Search Poster #: 1 Campus: San Francisco State University Poster Category: Agriculture/Biofuels/Environment Keywords: Nurdles, Antibiotic Resistance, Biofilm-forming Project Title: Profiling Nurdle-colonizing Bacteria Found on California’s Coastlines Author List: Zoubeidi, Nabil; Graduate, Biology, San Francisco State University, Presenting Author
Chen, Lily; Faculty, Biology, San Francisco State University Abstract  Nurdles are small microplastic beads formed as a byproduct from industrial manufacturing and packaging that end up in the environment via run-offs, spills from shipment vehicles, and erosion. The pressing concern regarding nurdles and the bacteria that colonize them is the potential of antibiotic resistant or pathogenic bacteria using nurdles as vehicles of transmission. Nurdles due to their small size could also be viewed as food by marine life and consumed, potentially allowing antibiotic resistant or pathogenic bacteria to enter the human food chain through commercial fishing. The study aims to examine what bacterial species colonize the collected nurdles from three California public beaches as well as to explore how biofilms may impact the resistome of bacteria over time. From the 3 nurdle collection sites (Ocean beach in San Francisco, Long beach and Seal beach in Orange County), a total of 32 bacterial isolates were selected and characterized phenotypically initially. Of those 32 isolates, 10 isolates were gram-negative and 22 were gram-positive. Nine gram-negative isolates were selected for 16s rRNA gene sequencing due to our research interests in endotoxin encoding gram-negative animal/human pathogens. Of the nine gram-negative isolates, 1 Sphingobacterium isolate (L5A) and 2 Vibrio isolates (3L3A and 3L4A) were examined for antibiotic susceptibility testing using Kirby-Bauer disk diffusion and broth dilution MIC assays. The results show L5A is more resistant to antibiotics tested (ampicillin, tetracycline and chloramphenicol) than 3L3A and 3L4A (two common marine bacteria). Sphingobacterium, formerly part of the genus Pseudomonas, is ubiquitous in nature that may, rarely, cause human infections in immunocompromised individuals and cystic fibrosis patients. The salt tolerance test and biofilm-forming experiments are currently in progress. This study will gleam insight into microbiota in marine nurdles and the resistance profiles that correlate the potential health impacts of nurdle-derived bacteria. | Poster #: 2 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: Mutagenesis, Chalcones, Genetics Project Title: Identification and Isolation of Chalcone Mutants of Caenorhabditis elegans Author List: Alsubhi, Arwa; Graduate, Biology, California State University, Fresno, Presenting Author
Calderon-Urrea, Alejandro; Faculty, Biology, California State University, Fresno Abstract Plant parasitic nematodes cause massive annual losses in crops and revenue, and traditional methods of control are environmentally hazardous or are becoming less effective. Organic compounds Chalcone 17 and Chalcone 30 have proved efficacious, but their molecular mechanism for killing nematodes is unknown. This study hypothesizes that these chalcones affect an enzyme or protein, which disrupts the biochemical pathways of nematodes, leading to their death. The nematode Caenorhabditis elegans has been used before to identify mutations that nullify the lethal effects of Chalcone 17, but these mutations were lost. This study seeks to isolate additional mutations that can survive in high concentrations of Chalcone 17 and 30. This process begins by inducing mutations in C. elegans via ethyl methanesulfonate (EMS), a mutagenizing agent, and growing the mutants in the presence of Chalcone 17 and 30 separately. Surviving nematodes are isolated and backcrossed to assess whether mutations are transmissible. In this way, the study will pave the way for exploring how these chalcones kill nematodes. At the time of submitting this abstract, we have established the mutagenesis protocols and have conducted a pilot screen on ~500 mutagenized worms, which generated approximately 4500 M2 worms, but have not identified any putative mutants yet. We will continue mutagenizing and screening nematodes and hope to have identified mutants by the time of the symposium. | Poster #: 3 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: Arabidopsis thaliana, glutaredoxin, nitrogen Project Title: Roles of Class III Glutaredoxins in Plant Nitrogen Use Efficiency Author List: Massaro, Isabelle; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Carpinelli, Sophia; Undergraduate, Biological Sciences, California State University San Marcos
Escobar, Matthew; Faculty, Biological Sciences, California State University San Marcos
Abstract Glutaredoxins (GRXs) are small oxidoreductase enzymes that reduce disulfide bonds in target proteins. GRXs are divided into three major classes, where Class III GRXs are found exclusively in land plants. We previously found that a small cluster of Class III GRX genes (AtGRXS3/4/5/7/8) in the Arabidopsis thaliana genome are transcriptionally activated by nitrate, and that they may play important roles in plant nitrogen signaling and transport. The focus of this study was to determine how the AtGRXS3/4/5/7/8 gene cluster influences patterns of gene expression and overall plant nitrogen use efficiency (NUE). Two AtGRXS3/4/5/7/8 knockout lines, one transgenic line that ectopically overexpresses AtGRXS8, and wild-type plants were grown hydroponically and supplied with different concentrations of nitrate. To identify changes in gene expression in our transgenic lines, plants were nitrogen-starved for 26 hours and then supplied with 5 mM KNO3 for either 4 hours or 24 hours before harvest. RNA was then isolated from the shoots to quantify gene expression through real-time PCR. Separately, plants were grown continuously under low nitrate (0.4 mM KNO3) or high nitrate (9 mM KNO3) conditions to determine how efficiently these plants take up and utilized nitrate in the growth medium. Biomass, total protein, and nitrate content were quantified in these plants, allowing the indirect measurement of NUE. We found no consistent alterations in patterns of gene expression of the target genes for our transgenic plants in relation to nitrate. However, our AtGRXS3/4/5/7/8 knockout plants showed significant increases in shoot:root ratio when grown under high nitrate, but no change in nitrate or protein concentration in the shoot. Our AtGRXS8 overexpression plants showed a reduction in shoot:root ratio when grown under both high and low nitrate conditions and a reduction in shoot nitrate concentration, but no change in protein concentration in the shoot. These results suggest that AtGRXS3/4/5/7/8 may play a role in determining how plants allocate energy between their root and shoot systems in nitrogen-rich soils. In the long term, these findings could have agricultural impacts to help reduce fertilizer runoff and eutrophication of surrounding environments by helping to increase NUE in crops. This research was funded by the NSF Research Experiences for Undergraduates program site at California State University San Marcos and by the NSF IOS Grant 1651584 to M. E. | Poster #: 4 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: Vinyl Chloride Biodegradation, Saline Concentration, Bacteria Project Title: Biological Degradation of Vinyl Chloride Under Extreme Environment of High Saline Concentrations Author List: Robles, Monica; Graduate, Chemical Engineering, California State University, Long Beach, Presenting Author
Asvapathanagul, Pitiporn; Faculty, Civil Engineering, California State University, Long Beach
Haddad, Maryam; Faculty, Chemical Engineering, California State University, Long Beach
Abstract Vinyl Chloride, a known human carcinogen, yielded from the reductive dechlorination of perchloroethylene (PCE), trichloroethene (TCE), and dichloroethane, has vastly contaminated surface water, groundwater, and soil. Freshwater polluted or spiked with vinyl chloride studies have been widely applied, but studies of vinyl chloride removal near coastal regions, where salinity plays a significant role, are absent. Our hypothesis states that “Extreme conditions of salinity interfere with vinyl chloride degradation in various settings.” Our aims of the project are to discover salt-tolerant vinyl chloride degrading bacteria and to understand their growth mechanisms and metabolic activities. Our study utilizes five bacteria genera, including Dehalococcoides, Mycobacterium, Nocardioides, Clostridium, and Pseudomonas, to study their ability to remove vinyl chloride under extreme salt concentrations at different temperatures, moisture level content, oxygen levels and substrate concentrations. Batch experiments are employed to test the hypothesis. Gene quantification using polymerase chain reaction (qPCR) along with optical density are applied to assess cell abundance, and a spectrophotometer (Aqualog/Horiba) is utilized for the detection of vinyl chloride concentrations. Furthermore, environmental samples from a water reclamation plant (e.g., aeration tank and anaerobic digestion samples), were spiked with vinyl chloride to monitor their bacteria population sizes and to identify the vinyl chloride tolerant bacteria present. The next-generation DNA sequencer (Illuminar-Iseq100) is used to identify vinyl chloride resistant bacterial groups. The bacteria from environmental samples were also tested for their growth rate during vinyl chloride removal. | Poster #: 5 Campus: California State University, Fullerton Poster Category: Agriculture/Biofuels/Environment Keywords: SELEX, Pesticides , Biosensors Project Title: In vitro Development of DNA Based Biosensors for Pesticide Imidacloprid Author List: Azusada, Tiffany; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Hout Ty, Veng; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Jue, Nathaniel; Faculty, Biology and Chemistry , California State University, Monterey Bay
Pecic, Stevan; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Imidacloprid is a systemic insecticide used to control termites, fleas, and sucking insects by disrupting their central nervous system. This chemical is found in many products and is beneficial in people’s homes, pets, and crops. However, an increased amount of exposure to imidacloprid is toxic to humans and detrimental to the environment and wildlife. According to the National Pesticide Information Center, brief exposure to imidacloprid may cause eye irritation, confusion, or vomiting. Additionally, this insecticide is traced in groundwater due to the drainage from soil which subsequently terminates fish and other sea animals. Thus, it is important to continuously detect levels of imidacloprid in soil and water to manage this public health issue. The goal of our lab is to create DNA-based biosensors for imidacloprid using the method called Systematic Evolution of Ligands by Exponential Enrichment, or SELEX. This process identifies specific DNA aptamers from a random DNA library that will bind to the target of interest (imidacloprid), with high affinity and high specificity. In every round, we introduced the imidacloprid target to collect all the specific DNA that binds to it. We lowered the concentration of the target every few rounds in order to isolate high binding affinity aptamers. We have completed 25 rounds of SELEX and the target concentrations were decreased from 100 uM to 25 nm. The PCR amplification yielded 80-100 ng/uL of DNA products, and the double-stranded DNA was separated into single-stranded DNA. The concentration of the single-stranded DNA was measured and 100 pmol was used as the starting library for the next SELEX round. Finally, we incorporated the purified PCR product (750 ng/uL) directly into the plasmid vector. The plasmids were then transformed into competent cells, and positive colonies containing recombinant plasmid DNA were screened via blue/white screening. We randomly picked 22 colonies and cultured them in LB media. We isolated plasmids from the cultured cells and sent the samples to the DNA sequencing service facility. We received 21 sequences back and are currently testing the biosensors in fluorescence assay. These findings will help us to design and develop a DNA aptamer-based biosensor for the detection of imidacloprid. This project is supported in part by COAST research award. | Poster #: 6 Campus: California State University, Fullerton Poster Category: Agriculture/Biofuels/Environment Keywords: Chlamydomonas reinhardtii, Aquaculture, Classical Strain Improvement Project Title: Classical Strain Improvement of Chlamydomonas reinhardtii with an Increased Protein and Lipid Content for Use as an Aquaculture Fishmeal Supplement Author List: Salman, Tanya; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Xia, Angela; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cohen, Amybeth ; Faculty, Biological Science, California State University, Fullerton
Abstract The aquaculture industry regularly uses fishmeal (FM) composed of small, wild fish, such as sardines and anchovies, as it supplies high levels of protein and omega-3 fatty acids. The use of ocean resources for FM is unsustainable and damages marine ecosystems, creating a biological imbalance, as large numbers of smaller fish are regularly depleted from coastal environments. The single-celled green alga, Chlamydomonas reinhardtii, is a potential replacement for the wild fish used in FM, because of its adaptability, ease of cultivation, and standing FAO approval of Generally Recognized As Safe (GRAS) for human consumption. Classical Strain Improvement (CSI) techniques have successfully modified production rates of chloroplast proteins in C. reinhardtii (Fields et al. 2019). This research project is utilizing similar CSI techniques, including UV mutagenesis and nitrogen-deficient induced gametogenesis with mating, on five pairs of wild-type C. reinhardtii laboratory strains, in order to develop genetically diverse offspring with high protein and lipid levels. Crude protein assays were performed using BugBuster Protein Extraction Reagents, followed by Bradford assays to quantify protein levels. After four successive rounds of mating offspring to offspring, a 21.2% and 27.6% increase in crude protein content was recognized in the offspring generated from cc407(+) x cc408(-) and cc1010(+) x cc1009(-) parents, respectively. Preliminary BODIPY 493/503 lipid-based fluorescence microscopy data supports a qualitative increase in neutral lipid body size in 2344(+) and 2342(-) offspring after one round of UV mutagenesis. Quantitative lipid data will be gathered on all of the offspring generated using ImageJ. Once five successions of mating and UV mutagenesis have been conducted, and the most significant percentage change in protein and lipid content from parent to offspring has been assessed, the top-producing individuals will be identified. Fluorescence-Activated Cell Sorting (FACS) will be utilized to select the highest lipid producers out of the entire population using BODIPY 493/503 staining. The predicted outcomes are a more significant increase in the protein and lipid percentage in offspring relative to the parents.
Acknowledgments: This project is supported by the Department of Biological Science at CSU Fullerton and the CSU Council on Ocean Affairs, Science & Technology (COAST) Undergraduate Student Research Support Program.
| Poster #: 7 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: Bacteria, Genomics, Bioremediation Project Title: Bioremediation of pesticides: Applying molecular genomic techniques to uncover efficient, eco-friendly methods of reducing agricultural chemical waste Author List: Surratt, Avalon; Undergraduate, Biology and Chemistry, California State University, Monterey Bay, Presenting Author
Ryder, Rahil; Graduate, Environmental Science, California State University, Monterey Bay
Garcia, Vanessa; Undergraduate, Biology and Chemistry, California State University, Monterey Bay
Lee, Liliana; Undergraduate, Biology and Chemistry, California State University, Monterey Bay
Haffa, Arlene; Faculty, Biology and Chemistry, California State University, Monterey Bay
Jue, Nathaniel; Faculty, Biology and Chemistry, California State University, Monterey Bay Abstract Bioremediation, the process of microorganisms breaking down pollutants, overcomes the limitations of traditional pollution remedies. Preferred for its efficiency and reliance on natural product, it relies on naturally-occurring organic conversions by locally-adapted microbes as opposed to induced physicochemical reactions. Therefore, it is a desirable method for cleaning toxic agricultural waste. Our research focuses on these bacteria microorganisms that can remediate pesticide pollutants. We are identifying the functional genetic mechanism that enable the bacteria to metabolize carbon sourced from pesticides in order to divulge the biological mechanisms of bioremediation. To identify these mechanisms, we culture bacteria isolated from lands adjacent to pesticide-treated agricultural areas and grew them in a minimal media environment where the pesticide is their only carbon-source, extracted and isolated DNA from these cultures, generated DNA sequencing for these samples on Illumina and MinION sequencing platforms, and assembled their genomes to establish the genomic references for these organisms. Using these genome references, we will use transposon-mediated genetic mutational screening (Tn-seq) in minimal media on electrocompetent bacterial cultures to identified genes essential to pesticide bioremediation. This data will be used to advance the development of a biotechnological approach to remediating agricultural chemical waste. | Poster #: 8 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Therapeutic design, triple negative breast cancer, protein simulations Project Title: Simulation Based Analysis of HerPBK10 Drug Delivery Molecule Targeting Chemo Resistant and Metastatic Breast Cancer in Varying pH Conditions Author List: Crisostomo, Isabella; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Sanchez, Romny; Graduate, Chemistry and Biochemistry, California State University, Northridge
Medina-Kauwe, Lali; Cedars-Sinai Medical Center
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract The median survival for patients with metastatic breast cancer is ~2-5 years and even lower for metastasis to the brain. This drastically reduces the therapeutic options as most targeted therapies cannot cross the blood-brain barrier (BBB). Transferring systemic targeted therapies across the BBB and into brain-localized tumors remains a major challenge. Increased cell surface density of HER3 has been shown to be associated with tumor progression, therapeutic resistance, and metastasis including the brain-metastasis of HER2+ and triple-negative breast tumors. Medina-Kauwe lab at Cedars-Sinai Medical Center has developed a selective drug delivery molecule named HerPBK10, which contains a pentameric human adenovirus serotype-5 penton base protein along with the heregulin ligand to target HER3 overexpression in chemo resistant and metastatic breast cancers. This nanoparticle can carry a diverse set of cargo like siRNA and corrole compounds to cause tumor cell death. Adenovirus penton base proteins intrinsically possess the ability to lyse endosomes via interaction with endogenous alpha-V/beta-5 integrin, rendering them excellent candidates for integration into nanoparticles. To deduce the effect of ionic residues on the stability and experimentally observed endosomolysis of HerPBK10 at pH of ~5, we generated multiple pH-specific structural models of a truncated version of the pentameric particle at pH of 7, 5, 3, and 1. These were relaxed in their physiological environment by implicit solvent molecular dynamics simulations. Endosomal lysis was observed at pH of 3 and 1 but not at pH of 5, probably due to long simulations needed to observe this lysis at pH=5. These studies are being expanded to model the full-length bioparticle using enhanced conformational sampling methods at specific biological pHs 7,6,5,4, and 3 using serotype-5 and serotype-2 penton base protein sequences. The structural models will be analyzed in terms of their stability at different pHs and effect of different ionic residue distributions from serotype-5/2 protein sequences. These studies will provide insights on the optimization of the therapeutic bioparticle structures for effective drug/siRNA delivery to chemo resistant and metastatic tumors. This project is funded in part by the Department of Defense grant W81XWH-19-1-0592. | Poster #: 9 Campus: San José State University Poster Category: Biochemistry Keywords: SIRT1, enzyme kinetics, allosteric regulation Project Title: Dissecting the Mechanism of Allosteric Regulation of SIRT1 Author List: Leong, Emily; Undergraduate, Chemistry, San José State University, Presenting Author
Dosanjh, Reena; Undergraduate, Chemistry, San José State University
Huynh, Johnson; Undergraduate, Chemistry, San José State University
Hur, Yujin; Undergraduate, Chemistry, San José State University
Wang, Ningkun; Faculty, Chemistry, San José State University
Abstract SIRT1 is a lysine deacetylase enzyme that plays a part in various pathways in the body, including insulin secretion and neurodegeneration, identifying it as a potential drug target for common diseases such as Type II diabetes and Alzheimer's disease. Our goal is to dissect the activity of SIRT1 with and without a small molecule regulator against different peptide substrates. Although a number of small molecules have been found to regulate SIRT1 activity, the exact mechanism of this regulation remains unclear, hindering further development of specific SIRT1 regulators suitable for therapeutic use. Previous studies have found that resveratrol, a well-studied SIRT1 modulator, is not only an activator of SIRT1, but also can be an inhibitor or have no effect on its activity depending on different substrate sequences. However, these studies are end-point assays with limited information, and more detailed work must be done to elucidate the mechanism of this substrate-sequence specific modulation of SIRT1. We aim to elucidate the mechanism of how SIRT1 activity can be allosterically regulated using resveratrol as a chemical tool. We hypothesize that resveratrol alters SIRT1 enzyme activity by changing the conformation of the enzyme, thus changing the efficiency of substrate recognition, leading to a larger change in KM than in kcat. To test this we examined the enzyme kinetics parameters of SIRT1 with and without resveratrol with different peptide substrates. We have successfully expressed and purified SIRT1 with Ni-NTA and SEC columns. The Michaelis-Menten parameters for SIRT1 against substrates Ac-p300 and Ac-H4 with and without resveratrol have been determined, where SIRT1 activity has been inhibited by resveratrol. The KM for Ac-p300 showed a 2-fold increase after the addition of resveratrol ( from 30.2 µM to 59.2 µM), and the kcat did not significantly change (from 0.068 M-1s-1 to 0.069 M-1s-1). The KM for Ac-H4 also showed a 2-fold increase (from 107.9 µM to 217.2 µM) while kcat slightly decreased (from 0.088 M-1s-1 to 0.070 M-1s-1 ). Our research increases our understanding of the mechanism for the regulation of SIRT1 activity, which can be beneficial to the development of drugs.
| Poster #: 10 Campus: San José State University Poster Category: Biochemistry Keywords: intracellular pH, cell death, microscopy Project Title: Regulation of Proliferation and Cell Death by Increased Intracellular pH Author List: Morales, Emilio; Undergraduate, Biological Sciences, San José State University, Presenting Author
Montan, Carly; Graduate, Biological Sciences, San José State University, Presenting Author
Gates, Hillary; Graduate, Biological Sciences, San José State University
Grillo-Hill, Bree; Faculty, Biological Sciences, San José State University Abstract Background:
Dysregulated cell death is associated with diseases including neurodegeneration (increased cell death) and cancer (reduced cell death). Dysregulated intracellular pH (pHi) is also associated with these diseases, where degenerating neurons have lower pHi, and cancer cells have constitutively higher pHi than normal cells. Our lab published that higher pHi is sufficient for increased proliferation in the developing Drosophila eye, which should cause a larger adult eye, however the eye was smaller. Cell counts showed a significant decrease in cell number. We hypothesize that reduced cell number at higher pHi occurs through activation of a cell death pathway. The goal of our project is to determine if decreased cell number at higher pHi is conserved in mammalian cells, and to then determine the mode of cell death.
