that are removed and exchanged with shorter molecules to allow for better electron coupling between individual QDs. We confirm the exchange of oleate ligands with alkanedithiols (# carbons = 2,6 & 8) and carboxyclic acids and observe optical redshifts of 100-260 cm-1 using attenuated total reflection-FTIR. Optical redshifts are consistent with a change in the dielectric constant surrounding the QDs. Alkanedithiol treated QDs show increased oxidation and an optical blueshift due to the reduction in QD diameter when exposed to oxygen. Air-free FTIR only revealed a red-shift after the ligand exchange chemistry. We used X-ray absorption spectroscopy to show that alkanedithiol treated QDs have dangling bonds that result in an increased oxidative rate. Conversely we show that short chain carboxylates reduce the oxidation mechanism. We also probed the conductivity of PbSe solids using interdigitated electrodes and found low currents (10-12 A/cm2) when PbSe-oleate QDs were used. Our work focusing on the surface of PbSe will aid applications in infrared emission studies (biolabelling) as well as energy harvesting studies (photovoltaics).

Poster #: 280
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Anti-cancer, peptide synthesis, organic chemistry
Project Title: Structural Revision and Total Synthesis of the Correct Enantiomeric Form of Micromide, a Highly Potent Anti-Tumor Agent from Cyanobacteria
Author List:
Miner, Devin; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Mahoney, Mark; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Wang, Lee; 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: Background: Marine cyanobacteria have provided a bountiful array of biologically active products, among which, is a secondary metabolite isolated from the benthic cyanobacteria Moorea producens, formerly classified as Lyngbya majuscula. Lipophilic extracts that showed activity against solid tumors were fractionated using bioassay-guided fractionation, leading to the discovery of micromide in 2004. Micromide displays an impressive potency against KB cells (IC50 = 260 nM). Micromide is a linear lipopeptide consisting of a modified thiazole head, a five residue peptide body and an aliphatic tail. Lipopetide natural products have shown cytotoxicity through various mechanisms such as membrane disruption and microtubule inhibition, however, the mechanism of micromide has yet to be elucidated. Natural products from marine cyanobacteria provide the challenge of accessibility, as they are difficult to harvest and provide little material to work with. In addition, these secondary metabolites vary from strain to strain, and may not be present in a harvested sample. Total synthesis also determines the accuracy of proposed structures, which are prone to error. Therefore, synthetic versions are critical to the elucidation of natural products.
Results: Based on the structure published by Williams, we synthesized micromide using highly enantioselective methods to find that the NMR spectra did not match that of the natural product. Recently, the Tian group at the Hebei University independently synthesized micromide and published their findings. Both the spectra published by the Tian group and ours were identical. However, Tian erroneously concluded the synthetic product was micromide without further testing. The NMR spectrum did not match that of the natural product. In addition, our testing showed absolutely no biological activity against KB cells. In order to correct the structure of micromide, we have found evidence to suggest that the synthetic product is an epimer of the natural product. Due to the lengthy nature of solution-phase chemistry, we have adapted our synthesis using solution phase peptide synthesis methodology. We report the successful total synthesis of epi-micromide, as well as current efforts to correct the structure of micromide. We also report the biological data of epi-Micromide. Microanalyses confirmed that the product is identical to the solution-phase product, reaffirming that our prior solution-phase protocol was enantioselective.

Poster #: 281
Campus: CSU San Marcos
Poster Category: Synthetic Chemistry
Keywords: Gold(I) catalysis, Friedel–Crafts, microwave
Project Title: Gold(I)-Catalyzed Friedel–Crafts-like Arylation of Benzylic Alcohols to Afford 1,1-Diarylmethanes
Author List:
Oakley, James; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author, Nagel Award Finalist
Mendez, Sharai; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Stanley, Tyler; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Cain, Stephanie; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Iafe, Robert; Chemistry & Biochemistry, California State University San Marcos

Abstract: Applications of 1,1-diarylmethanes and their syntheses have attracted significant interest within the past few decades. The 1,1-diarylmethane moiety is a ubiquitous structural constituent of calixarenes, pillararenes, and synthetic receptors. This motif is also common in many biologically active compounds, of which have anticancer, antidepressant, antifungal, anti-inflammatory, antimicrobial, antiviral, and cytotoxic activity. Cyclic derivatives of these molecules, fluorenes, have unique properties in materials science, pharmaceuticals, and organic synthesis. The Friedel–Crafts benzylation (FCB) reaction represents one of the most accessible and widely used methods to obtain 1,1-diarylmethanes. During our prior investigation using gold(I) salts to catalyze etherification reactions, we discovered that phenols react via a Friedel–Crafts reaction mechanism in preference to the etherification pathway. To our satisfaction, we were successfully able to cleanly promote the Friedel–Crafts reactivity using microwave conditions. From this, our lab has developed the first microwave-assisted, gold(I)-catalyzed Friedel–Crafts reaction of electron-rich benzenes with benzylic alcohols to afford 1,1-diarylmethanes, a common structural motif found in many pharmaceuticals including breast cancer treatment drugs. To our knowledge, while examples of gold(III)-catalyzed Friedel–Crafts reactions are well-known, a microwave assisted, gold(I)-catalyzed Friedel–Crafts reaction is currently unprecedented in the literature. The long-term objective is the synthesis of bioactive natural products and other various building blocks useful in the synthesis of bioactive molecules
Results: The gold(I)-catalyzed Friedel–Crafts reaction has been optimized using phenol as the nucleophile, indanol as the electrophile, 5 mol % of the gold(I) and silver(I) salts, and slightly elevated temperatures to afford 90% of the target 1,1-diarylmethane. Using these conditions, the substrate scope for this reaction was explored using various electron-rich nucleophiles, such as anisol, p-methylanisol, sesamol, 2-naphthol, and p-tert-butylphenol. Excellent yields (34–99%) were also obtained when the electronics were varied on the electrophile; however, strongly electron withdrawing groups shut the reaction down. The gold(I)-catalyzed reaction also worked well in the preparation of four unique fluorenes (72–98%). Also, a gold(I)-catalyzed FCB reaction using a heteroaromatic nucleophile will be presented.

Poster #: 282
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Organic Chemistry, Biophysics, Fluoresence
Project Title: tCc – A New Family of Fluorescent Nucleosides
Author List:
Ngo, Katrina ; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Andersen, Susan; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Samaan, George; Graduate, Chemistry and Biochemistry, San Diego State University
Purse, Byron; Chemistry and Biochemistry, San Diego State University

Abstract: Fluorescent nucleotides can act as useful molecular probes as well as fluorescent markers for the study of nucleic acids, but brighter probes are needed for new applications including fluorescence microscopy. Previous work done in our group on nucleoside analogues has led to the development of a new generation of modified fluorescent analogues known as tricyclic cytosines (tC), which are able to participate in normal G:C base pairs in DNA while functioning as molecular probes. The properties of these tC compounds led us to propose a new structure for fluorescent nucleotide design, tCc. We hypothesize that tCc compounds will be the most fluorescent of the cytidine analogues because of the stronger conjugated system that exists within the molecule. We used an efficient series of synthetic reactions to construct the molecule, and we characterized the intermediates and products using 1H NMR spectroscopy and mass spectrometry. The main ring system of the molecule was built using a series of reactions that included the Vilsmeier reaction and the Knoevenagel condensation for critical C–C bond forming steps. The unique carbon-carbon bond linking the sugar to the nucleobase was achieved by utilizing the Heck reaction and our nucleoside analogue was confirmed via NMR spectroscopy and mass spectrometry. Initial observations show that the parent tCC compound is fluorescent, but further tuning will be needed to maximize its brightness. In addition, synthesis of an 8-DEA tCc derivative has shown that an addition of an EDG increases fluorescence substantially. This was observed through preliminary photophysical studies as well as molar absorptivity calculations. Future steps include transforming our nucleosides into nucleoside phosphoramidites and incorporating them into DNA using solid-phase synthesis.

Poster #: 283
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: fisetin, prostate cancer, medicinal chemistry
Project Title: 7-O-Aminoalkyl-3,3′,4′-O-Trimeylfisetins: Synthesis and Evaluation in Prostate Cancer Cell Models
Author List:
Lee, Maizie; Undergraduate, Chemistry, California State University, Fresno, Presenting Author, Nagel Award Finalist
Muthima, Kevin; Graduate, Chemistry, California State University, Fresno, Presenting Author
Li, Xiang; Graduate, Chemistry, California State University, Fresno
Chen, Guanglin; Chemistry, California State University, Fresno
Chen, Qiao-Hong; Chemistry, California State University, Fresno