Methods:
A published method was used to chemically increase pHi in Madin-Darby Canine Kidney (MDCK) cells. MDCK cells were treated with 5mM ammonium chloride (NH4Cl), and cells were counted after 24 and 48 hours. We next used phase holographic imaging to visualize control and 5mM NH4Cl treated MDCK cells every 10 minutes over 24 hours. We analyzed movies and images taken of our control and NH4Cl-treated cells. First, a built-in analysis module determined the confluence of cells grown in both conditions. We next performed manual counts of proliferating cells through a FIJI (NIH ImageJ) workflow.
Results:
We found decreased cell number at higher pHi, suggesting this is conserved in MDCK cell lines. Automated analysis of the holographic movies showed no difference in confluence between control and NH4Cl treated MDCK cells, suggesting that the area occupied by cells does not change. Manual counts of proliferating cells found increased proliferation in cells treated with NH4Cl compared to the control cells. These data are consistent with our lab’s previous findings in the developing fly eye.
There are two potential mechanisms that could cause increased proliferation with no change in confluence. First, cell size could be decreased; we will test this by measuring volume in the HoloMonitor software. Second, increased cell death at higher pHi could reduce cell number directly. To test this, we will determine expression of cell death markers through FACS and Western Blot. Our project will identify how increased pHi regulates cell number, which has implications for novel cancer therapeutic approaches.
Funding: NIH SC3GM132049 and CSUPERB NI | Poster #: 11 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: heat-shock proteins, lipids, pharmacology Project Title: How HSPA1A, a stress-inducible 70-kDa heat shock protein, travels to the cell surface of stressed and cancer cells Author List: Altman, Rachel; Graduate, Biological Science, California State University, Fullerton, Presenting Author, Eden Award Finalist
Nikolaidi, Nikolas; Faculty, Biological Science, California State University, Fullerton Abstract HSPA1A is a stress-inducible seventy-kilodalton heat shock protein (Hsp70) that regulates cell survival. This protein also localizes at the plasma membrane (PM) of 90% of stressed and tumor cells. In cancer cells, the presence of HSPA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, abolishing HSPA1A from the surface of tumors is a promising therapeutic. However, the PM-translocation pathway of HSPA1A to the cell surface remains enigmatic because this protein lacks membrane localization signals. Considering that HSPA1A binds with high selectivity to negatively charged lipids, like phosphatidylserine (PS) and mono-phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HSPA1A with these lipids allows the chaperone to translocate to the PM. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A’s PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that depletion of PS, PI(4)P, and PI(3)P by co-transfecting HSPA1A with known lipid-biosensors, significantly reduced HSPA1A’s surface presentation. In contrast, reducing PI(4,5)P2 availability by overexpression of the PLCδ-PH biosensor had minimal effects on HSPA1A’s PM-localization. Next, we manipulated the cellular lipid content using ionomycin (a PLC activator), phenylarsine oxide (PAO) and GSK-A1 (general and a selective PI4-Kinase inhibitors, respectively), wortmannin (a PI3-Kinase inhibitor), and fendiline (a repurposed FDA approved drug that reduces PS) using lipid-biosensors as positive and negative controls. These experiments revealed that the HSPA1A’s PM localization was unaffected by ionomycin but was greatly reduced in the presence of PAO, GSK-A1, wortmannin, and fendiline, corroborating the findings obtained by the co-transfection experiments. We further verified these results by selectively depleting PI(4,5)P2 and PI(4)P using a rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A’s surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and plasma membrane lipids. These findings provide the basis for future interventions to render tumor cells sensitive to radiation therapy. | Poster #: 12 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: copper transport, blood, urine, , Project Title: Chemical and biological characteristics of small copper carriers (SCC) found in blood fluid and urine Author List: Linder, Maria; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Ma, Sandy; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Giolli, Brian; Graduate, Chemistry and Biochemistry , California State University, Fullerton
Roque, Theodore; California State University Fullerton
Carilllo, Heather; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Abstract In mammals, levels of Cu in tissues and fluids are maintained at very constant levels. This homeostatic state is mainly regulated by the rate at which Cu is excreted from the body from the liver via the bile into the GI tract, exiting with the feces. In certain not uncommon genetic states, biliary Cu excretion is compromised, which results in copper overload and damage to the liver and other organs. In partial compensation, there is a large increase of Cu excretion through the urine, which normally plays a very minor role. A small copper carrier (SCC) was first detected by Gray et al. in the urine of mice modeling the genetic Cu overload of Wilson disease, and our lab then detected it also in blood plasma, and in the urine and plasma of Labrador retrievers with the same genetic mutation as in Wilson disease. SCC is less than 3 kDa in mass, and present in uM concentrations. The studies to be reported were initiated to purify and characterize SCC chemically and physically, and to investigate a potential role in Cu distribution via the circulation.
Various ways of purifying SCC were examined, ranging from ultrafiltration; dialysis; size exclusion, hydrophilic interaction and anion exchange chromatography; as well as lectin binding. Starting material was porcine blood plasma, which has higher amounts of SCC than in other species. 67Cu-radiolabeled SCC secreted by cultured rat hepatoma cells was used to determine whether SCC could deliver its Cu for uptake by cells. The pH stability and effects of reducing and chelating agents on removing its Cu were also examined, using dialysis in tubing with a 0.1-0.5 kDa cutoff.
A combination of 10 and 3 kDa cutoff ultrafiltration, thorough dialysis into nanopure water with small pore tubing, concentration by lyophilization, followed by small pore size exclusion chromatography produced a markedly purified product, with an apparent mass of about 800 Da, and with excellent yield, though still without contaminants. The SCC-Cu complex contained Cu2+, and EPR indicated the Cu was bound to O and N ligands. The complex was stable in dialysis against buffers rang from pH 4 to 8, but came off at pH 2 and 9, and when reducing agents were applied. The Cu in 67Cu-SCC was readily absorbed by all the cultured cells tested, which included the hepatic cells that had secreted it. Uptake rates were inhibited by an excess of non-radioactive Cu(I), as well as by Ag(I), both of which are substrates for the only as yet identified Cu uptake transporter in the plasma membrane (CTR1). CTR1 knockout cells had 60% less Cu uptake from SCC, but still took it up, and endocytosis inhibitors did not reduce uptake rates. The results imply that SCC may represent an additional means by which Cu is distributed from the liver to other cells and to the blood fluid, where an excess production – as in Cu overload – results in its filtration by the kidney for urinary excretion.
| Poster #: 13 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: DNA Repair, biochemistry, fluorescence micoscopy Project Title: Probing the localization of Rad1-Rad10 and Mus81-Mms4 at Ultra-fine Bridges in Damaged DNA and the Necessity of Slx4 in Single Strand Annealing Repair Author List: Hadi, Hafridha; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Guzmán, Jr., Jimmy ; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract Double-strand breaks (DSBs) in DNA can be deleterious if left unrepaired. Eukaryotic cells have developed different repair mechanisms to counter the harmful effects of DSBs. In S. cerevisiae, the cell may utilize the classical Double-Strand Break Repair (DSBR) pathway as a conservative process to repair DSBs, or any of several others, including Single Strand Annealing (SSA), which is a non-conservative process. The DSBR pathway uses a sister chromatid or a homologous chromosome for repair, and is favored over SSA in G2 and M phases. SSA may be favored in G1 phase and requires the scaffold protein Slx4, but the details are not clear.
In part one of this project, we investigated ultra-fine bridges (UFBs), a form of intertwined chromatids that form during DSBR and must be resolved prior to cytokinesis. It is unknown whether endonucleases Rad1-Rad10 and Mus81-Mms4 localize to UFBs. Our aim was to track the localization of Rad1-Rad10 and Mus81-Mms4 throughout the cell cycle via a yeast strain containing fluorescently-labeled versions of Rad10, Mus81, and Dpb11 genes. We arrested yeast cultures at the G1/S boundary with α-factor and released them back into cell cycle. Aliquots were collected at eleven time points over three hours for analysis by flow cytometry and fluorescence microscopy. Preliminary data confirms that the cells were successfully arrested and released. It also reveals the presence of Rad1-Rad10 and Mus81-Mms4 at UFB-positive cells, and the localization of Mus81-Mms4 at specific sites within the UFBs.
In part two of this project, we aim to develop an assay system utilizing Spinach as a reporter of successful SSA repair. This reporter system would determine if Slx4 is required for SSA repair in G1 phase. Toward that goal, we cloned the Spinach fluorescence RNA aptamer gene under control of a galactose-inducible promoter into a plasmid that can be propagated in yeast. A yeast strain was transformed with the Spinach plasmid, induced with galactose, and treated with 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), the ligand that binds Spinach to create the green fluorophore. Results showed green fluorescent focus formation throughout cells, but considerable variability in signal. Cells sometimes fluoresced without galactose induction or lost all fluorescence following induction. To use Spinach as a viable assay system, experimental conditions must be found that reproducibly yield increased green fluorescence upon galactose induction. | Poster #: 14 Campus: California State Polytechnic University, Pomona Poster Category: Biochemistry Keywords: Acinetobacter , cultivations, planetary protection Project Title: Impact of trace elements on the cultivation of spacecraft-associated Acinetobacter Author List: Mogul, Rakesh; Faculty, Chemistry and Biochemistry, California State Polytechnic University, Pomona
Miller, D; Staff, Chemistry and Biochemistry, California State Polytechnic University, Pomona
Jaramillo, Daniel ; Graduate, Chemistry and Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Aldaco, Juan; Graduate, Chemistry and Biochemistry, California State Polytechnic University, Pomona, Presenting Author Abstract The cleanroom facilities used during spacecraft assembly are routinely cleaned with alcohol reagents in efforts to minimize biological and organic contamination. Similarly, alcohols such as ethanol and 2-propanol are commonly used in clinical facilities and laboratories to clean benchtops and surfaces. Among the microorganisms commonly found in spacecraft assembly facilities and clinical settings are the Acinetobacter, which have been shown to grow on ethanol as a sole carbon source under both nutrient rich and poor conditions. In this study, we focus on the spacecraft-associated Acinetobacter and identification of cultivation conditions that promote rapid growth on ethanol. Namely, we are assaying the impacts of trace elements and pH on the incorporation of ethanol as a sole carbon. Our results show that the Mars Odyssey strain of A. radioresistens 50v1 exhibits similar growth rates in diluted minimal media when supplemented with either just iron (25 µM) or a trace elements mixture (containing Fe, Co, Cu, Zn, Mo, and B at 50 µM each). However, cultivations with ferrous ion, as the sole defined trace element, yielded lag phase times that were ~2-fold higher than those measured in media containing the trace elements mixture. Further, growth rates were slightly higher for the 50v1 strain at pH 7.2 (0.2x M9 minimal media with trace elements) when compared to pH 7.8 (0.2x modified M9). Currently, cultivations on A. johnsonii 2P08AA, A. johnsonii 2P07AA, A. guillouiae 2P07PC, and A. guillouiae 2P07PB are being performed. The combined results for this study will be discussed. | Poster #: 15 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Akt isoforms, Unfolded Protein Response, Endoplasmic reticulum stress Project Title: Identification of the Akt Isoform that Regulates the Unfolded Protein Response Author List: Palma, Miguel; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Eden Award Finalist
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract The endoplasmic reticulum (ER) is an important cellular organelle that is a site of several important functions including protein folding and secretion. Under conditions that generate ER stress such as imbalance in the protein folding load and capacity, cells begin a signaling program called the unfolded protein response (UPR). The UPR serves to restore homeostasis by triggering transcriptional pathways through stress sensing proteins in the ER membrane. In recent years, several groups including ours have shown that the UPR is regulated by Akt. Akt is a serine/threonine protein kinase that plays key roles in many important cellular processes including survival, metabolism and migration. There are three isoforms of Akt - Akt1, 2, 3 - with unique functions. However, most of the studies on Akt including the ones focused on ER stress, use reagents that do not distinguish between the isoforms. Our study aims to identify the specific Akt isoform that regulates the UPR. To do so, we designed an experimental strategy whereby the cells’ endogenous Akt isoforms were inhibited using a small molecule inhibitor and the role of individual isoforms was assessed by expressing the inhibition-resistant constructs one at a time. Specifically, we used human embryonic kidney cells which are a good model cell line to study UPR and express all three Akt isoforms endogenously. A potent pan-Akt inhibitor, MK2206, was used to achieve inactivation of the endogenous forms of Akt. The inhibition resistant constructs of Akt1, 2 and 3 were generated using polymerase chain reaction based site-directed mutagenesis to create a tryptophan to alanine mutation at position 80 (W80A). This tryptophan is important for MK2206 to bind to Akt, thus the W80A mutation renders the cloned Akt constructs resistant to inhibition while not affecting the overall protein structure or function. ER stress was triggered using tunicamycin, which inhibits N-linked glycosylation of newly synthesized proteins in the ER thereby promoting their misfolding. Cell lysates were analyzed by western blotting to detect changes in the UPR. My current data show a prominent role of the Akt2 isoform in regulation of UPR. My future experiments entail investigating interactions between Akt2 and the UPR effector proteins, and the mechanism of activation of Akt2 during ER stress. | Poster #: 16 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: apolipoprotein, TEV protease, chimera Project Title: Production of TEV protease for histidine-tag removal of apolipoprotein A-I chimeric proteins Author List: Rodriguez, Judy; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Weers, Paul; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Human apolipoprotein A-I (apoA-I) is the major protein in high-density lipoprotein and plays a critical role in cholesterol transport from peripheral tissue to the liver, a process known as reverse cholesterol transport. ApoA-I (28 kDa, 243 amino acid residues) is composed of an N-terminal (NT) helix bundle domain and a small and less structured C-terminal domain. It has been proposed that the NT helices play a role in lipid binding, however, sequence analysis predicted a low lipid binding affinity of the first helix of apoA-I. To better understand the precise role NT helices of apoA-I in lipid binding, chimeric proteins were designed, which were successfully expressed in a recombinant E. coli system. The chimeras were composed of helix 1 or 2 of apoA-I, followed by insect apolipophorin III, which served as a scaffold protein and was inactivated by a disulfide bond. Since the chimera sequence starts with a His6-tag to facilitate purification from E. coli lysates, this may potential interfere with the lipid binding function of the NT helices and thus needs to be removed. Therefore, a Tobacco Etch Virus (TEV) protease site was inserted between the His6-tag and the chimera. TEV protease was also expressed recombinantly in E. coli with a yield of approximately 20 mg per liter culture medium. To verify His6-tag removal by TEV, wild-type apoA-I was incubated at room temperature with the purified protease at a mass ratio of 20:1. SDS-PAGE analysis showed that after 2 h of incubation a smaller band appeared just below apoA-I, indicative of His6-tag removal. When the incubation was continued for 16 h, approximately 75% cleavage was achieved. Upon optimization of TEV proteolysis conditions, the chimeric apolipoproteins will be treated with TEV, and cleaved protein will be separated from uncleaved proteins by Ni-affinity chromatography. The chimera proteins lacking the His6-tag will then be assessed for lipid binding activity using model phospholipid bilayer vesicles. In summary, the TEV protease bacteria expression system produced an active enzyme, which, after optimization of proteolysis conditions, will be used to remove the His6-tag from chimera apolipoproteins. This will allow lipid binding analysis of the apolipoprotein chimeras and provide important insight in the role of the first two helices of apoA-I.
Research is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number GM089564.
| Poster #: 17 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Orphan GPCRs, G Protein Signaling, Molecular Dynamics Project Title: Structural Characterization of the G12 Selective Designer Receptor Exclusively Activated by Designer Drugs in Complex with G12 and G13 Proteins Author List: Cruz, Christian; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Abrol, Ravinder; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract G-protein-coupled receptors (GPCRs) are the largest superfamily of integral membrane proteins activated by extracellular ligands that enable intracellular signaling pathways through coupling to G proteins and 𝝱-arrestins. Orphan GPCRs are receptors where ligands and/or G protein coupling has not yet been identified. DREADDs (Designer Receptors Activated by Designer Drugs) are being used with synthetic ligands to characterize orphan receptor signaling and to control G protein coupled signaling in different physiological situations. Currently DREADD receptors are available for coupling to Gs, Gi/o,Gq/11 and G12 proteins. The G12 coupled DREADD receptor (M3D-GPR183/ICL3) designed through a machine learning model does not couple to the homologous G13 protein and there is a need for a G13-specific DREADD receptor. We are structurally characterizing the G12 selective DREADD receptor (DRE12) in complex with G12 protein (DRE12:G12 complex) and G13 protein (DRE12:G13 complex) by relaxing their homology-based structures in the membrane environment through atomistic molecular dynamics (MD) simulations. These simulations allow for analysis of the movement of residues and structural motifs of the G-protein and receptor as they interact with each other and with the lipid bilayer environment. Trajectories of protein complex snapshots for the DRE12:G12 and DRE12:G13 complexes has been generated that provides a first glimpse into the differences between these two complexes. The protein-protein interactions were characterized through hydrogen-bond analysis and the structural flexibility of protein residues in both complexes were analyzed through root mean square fluctuation (RMSF) calculations. The hydrogen-bond analysis showed some DRE12 receptor residues interacting with both G12/G13 and some residues exclusively interacting with G12 or G13 proteins. The G13 protein in DRE12:G13 complex appears to be more dynamic than the G12 protein in the DRE12:G12 complex, providing hints on why DRE12:G13 coupling is not observed experimentally. This is being currently examined by binding free energy analysis. These analyses will assist in the design of a G13-selective DREADD to enable characterizing G13 signaling in different physiological contexts. Research is funded through BUILD PODER NIH Research fellowship grant #5TL4GM11897707 and #SRL5GM118975-07 as well as in part by NIH grant SC2GM130480. | Poster #: 18 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Cancer, Biochemistry, Glyoxalase Project Title: Investigating the role of Nrf2 translocation to the nucleus and to elevated AKR expression cancer cells treated with a Glo 1 inhibitor Author List: Reque, Leticia ; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Anderson, Jennifer; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Feldman, Alexander ; Graduate, Chemistry and Biochemistry, California State University, Northridge
Tamae, Daniel; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract Breast cancer and prostate cancer are the two of the most frequently diagnosed cancers in the developed world. In breast tumor biopsies, Glyoxalase 1 and 2 (Glo 1/2) are over-expressed relative to adjacent tissue. In prostate tumors, high levels of Glo 1 expression correlates with poor prognosis. The Glo 1/2 system is critical for the detoxification of metabolically derived dicarbonyl species such as methylglyoxal and glyoxal. The inhibition of Glo 1 by a glutathione analogue, results in cell death in a panel of breast, prostate, and ovarian cancer cell lines (EC50 = 9.0-29 uM). We hypothesize that Glo 1 inhibition leads to the accumulation of reactive carbonyl species and that members of the aldo-keto reductase (AKR) superfamily may be a potential compensatory response as they have been found to reduce dicarbonyls to corresponding alcohols. We also hypothesize that Glo1 inhibition may lead to elevated AKR expression via the Nrf2 transcription factor. Recent studies have shown that methylglyoxal (MG) stabilizes Nrf2 via covalent modification of KEAP1. Furthermore, Nrf2 is well-established to drive the expression of many members of the AKR family. We used qRT-PCR and observed 2- to 3-fold increases in AKR1C1 in the MCF-7 and MDA-MB-231 cell lines and 2-fold increase in AKR1C3 in the MCF-7 and 22Rv1 cell lines. We have used nuclear fractionation of cell lysates to observe enrichment of Nrf2 in these cell lines within 6-12 hours of treatment with Glo 1 inhibitor. Taken together, these findings corroborate that elevated MG in the cell results in translocation of Nrf2 and elevated AKR expression.
| Poster #: 19 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Enzyme inhibition, Structure-activity relationship, Alzheimer’s disease Project Title: A polypharmacology approach: Development of dual FAAH/AChE inhibitors for the treatment of chronic pain in patients with Alzheimer’s disease Author List: Gonzalez, Michael; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Pecic, Stevan; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Alzheimer’s disease (AD) is a continuously growing degrative disease that has pushed for medicinal research and pharmaceuticals to advance the replacement of conventional drug treatments utilized today. Currently, there are five different hypothesis that are proposing to the development of AD in the brain, and one, Cholinergic hypothesis, involves acetylcholinesterase (AChE) enzyme and its breakdown of neurotransmitter acetylcholine (ACh), an important factor in neuronal communication. Inhibition of AChE increases ACh concentrations, which has beneficial therapeutic effects on AD patients, such as improved cognition and global function, and spawned treatments such as donepezil. Fatty Acid Amide Hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes endocannabinoids. The pharmacological inactivation of FAAH produces analgesic and anti-neuroinflammatory effects, reduces neuronal death and memory retention loss, and shows to improve neuronal transmission. Therefore, inhibition of FAAH represents a promising therapeutic strategy for the treatment of AD. Previous literature has also shown a link between AD and pain via the central nervous system. In our lab, we identified several potent AChE inhibitors which utilize a donepezil scaffold. Our detailed kinetic evaluations of the most potent inhibitor (IC50 = 51 nM) showed a mixed inhibition mode, same as donepezil. In a separate study, we investigated a series of viable 4-phenylthiazole FAAH inhibitors. These FAAH inhibitors, developed in our research group, ranged from IC50 values of 30.8 nm to competitive values of 8.4 nm. A new developing avant-garde approach to drug design is utilizing the concept of polypharmacology to develop single multi-target directed ligands; one drug that will affect multiple binding targets. Our aim is to combine selective parts of AChE and FAAH inhibitors identified in our lab into a single small molecule, enhancing its AD therapeutic potential. All dual AChE/FAAH compounds were synthesized via environmentally friendly microwave-assisted synthetic route and evaluated against AChE enzymes and FAAH enzymes. This dual inhibitor series was developed merging the 4-phenylthiazole moiety from FAAH inhibitors and piperidine-pyridyl moiety from AChE inhibitors. We also performed some selected absorption, distribution, metabolism, excretion, and toxicology (ADMET) predictions to assess their drug-like properties. | Poster #: 20 Campus: California State University, Los Angeles Poster Category: Biochemistry Keywords: ice-binding protein, alcohol dehydrogenase, enzymatic activity Project Title: Cryoprotective effect of an ice-binding protein on the enzymatic activity of alcohol dehydrogenase Author List: Aguirre, Daisy; Graduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wen, Xin; Faculty, Chemistry and Biochemistry, California State University, Los Angeles Abstract The long-term storage of freeze-labile enzymes in solution is challenging as these enzymes lose their function under repeated freezing and thawing. Additives are often used as co-solutes in order to protect freeze-labile proteins for the long-term storage. Ice-binding proteins (IBPs) can inhibit ice crystal growth enabling the survival of the organisms under freezing temperatures, a characteristic that is of high importance to organisms that live in cold environment conditions and have thus been considered as potential protective agents for cryopreservation naturally. This study aims to investigate the cryoprotective effects of a recombinant IBP (rIBP) on a model freeze-labile enzyme, alcohol dehydrogenase (ADH), under freezing and thawing conditions. The ADH samples (0.20 mg/mL), prepared in the presence of rIBP (0.50 mg/mL) and a protein control that does not have ice-binding properties, bovine serum albumin (BSA), respectively, were subjected to multiple freezing (-20 ºC) and thawing treatments (n = 4-7). The enzymatic activity of the ADH samples were assayed after each freezing-thawing cycles. In the absence of any additives, ADH lost more than 20% of its activity after a single freezing-thawing treatment and more than 50% of its activity after three freezing-thawing treatments. The presence of rIBP significantly protected the activity of ADH that about 80% of its enzymatic activity remained after four repeated freezing-thawing treatments. The presence of BSA does not have any protective effects on ADH under the same freezing-thawing treatments. Contacts between ice and ADH have been identified as a destabilizing factor for this enzyme. Our results suggest that interactions between ice and rAFP minimize the potential contacts between ice and ADH and thus protect the activity of the enzyme. This finding also suggests a novel approach into potential applications in enzyme protection in food, medical, and pharmaceutical industries. | Poster #: 21 Campus: Sonoma State University Poster Category: Biochemistry Keywords: Sustainability, Bioinorganic , Energy Project Title: New Catalysts for Production of Energy from Hydrogen Author List: Lerue, Christian; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Nguyen, Marilyn ; Undergraduate, Chemistry , Sonoma State University, Presenting Author
Davenport, Johanna ; Undergraduate, Chemistry , Sonoma State University
Pyle, Tristan ; Undergraduate, Chemistry , Sonoma State University
Gonzalez, Rosario ; Undergraduate, Chemistry , Sonoma State University
Bustos-Works, Carmen ; Faculty, Chemistry , Sonoma State University Abstract The general goal of this project is to elucidate the mechanism of enzymatic hydrogen utilization and extend this knowledge to the development of iron based catalysts. This is important to further the use of hydrogen as an alternative fuel source. Our innovative approach uses photochemistry as a tool to generate and probe the reactivity of small inorganic model compounds that have the potential as inexpensive environmentally friendly catalysts. Our compounds are based on iron-iron hydrogenase enzymes. Iron-iron hydrogenase is an enzyme found in anaerobic bacteria and catalyzes the reversible oxidation of molecular hydrogen and our goal is to mimic what nature has done.
Specific aims:
1. Determine how changes in the structure of the model compounds change the photochemical reactivity of the molecules.
2. Elucidate the catalytic species in solution that function as the catalysts for hydrogen generation and activation using both experimental and theoretical techniques.
3. Determine the catalytic mechanism.
To achieve the specific aims above, we have successfully improved the synthesis process of these model compounds and characterized the photochemistry through various spectroscopic techniques (how molecules interact with light) to confirm the molecules are photochemically active and potential catalysts for the production of energy from hydrogen.
Results include:
Clearly established synthetic protocols
Confirmation of photochemical activity both qualitatively and quantitatively
Quantitatively our molecules are 4.4-2.3% photochemically efficient
Theoretical calculations have shown intermediates that might be important in the catalytic process
Our current work focuses on experimentally confirming the theoretical resources, making new molecules and measuring the activation of hydrogen with our current
Acknowledgements
We would like to acknowledge the National Science Foundation for funding this project and Sonoma State University Chemistry Department for their contributions to this project.
| Poster #: 22 Campus: San José State University Poster Category: Bioengineering Keywords: biorecycling, rare earth elements, electronic waste Project Title: Engineering e-waste resistance in Methylorubrum extorquens for enhanced bio-recycling of rare earth elements from postconsumer electronics Author List: Hussain, Tanzeena; Graduate, Biological Sciences, San José State University, Presenting Author
Huang, Alice; Undergraduate, Biological Sciences, San José State University
Tani, Jason; Undergraduate, Biological Sciences, San José State University
Grace, Joseph M.; Graduate, Biological Sciences, San José State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University
Abstract Rare Earth Elements (REEs) are widely used in electronics, green energy technologies, and medical devices. Worldwide demand for REEs is expected to reach 3 million metric tons by 2025. However, current REE mining methods are costly and environmentally destructive. We are genetically engineering Methylorubrum extorquens as a cost-effective, environment friendly REE bio-recycling platform to recover REEs from electronic waste (e-waste). M. extorquens is a methylotrophic bacterium which uses REEs as cofactors for methanol oxidation and can acquire REEs from hard drive magnets and mining ores. Excess REEs are stored in polyphosphate granules which can be extracted to recover REEs. One challenge in using a biological approach to recover REEs is the toxicity of complex e-waste streams due to metals like Hg, Li, Cd, Cr and Pb. These metals compete with metal cofactors to inhibit enzyme activities and promote oxidative stress. Here we show that M. extorquens can use 0.5% pulverized smartphones as REE source, but 5% smartphone causes cell death. We isolated a spontaneous mutant (EWR) that can grow with 8% smartphone. Oxygen consumption and disc diffusion assays determined that resistance to e-waste in the EWR strain is a specific response and does not allow resistance to heat, solvent, oxidative, or osmotic stresses. Whole genome sequencing of the EWR strain revealed deletions in two transporter genes and a point mutation in a transcriptional regulator gene. Strain reconstruction and phenotypic testing identified a deletion in the oxlT1 oxalate formate antiporter gene as the mutation responsible for e-waste resistance. This antiporter exchanges oxalate and formate and can operate in either direction depending on concentration gradients. We hypothesize that excreted oxalate binds to toxic metals in e-waste which is then transported into the cell through OxlT1; deletion of oxlT1 therefore reduces toxic metal import. Consistent with this hypothesis, exogenous oxalate decreased e-waste resistance of the wild type and oxlT1 strains, while exogenous formate increased e-waste resistance. Five putative oxlT genes are encoded in the M. extorquens genome. Future studies will determine if deletion of additional oxlT genes further increases e-waste resistance. This work will advance the development of a REE bio-recycling platform that will decrease U.S. reliance on foreign REE suppliers. | Poster #: 23 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Bioengineering Keywords: tissue engineering, blood vessels, microfluidics Project Title: Injectable 3-D Human Microvasculature Author List: Jackson, Madeline; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo, Presenting Author
Kahn, Elias; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Heylman, Christopher; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract The aim of preclinical testing is to develop accurate preliminary toxicity profiles of new therapies, but existing in vitro models do not recapitulate the human physiology accurately enough to do so, leading to low rates of success in clinical trials. To increase the accuracy and reproducibility of these in vitro models, culture conditions should mimic human biology as closely as possible. This can be done by introducing three-dimensional and dynamic culture conditions, as well as incorporating a microvascular network that supports the model by mimicking physiological distribution of oxygen, nutrients, waste products, and therapies. To serve this purpose, an injectable, three-dimensional microvasculature was developed via the co-culture of human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF) in a fibrin matrix. Under static conditions, the optimal ECM composition was determined to be pure fibrin based on reduced vessel formation and inconsistent curing with fibrin/collagen co-gels. The optimal cell type ratio was determined to be 10:1 HUVECs:HDFs due to overcrowding with fibroblasts in cultures with lower ratios. Static, three-dimensional cultures in fibrin performed best when plated in 80 µL, as demonstrated by an even spread of vascular structures across the edges, corners, and center of the chamber. An Anti-VE Cadherin primary antibody and a FITC secondary antibody were utilized in an immunostaining protocol to identify and quantify vessel morphology using fluorescent microscopy. Characterization of the network growth rate showed that at days 7 and 9, the overall number of bifurcations and overall network length significantly increased, while the overall connectedness of the network significantly decreased, as compared to day 3. In addition, cobalt ions were used to mimic hypoxia to control the rate of endothelial network formation. Direct cobalt exposure limited proliferation and network formation, but cobalt conditioned media increased the total length of the network. Upon transitioning to the dynamic platform, intermittent success in forming a vascular network was found when using hydrostatic pressure-driven flow, but was not repeatable. Switching to syringe pump-mediated flow allowed for more repeatable flow characteristics and culture conditions. Future work will investigate flow rates, ECM composition, and microfluidic device design to achieve an optimal dynamic model that promotes vascular network formation. | Poster #: 24 Campus: California State University, Chico Poster Category: Bioengineering Keywords: plant-growth promoting rhizobacteria, phytohormones, heat-stress Project Title: Isolation of plant-growth promoting rhizobacteria from mixed-conifer forest in Sierra Nevada, California Author List: Thorup, Kelli; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Blee, Kristopher; Faculty, Biological Sciences, California State University, Chico Abstract Climate change enhances the occurrence of extreme weather: wildfires, drought, rising summer temperatures—all of which dramatically decline forest growth and increase tree mortality in the mixed-conifer forests of Sierra Nevada, California. However, microbiota living in mutualistic relations with plant rhizospheres have been found to mitigate the effects of suboptimal environmental conditions. It is the goal of this research is to isolate native beneficial bacteria—plant-growth promoting rhizobacteria (PGPR)—that can alleviate heat stress in Pinus ponderosa and Pseudotsuga menziesii seedlings. Bacteria was isolated from the rhizosphere of P. ponderosa juveniles located in mixed-conifer stand, and further characterized for PGP potential based on ability to produce key growth regulatory phytohormones including auxin, cytokinin, and gibberellic acid. Out of ten soil samples taken, sixteen colonies were isolated and qualitatively confirmed to produce indole-3-acetic acid (auxin) using Salkowski’s reagent. These bacterial isolates were further analyzed to quantitatively assess auxin, cytokinin, and gibberellic acid production through a variety of spectrophotometric assays. Furthermore, bioassays will be performed to determine isolates abilities to increase tolerance in heat-stressed P. ponderosa and P. menziesii seedlings. Upon completion of this research, a PGPR could be utilized to support the growth and transplantation of conifer seedlings as summer temperatures continue to rise due to the effects of climate change. | Poster #: 25 Campus: California State University, Long Beach Poster Category: Clinical Keywords: mobile health, heart rate, cardiometabolic disease Project Title: Evaluating adherence in a mobile health exercise intervention for cardiometabolic disease Author List: Phan, Madeleine; Undergraduate, Biochemistry, California State University, Long Beach, Presenting Author
Dawson, Jacqueline; Faculty, Physical Therapy, California State University, Long Beach Abstract Attendance is used to evaluate adherence in health and exercise interventions but does not adequately report exercise intensity and duration. Training dose is total exercise exposure reflecting actual duration, frequency, and intensity of a workout session. Quantifying training dose-based adherence may be useful in mobile health (mHealth) exercise interventions to obtain a more precise measure of exercise effort. Greater exercise effort corresponds to improved health benefits. Thus, we aim to examine adherence in sedentary individuals based on (1) attendance calculated as weekly session completion and (2) training dose calculated from heart rate (HR) zones. Fifty-four sedentary (< 30 min/week of exercise) adults (32.1 ± 11.0 yr) were randomized (1:1:1) to 8 weeks of synchronous videoconference exercise (SYNC, n=22), asynchronous exercise videos (ASYNC, n=19), or attention control (CON, n=13). SYNC and ASYNC groups performed instructor-led, 35-min high-intensity functional training sessions 3x/wk and were encouraged to perform self-directed exercise. Participants wore a chest strap sensor (Myzone MZ-3) during activity to capture HR duration and frequency. Training dose was found by classifying minutes of moderate-to-vigorous physical activity (MVPA) using 2 methods. Myzone MVPA was calculated as total minutes of MVPA by the MZ-3. HR reserve (HRR) MVPA was calculated as total minutes of MVPA using the HRR equation of HRR = [(max HR – resting HR) x target zone] + resting HR). Adherence was evaluated as session completion, Myzone and HRR MVPA. Differences between groups were tested using one-way ANOVA with Bonferroni-adjusted t tests in post hoc analyses. Mean weekly session completion was significantly greater in SYNC (24.7 ± 14.2%; p=0.006) and ASYNC (21.0 ± 16.74%; p=0.043) compared to CON (6.9 ± 10.9%). Myzone MVPA mean weekly training dose was significantly greater in SYNC (785.3 ± 598.0 min) compared to CON (224.1 ± 470.8 min; p = 0.023), but no significant differences were observed between ASYNC (588.9 ± 508.9 min) and CON (p = 0.244), or ASYNC and SYNC (p = 0.771). No significant differences were observed between groups (p>0.05) for HRR MVPA mean weekly training dose. Weekly attendance seems to overestimate adherence in the asynchronous and control delivery of exercise. Training dose is a more narrow measure than attendance for observing adherence in digital exercise interventions. | Poster #: 26 Campus: California State University, Northridge Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Automated Workflow , Molecular Dynamics , Double Decoupling Method Project Title: Automated, Efficient and Rigorous Absolute Binding Free Energy Calculations with 3D-RISM Author List: Ayoub, Steven; Graduate, Chemistry and Biochemistry , California State University, Northridge, Presenting Author
Barton , Michael ; Graduate, Physics and Astronomy, California State University, Northridge
Luchko, Tyler ; Faculty, Physics and Astronomy, California State University, Northridge
Abstract There is a growing need for fast, accurate prediction absolute binding free energies estimations (ABFE) in many aspects in drug discovery, such as structure-based drug design and thermodynamics of biomolecular recognition. However, calculating ABFEs is a challenging task which requires several thermodynamic steps and the introduction of geometric restraints. Here we have implemented a new approach that combines the computational efficiency of the generalize Born (GB) and accuracy of the 3D-reference interaction site model (3D-RISM) implicit solvent models with the rigor of a double decoupling method (DDM) thermodynamic cycle. This approach uses conformational restraints and GB solvent to enhance convergence, bookended with 3D-RISM correction. Although this procedure requires fewer than 30 simulations per molecule (relatively small number for DDM methods), carrying out the simulations and post-processing can be tedious and prone to errors. Due to this we are in the process in automating and streamlining a python workflow package to perform all calculations of the thermodynamic cycle, post-processing and data analysis. Our implementations of DDM with 3D-RISM and GB shows ABFEs can be improved with 3D-RISM. We tested the method on CB7 from SAMPL4 challenge and a range of restraint forces. The incorporation of 3D-RISM reported an RSME of 3.1 kcal/mol and a slope of 1.3. A mean average error of 2.6 kcal/mol and mean squared value of -1.9 kcal/mol once 3D-RISM correction was applied. Overall, this method allows more extensive sampling and avoids the steric clashes that occur during annihilation creation of the ligand that are require with explicit solvent models. | Poster #: 27 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: phenylureas, excited states, density functional theory Project Title: Observing the photoexcited states of phenylurea herbicides Author List: Raj, Varun; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Closser, Kristi; Faculty, Chemistry and Biochemistry, California State University, Fresno Abstract Photodegradation is one of the primary abiotic mechanisms by which organic molecules can break apart. In the Central Valley, a large quantity of these herbicides are used every year and can affect the environment, particularly through the water supply. After they serve their purpose, the herbicides themselves can cause soil and water contamination, specifically worsening the quality of drinking water for humans. They also degrade through many different pathways forming products which are potentially more toxic than the parent compound, presenting health risks to humans and the environment. The use of photodegradation may allow us to see the herbicide’s product be less harmful to the environment. In this project, we calculated the excited states, initial forces, and state derivative forces of several different phenylurea herbicides using quantum chemical methods to help predict how these molecules degrade. The calculations were done using Q-Chem, a commercially available software package using density functional based methods with Gaussian basis sets (6-311G** and 6-311++G**). Various classes of functional methods (B3LYP, CAM-B3LYP, B88-LYP, and w-B96) were used to compare the structure and properties of the different phenylurea herbicide molecules. As a result of comparing molecules’ excited states, we decided on CAM-B3LYP out of the four functional methods to move forward with the state derivative forces calculations and future works. We found many similarities between these molecules but also some very interesting differences. If the photodegradation products for a given class of molecules can be accurately predicted, this can potentially lead to developing safer and effective alternatives for widely used pesticides.
| Poster #: 28 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: pyrethroids, density functional theory, insecticides Project Title: Comparative computational study of Lambda-Cyhalothrin and Cinerin II Author List: Kepler, Kelly; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Closser, Kristi; Faculty, Chemistry and Biochemistry, California State University, Fresno Abstract Lambda-Cyhalothrin is one of many pyrethroid insecticides that are synthetically derived based on the structure of pyrethrin compounds found in chrysanthemum flowers, Cinerin II is one example of such a pyrethrin. Both pyrethroids and pyrethrins function as nervous system disruptors on small insects, however pyrethroids are known to have signficantly increased stability in contrast to pyrethrins. With these characteristics in mind, we studied the ground state conformation of Lamda-Cyhalothrin and Cinerin II using Density Functional Theory. Low energy structures for Lambda-Cyhalothrin and Cinerin II have been isolated using B3LYP with increasing basis sets and accounting for environmental effects through the polarizable continuum model for solvation. Using these structures, the electron orbitals were compared to determine potential differences in reactivity. Future work will include the determination of possible photodegradation pathways based on the excited state electronic structure to illustrate Lamda-Cyaholthrin and Cinerin II’s contrasting stability. | Poster #: 29 Campus: California State University, Chico Poster Category: Diagnostics/Imaging/Analytical Keywords: Optical Coherence Tomography (OCT) Imaging, Deep Learning and Convolutional Neural Network (CNN), Image processing for early dental caries detection Project Title: Comparing Deep Learning Models using the Grad-CAM Heat-map Algorithm and Augmented OCT Images for Early-Stage Dental Caries Diagnosis. Author List: Romero, Andrew; Undergraduate, Department of Electrical and Computer Engineering, California State University, Chico, Presenting Author
Granados, Andreina; Graduate, Department of Electrical and Computer Engineering, California State University, Chico
Salehi, Hassan; Faculty, Department of Electrical and Computer Engineering, California State University, Chico
Abstract Dental caries are largely preventable yet remain the most prevalent chronic disease in children and adults, affecting over 91% of adults. Clinical evidence shows that early-stage demineralization of dental tissue can be reversed but the current conventional approach for diagnosing dental caries has low sensitivity. Deep learning models, particularly deep convolutional neural networks (CNN), can be employed along with optical coherence tomography (OCT) imaging system to more accurately identify early dental caries. In this study, 31 human tooth samples were imaged ex vivo using OCT imaging system. These cross-sectional scans are then augmented using digital image processing techniques, including rotation in increments of fifteen degrees and flipping. Five deep learning architectures were developed to propose an improved diagnostic approach. The combination of OCT imaging system supplemented with deep learning-based classification for improved diagnostic accuracy of early-stage dental caries can be effective but lacks interpretability. Applying the Grad-CAM heat-map generation algorithm to the last convolutional layer of a CNN model allows users to visualize important features in the OCT image, demonstrating why a certain prediction is made. The deep learning models include a basic CNN, VGG16, and VGG19, with the VGG models including transfer learning and fine tuning. The basic CNN and VGG16 models currently support images of size 300 x 300, where each class contains 40,000 images. The heat-maps are successfully generated for these models, highlighting important features of the OCT image. Validating the decisions of various machine learning algorithms is critical for a safe implementation of artificial intelligence (AI) in the biomedical industry.