Abstract: Prostate cancer is the most common cancer among men. Early stage prostate cancer is localized and treatable with hormone therapies. So far no effective treatment is available for metastatic, advanced prostate cancer. Fisetin, a naturally occurring plant flavonoid, has been revealed to possess potential in treating prostate cancer according to in vitro and in vivo studies. However, fisetin is known to merely have moderate potency towards prostate cancer cells. Another drawback to fisetin is its low bioavailability. Structurally, fisetin is characteristic of four phenolic hydroxyl groups. Previous studies indicate the chemical modifications on the hydroxyl groups (OH) of flavonoids can overcome, at least partially, the drawback. This study aimed to explore the potential of 7-O-substituted-3,3′,4′-O-trimethylfisetins as anti-prostate cancer agents. To this end, a synthetic method to 7-O-aminoalkyl-3,3′,4′-O-trimethylfisetins has been successfully developed. Five desired fisetin derivatives have been achieved through this six-step synthetic approach, including aldol condensation, Algar-Flynn-Oyamada (AFO) reaction, O-methylation, debenzylation, O-alkylation, and N-alkylation. All synthesized compounds have been characterized by interpreting the 1H and 13C NMR data. The anti-proliferative activity of two derivatives, as well as fisetin and 3,3′,4′-O-trimeylfisetin, towards three human prostate cancer cell lines has been assessed via WST-1 cell proliferation assay. Our results indicate that 7-O-Aminoalkyl-3,3′,4′-trimethoxyfisetins are significantly more potent than fisetin and 3,3′,4′-O-trimeylfisetin in inhibiting the cell proliferation in the three cancer cell models.
M Lee was grateful to the support from the Undergraduate Research Grant program from CSU-Fresno. K Muthima is supported by CSU-Fresno Bridge to Doctorate Program.

Poster #: 284
Campus: CSU Fresno
Poster Category: Synthetic Chemistry
Keywords: quercetin, prostate cancer, medicinal chemistry
Project Title: Nitrogen-Containing Derivatives of Tertramethylquercetins: Synthesis and Antiproliferative Evaluation in Prostate Cancer Cell Models
Author List:
Rajaram, Pravien; Undergraduate, Chemistry, California State University, Fresno, Presenting Author, Nagel Award Nominee
Jiang, Ziran; Undergraduate, Chemistry, California State University, Fresno, Presenting Author
Prasakda, Alison; Bullard High School
Rivera, Alyssa; Undergraduate, Chemistry, California State University, Fresno
Chen, Guanglin; Chemistry, California State University, Fresno
Chen, Qiao-Hong; Chemistry, California State University, Fresno

Abstract: As part of our ongoing research program to develop natural product-based anti-prostate cancer agents, twenty-four new 3-O-aminoalkyl-3′,4′,5,7-O-tertramethylquercetins and twenty four new 5-O-aminoalkyl-3,3′,4′,7-O-tertramethylquercetins have been designed and synthesized for the evaluation of their antiproliferative potency in three human prostate cancer cell models. The twenty-four 3-O-aminoalkyl-3′,4′,5,7-O-tertramethylquercetins have been prepared from commercially available rutin through a four-step procedure including global methylation, glucoside hydrolysis, O-alkylation, and N-alkylation. The twenty-four 5-O-aminoalkyl-3,3′,4′,7-tertramethylquercetins have been achieved from commercially available quercetin through a three-step transformation, including selective tetramethylation of quercetion, O-alkylation with the appropriate dibromoalkane, and N-alkylation. The structures of all these forty-eight quercetin derivatives have been characterized by 1H and 13C NMR, as well as HR-MS data. Our WST-1 cell proliferation assay data indicate that these 48 derivatives are more potent than quercetin in the three prostate cancer cell models; that most derivatives are more potent towards the androgen-sensitive prostate cancer cell line (LNCaP) than androgen-insensitive cell lines (PC-3 and DU145), and that modification on 5-OH of tetramethylquercetin led to the optimal derivatives with low micromolar to submicromolar IC50 values. We are grateful to CSU-Fresno for an Undergraduate Research Grant to P. Rajaram and the support from ACS seed project 2017 to A. Phasakda.

Poster #: 285
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Anti-cancer, synthesis, angiogenesis
Project Title: Our Adventurous Syntheses of Azaspirene, a Powerful Angiogenesis Agent against Cancer
Author List:
Miner, Devin; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Smith, Paul; Graduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Perez, Adam ; Undergraduate, Chemistry and Biochemistry, San Diego State University
Walsworth, Kevin; Graduate, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; 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 #: 286
Campus: San Diego State University
Poster Category: Synthetic Chemistry
Keywords: Anti-Cancer, Anti-Malarial, Natural Product
Project Title: Total Synthesis of Lagunamide A: A Highly Stereoselective Approach for a Powerful Anti-Malarial and Anti-Cancer Agent
Author List:
Baac, Ann; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Cox, Stephanie; Undergraduate, Chemistry and Biochemistry, San Diego State University, Presenting Author
Singh, Simranjeet; Chemistry and Biochemistry, San Diego State University
Wang, Lee; Graduate, Chemistry and Biochemistry, San Diego State University
Bergdahl, Mike; Chemistry and Biochemistry, San Diego State University