Acknowledgements:
This research was supported by the National Science Foundation (NSF MRI-1920345). The authors would like to thank Dr. Mina Mahdian at Stony Brook University, School of Dental Medicine.
| Poster #: 30 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: Prussian blue nanoparticles, plasmonic electrochemical microscopy, hydrogen peroxide Project Title: Prussian Blue Nanosensors for Detection of Hydrogen Peroxide Author List: Wang, Kinsley; Undergraduate, Chemistry and Biochemistry , California State University, Los Angeles, Presenting Author
Abstract Hydrogen peroxide is a chemical that has harmful environmental and health impacts at even low concentrations. As the body’s strongest natural oxidant, the development of a sensing system for hydrogen peroxide at low concentration levels would be advantageous. Prussian blue, an iron-cyanide-based pigment, has steadily became a widely used electrochemical sensor. Previously, the reduction of hydrogen peroxide at Prussian blue nanoparticles (PBNPs) has been studied using traditional ensemble methods, which only provide averaged information. Investigating PBNPs at a single entity level is paramount for correlating their ability to reduce hydrogen peroxide to their particle structures and will shed light on the major factors governing the reduction activity of these nanoparticles. Here we report on the use of plasmonic electrochemical microscopy (PEM) to study the reduction of hydrogen peroxide at the individual Prussian blue nanoparticle level. First, two types of PBNPs were synthesized; type I was synthesized with the double precursors method and type II was synthesized with polyvinylpyrrolidone (PVP)-assisted single precursor method. Second, both PBNPs types were compared on their electrochemical reduction to form Prussian white, and the effect from the different particle structures was investigated. Type I PBNPs provided better PEM sensitivity and were used to study the catalytic reduction of hydrogen peroxide. Third, progressively decreasing plasmonic signals with respect to increasing hydrogen peroxide concentrations were observed, demonstrating the capability of PBNPs to sense hydrogen peroxide at a single nanoparticle level by utilizing this optical imaging technique. This work has been funded by National Science Foundation (NSF) Major Research Instrumentation (MRI) grant, NSF CAREER award, and NSF Partnership for Research and Education in Materials (PREM). | Poster #: 31 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: Alpha synuclein, Parkinson’s disease, live cell imaging Project Title: Real-time imaging of neuroblastoma cell membranes exposed to alpha-synuclein pre-formed fibrils Author List: Feng, Christina ; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wang, Marisol; Undergraduate, Chemistry and Biochemistry, California State University, Los Angeles, Presenting Author
Wang, Yixian; Faculty, Chemistry and Biochemistry, California State University, Los Angeles
Abstract Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder in the US, behind Alzheimer’s. In PD, aggregates of the natively unstructured protein alpha-synuclein (alpha-Syn) are known to induce the death of dopamine-producing neurons in the substantia nigra, ultimately hindering motor function. Injection of alpha-syn pre-formed fibrils (PFFs) into mice has been demonstrated to induce the alpha-Syn aggregate pathology in neuronal populations proximal to the injection site, and in more distant regions with neuronal connections to the site of injection, demonstrating the important role of PFFs in the progression of PD, although the nature and mechanism of fibril disruption has yet to be characterized. We have previously demonstrated using a new analytical imaging technique, scanning ion conductance microscopy (SICM) to real-time monitor the interaction between alpha-syn oligomers and live cell membranes. This work used SICM to image in real-time the topographies of SH-SY5Y neuroblastoma cell membranes after the addition of alpha-Syn PFFs. No membrane disruption was observed in fixed cells within 72 hours of treatment, although small protrusions were revealed. This indicates that fibrils interact with membranes differently than oligomers. In the future, we plan to use fluorescence imaging to confirm the presence of alpha-syn on the cell membrane, as well as test different concentrations and sources of alpha-syn fibrils. This project is funded by the National Institutes of Health (NIH) Research Enhancement Award (R15NS120157) and CSUPERB President’s Commission Scholar Award. | Poster #: 32 Campus: California State University, East Bay Poster Category: Disease (Pathogens) Keywords: Toxoplasma gondii, SR proteins, Alternative splicing Project Title: How do Serine-Arginine-Rich Proteins contribute to the regulation of alternative splicing during Toxoplasma gondii developmental differentiation? Author List: Martinez, Cynthia; Graduate, Biological Sciences, California State University, East Bay, Presenting Author, Eden Award Finalist
Guiton, Pascale ; Faculty, Biological Sciences, California State University, East Bay Abstract Toxoplasmosis is caused by the obligate intracellular parasite Toxoplasma gondii and is thought to be prevalent in one-third of the world’s human population. Infection with T. gondii is usually asymptomatic in healthy individuals but can result in a lifelong chronic disease for which there is no cure. Thus, it is imperative to identify novel therapeutic targets in the fight against this ubiquitous parasite.
T. gondii alternates between different forms, including the fast replicating tachyzoites responsible for the acute phase of the infection and the encysted bradyzoites that persist in the skeletal muscles and the brain. Although several regulatory mechanisms of gene expression, such as alternative splicing, have been implicated in developmental differentiation in T. gondii and other Apicomplexans, very little is known about the regulation of alternative splicing in T. gondii. Serine-Arginine-Rich (SR) proteins are known regulators of alternative splicing in eukaryotic organisms. There are at least 4 developmentally regulated and poorly characterized SR proteins in T. gondii, namely TgSR1, TgSR2, TgSR3, and TgSR4. Previous studies revealed that, unlike SR1, SR2, and SR4, SR3 is essential for in vitro fitness of Type I tachyzoites and contributes to tachyzoite-to-bradyzoite conversion when overexpressed. Given their expression patterns and crucial function in development, I hypothesize that disruption of these SR proteins will compromise gene products needed for T. gondii differentiation. I successfully generated Type I tachyzoites deficient in SR2 and SR4; however, I was unable to do so for any of the four SR genes in the Type II strain despite several attempts. Together, these results suggest that SR proteins act in a strain-dependent manner, which is notable given the differences in virulence among T. gondii strains, with type I parasites being more virulent than their Type II or Type III counterparts in mice. I will perform RNA sequencing to identify spliced variants resulting from the activity of SR2 and SR4 in parental and mutant parasites under normal and nutrient-poor conditions. The latter is known to induce tachyzoite to bradyzoite differentiation. My findings will uncover new genes critical for T. gondii differentiation as well as potential targets of novel therapeutics to control the chronic stage of toxoplasmosis. This work will also broaden our understanding of conserved regulatory mechanisms of alternative splicing in Apicomplexa.
| Poster #: 33 Campus: California State University, East Bay Poster Category: Disease (Pathogens) Keywords: Toxoplasma , protein trafficking, gene expression Project Title: How are proteins targeted to specialized secretory organelles in Toxoplasma gondii Author List: Babacarkhial, Edres; Graduate, Biological Sciences, California State University, East Bay, Presenting Author
Kuchibhotla, Sahana; Mission San Jose High School
Guiton, Pascale ; Faculty, Biological Sciences, California State University, East Bay, Presenting Author
Abstract Toxoplasma gondii is an obligate intracellular coccidian in the phylum Apicomplexa, which encompasses pathogens of medical and veterinary importance. One third of the global human population is believed to be infected with Toxoplasma. As a member of the Apicomplexa, Toxoplasma possesses at its apex specialized secretory organelles, rhoptries, micronemes, and dense granules. Their contents are released during infection to promote invasion, intracellular replication, and modulation of host immune processes. The subcellular localization of these secreted proteins determines when and how they access the host cell. Specifically, rhoptry proteins are only expressed during cell division, loaded into the rhoptries, and secreted directly into the host cytosol during invasion. By contrast, dense granule proteins are constitutively expressed, secreted in the parasitophorous vacuole (PV), and some of them eventually cross the PV membrane into the host cytosol. Yet, the targeting and trafficking of these proteins to the respective secretory organelle is not well understood. We hypothesized that the time of expression of these proteins may contribute to their targeting. Here, we showed that expression of an epitope-tagged version of a known dense granule protein (GRA7-HA) under a rhoptry promoter directs this protein to the rhoptry, as detected by immunofluorescence microscopy. Similarly, the expression of a known rhoptry protein (ROP16-HA) under a dense granule promoter results in its secretion into the PV. Together, our findings indicate that promoter activities (i.e. the time of expression) are crucial for their targeting and loading into the respective organelles. Further studies will determine the molecular mechanisms of this targeting, which can be a result of direct interactions with other rhoptry proteins or with elusive regulatory proteins. Finally, our study also serves as a cautionary tale for the expression of proteins under non-endogenous promoters, as is commonly done in the field. The findings from such studies may provide a misleading foundation for future hypotheses. | Poster #: 34 Campus: California State University, Los Angeles Poster Category: Disease (Pathogens) Keywords: Tuberculosis, epithelial cells, gDNA Project Title: Development of a qPCR-based protocol to quantitate M. smegmatis proliferation Author List: Wesley-Cardwell, India; Undergraduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Martinez, Erik; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author
Dzul, Janette; Undergraduate, Biological Sciences, California State University, Los Angeles
Porter, Edith; Faculty, Biological Sciences, California State University, Los Angeles Abstract Mycobacterium tuberculosis (Mtb), an airborne pathogen, infects people on a global scale. Increasing rates of multidrug-resistant Mtb and limited efficacy of the current tuberculosis vaccine warrant novel approaches to combat tuberculosis. Epithelial cells lining the airways are the first to encounter Mtb. Epithelial cells increase the production of antimicrobials after stimulation with certain cytokines. T helper cells of the subtype TH1 are the specialized immune cells targeted in the current tuberculosis vaccine. Our lab hypothesizes that targeting the subtype TH17 for tuberculosis vaccines may offer enhanced protection because TH17 cells are known to activate epithelial cells. As a first step, we aim to test the antimycobacterial activity of epithelial cells stimulated with TH17-derived cytokines using Mycolicibacterium smegmatis (Ms), an avirulent model organism for Mtb. Mycobacteria have lipid-rich cell walls that promote aggregation and protect against lysis, which makes it unreliable to measure bacterial proliferation by colony forming unit assays. Here, our objective is to develop a protocol for lysing Ms cells and extracting gDNA for a PCR-based quantification of bacteria. Various extraction kits from Qiagen and Zymo, lysis buffers containing Triton-X-100 or Tri-reagent, temperatures, and mechanical lysis approaches were compared. We found that incubation with Tri-reagent combined with intermittent high-speed homogenization with 0.1- and 0.5-mm diameter beads liberated bacterial gDNA which could be partially purified with Direct-zol™ DNA/RNA Miniprep. gDNA quantity and purity were determined by Nanodrop and ethidium bromide-stained agarose gel electrophoresis. This research will continue with the use of qPCR on gDNA to assess Ms proliferation. | Poster #: 35 Campus: California State University, San Bernardino Poster Category: Disease (Pathogens) Keywords: viral pathogens, cell lines, immortalization Project Title: Generation of Feline Macrophage Cell Line to Study Feline Viral Pathogens Author List: Fraser, Cameron; Graduate, Biology, California State University, San Bernardino, Presenting Author
Espinoza , Adam; Graduate, Biology , California State University, San Bernardino, Presenting Author
Newcomb, Laura; Faculty, Biology, California State University, San Bernardino
Abstract Immortalized and well characterized cell lines serve as important models for basic biomedical research. The advent of modern in vitro cell culture techniques and cryobanking represents a significant opportunity to save, characterize, and catalogue a wide variety of cells, including immortalization to continually propagate a cell line for controlled laboratory studies. However, because much focus is on human biomedical research, there is a lack of animal cell lines developed for studying veterinary medicine. This deficiency contributes to a wide gap in knowledge whose understanding may be critical to conservation efforts. A. jubatus, or the African cheetah, is a striking example of a need for more biomedical knowledge to save the species due to their uniquely high susceptibility to viral pathogens such as feline immunodeficiency virus (FIV) and feline coronavirus (fCoV). Here we demonstrate the feasibility to produce immortalized macrophage cell lines from feline whole blood samples. We intend to optimize this method for subsequent use to generate cheetah cell lines for cheetah conservation research, with particular focus on studying viral life cycles of FIV and fCoV. Here we report the isolation of domestic feline monocytes as adherent cells from whole blood samples. We successfully transduced these cells with SV40 Large-T antigen immortalizing gene and confirmed transduction by antibiotic selection. Immortalization was achieved as the cells have been passaged over 20 times and are cryopreserved for storage. RNA was isolated from these cells and RT-qPCR is being carried out to confirm expression of SV40 T-antigen and assess expression of macrophage specific genes CD14, fCoV receptor CD13, and FIV receptor CD4. Next, we will establish clonal populations and confirm gene expression as with our starting cell stock. Lastly, we will infect these cell lines with pathogenic feline viruses, including FIV and fCoV. Future work will include collaboration with the Living Desert Zoo to obtain whole blood samples from their resident cheetahs and generate a cheetah macrophage cell line susceptible to infection by important cheetah viral pathogens. We hope to establish cell lines that may be useful in addressing the gaps in knowledge critical to understanding the root cause of cheetah viral susceptibility. | Poster #: 36 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Metabolism, Cancer Biology, Bioinformatics Project Title: Characterizing AK4 Gene Co-Expression Networks Within Lung Adenocarcinoma Tumor Stage Progression Author List: Chacon, Jonathan; Graduate, Biological Sciences, California State University, Los Angeles, Presenting Author, Eden Award Finalist
Lanning, Nathan; Faculty, Biological Sciences, California State University, Los Angeles Abstract Within a developing tumor microenvironment, the ability to detect and respond to low oxygen availability, or hypoxia, appears to be a common feature amongst most cancers. This hypoxic response has many molecular drivers, but none widely studied as HIF1, which initiates expression of genes that promote cancer growth and metastasis. Recent evidence has revealed HIF1 stability within lung adenocarcinoma (LUAD) to depend on the levels of the mitochondrial adenylate kinase AK4–which itself negatively correlates with LUAD patient outcomes. Using LUAD patient transcriptome data within TCGA, we sought to characterize additional AK4 co-expression networks across LUAD tumor stages 1-4 in order to identify conserved biological themes across LUAD tumor progression. Over-representation analyses revealed these networks to comprise of genes involved in pyrimidine metabolism across all LUAD tumor stages. Furthermore, differential gene expression analyses revealed upregulation of transcripts across numerous pathways associated with nucleotide metabolism as a whole. To further discriminate potential drivers of LUAD tumor progression within AK4 co-expression networks, a supervised classification approach using partial least squares discriminant analysis was used at LUAD stage-stage interfaces, identifying candidate genes that may promote LUAD tumor growth or regression. Collectively, these results characterize regulatory gene networks that may contribute to underlying metabolic perturbations within LUAD, and potentially reveal mechanistic insight into the complementary role of AK4 in LUAD tumor development. | Poster #: 37 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: microRNA, Promoter, Diabetes Project Title: Investigating the rat miR-375 and human miR-375 promoter Author List: Hernandez, Leslie; Undergraduate, Biology, California State University, Chico, Presenting Author
Keller, David; Faculty, Biology, California State University, Chico Abstract Pancreatic specific microRNA-375 (miR-375) is a short non-coding RNA that is important for regulating the expression of several hormones. Mir-375 is over-expressed in people who have been diagnosed with Type 2 diabetes (T2D) and has also been tied to insulin secretion and beta-cell proliferation. The overexpression of mir-375 in people with T2D leads to my question, how is mir-375 regulated. Previous data from the Keller lab shows that mir375 is regulated differently in rats than in humans. My hypothesis is that there are differences in the promoters between human and rat mir-375 genes. I conducted a luciferase transcription assay to test the activity of the rat promoter in Human Embryonic Kidney cells (HEK-293T). I transfected the cells with a control plasmid and the mir-375 promoter plasmid. The mir375 promoter stimulated the plasmids and the average results between two luciferase reporter assays showed a 9.48 fold increase comparing the mir-375 promoter to the control. I began the process of cloning the human mir-375 promoter via PCR to amplify specific segments of DNA using primers to determine which promoter has more activity. Using the negative control vector, I was able to provide a basis for quantitive analysis to regulate gene expression. With the usage of the negative control vector, I carried out a whole bug PCR colony screening and was able to verify that the promoter sequence was present. The future direction of this project would continue the investigation of the mir-375 promoter for future discovery if ICER would repress the human promoter to bring levels of mir-375 to a regular position. If there is identification on how the hmiR-375 and rmiR-475 promoters are regulated, this revelation will contribute to the advancement of personalized diabetic therapies.
| Poster #: 38 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Kaposi’s Sarcoma Herpesvirus, cell biology, innate immunity Project Title: Elucidating Viral G-Protein Coupled Receptors’ Role in Innate Immunity in Bystander Host Cells Author List: Verzosa, Amanda; Graduate, San Francisco State University, Presenting Author
Sanchez, Dr. Erica; Faculty, Biology, San Francisco State University Abstract Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is a human herpesvirus and the infectious cause of Kaposi’s Sarcoma; an angioproliferative, endothelial cell neoplasm that predominantly affects patients with HIV/AIDS. KSHV encodes for a viral G protein-coupled receptor (vGPCR), a receptor with homology to human chemokine receptors and a prominent gene expressed during KSHV lytic infection. KSHV infected cells secrete extracellular vesicles (EVs), that are known to activate the complement system, an innate immune response. vGPCR and its role in inducing specific innate immune responses has not been fully elucidated. We determine if vGPCR plays a role in EV-induced complement activity. We hypothesize that vGPCR alters the composition of host cell EVs and induces the complement system in bystander cells. We have successfully generated cells stably expressing vGPCR using an expression vector, which has been shown through immunofluorescence assays and flow cytometry analyses. Currently, cells overexpressing vGPCR are being used to test for complement activity in bystander cells through an immunofluorescence and flow cytometry approach. C5b-9, a complement protein, is expected to deposit onto the surface of bystander cells when treated with conditioned media from cells overexpressing vGPCR. Additionally, bystander cells will be treated with EVs from cells overexpressing vGPCR to test for complement activity. Results will determine whether vGPCR plays a role in EV-induced complement activity in bystander cells. This research will contribute to the understanding of the molecular and cellular mechanisms of vGPCR, which can lead to preventative therapeutic treatments for KSHV oncogenesis. | Poster #: 39 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, stem cells, genetics Project Title: Analyzing the regulatory cross-talk between two genetic master regulators of intestinal stem cells in Drosophila melanogaster Author List: Lemus, Aaron; Graduate, Biology, California State University, Northridge, Presenting Author
Cano, Ithan; Undergraduate, Biology, California State University, Northridge, Presenting Author
Hossine, Marziiah; Undergraduate, Biology, California State University, Northridge
Faramarzi, Sandra; Undergraduate, Biology, California State University, Northridge
Loza-Coll, Mariano; Faculty, Biology, California State University, Northridge
Abstract Multicellular organisms can maintain organ homeostasis in great part to the activity of adult stem cells (ASCs), whose function is to replace cells that were lost to damage, disease or normal tissue turnover. Like embryonic stem cells, ASCs can divide asymmetrically, giving rise to a new copy of themselves (i.e. self-renewal) and a sister cell that commits to differentiation into a specific cell type.