Abstract: Background: Lagunamide A is a natural product with exceptional biological activity showing great promise for being a future therapeutic agent. Current data shows this depsi-peptide mode of action an intrinsic apoptotic pathway by cleavage of capsase-9 which activates a series of cascades resulting in mitochondrial assisted apoptosis, a common approach for therapeutics. Lagunamide A has many biological activities and exceptional IC50 values. Amazing cytotoxicity is present against P388 murine leukemia cell lines (IC50 6.4-20.5 nano-M) and Ileocecal colen cancer (IC50 1.6 nM). This possible therapeutic also has impressive anti-malarial properties (IC50 0.19-0.91 micro-M). With such noteworthy activities, Lagunamide A shows great promise as be a new powerful therapeutic agent and brings the need for a direct total synthesis of this macrocyclic depsi-peptide.
Results: We will present our total synthesis of Lagunamide A. This encompasses of a highly convergent asymmetric route which installs 5 of the 10 critical stereocenters integrated heavily along the backbone of the natural product. Four stereocenters are installed with high accuracy using two novel iterative Vinylogous Mukaiyama Aldol Reactions with high yields and excellent diastereomeric ratios. We will also present a new optimized method for coupling unique N-methylated unnatural peptide fragments completed via solid phase synthesis. This produces the pentapeptide fragment which completes the northern hemisphere of Lagunamide A. All data for all fragments will be presented including full characterization via NMR spectroscopy, X-ray crystallography, FTIR, HPLC, and LCMS. Our presentation of these methods for creating each fragment include modifications of the total synthesis which will create divergence for modifications of the natural product. This sets the stage for future work of Lagunamide A to be the syntheses of analogs for structure-activity relationship (SAR) studies which will be tested against various malarial and cancer cell lines.

Poster #: 287
Campus: CSU Fullerton
Poster Category: Other
Keywords: Heart Catheter, Bio-medical, Implantable cloth
Project Title: Automatic Sewing Process of an Implantable Cloth Used in Heart Valve Surgery
Author List:
Mashni, Michael ; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Estelle, David; Undergraduate, Mechanical Engineering, California State University, Fullerton, Presenting Author
Limsakoune, Chacphet; Mechanical Engineering, California State University, Fullerton
Robson, Nina; Mechanical Engineering, California State University, Fullerton

Abstract: With an increase in demand for heart trans catheters, we have teamed up with Edwards Lifesciences to develop a more efficient process in their current manufacturing of heart valves. The current development of one trans-catheter heart valve (THV) consists of a multi-stage operation that takes over 15 hours to complete. One of these stages consists of an approximately 41-minute, ground-level sewing operation in which a highly skilled operator will sew an implantable cloth onto a valve frame template. Due to the immense dexterity required to operate this process and the resulting stress inflicted on operators’ backs, hands, and eyes, automation of a standardized sewing process has been identified as a must in order to effectively meet both rigorous valve definition standards as well as rising demand for THV replacements. Our goal is to develop and integrate a device that will interchangeably mass produce the different models of the heart trans catheters. This will dramatically reduce the time it takes to complete equipment changeovers and simplify the streamline steps, which will result in a significant lower manufacturing cost. To achieve these objectives, our project relates to the development of an automated cost-effective manufacturing platform for the ground-level sewing operation of a THV.
During the past year we were able to design a robotic sewing device, based on the experimental hand trajectory from a human operator sewing an implantable cloth using motion capture system. The data from the operators’ hand movements were then exported and analyzed in MATLAB and each fingertip trajectory point was plotted to develop a pattern. The data analysis from the motion capture system of the manual sewing process showed that the workspace of the manual sewing process can be reduced to planar. As a next step, a prototype of the sewing device was developed and tested. The tests compared the human manual sewing trajectory with the one, obtained from the robotic device. The tests showed that the sewing robotic device was able to closely follow the experimentally obtained trajectory of the humans’ operator; with a minimal error between the two trajectories.
We hope in the future to be able to perfect the trajectory of our sewing device so that it will be as precise as possible. In addition, we also want to be able to record and measure the tension in the thread that the sewers exert. Once we are able to quantify this force we can then implement it in our sewing machine so that our sewing machine can be as practical as possible.