A healthy ASC needs to constantly make several decisions throughout its lifetime: 1) to divide or not; 2) if it divides, to self-renew or to differentiate; 3) if it differentiates, into which cell type to differentiate. Over the years, in vivo research on several model organisms has revealed the existence of genetic master regulators that control several aspects in the biology of ASCs. When these genetic master regulators are manipulated, ASCs will often show simultaneous alterations in the division and/or differentiation potential of ASCs and their progeny. While experimental work has mainly focused on manipulating master regulators and elucidating their downstream effects on an individual basis, a lot less is known about how master regulators may regulate one another.
To address this important outstanding question in the field, we use intestinal stem cells (ISCs) in the posterior midgut of the fruit fly (Drosophila melanogaster). We focused on two known master regulator genes of these cells: the Snail family transcription factor Escargot (Esg) and the signal transducer protein STAT. We first genetically manipulated the expression of Esg in flies carrying a STAT-GFP activity reporter, to determine how changes in Esg function may affect the function of STAT. Then, we carried out the converse experiment, manipulating the activation of the STAT pathway in flies carrying an esg-GFP reporter, to determine if changes to STAT activity could affect the expression of Esg. Through a high-throughput and automated analysis of immunofluorescence microscopy images, we quantified changes in levels of expression and activity of one master regulator in response to the experimental manipulation of the other. Here we discuss our preliminary results related to a functional cross-talk between these two important regulators of ISCs in flies.
(This study was entirely supported by a NIH Score-2 grant).
| Poster #: 40 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: antibiotic resistance, antisense, sRNA Project Title: Usage of Antisense Molecules to Curb MicF Related Antibiotic Resistance Author List: Susas, Jeremiah Lyn; Undergraduate, Biology, California State University, Northridge, Presenting Author
Zambrano, Raphael Angelo; Undergraduate, Biology, California State University, Northridge, Presenting Author
Takahashi, Melissa; Faculty, Biology, California State University, Northridge
Abstract Antibiotic resistance is an ongoing, global issue affecting multiple industries. Due to overexposure of antibiotics, many bacteria develop mechanisms to prevent antibiotics from destroying or entering them. In Escherichia coli and related bacteria, there is a large pore called outer membrane protein F (OmpF) that antibiotics can enter through. However, in a stressful environment, one of these mechanisms blocks the production of OmpF, preventing large amounts of antibiotics from entering the bacteria. This mechanism involves a small RNA called MicF that binds to ompF mRNA and inhibits the translation of OmpF. Our project targets MicF by creating antiMicF, a synthetic, antisense RNA molecule that complements and binds to MicF’s nucleotide bases allowing OmpF’s translation and production. Previously, we have shown that an antisense RNA can inhibit MicF binding and when expressed in Escherichia coli can improve antibiotic efficacy. However, instability with RNA is a concern when delivering these antiMicF candidates into the cells. Antisense peptide nucleic acids (PNA) provides a promising solution, but PNAs have size constraints that requires the molecule to be 10-12 nucleotides (nt) in length. The previous antiMicF candidate was 33 nt long, thus the project currently focuses on designing shorter antiMicF candidates that can efficiently sequester MicF. To test antiMicF candidates, they are placed in cell-free assays, along with MicF and a reporter construct, ompF attached to a gene encoding green fluorescent protein (GFP). Cell-free assays are reactions that can perform transcription and translation without the use of cells. A high rate of GFP fluorescence indicates that antiMicF to MicF binding is successful in preventing MicF from binding to ompF. Despite shorter antiMicF designs showing decreased fluorescent rates compared to previous candidates, the data reveals that sequestering the region of MicF that binds between the ompF ribosome binding site and start codon is crucial for interfering with OmpF downregulation. Moving forward, we continue to improve the efficiency of these shorter antiMicFs by exploring other regions in MicF, lengths of antiMicF, and strategies for delivery in hopes to successfully inhibit MicF-regulated antibiotic resistance. | Poster #: 41 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: sirtuin, testes, fertility Project Title: The effects of sirtuin 4 on the structure and function of testes in mice Author List: Hamzehpour S., Arshia; Undergraduate, Biological Sciences, San José State University, Presenting Author
Nguyen, Albert; Undergraduate, Biological Sciences, San José State University, Presenting Author
Coakley, Aeowynn; Undergraduate, Biological Sciences, San José State University
Nguyen, Thi-Tina; Undergraduate, Biological Sciences, San José State University
Huynh, Frank; Faculty, Biological Sciences, San José State University
Abstract Sirtuin 4 (SIRT4) is an enzyme that regulates protein function by removing post-translational acyl modifications. However, the full biological role of SIRT4 is still emerging. To uncover novel biological roles of SIRT4, we previously used an unbiased, bioinformatics approach to determine which tissues SIRT4 is most highly expressed in. By analyzing large-scale datasets across a plethora of databases, we found that the testes were consistently found to be the highest expressors of SIRT4. Interestingly, by performing a gene correlation analysis and functional cluster analysis, we found that SIRT4 gene expression correlated with many genes involved in spermatogenesis. Therefore, we hypothesized that SIRT4 may have a critical effect on testicular structure and function. To address this hypothesis, we performed a gross examination of the testes and evaluated fertility in wild-type mice compared to mice with a loss of SIRT4 (SIRT4KO) mice. Our data showed a trend for the testes from SIRT4 heterozygous and SIRT4KO mice to be smaller in length, width, and weight than wild-type mice. To see if this reduced testicular size in SIRT4KO males led to impaired fertility, we bred SIRT4KO males with wild-type female mice. While the resulting litters appeared to have normal gestation periods and litter sizes, 2 out of 12 pups born displayed signs of birth defects, including short, thin tails and greatly decreased size and weight. These mice appeared malnourished and had considerably reduced physical activity compared to their littermates. These results suggest that loss of SIRT4 in males may lead to increased birth defects in their offspring, perhaps revealing a role for SIRT4 in sperm quality. Taken together, our data suggest that SIRT4 may play a vital role in the development of testicular structure and function, which justifies further studies into the role of SIRT4 in the testes.
| Poster #: 42 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Photosynthesis, Yellow-light, Cryptochrome Project Title: Nuclear- and Chloroplast-encoded Gene Responses to Yellow Light in Chlamydomonas reinhardtii Author List: Gonzalez, Adriana; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Horne, Ronald; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Arce, Laura; Staff, Biological Science, California State University, Fullerton
Cohen, Amybeth ; Faculty, Biological Science, California State University, Fullerton Abstract The photosynthetic D1 protein in the unicellular green alga Chlamydomonas reinhardtii is translationally regulated by a set of nuclear-encoded RNA binding (RB) proteins (RB38, RB47, and RB60). The RB complex interacts with a stem-loop structure in the 5’-untranslated region of the chloroplast-encoded psbA mRNA to initiate translation of D1, the primary electron acceptor in photosystem II. White, red, and blue light are known to regulate the accumulation of the rb38, rb60, and psbO (encodes the Oxygen-Evolving Enhancer protein 1) mRNAs (Alizadeh and Cohen, 2010), but the effects of yellow light are unknown. The animal-like cryptochrome (aCRY), absorbs red, blue, and yellow light and regulates the expression of biosynthetic and metabolic genes in C. reinhardtii (Beel et al, 2012). Thus, the expression of the rb and psb genes in response to yellow light was investigated. C. reinhardtii cells were cultured to a density of 5x106 cells/mL before exposure to yellow light (590 nm) for 0 to 120 minutes. Total RNA was isolated, and reverse transcribed into cDNA. Maximal induction of rb60 and psbO was visible after 90 minutes of yellow light exposure, as revealed by RT-PCR analyses. This result suggests an involvement of aCRY in the regulation of these genes. rb38 is not influenced by yellow light, despite being induced by other light qualities. Comparable with exposure to white, red, and blue light, psbA and rb47 are constitutively expressed in the presence of yellow light. Given these data, RT-PCR and western blot analyses will be used to further assess the role of aCRY in regulating the rb and psb genes using aCRY knockout and rescue strains. Understanding the role of aCRY in the regulation of photosynthetic genes is critical to dissecting several cellular pathways, as the aCRY photoreceptor is highly conserved across animal and plant species.
Acknowledgments: This project is supported by the Department of Biological Science at CSU Fullerton, a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [T34GM008612-26], and a California Institute for Regenerative Medicine grant awarded to CSUF.
| Poster #: 43 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: inflammation, macrophage, complement Project Title: Innate immune protein C1q modulation of gene expression in M0, M1 and M2 macrophages Author List: Choi, Kevin; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Garcia-Paez, Mario; Undergraduate, Biological Sciences, California State University, Long Beach
Haque, Mehernaz ; Graduate, Biological Sciences, California State University, Long Beach
Fraser, Deborah; Faculty, Biological Sciences, California State University, Long Beach Abstract Innate immune protein C1q is known for its ability to activate the classical pathway of complement, a proinflammatory process necessary for protection against foreign pathogens. However, C1q can play a dual role, and previous studies have shown that exogenously added C1q modulates gene expression in inflammatory (M1) and resting (M0) macrophages and polarizes them towards a resolving M2-like phenotype. Macrophages produce C1q constitutively. Therefore, this study tests the hypothesis that endogenous C1q also modulates macrophage gene expression. RNA sequencing was performed on mRNA from murine bone marrow derived macrophages (BMDM) from wild-type or C1q deficient mice that were cultured under M0, M1, or M2 conditions. Our analysis showed that 1884 genes were modulated by C1q in M0, 65 in M1 and 307 in M2 BMDM. There were also 40 genes that were modulated by C1q in BMDM regardless of polarization state. In M0 resting macrophages, GO process analysis indicated that C1q modulated numerous cellular processes including those involved in myeloid cell differentiation. KEGG pathway analysis also indicated that C1q modulates genes in multiple disease pathways including chronic myeloid leukemia and mTOR signaling pathways. In the overlapping C1q-modulated genes in M0 and M2, 8 out of the first top 10 modulated GO processes were metabolic processes. Additionally, oxidative phosphorylation and neurogenesis were shown to be modulated in the same overlapping gene set. Gene ontology analysis of overlapping C1q-modulated genes between M0 and M1 revealed C1q modulates transferase activity, indicating a possible role in signal transduction. C1q modulated transcription of 29 phosphoproteins in all three macrophage phenotypes tested. These data suggest that not only is C1q an essential component in the immune system, but also is instrumental in a wide range of other internal processes and signal transduction. These data can be used to further elucidate roles of complement outside of its traditional place in the innate immune system, and it is exciting to see the non-complement roles of locally synthesized C1q.
This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T34 GM 008074 and SC3GM111146. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
| Poster #: 44 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: chromosome breakage, Schizosaccharomyces pombe, flow cytometry Project Title: When do fission yeast break their chromosomes during the cell cycle? Author List: Luo, Jiayu; Graduate, Department of Biology, San Francisco State University, Presenting Author
Pasion, Sally G; Faculty, Department of Biology, San Francisco State University Abstract The stability of chromosome integrity is essential for cell survival, tissue homeostasis and organ development. Disruption in chromosome stability often leads to cell death or increased susceptibility of cancers and tumors. Chromosomes of fission yeast (Schizosaccharomyces pombe) share many common features with human chromosomes, such as large replication origins, large centromeres, and conserved telomere proteins. Therefore, fission yeast is a good model organism to study chromosome features in higher eukaryotes. Fission yeast cdc24+ plays essential roles during DNA replication. Although the exact function of cdc24+ is not clear yet, Cdc24p interacts with many highly conserved replication proteins, such as DNA ligase and the Dna2 helicase. Previous studies suggest that, after 4 hours at the restrictive temperature (34℃), asynchronous cdc24 truncation mutants (cdc24-M38 and cdc24-G1) break chromosomes, while the cdc24 point mutant (cdc24-M81) is able to maintain chromosome integrity similar to the wild type. This chromosome breakage has since been reported in mutants of the conserved replication proteins, Dna2 and DNA ligase. Intriguingly, our studies reported here demonstrate that a longer 12-hour incubation at the restrictive temperature results in the cdc24 point mutant phenocopying the truncation mutants; the point mutant shows both loss of cell viability and chromosome breakage. It is not known whether this breakage occurs specifically during S phase or simply at any point during the cell division cycle, or why there is a delay in the viability loss and breakage phenotypes compared to the truncation mutants. To address the cell cycle kinetics of chromosome breakage, we used the cold-sensitive nda3 mutant. nda3+ encodes beta tubulin, and the nda3 mutant blocks spindle formation and chromosome movement during mitosis. We used the cdc24nda3 double mutant to synchronize cells. Our results suggest that, after 8 hours at 19℃, about 37 – 43% of the cells are in mitosis. After releasing the cells to 25℃ for 100 minutes, 32-43% of the cells are in S phase. We are using flow cytometry, microscopy, and pulsed field gel electrophoresis to determine whether breakage occurs during S phase or during mitosis in these synchronized cdc24nda3 mutants. These studies will contribute to understanding how cells survive replication stress and genome instability. | Poster #: 45 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Endometrial cancer, ADAM17, fibroblast growth factor receptor Project Title: Targeting ADAM17 in FGFR2-mutant endometrial cancers Author List: Kala, Maria; Undergraduate, Chemistry & Biochemistry, California State University, Los Angeles, Presenting Author
Dixit, Garima ; University of Iowa, Iowa City
Maretzky, Thorsten; University of Iowa, Iowa City
Abstract Endometrial cancer (EC) is the most common form of uterine cancer. Recent studies have shown that approximately 15% of endometrial cancer patients harbor somatic mutations in the receptor tyrosine kinase (RTK) fibroblast growth factor receptor 2 (FGFR2). However, little is known about those mutations associated with advanced and recurrent diseases in ECs. Previous studies have shown that A disintegrin and metalloprotease (ADAM17) is the key regulator of epidermal growth factor receptor (EGFR) signaling and mediates the crosstalk between EGFR/ERK1/2 and FGF7/FGFR2 in keratinocytes. Our preliminary data suggested that FGFR2-dependent activation of epidermal growth factor receptor (EGFR) signaling in endometrial cells require stimulation of ADAM17 and the release of heparin-binding epidermal growth factor (HB-EGF). Additionally, we found that FGFR2 mutations were not constitutively active and required FGF7 stimulation; their mediated-tumor formation in endometrial cancers include members of EGF-receptor family, NOTCH pathway and AKT/PI3K pathway. The objective of this study was to investigate the role of ADAM17 in progression of endometrial tumors in mice that are carrying mutations on FGFR2. Cell-line derived xenograft (CDX) mouse model was used to grow endometrial tumors in NOD scid gamma (NSG) mice over a period of 6-8 weeks. The mice were injected with DPC333- a broad spectrum metalloprotease inhibitor to evaluate the role of ADAM17 in tumor formation. The data suggested that metalloprotease inhibition potentially leads to cell-apoptosis and reduced ERK phosphorylation thereby reducing tumor growth of FGFR2 endometrial cancer cells in the CDX mouse model. These findings provide new insights into the role of ADAM17 and FGFR2 in progression of EC and may lead to clinical approaches in the study of FGFR inhibitors and significance of somatic mutations in EC. | Poster #: 46 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: nitrogen fixation, diazotroph, cnidarian Project Title: Nitrogen fixation in the sea anemone Aiptasia, a model system for coral symbiosis Author List: Lopez, Jaime; Graduate, Biological Sciences, California State University, Chico, Presenting Author
Phillips, Ryan; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Cabrera, Benjie; Undergraduate, Biological Sciences, California State University, Chico
Tran, Cawa; Faculty, Biological Sciences, California State University, Chico Abstract Collectively known as a holobiont, the coral host and its diverse community of microbes (photosynthetic algae, prokaryotes, fungi, and viruses) can flourish under oligotrophic conditions that are limited in essential nutrients like nitrogen. Bacteria known as diazotrophs can fix atmospheric nitrogen for organismal use and are associated with various coral species, suggesting they are key members of the coral microbiome. Environmental changes stimulate diazotrophic activity and elevate nitrogen levels, resulting in different impacts on different coral species, in which some experience improved photosynthetic and calcification rates, while others lose their algal symbionts (i.e., bleach). Hence, our comprehension of diazotrophs and their activity in corals requires further investigation. To this end, we identified diazotrophs and investigated their nitrogen-fixation activity in the sea anemone Exaiptasia pallida (or ‘Aiptasia’), a laboratory model for coral studies. The biomarker gene for diazotroph identification, nifH, was targeted in symbiotic (with algae) and aposymbiotic (without algae) anemones using PCR. Next, PCR products containing nifH were cloned and Sanger-sequenced to identify diazotroph species. Lastly, nitrogen-fixation rates were indirectly assessed in symbiotic and aposymbiotic anemones by an acetylene reduction assay. We confirmed 12 different diazotroph species in symbiotic and 8 different species in aposymbiotic Aiptasia. There was little overlap of diazotroph species between the two groups: Whereas Sulferivermis fontis and Rhodococcus spp. were found in both groups, other species were unique to each group. For example, Nitrincola sp. appeared exclusively in symbiotic Aiptasia, making up 42% of its diazotrophic community. Interestingly, no detectable nitrogen-fixation activity was found in either group via acetylene reduction assay. These results suggest that anemones may already be acquiring sufficient nitrogen without the assistance of diazotrophs under normal conditions, but activity may change if the environment changes. Future work will focus on determining diazotroph-community changes and nitrogen-fixation rates in anemones subjected to heat stress, which may stimulate diazotrophic activity. Potential differences between symbiotic and aposymbiotic Aiptasia may provide insight into nitrogen-cycling patterns between healthy and bleached corals respectively. The authors would like to acknowledge CSUPERB for support of this research. | Poster #: 47 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Kinase, Saccharomyces cerevisiae, Molecular Biology Project Title: Characterization of Saccharomyces cerevisiae pah1∆ strain and studies on Pah1 protein phosphorylation. Author List: Padilla, David; Undergraduate, Biological Sciences, California State University, Long Beach, Presenting Author
Gharakhanian, Editte; Faculty, Biological Sciences, California State University, Long Beach Abstract Vacuoles in Saccharomyces cerevisiae serve as a model for membrane fusion and fission events. We have established Env7 as a vacuolar membrane kinase. Env7 is a conserved kinase that negatively regulates vacuolar fusion. However, Env7 substrate(s) remain unknown. Therefore, we hypothesized that Pah1 might be a direct or indirect substrate of Env7. Pah1is a phosphatase implicated in diacylglycerol (DAG) generation, which is essential in membrane fusion events; previous studies established a negative genetic interaction between ENV7 and PAH1. Also, a small fraction of Pah1 localizes to the vacuole membrane. Here, we characterized pah1∆ with respect to fitness and vacuolar morphology. To date, we have confirmed that pah1∆ has severe fitness defects; vacuole morphology studies with confocal microscopy are in progress. Towards Pah1 phosphorylation studies, we have introduced protein A-tagged wild-type and phosphorylation defective Pah1 encoding plasmids into wild-type and env7∆ strains. We will be carrying out subcellular fractionation and detection of the proteins by western blots. It has been established that phosphorylated Pah1 shows upshift bands in such assays. | Poster #: 48 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Genomics, Nanopore, DNA Project Title: Genomic Mapping with Single-Strand Binding Protein in a Solid-State Nanopore Author List: Howald, Nathan; Graduate, Physics and Astronomy, California State University, Long Beach, Presenting Author
Klotz, Alexander; Faculty, Physics and Astronomy, California State University, Long Beach Abstract Nanopore sensors have demonstrated the capacity for miniaturized, long-read-length genomics with minimal sample preparation. Current nanopore sequencing devices rely on biological pores which suffer from low throughput. Solid-state nanopores lack the sensitivity of biological pores but are considerably faster, more robust, and more tunable. Here, we develop a technique for genomic mapping using solid state nanopores in which the local AT-richness along a double-stranded DNA molecule is detected electrically by binding single-strand binding protein (SSB) to denaturation bubbles formed in the molecule at high temperature, exploiting the fact that AT bonds dissociate at a lower temperature than CG bonds. After being heated in the presence of SSB and cooled to room temperature, DNA molecules detected in a solid-state nanopore sensor are observed with large spikes in their current blockage signal, corresponding to the blockage of ions by bound SSB. By tracking the location of SSB-associated spikes along the molecule, a map of the local AT or CG content may be produced. | Poster #: 49 Campus: California State University San Marcos Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: metagenomes, antibiotic resistance, next-generation sequencing Project Title: Taxonomic and Antibiotic Resistance Changes to Coastal Microbiomes in Response to Rainstorm Runoff Author List: Murguia, Elizabeth; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Lopezguerra, Christian; Undergraduate, Biological Sciences, California State University San Marcos
Pylant, Mackenzie; Undergraduate, Biological Sciences, California State University San Marcos
Stretch, Ebony; Undergraduate, Biological Sciences, California State University San Marcos
Zanesco, Dennis; Undergraduate, Biological Sciences, California State University San Marcos
Cope, Matthew; Undergraduate, Biological Sciences, California State University San Marcos
Rivera, Uriel; Undergraduate, Biological Sciences, California State University San Marcos
Rahimi, Anahita; Undergraduate, Biological Sciences, California State University San Marcos
Becket, Elinne; Faculty, Biological Sciences, California State University San Marcos
Abstract Antibiotic resistance (AR) is a global healthcare problem driven by the overuse of antibiotics in clinical, agriculture, and aquaculture applications. Urban and agricultural runoff introduce antibiotic-resistant bacteria and antibiotic contamination to recipient environments. Antibiotics change microbial community compositions in favor of resistant species and can trigger the exchange of DNA carrying antibiotic resistance within a given community. However, mapping the changes in microbial community compositions and AR gene abundance is yet to be elucidated in response to rainstorm runoff. We therefore analyzed the taxonomic and antibiotic resistance gene abundance changes to coastal microbiomes in response to rainstorm runoff. Sampling at the Batiquitos lagoon outlet in Carlsbad California occurred over 14 days; before, during, and after the first two rainstorms of the 2019-2020 season. Coastal water was captured on-site on a 0.22 µm mixed cellulose ester (MCE) membrane filter. The lab performed total DNA isolation and shotgun library preparation on the isolated microbiomes followed by 2 x 150 base paired-end sequencing. Microbial composition and antibiotic resistance gene identification was performed on the resulting metagenomes, to determine a time-course profile of relative microbial abundance and antibiotic resistance profiles. To normalize microbial abundances across samples, DNA extraction was repeated using a quarter of the original sample MCE filters. Five nanograms of extracted Thermus Thermophilus DNA, an extremophilic bacteria native to Japan, was added to each sample post lysis. With the T. Thermophilus added to each sample, the lab performed shotgun library preparation and analyzed the libraries followed by 2 x 150 base paired-end sequencing using an Illumina iSeq 100. We observed an overall bimodal increase in alpha diversity and AR gene counts in the 24-72-hour period following each rainstorm. This is reflected by a relative depletion of Cyanobacteria and relative increase in Proteobacteria and Bacteroidetes. Increases in Proteobacteria appear to be predominantly marine eutrophication-associated microbes while Bacteroidetes increases were predominantly freshwater- and soil-associated microbes commonly implicated in fish and human disease. The microbial community profile returned to a pre-storm composition after approximately five to six days contrary to the three day recovery time commonly referenced. | Poster #: 50 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: DNA Metabarcoding, American pika, diet Project Title: DNA Metabarcoding Reveals Differences in American Pika Diets Author List: Villaseñor, Monica; Undergraduate, Biological Sciences, San José State University, Presenting Author
Luong, Yvonne; Undergraduate, Biological Sciences, San José State University, Presenting Author
Martinez, Salvador; Undergraduate, Biological Sciences, San José State University
Quezada, Giovanni; Undergraduate, Biological Sciences, San José State University
Castillo Vardaro, Jessica; Faculty, Biological Sciences, San José State University
Abstract The American pika (Ochotona princeps) is a small, climate-sensitive mammal that is restricted to cool microclimates found within the crevices of fractured rock habitats (e.g., talus and lava). Documented local extinctions of populations of American pikas have occurred over the past century, particularly within the Great Basin ecoregion, presumably as a result of a rapidly changing climate. While much attention has been given to climatic variables (e.g., temperature, precipitation), few studies have looked at the association between diet and population vulnerability. As the climate changes, so does the vegetation. American pikas are generalist herbivores, but they are known to selectively consume some plant species more than others. We are quantifying pika diet through DNA metabarcoding, whereby we identify all the plants present within a fecal sample. We are targeting two gene regions within the plants’ chloroplast genome: trnl and ITS. We then compare the sequences to reference databases to identify the species of plants present. This high-throughput sequencing approach will enable us to compare populations from the low elevation, hot, dry, Great Basin to those in the high elevation, cool, wet, Sierra Nevada mountains. We predict that the quality of food (e.g., protein and nitrogen content) will be greater in the Sierra Nevada than the Great Basin. | Poster #: 51 Campus: San Diego State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Symbiosis, chlorophyll fluorescence , Project Title: Significance of symbiotic nitrogen-fixing cyanobacteria during the early colonization of the land plants Author List: Picou, Race ; Graduate, Biology, San Diego State University, Presenting Author
Abstract The symbiosis between cyanobacteria of the order Nostocales and the land plants (Embryophytes) perhaps played a pivotal role in the terrestrialization of life on Earth.
Approximately 500 million years ago, life was beginning to colonize the land. Among the eukaryotes, it is likely that the first truly terrestrial taxa were the Embryophytes and their algal relatives (Charophytes). Despite evolving many mechanisms for surviving the challenges of living on land, such as increased heat, desiccation, more UV radiation and greater gravity; the Embryophytes did not individually evolve to withstand nitrogen limitation. Early Earth paleosol was not saturated with soluble nitrogen, such as ammonia, which is one of the most important nutrients for plants, and only prokaryotes are capable fixing atmospheric nitrogen into soil.
Today, there are abundant mutual symbioses based on nitrogen exchange between one such prokaryote, the Nostocales, and the most ancient lineages of the Embryophytes. I hypothesize that this symbiosis allowed the ancestor of the Embryophytes to withstand nitrogen limitation, allowing its descendants to fully colonize and terraform the land. To begin testing this grand hypothesis, the range of extant taxa which are symbiotically competent with the Nostocales must be investigated, and new experimental methods will be required to identify those taxa whose symbioses are less charismatic.
Obvious and well-known symbioses between Embryophytes and Nostocales are marked by conspicuous structures to house the cyanobacterial symbionts, such as the slime pits of the hornwort Anthoceros; or inseparable associations, such as the obligate symbiosis of the fern Azolla. Subtle symbioses, such as Pleurozium, are harder to identify.
I propose that the collection of chlorophyll fluorescence data from populations of plants axenically grown on nitrogen-deprived defined media in the presence or absence of Nostocales (Nostoc punctiforme) will be a suitable means of determining potential symbiotic competency of study taxa. Furthermore, I propose that the model plant Physcomitrium patens will prove potentially symbiotically competent with Nostoc punctiforme using this method. I anticipate my results will positively correlate with rigorous isotopically labeled nitrogen exchange and differential gene expression experiments, which are established methods of quantifying mutual symbioses between plants and Nostoc punctiforme.
| Poster #: 52 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Manganese oxidation, Pseudomonas psychrophila, Bioremediation Project Title: The identification, characterization and genetic analysis of novel Manganese oxidizing bacteria from the species Pseudomonas psychrophila Author List: Jones, Ian; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Tracy, Chase; Undergraduate, Biological Sciences, California State University, Chico, Presenting Author
Geszvain, Kati; Faculty, Biological Sciences, California State University, Chico
Abstract Manganese (Mn) is an abundant transition metal which can be oxidized by diverse bacterial species from the soluble Mn(II) state to the insoluble Mn(IV) state. These Mn oxidizing bacteria (MnOB) take part in the biogeochemical cycling of manganese and can form extracellular, solid, manganese oxides. Biogenic Mn oxides are powerful oxidizing agents and possess absorptive properties. As a result, MnOB are actively studied for bioremediation applications including the removal of environmental contaminants such as arsenate [As(III)] and dibutyl phthalate (DBP). Pseudomonas putida GB-1 is a model MnOB that forms solid Mn oxides in late exponential and early stationary phase under nutrient deficient conditions. Because Mn oxidation is proposed to play a role in degradation of complex organics, samples from a compost pile were screened for MnOB. Two novel strains of MnOB were isolated and identified. Genome sequencing revealed that both organisms were strains of the species Pseudomonas psychrophila (strains DSV001a and MS-1). Previous strains of this species tolerate growth at low temperatures. Therefore, preliminary work has compared the growth and Mn oxidation at low temperature of the P. psychrophila strains to that of P. putida GB-1. Both P. psychrophila strains grow well and oxidize Mn to temperatures as low as 4C. Using triparental conjugation, we have shown that it is possible to move foreign DNA into these P. psychrophila strains, including a suicide vector carrying the Tn5 transposon. This has made it possible to isolate transposon insertion mutants with altered Mn oxidation ability. In this study, we have characterized the MnOB species P. psychrophila DSV001a and MS-1 by comparing their growth and manganese oxidizing capabilities to that of P. putida GB-1 and have shown that these strains are amenable to genetic manipulation. In future work, we plan to identify the Tn5 insertion sites in the mutants with altered Mn oxidation to better understand the mechanism of Mn oxidation in the Pseudomonad genus. Finally, these data suggest that the novel strains could be used for low-temperature bioremediation, for example in wastewater treatment plants. | Poster #: 53 Campus: San José State University Poster Category: Other Keywords: motor control, electrophysiology, muscle spindle Project Title: Stimulation of gamma motor neurons in vitro using optogenetics Author List: Mandawe, Remie ; Graduate, Biological Sciences, San José State University, Presenting Author
Karekal, Apoorva; Graduate, Biological Sciences, San José State University
Byri, Sai Kiran; Undergraduate, Biological Sciences, San José State University
Cheline, Danitza; Undergraduate, Biological Sciences, San José State University, Presenting Author
Occiano, Alyssa ; Undergraduate, Biological Sciences, San José State University
Ortiz, Serena; Undergraduate, Biological Sciences, San José State University
Hochman, Shawn; Emory University
Wilkinson, Katherine A.; Faculty, Biological Sciences, San José State University Abstract Gamma motor neurons innervate the intrafusal fibers of the muscle spindle and control muscle spindle length and sensitivity of the muscle spindle afferents. It is challenging to independently stimulate the gamma motor neurons without also recruiting the alpha motor neurons that control the force generating extrafusal fibers. Optical stimulation in mice expressing the blue light gated Channel rhodopsin 2 (ChR2) in motor neurons led to alpha motor neuron recruitment from small to large diameter with increasing optical intensities (Llewellyn, et al., 2010). We hypothesized that gamma motor neurons, which are smaller than alpha motor neurons, will be recruited first in mice expressing ChR2 in ChAT-positive motor neurons. The extensor digitorum longus muscle and sciatic nerve were dissected and placed in a tissue bath with oxygenated synthetic interstitial fluid. The sciatic nerve was attached to an extracellular suction electrode that recorded muscle spindle afferent activity. We used a light guide to deliver blue LED light (470nm; 0.5mW-5 mW) to the end of the nerve. We found that lower levels of optical stimulation recruited the gamma motor neurons as evidenced by an increased muscle spindle afferent firing rate. Higher optical intensities recruited alpha motor neurons to produce a twitch muscle contraction. Blocking neuromuscular transmission with alpha bungarotoxin blocked both the LED-induced twitch contractions and the increases in muscle spindle afferent firing, eliminating the possibility of direct optical recruitment of muscle spindle afferents. In some muscle spindle afferents with baseline firing, higher frequencies of optical stimulation led to greater increases in afferent firing rates as expected. Gamma motor neuron stimulation could recruit muscle spindle afferent firing in units that only fired during stretch, however, the recruitment was not always predictable and long lasting changes including the development of baseline firing was sometimes observed. We predict that a gamma motor neuron specific driver for ChR2 expression will decrease variability and are currently testing mice with ChR2 expression in Npas1 expressing cells, a recently identified gamma motor neuron marker (Blum, et al., 2021). The ability to stimulate gamma motor neurons in vitro will be valuable for studying gamma motor neuron function and screening for intrafusal fiber dysfunction in disease models. | Poster #: 54 Campus: San Francisco State University Poster Category: Other Keywords: xenopus , developmental biology, confocal microscopy Project Title: The Role Somite Morphology Plays in Proper Nerve Development in Xenopus laevis Author List: Alvarado, Salvador; Graduate, Biology, San Francisco State University, Presenting Author
Domingo, Carmen; Faculty, biology, San Francisco State University Abstract The model organism, Xenopus laevis (African clawed frog), provides an interesting model for studying the formation of the early embryonic nervous system. In particular, the transient neurons known as Rohon Beard (RB). RB neurons are formed during embryogenesis of amphibians and fish, and later degenerate as the adult nervous system forms. RB neurons form at the dorsal-most part of the neural tube. They send their axonal projections in both a rostral and caudal direction as well as ventrally between the intersomitic boundaries that separate each somite. For the purpose of this study, we examined the positioning of the RB projections with respect to the intersomitic boundary. Using the antibody, HNK-1, we examined the location, length and angle of RB axonal projections as they exit the neural tube at various stages of development. Preliminary data suggests that RB axons aggregate near the intersomitic cleft. This provides an early indication of the important role that the intersomitic boundaries may play in establishing the early embryo’s mechanosensory system. | Poster #: 55 Campus: California State University, Sacramento Poster Category: Other Keywords: Drosophila melanogaster, gut-microbiome-brain axis, gut biomechanics Project Title: Kismet affects gut biomechanics, the gut microbiome, and gut-brain axis in Drosophila melanogaster Author List: Penn, Aliyah; Undergraduate, Biological Sciences, California State University, Sacramento, Presenting Author
Sundaramurthy, Punithavathi; Graduate, Computer Science, San José State University, Presenting Author
Niosi, Angelo; Graduate, Biological Sciences, California State University, Sacramento
Yadav, Prince; Undergraduate, Physics, California State University, Sacramento
Cavanaugh, Jeffrey; Undergraduate, Physics, California State University, Sacramento
Vo, Nguyen Henry; Undergraduate, Biological Sciences, California State University, Sacramento
Welch, Chloe; Graduate, Biological Sciences, California State University, Sacramento
Morris, Eliza; Faculty, Physics, California State University, Sacramento
Jensen, Mikkel; Faculty, Physics, California State University, Sacramento
Lee, Wendy; Faculty, Computer Science, San José State University Abstract INTRODUCTION: The gut microbiome may contribute to the pathophysiology of neurodevelopmental disorders (NDDs), yet it is unclear how NDD risk genes affect gut physiology in a manner that alters bacterial colonization. We aimed to address this question using Drosophila melanogaster with a mutation in kismet (kis), the ortholog of the human autism risk gene Chromodomain helicase DNA binding protein 8 (CHD8). Kis/CHD8 encodes a chromatin remodeler that affects gene expression in the brain and gut. We used flies with heterozygous loss of kis to examine gut biomechanics, the gut microbiome, and the connection between gut microbiota and behavior within the so-called “gut-brain axis.”
METHODS & RESULTS: To quantify changes in gut tissue mechanics, we dissected kis mutant and control flies and affixed whole guts between two clips mounted on a high-precision force transducer and length controller, capable of measuring forces to micro-Newton precision. Our measurements revealed that there are significant differences in the mechanics of kis mutant guts compared to control guts, in terms of elasticity, strain stiffening, and ultimate tensile strength. To characterize the gut microbiomes of kis mutant and control flies, we isolated genomic DNA from midguts and used 16S metagenomic sequencing. Analysis of the sequencing data showed that loss of kis profoundly altered the abundance of multiple bacterial taxa in the midguts, including opportunistic pathogens that were present in kis mutants but not controls. To investigate the putative connection between the gut microbiome and behavior, we treated kis mutant and control flies with an antibiotic and then evaluated their courtship behavior. Depletion of the gut microbiome rescued courtship behavior defects of kis mutant flies and, in striking contrast, induced courtship defects in the control strain. This data demonstrates that (1) the gut-brain axis plays a prominent role in fruit fly behavior, and (2) antibiotic treatment can have either positive or negative impacts on behavior, depending on the status of gut dysbiosis prior to treatment. FUTURE DIRECTIONS: We are currently analyzing RNA-sequencing data to determine how kis affects gene expression in the gut epithelium to influence gut biomechanics, as well as further examining the connection between gut biomechanics, microbial colonization, and behavior. FUNDING: CSUS Research and Creative Activities Award and G2E Award.
| Poster #: 56 Campus: San Francisco State University Poster Category: Other Keywords: Vision, Retina, Convergence Project Title: Cell Structure and Synaptic Contacts in a Unique Vertebrate Retina Author List: Wagh, Abhiniti; Graduate, Biology, San Francisco State University, Presenting Author
Magaña Hernandez, Laura; Graduate, Biology, San Francisco State University
Fathi, Jessamyn; Undergraduate, Biology, San Francisco State University
Anastassov, Ivan ; Faculty, Biology, San Francisco State University Abstract Bipolar cells are integral to vision, as they are interneurons in a pathway that relays electrical signals to the brain. Individual bipolar cells are also a convergence point, because they each receive inputs from many photoreceptors. Skates (Leucoraja erinacea) have ‘simplex’ retinas with only one kind of photoreceptor - rods. Duplex retinas have both rods and cones and use them for dim and bright light detection, respectively. Uniquely, skate rods can function in the domain of cones. Cells in the rod pathway are usually separate from those in the cone pathway. Therefore, only ON bipolar cells are expected to exist in the rod pathway in skate. However, earlier studies have suggested possibly OFF bipolar cells exist in the skate retina. These cells, usually found in the cone pathway of vertebrates, only make indirect connections to rods, via another cell type, called AII amacrine cells. The possible existence of OFF bipolar cells with direct contacts to skate rods supports our hypothesis that the simplex skate retina may exhibit various hybrid morphology features mediating its functional plasticity, or ability to respond to bright light in the absence of cone structures.
Scanning electron microscopy (SEM) images were analyzed in the programs Reconstruct and Amira. A 3-D model of a putative bipolar cell was constructed from the SEM images in Reconstruct. Preliminary results show a cell with many dendritic processes that branch extensively. In this project, about 35 putative bipolar cell dendritic processes have been quantified. This provides a preliminary estimate of convergence of ~35 rods: 1 bipolar cell. Further confirmation of whether each bipolar cell dendritic process represents an actual contact with a rod will be done by confirming proximity to rod ribbons, crescent-shaped structures, where synaptic vesicles line up, located within rod terminals. Ribbons will be used as a proxy for rod terminals, thus if there are bipolar cell processes resting within a predefined distance under a ribbon, contact is likely. Since ON and OFF bipolar cells make different kinds of synaptic contacts, if both kinds of contacts are seen being made by one bipolar cell structure, then this will be further evidence for the hybrid morphology of the skate retina. Preliminary results show a single bipolar cell in the skate retina putatively making both kinds of contacts with a photoreceptor.
| Poster #: 57 Campus: California State University, Dominguez Hills Poster Category: Other Keywords: Neuroscience education, STEM pedagogy, CUREs Project Title: Supporting First-Year STEM Student Success through a Combination of High Impact Educational Practices and CUREs Author List: Camarillo, Alex; Undergraduate, Clinical Sciences, California State University, Dominguez Hills, Presenting Author
Zamora, Stacy; Graduate, Biology, California State University, Dominguez Hills
De La Torre, Maria; Undergraduate, Chemistry & Biochemistry, California State University, Dominguez Hills
Vieira, Philip A.; Faculty, Psychology, California State University, Dominguez Hills Abstract A disparity exists for first-generation minority students pursuing degrees and careers in STEM when compared to their white majority counterparts. Currently, there is a barrier between accomplishing goals in STEM due to the lack of availability of resources and opportunities such as: access to mentorship, quiet areas to study, financial support for exams and applications, amongst others. Additionally, many students feel disengaged with their coursework, particularly those from minority households who are the first in their family to pursue higher education. By students not being supported with proper resources and opportunities we observe a decrease retention rate of minority freshmen in STEM majors. With a decrease of minorities staying in STEM during their academic career, this directly impacts the STEM workforce. A study by Excelencia in Education showed between 2012 and 2022 there will be a 11% projected growth in the STEM occupations, concurrent with the projected growth of the Hispanic population in the United States. Consequently, Hispanics students obtaining certificates and degrees in STEM will be vital for the STEM workforce. To address the need, a pedagogical study using transformative exploratory sequential mixed methods was utilized. This included designing a first-year seminar for the career development of entering undergraduate students at California State University Dominguez Hills, a Hispanic-serving institution. The seminar helped determine student interest in STEM careers and retention in STEM education and included a variety of pedagogical strategies including Design Thinking (DT) training and Course-based Undergraduate Research Experiences (CUREs). Additionally, low-cost electrophysiological tools from Backyard Brains were used for students to explore their research interests in the field of neuroscience. We examined whether students pursue extracurricular activities related to their career interests, made connections with a STEM mentor, joined STEM related clubs, and applied to entry level STEM jobs. The impact of this ongoing study includes providing a framework from which STEM departments can create a survey course to recruit incoming first-year students from underrepresented backgrounds and encourage retention in STEM majors and careers. Lastly, this develops a pipeline for students to enter and graduate as a STEM major at this university and across the CSU-system to retain interest in STEM and join the STEM workforce. | Poster #: 58 Campus: California State University, Dominguez Hills Poster Category: Other Keywords: neurobiology, addiction neuroscience, immunohistochemistry Project Title: Neurobiological correlates of substance use disorder: disrupted ΔFosB protein expression in the PFC and NAc of cocaine-exposed male and female rats Author List: Zamora, Stacy; Graduate, Biology, California State University, Dominguez Hills, Presenting Author, Eden Award Finalist
Lugo, Tania; Undergraduate, Psychology, California State University, Dominguez Hills
Yang, Sabin; Graduate, Biology, California State University, Dominguez Hills
Urstadt, Kevin R.; Occidental College, Psychology, California State University, Dominguez Hills
Zavala, Arturo R.; Faculty, Psychology, California State University, Long Beach
Vieira, Philip A.; Faculty, Psychology, California State University, Dominguez Hills Abstract Substance Use Disorder (SUD) is a chronic relapsing disease associated with long-lasting neurobiological alterations in the brain’s mesolimbic reward pathway. The rate of cocaine abuse, a specific SUD, has been rising in the USA, creating an economic and healthcare burden. To understand the pathogenesis of SUD, we focused on a neuronal transcription factor implicated in cocaine abuse called ΔFosB because its activity can lead to long-lasting changes in the brain that may support the chronic nature of SUD. To measure ΔFosB, we first obtained brain tissue from adult male and female Sprague-Dawley rats that were exposed to cocaine under four different conditions: control (saline), acute intoxication, short-term withdrawal, and long-term withdrawal. We isolated two subregions of the reward pathway, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), by taking 50µm coronal sections of the brain. We then performed immunohistochemistry on these sections to label neurons possessing ΔFosB. Sections were then imaged under epifluorescent microscopy and automated cell counting was performed on the images taken using ImageJ. In each group, the number of ΔFosB -positive neurons was assessed within the ventrolateral PFC, ventromedial PFC, and the NAc. When performing a single factor ANOVA, we observed a difference in the amount of ΔFosB -positive cells in all experimental groups (p = 0.05). Additionally, when comparing the amount of ΔFosB – positive cells in the regions themselves, there was a statistical difference observed only in the ventromedial PFC (p < 0.01). Post Hoc testing was performed and confirmed statistical differences for amount of ΔFosB -positive cells in the ventromedial PFC. These preliminary results indicate that there is an association between cocaine use and the amount of ΔFosB present in the mesolimbic pathway. Further analysis will be performed to compare sex differences in the number of ΔFosB -positive cells in the different treatment groups. Overall, these new insights can lead into possible treatments that target transcription factors in those suffering from SUD. | Poster #: 59 Campus: California State University San Marcos Poster Category: Other Keywords: Immunology, Immunogenetics, Flowcytometry Project Title: The Evaluation of Major Histocompatibility Complex (MHC) Class I Expression from Different induced Pluripotent Stem Cell (iPSC) lineages Author List: Suanico, Ken; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Nguyen, Kayla; Undergraduate, Biological Sciences, California State University San Marcos
Mothé, Bianca; Faculty, Biological Sciences, California State University San Marcos
Abstract Stem cells hold the promise of being the future of therapeutics for cardiovascular, metabolic, and neurological diseases. Induced pluripotent stem cells (iPSCs), like embryonic stem cells (ESCs), can differentiate into cells of the three germ layers that make up all the tissue and organs in the human body. iPSCs are reprogrammed from somatic cells such as skin or blood cells. With the capability of culturing an unlimited supply, iPSC research has been expanding the frontiers of medicine. Technology combining stem cell biology and genomics aims to provide an autologous iPSC-derived neuron replacement therapy for Parkinson’s disease, for example. Stem cell therapies targeting ocular disease, heart failure, and diabetes are currently in research and development. Before these therapies can be administered parameters such as tumorigenicity, dose toxicity, and immunogenicity must be evaluated. Immunogenicity is defined as the ability of cells or tissues to provoke an immune response. A component of immunogenicity is Major histocompatibility complex (MHC) molecules, which are surface bound molecules expressed on all nucleated cells and present intracellular peptide fragments. If these fragments are recognized as foreign by T cells or B cells, then an immune response is triggered leading to destruction of the cell. It is presumed that iPSCs are like ESCs and do not express MHC I. My research aims to test the hypothesis that MHC class I molecules are not expressed on iPSCs derived from different germ layers. Specifically, we hypothesize that iPSCs derived from immune privileged cell types will have lower expression of MHC I. We used flowcytometry to measure surface bound MHC molecules and RT-PCR analysis to measure expression of MHC molecules. Analysis of MHC I marker W6/32 by flow cytometry yielded data that suggests that the fibroblast-derived iPSC Gibco line is positive for MHC I. RT-PCR analysis with specific human MHC designed primers also provides evidence that the fibroblast-derived iPSC Gibco line expresses MHC I. The next steps are to perform analysis on cell types from ectoderm and endoderm. These studies could help identify a potential cell type ideal for therapeutic purposes and explore the immunological state of iPSCs from different germ layers. | Poster #: 60 Campus: California State University, Long Beach Poster Category: Other Keywords: food waste charcoal, bacteria, self-healing concrete Project Title: Application of Immobilized Bacteria on Food Waste Biochar as Sustainable Solutions for Construction Concrete Author List: Ly, Maggie; Graduate, Civil Engineering & Construction Engineering Management, California State University, Long Beach, Presenting Author
Asvapathanagul, Pitiporn; Faculty, Civil Engineering & Construction Engineering Management, California State University, Long Beach
Sanchez-Vazquez, Oscar; Graduate, California State University, Long Beach
Tran, Tung; Graduate, California State University, Long Beach
Calabrese, Andrea; Faculty, Civil Engineering & Construction Engineering Management, California State University, Long Beach
Abstract The lifespan of modern concrete ranges between 50 to 100 years before it needs to be renewed in order to keep the concrete in the best conditions. The compressive strength in concrete is high, but the tensile strength is low, and as more cracks develop within infrastructures, the quality of concrete deteriorates, which can become a safety hazard. The costs to repair concrete are high, thus it is crucial to find alternative ways to maintain concrete at lower costs. This study focuses on implementing food waste-based charcoal with bacteria such as Sporosarcina pasteurii—which can hibernate up to 200 years—into concrete in order to fill cracks formed in infrastructures over time. The food wastes are turned into charcoal, which is then mixed with the bacteria and concrete. When the bacteria is activated, it feeds on the food in the concrete and then fills the cracks of the concrete. In the United States, 40 percent of food goes to waste without being consumed, so food waste will be the base of the charcoal to not only creating concrete that can self-heal but also to put food waste to use before it can be discarded to landfills.
Four bacteria, including Bacillus megaterium, Bacillus subtilis, Pseudomonas stutzeri and Sporosarcina pasteurii, have been claimed for their calcite precipitation ability. The bacteria were dormant on lab-manufactured food waste biochar prior to integrating into concrete mixture. Three different amounts of bacteria and/or food waste biochar (3%, 5%, and 10%) were tested for each microorganism. The concrete specimens—control set—were tested for their strength at 28 and 90 days after preparation, while others were cracked and stored in an incubation room at a constant humidity level and temperature for 28 and 90 days, respectively. During the incubation period after crack, moisture activated hibernated bacteria embedded in concrete. Bacteria obtained nutrients from biochar and supplements provided in the concrete mixture to metabolically generate calcite to repair the concrete cracks. Consequently, the healed concrete specimens were tested for their compression strength in order to determine percent stress-inducing cracks. | Poster #: 61 Campus: San Francisco State University Poster Category: Product-focused Innovation Keywords: biopolymer, mutualistic bacteria, rheology Project Title: Developing new hydrocolloids from bacteria for commercial use Author List: Madras, Shanmukha; Graduate, Biology, San Francisco State University, Presenting Author
Huang, Jason; Graduate, Biology, San Francisco State University
Chen, Joseph; Faculty, Biology, San Francisco State University
Abstract Hydrocolloids are polymers derived from plants, animals, or microorganisms that can form viscous suspensions in water. They have many industrial applications, including serving in food production as thickeners, stabilizers, emulsifiers, and water-retention and gel-forming agents. These additives are commonly used to manipulate the textures and rheological properties of food products such as salad dressings and ice cream, as well as to extend shelf life. For example, xanthan gum is used as a thickening agent and sometimes as a substitute for fat. Due to an increasing demand in healthy food options and ready-to-eat meals, companies are looking for hydrocolloids with different properties to meet consumer demand. ExoPolymer, a biotech company based in the Bay Area, was recently founded to develop new hydrocolloids from bacteria that are superior to existing, commercially available products. In collaboration with ExoPolymer, we are currently purifying and characterizing exopolysaccharides (EPS) produced by different bacterial strains. The goals of the project are to evaluate the potentials of such EPS for industrial use and to find the optimal conditions for purifying EPS while maximizing yield. We have assessed the growth of five different bacterial strains in various culture media. Having determined that at least one strain grows optimally in a preferred medium, we plan to isolate different forms of the EPS for characterization with a rheometer. Such analysis will help determine whether this EPS of interest will meet potential market needs of the food and pharmaceutical industries.
This project is supported by a CSUPERB Industry Partnership Initiative Grant. | Poster #: 62 Campus: California State University, Sacramento Poster Category: Proteins (Include Proteomics) Keywords: amyloid, lipoprotein, protein folding Project Title: Amyloidogenic consequence of methionine oxidation in canine apolipoprotein A-I Author List: Lee, Tim; Graduate, Chemistry, California State University, Sacramento, Presenting Author
Roberts, Linda; Faculty, Chemistry, California State University, Sacramento Abstract Apolipoprotein A-I (ApoA-I) is the main protein of high-density lipoprotein (HDL), playing a key role in reverse cholesterol transport by scavenging cholesterol from peripheral cells and transporting it to the liver for disposal as bile. High serum ApoA-I is associated with decreased risk for cardiovascular disease. Misfolding of ApoA-I destroys its cardioprotective properties and contributes to amyloid formation in both vascular and cardiac tissue. For amyloid formation to occur, the protein’s normal self-association properties must be disrupted. Methionine oxidation has been shown to disrupt normal self-association and to lead to amyloid formation in human Apo-AI. Amyloid ApoA-I has also been found in the arteries of aged dogs despite that canine ApoA-I contains only 1 of 3 methionines found in the human protein. The goal of this research was to examine the influence of methionine oxidation on normal self-association, structure, and amyloid formation in canine ApoA-I compared to human ApoA-I. Self-association was determined by crosslinking protein at different concentrations using bis(sulfosuccinimidyl)suberate (BS3) followed by SDS-PAGE. Structural changes and amyloid formation were determined by ANS binding and Thioflavin T fluorescence, respectively.
Methionine-reduced (Met-R) canine ApoA-I exhibited less ability to self-associate than Met-R human ApoA-I and oxidation caused loss of self-association in both proteins. ANS binding decreased significantly in both Met-R and Met-O human ApoA-I in samples incubated at 37 degrees C for 28 days. In contrast, ANS binding in both Met-R and Met-O canine Apo-AI exhibited little change. ThT binding was highest in Met-O human ApoA-I; Met-O canine ApoA-I did not exhibit an increase in ThT binding. These results indicate that amyloid formation in Met-O canine ApoA-I is reduced compared to Met-O human Apo-A-I and suggest that additional factors are involved in producing ApoA-I amyloid deposits in the vasculature of aged dogs.
| Poster #: 63 Campus: California State University, Northridge Poster Category: Proteins (Include Proteomics) Keywords: chaperone protein, NMR spectroscopy, structure-function studies Project Title: Characterization of HdeB chaperone function from different vantage points Author List: Nguyen, Hiep; Graduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge Abstract HdeB and its homolog, HdeA, are acid-induced periplasmic chaperones that help protect pathogenic bacteria from denaturation and aggregation in their path through the acidic environment of the human stomach. While HdeA is active as a chaperone at acidic pH (pH <3), HdeB is active at near-neutral pH (pH ~4). Due to the continuous range of pHs in which these two chaperones operate, it has been hypothesized that HdeA and HdeB might work in relay to protect bacterial periplasmic client proteins from acid stress. The overall goal of this research is to characterize the HdeB chaperone mechanism and its potential synergistic cooperation with HdeA using NMR spectroscopy. ModA, a mutual client protein of both HdeA and HdeB, was chosen to investigate the mechanism of chaperone function due to its relatively small size, making it suitable for investigation by NMR.
The interaction between HdeB and ModA was probed using the 1H-15N correlation NMR experiment (1H-15N HSQC); peaks corresponding to each backbone amide (-NH) group were monitored using a sample of HdeB labeled with the NMR-active isotope 15N. Each peak in the HSQC was then correlated to its corresponding residue in HdeB in a process known as chemical shift assignment, which required analysis of a suite of NMR experiments. The assignment of HdeB allowed us to point out specifically which residues participate in binding client proteins, by monitoring changes in chemical shift when we added client ModA. Our comparison of the HSQC spectrum of 15N-labeled HdeB with unlabeled ModA between pH 6 (inactive) and 4.5 (active) showed significant chemical shift changes in the C-terminal residues of HdeB, reflecting the change in chemical environment as ModA interacted with that region of HdeB at the lower pH.
We have also nearly completed chemical shift assignment of the client protein ModA; with these assignments, we can perform the reciprocal experiment with unlabeled HdeB and 15N-labeled ModA in the same pH range. This experiment will enable us to characterize the interaction between HdeB and ModA from the point of view of the client protein instead. The enhanced understanding of the HdeB-ModA binding interaction could then serve as the basis for the generalization of the chaperone mechanism of HdeB with other client proteins. Future experiments will include adding HdeA to the HdeB-ModA complex with a pH titration from neutral to acidic to understand the relay mechanism between HdeA and HdeB. | Poster #: 64 Campus: Humboldt State University Poster Category: Proteins (Include Proteomics) Keywords: cellulase, metagenome, biofuel Project Title: Evaluation of Putative Cellulases Enzymes at Various pHs from the Rumen Metagenome, for Cellulosic Biofuel Production Author List: Collins, Jasmine ; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Darlington, Aaron; Undergraduate, Chemistry, Humboldt State University, Presenting Author
Jensen, Annie; Undergraduate, Biology, Humboldt State University
Losee, Jayden; Undergraduate, Chemistry, Humboldt State University
Morales, David; Undergraduate, Chemistry, Humboldt State University
Calderon, Vincent; Undergraduate, Chemistry, Humboldt State University
Kishineff, Madison; Undergraduate, Chemistry, Humboldt State University
Tessa, Balkow; Undergraduate, Chemistry, Humboldt State University
Hess, Matthias; Dept. of Animal Science, University of California, Davis
Escobar, Matthew; Faculty, Biology, California State University San Marcos
Cappuccio, Jenny; Faculty, Chemistry, Humboldt State University Abstract Cellulose, the most abundant biopolymer on earth, can be broken down by enzymes classified as cellulases. The resulting glucose monomers can then be used as substrates for the production of biofuel. The purpose of this study was to evaluate the cellulolytic activity of putative cellulases identified from metagenomic data generated from fiber adherent microbes of the cow rumen, as part of a collaborative project with the CSU Cellulosic Biofuel CURE (course based undergraduate research experience) and the Joint genome institute (JGI). The JGI provided putative cellulase genes with histidine tags transformed into E. coli BL21(DE3). Four strains (CJD8-7,ME9-8,CJD8-11 and CJD9-38) were over-expressed and the putative cellulases purified with affinity chromatography and analyzed for their varying enzyme activity over a pH range of 5-8. To do this we utilized a semi-quantitative carboxymethylcellulose (CMC), plate assay with congo red detection of cellulolytic activity compared to a control Aspergillus niger cellulase. Results indicate cellulolytic activity in strains CJD8-7,ME9-8,CJD8-11 and CJD9-38 at pH 5-8. Moreover, the putative cellulases, ME9-8 at pH 7 and CJD8-11 at pH8 displayed activity comparable to or exceeding the control cellulase activity. Future steps will include a spectrophotometric kinetic assay utilizing ortho-nitrophenyl-beta-galactoside (ONPG) as a substrate monitoring the production of ortho-nitrophenol. Importantly, this study indicates that these putative cellulases have the potential to enhance biofuel production over a wide pH range. | Poster #: 65 Campus: California State University, Fresno Poster Category: Synthetic Chemistry Keywords: PROTAC, Androgen Receptor, Prostate Cancer Project Title: Combating Prostate Cancer via Non-Ligand-Binding-Domain Androgen Receptor PROTACs Author List: Munoz, Erick; Graduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Wu, Sitong; Graduate, Chemistry and Biochemistry, California State University, Fresno
Chen, Guanglin; Staff, Chemistry and Biochemistry, California State University, Fresno
Oceguera, Esveidy; Undergraduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Lee, Tong; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Hang, Jasmine; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Chen, Qiao-Hong; Faculty, Chemistry and Biochemistry, California State University, Fresno
Abstract There are approximately 248,000 estimated prostate cancer cases in the United States for 2021. Although most men diagnosed with early-stage prostate cancer do not die from it, there are still no effective treatments available for patients with aggressive and lethal castration resistant prostate cancer (CRPC). One current treatment for CRPC is through the inhibition of androgen receptors, a key protein for cell growth and survival, but there are two drawbacks from this method. The first is that, due to the mode of action, inhibitors tend to require larger doses and therefore leading to side-effects caused by off-target binding. The second drawback is these inhibitors tend to bind to the Ligand Binding Domain (LBD) of androgen receptors. Cancer cells tend to divide rapidly, therefore giving chance for compensatory secondary mutation(s) at the LBD of the androgen receptor triggering resistance to the cytotoxicity caused by inhibitors. To combat these two drawbacks, incorporating AR inhibitors into Proteolysis Targeting Chimeras (PROTACs) is critical due to their catalytic nature. This study aims to develop two groups of PROTACs which do not require the LBD to degrade androgen receptors. These hetero-bifunctional molecules consist of either Niclosamide, a potent AR-V7 inhibitor, or an EPI derivative, a first-in-class AR inhibitor, and Von Hippel Lindau (VHL) ligand, an E3 ligase ligand. The synthetic approach to two Niclosamide-PROTACs with different linkers have been developed, culminating in the achievement of two highly advanced intermediates that are one step away from our desired PROTACs. The synthetic approach to a group of EPI-PROTACs is being explored. Toward this end, one new EPI derivative has been synthesized with an appropriate carboxylic acid functional group that is ready for attaching different linkers. Chemical structures from each synthetic step have been characterized by 1H and 13C NMR data, as well as IR spectra. E. Munoz is supported by CSU-Fresno Bridge to Doctorate Program funded by the NIH under Grant # T32GM137948. E. Oceguera and T. Lee are supported by the CSU-Fresno NIH RISE Program funded by the NIH under Grant # R25GM131956. E. Oceguera is a 2021 Howell-CSUPERB Scholar and partially supported by the CSU-LSAMP program funded by NSF under grant # #HRD-1826490, CSU Office of the Chancellor, and Fresno State. | Poster #: 66 Campus: California State University, Northridge Poster Category: Synthetic Chemistry Keywords: catalysis, synthesis, selectivity Project Title: Ruthenium dimer catalysts for ketone transfer hydrogenation Author List: Yeremyan , David; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Paul, Tina; Undergraduate, Chemistry and Biochemistry, California State University, Northridge
Kelson, Eric; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract Many important pharmaceutical syntheses entail the hydrogenation of ketone groups often into chiral centers. This becomes challenging if intermediates have multiple ketone carbonyls and/or the products must have specific geometries. Though Noyori-type catalysts had initial promise in achieving this, reaction selectivities could not be generalized due to their dependence on weak (and unpredictable) non-covalent interactions between the catalyst and substrate. The Kelson group is investigating alternative catalysts that directly bind ketone substrates within a customizable environment. Rational design of the catalytic site could restrict binding to specific substrates and ensure production of the desired geometries. This work could eventually help streamline drug production and improve purity and safety. We report here investigation of the dimeric (X)2(PR3)Ru(Tppz)Ru(PR3)(X)2 catalytic system [where Tppz=tetra(2-pyradyl)pyrazine, R=phenyl, cyclohexyl, or phenoxy, and X=Cl, OH, or 2-pyrrolidinonate (Pyr)]. This system features two closely-bridged catalytic ruthenium atoms with phosphine ligands large enough to cooperatively influence the substrate binding sites (X). This system was first surveyed through in-situ reactions where Cl3Ru(Tppz)RuCl3 reacted with potassium t-butoxide base and phosphine before addition to ketone substrate in refluxing 2-propanol. Aromatic and aliphatic ketones rapidly hydrogenated while bulky tertiary carbons adjacent to the target carbonyl slowed the reaction (as expected for direct substrate coordination). Catalytic activity was phosphine dependent with triphenylphosphite being fastest and triphenylphosphine the slowest. The borneol/isoborneol product ratios from camphor hydrogenation also decreased from triphenylphosphite to triphenylphosphine as expected for increasing steric crowding of the catalytic sites. In ongoing efforts to directly prepare the active catalysts, (cymene)Ru(PR3)Cl2 starting materials react with Tppz in benzene to form Cl2(PR3)Ru(Tppz)Ru(PR3)Cl2 dimers. The PPh3 dimer reacts with Pyr in aqueous ethanol to form (HO)(Pyr)(PPh3)Ru(Tppz)Ru(PPh3)(Pyr)(OH) which exhibits activity identical to the in-situ system. These results demonstrate that this catalytic system could lead to customized transfer hydrogenation catalysts. 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 #: 67 Campus: California State University, Long Beach Poster Category: Synthetic Chemistry Keywords: metal-organic framework, electrodepostion, surface chemistry Project Title: Covalently attaching MIL-88B(Fe) onto stainless steel surfaces Author List: Ruiz, Angel; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Tian, Fangyuan; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Our study focused on studying and analyzing efficient methods for depositing MIL-88B(Fe) onto stainless steel (SS). Mechanical cleaning (i.e., 400-1200 grit sandpaper) to achieve uniform and leveled surfaces, followed using a solution containing 1:1:1 ratios of acetone, ethanol, and 40% nitric acid to thoroughly clean SS samples. Our improved solvothermal synthesis method included submerging the SS samples in a MIL-88B(Fe) “mother” solution prior to heating in an oven at 100 ℃ for 12 hours. This method allowed for the MOF particles to synthesize directly on the SS samples, following covalent attachment. Apart from this study, we utilized a quartz crystal microbalance (QCM) to study the drug loading qualities of MIL-88B(Fe). Here, Au QCM chips were employed to better comprehend the capacities of MIL-88B(Fe)’s porous framework for loading our model drug, ibuprofen. MHDA/MIL-88B(Fe) thin-films were used to functionalize Au surfaces, then the Sauerbrey equation helped to interpret our MOF’s drug loading ability. Further studies were conducted using IR to monitor the stability of our MOF across different lengths of time post-synthesis as well as in various solutions (i.e., acidic, organic, aqueous). The voltaic cell was used to synthesis MIL-88B(Fe) onto SS using an electric current. IR confirmed successful synthesis of our MOF onto SS, shortening the original submersion technique from 2 days to the EPD synthesis route of 8 minutes. | Poster #: 68 Campus: California State Polytechnic University, Pomona Poster Category: Synthetic Chemistry Keywords: organic synthesis, bioactive compound collections, drug discovery Project Title: Stereocontrolled Synthesis of Cyclobutane Fragments: A Compound Collection for Drug Discovery Author List: Lao, Kimchou; Undergraduate, Biotechnology, California State Polytechnic University, Pomona, Presenting Author
Rodriguez, Dani; Undergraduate, Biology, California State Polytechnic University, Pomona, Presenting Author Abstract Small molecule therapeutics play an important role in modern medicine and biotechnology, and chemical synthesis is a foundational technology for small-molecule drug discovery. Therefore, the efficient synthesis of collections of novel small molecule candidates, as well as the development and application of new reactions as tools for synthesis, remains a pressing challenge. Cyclobutane rings are important structural elements of small-molecule pharmaceuticals and other biologically active molecules. However, the synthesis of stereochemically defined cyclobutane compound collections remains a significant challenge, leading to cyclobutane structures being underrepresented in commercial and bioactive compound collections. This project focuses on the stereocontrolled synthesis of a cyclobutane-based fragment library. Starting from 3-oxocyclobutane carboxylic acid, amide formation was performed, followed by ketone derivatizations such as reduction, reductive amination, and Wittig reactions to construct a family of functionalized cyclobutane carboxamide compounds. The use of an 8-aminoquinoline amide enabled Pd-catalyzed C-H arylation on some cyclobutyl derivatives, leading to stereochemically defined, trisubstituted cyclobutane compounds. Our efforts thusfar have generated a colletion of over twenty molecules that have been characterized by NMR spectroscopy, mass spectrometry, and some by X-ray crystallography. We will move forward by screening for antimicrobial and further biological activities. | Poster #: 69 Campus: California State University, Fresno Poster Category: Synthetic Chemistry Keywords: Flavonolignan, Silybin, Prostate Cancer Project Title: Core Structure-Antiproliferative Activity Relationship Studies on 5,7,20-O-trimethylsilybins Author List: Wu, Sitong; Graduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Chen, Guanglin; Staff, Chemistry and Biochemistry, California State University, Fresno
Chen, Qiao-Hong; Faculty, Chemistry and Biochemistry, California State University, Fresno
Abstract Silybin (also known as silibinin) isolated from Milk Thistle (Silybum marianum). Silybin has been demonstrated to be a good lead compound due to its potential to prevent and treat prostate cancer. Its moderate potency and poor pharmacokinetic profile hindered it from moving forward to therapeutic use. As part of our ongoing project to search for optimized derivatives of silybin for the potential treatment for castration-resistant prostate cancer, this study aims to investigate the relationships between the core structure of 5,7,20-trimethylsilybin and their antiproliferative activities towards five different prostate cancer cell lines (PC-3, DU145, LNCaP, 22Rv1, VCaP). Towards this end, thirty-three derivatives with three different core scaffolds, including flavanonol-type flavonolignan (silybins), flavone-type flavonolignan (hydnocarpin-Ds) and chalcone, have been synthesized from 5,7,20-O-trimethylsilybin that was prepared from commercially available silybin. All chemical structures of these thirty-three derivatives have been well-characterized by NMR, IR, and HRMS data. Their antiproliferative activities towards three prostate cancer cell lines (PC-3, DU145 and LNCaP) have been evaluated through WST-1 bioassay. The cell proliferation data that we have already achieved indicated that silybin derivatives with flavanonol-type flavonolignan scaffold possess highest potency while the silybin derivatives with chalcone core structure have least potency against the three prostate cancer cell lines. The further evaluation of these derivatives on more prostate cancer cell lines (22Rv1 and VCaP) is currently in progress.
We are grateful to The California State University Program for Education and Research in Biotechnology (CSUPERB) for (i) the Graduate Student Research Restart award (to S. Wu) and the COVID-19 Research Recovery Microgrant (to Q. Chen). This work was also partially supported by the Scholarly and Creative Activity Award from the College of Science and Mathematics at California State University Fresno (Q. Chen).
| Poster #: 70 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: nearshore microbiome, climate change, horizontal gene transfer Project Title: The effect of climate change-associated extreme weather events on horizontal gene transfer in coastal microbiomes Author List: Sanders, Ciara; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Hunter, Brad; Undergraduate, Biological Sciences, California State University San Marcos, Presenting Author
Caceres, Lakme; University of California San Diego
Johansen, Haven; Western Washington University
Becket, Elinne; Faculty, Biological Sciences, California State University San Marcos
Abstract The nearshore coastal microbiome greatly contributes to a balanced ecosystem that is home to a diverse population of marine life. Important contributions of the microbiome include primary production, nutrient cycling, and protection from environmental stress and pathogens. Disrupting these microbial functions may be devastating to the ecosystem and other organisms inhabiting nearshore. A large disturbance experienced by nearshore coastal microbes is extreme weather events. The frequency of extreme weather events, like heat waves, wildfires, rainstorms, and floods are increasing in frequency due to climate change. Extreme weather events rapidly alter numerous environmental factors; here, we focus on temperature, pH, and salinity levels in the nearshore environment and how changes to these variables will affect the evolution of the nearshore microbiome. Extreme changes in these environmental variables may induce horizontal gene transfer (HGT) in nearshore bacteria as they attempt to adapt to unprecedentedly harsh environmental conditions. Because of this, we hypothesized that increases in temperature, decreases in pH, and decreases in salinity will increase the rate of HGT in the nearshore microbiome. If this is true, these disturbances may decrease the resilience of the nearshore microbiome and negatively impact the coastal ecosystem. To investigate this, we established methods for an in vitro HGT assay to quantify the baseline rate of HGT between a complex donor community and a recipient. Preliminary results reveal successful detection of baseline horizontal gene transfer events between environmental microbiome samples and a green fluorescent protein indicator lab strain of Escherichia coli. Future investigations will utilize these methods to investigate how changes in temperature, pH, and salinity affect the rate of HGT in nearshore microbiome samples. These methods will allow us to observe how each environmental variable can independently and in combination affect HGT in nearshore microbes as well as provide a better understanding of how nearshore coastal microbiomes will respond to future weather changes. | Poster #: 71 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: Bioenergy, Chlorella, Methylobacterium Project Title: A Co-Op Micro-cosm? Population Dynamics as Bacterial and Algal Species Interact in Unfavorable Condition Author List: Flores, Armando; Undergraduate, Department of Applied Environmental Science, California State University, Monterey Bay, Presenting Author
Haffa, Arlene; Faculty, Biology and Chemistry Department, California State University, Monterey Bay
Carr, Erin; Graduate, School of Biological Sciences, University of Nebraska-Lincoln
Riekhof, Wayne; Faculty, School of Biological Sciences, University of Nebraska-Lincoln Abstract Research on microalgae has shown that it is a promising alternative to 1st-generation biofuels, with several advantages over the traditional bio-derived fuels. However, several obstacles slow its commercial adoption, such as our project’s focus which is its limited biomass growth. Researchers have studied symbiotic bacteria to better facilitate algae growth but it is rarely discussed how exactly the bacterial candidates contribute to algae growth. Here we explore the relationship of Methylobacterium spp. with our model algae Chlorella sorokiniana in media cultures unfavorable to both candidates’ growth. The key objective of this project is to determine if our isolated bacterial species contribute to more significant algal growth and quantify the changes in populations through genetic sequencing. We understand that there is some interaction between Chlamydomonas reinhardtii and Methylobacterium spp. This co-culture allowed the algae to grow on a nitrogen source it could not process. We want to further explore this proposed mutualism with different symbiotic bacterial species and our model Chlorella algae from this previous study’s results. With this project, we are reapproaching the mutualism model idea, emphasizing the culture environment and the resources available. We want to determine if the limited nutrient sources can be managed by interspecies interaction and how much the proposed mutualism materializes with our candidates. This project incorporates co-culture trials, isolation of wild bacterial and algal species, and quantitative growth assays with our known algal and bacterial candidates to determine if and how much the interspecies interaction contributes to overall algal growth. From our preliminary results, we found that in environments where both the algae and bacteria could not be successful individually, the combination of the cultures allowed greater success in both populations. | Poster #: 72 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: Arabidopsis thaliana, glutaredoxin, CRISPR Cas9 Project Title: Functional characterization of AtGRXS6 and AtGRXC11 in Arabidopsis thaliana Author List: Preston Rubio, Christian; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Escobar, Matthew; Faculty, Biological Sciences, California State University San Marcos Abstract Nitrogen is an essential nutrient for plants, and also acts as a chemical signal causing major transcriptional and developmental changes. Recent evidence suggests that glutaredoxins (GRXs) may be involved in plant nitrogen signalling. GRXs are small redox enzymes that are found in all eukaryotes, and are generally involved in disulfide bridge reduction. Class III GRXs are unique to plants, but most remain functionally uncharacterized. This study focuses on two class III GRX genes, AtGRXS6 and AtGRXC11, that are strongly transcriptionally upregulated by nitrate in the soil. To study AtGRXS6 and AtGRXC11, we generated knockout lines for AtGRXS6 and AtGRXC11 individually, as well as a double AtGRXS6/AtGRXC11 knockout mutant, using CRISPR Cas9-mediated mutagenesis. We have now identified six independent AtGRXS6, AtGRXC11, and AtGRXS6/AtGRXC11 mutants with defined insertion and/or deletion mutation sites by DNA isolation, PCR amplification of the target gene, and sequencing. Additionally, we created AtGRXS6 overexpression lines, and demonstrated that these transgenic lines showed ~10-80-fold increases in AtGRXS6 transcript abundance by using real time RT-PCR. We are now in the process of determining the phenotypic effects of the knockout mutations or overexpression lines, with a specific emphasis on root system architecture and typical developmental responses to nitrogen in the soil. The long-term goal of this project is to better understand plant nitrogen use efficiency so that synthetic nitrate fertilizers can be applied more effectively in agriculture. This will minimize over-application of nitrogen fertilizers, which has significant detrimental effects on the environment. This project was funded, in part, by the CSUPERB: 2021 Graduate Student Research Restart Program to C.P.R. | Poster #: 73 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: microalgae, DNA, BODIPY Project Title: Axenic culturing and macronutrient analysis of a wild algae isolated from recharging basin water Author List: Xiong, Alina; Graduate, Chemistry and Biochemistry, California State University, Fresno, Presenting Author
Soghomonian, Angela; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Soghomonian, Mark; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Li, Yimeng; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Mujic, Alija; Faculty, Biology, California State University, Fresno
Maitra, Kalyani; Faculty, Chemistry and Biochemistry, California State University, Fresno Abstract Microalgae are one of the most promising alternative sources for renewable energy in the form of biofuel. The algal cells are also capable of storing a high amount of protein and starch making the biomass an attractive feedstock resource for the agricultural and the biotech industry. Algae are considered as the safer, noncompetitive, and rapidly growing organisms with high lipid content and nutrient value as compared to other sources of fuel that are harmful to the environment. We are interested in studying the phylogeny of locally growing native algae and analyzing its nutrient content for its potential use as a biofertilizer or food resource in agriculture.
A filamentous strain of wild algae was collected from storm drain basins. It was carefully isolated over many trials following inoculation with a single cell in plates containing sterile growth media. Different combinations of the treated basin water and BG11 media were used to facilitate the axenic growth of the wild algae under similar controlled conditions over a period of five weeks to produce healthy filaments of the native algae. DNA extraction, amplification, and purification for sequencing are currently ongoing for identifying the species of the isolated algae. A preliminary result of boron-dipyrromethene (BODIPY) dye staining indicated an abundance of lipid bodies in the filamentous cells. Our work will aid in identifying more native strains of algae and dissecting their potential to function as useful resources for biofuel and feedstock in agriculture.
| Poster #: 74 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: CRISPR/Cas-9 mutagenesis, Genetics, C. elegans Project Title: Using CRISPR to test gene products as possible targets of chalcones in Caenorhabditis elegans Author List: Sárközi, Zita; Graduate, Biology, California State University, Fresno, Presenting Author
Calderon-Urrea, Alejandro; Faculty, Biology, California State University, Fresno Abstract Plant-parasitic nematodes (PPNs) are among the biggest burden on modern agriculture, causing significant monetary, yield, and quality loss each year. Currently, synthetic chemical nematicides are used to control parasitic worms and protect crop plants. However, applying these chemicals can be challenging, as the temperature, soil water content, half-lives, and speed of microbial degradation can greatly affect their concentration in the soil. Additionally, chemical nematicides often show low selectivity toward PPNs and kill non-target organisms as well. Hence, the development of highly specific, sustainable nematicides is urgent. Previous research identified multiple chalcone analogs, which could be a safe and effective mode of nematode control. The study showed that Chalcone-17 and other chalcone analogs could effectively control Meloidogyne incognita, a primary PPN target while being safe for soil microbes. However, their mode of action is not yet known. Due to their specific structural attributes, they likely inhibit different proteins or enzymes. Previous research identified two putative Chalcone-17 target proteins, PIF1 helicase and MRPL15 large ribosomal protein, in resistant Caenorhabditis elegant mutant model nematodes. We hypothesize that mutations in one of these genes lead to a structural change in the expressed protein, making it inaccessible for Chalcone-17 without affecting its biological function. During the current research, I use CRISPR-based mutagenesis to induce alterations in the genes at specific locations and prove or negate their role in Chalcone-17 sensitivity. If resistance occurs in mutagenized animals, the presence of the mutations will be verified by targeted Sanger sequencing. As preliminary results, we have established in our laboratory ribonucleoprotein microinjection methodologies for C. elegans. We will soon begin microinjections of the various sgRNA sequences along with the Cas-9 protein. Identifying the target of Chalcone-17 could contribute to the development of effective chalcone cocktails, in which each component binds and inhibits a different protein or enzyme. Further research is necessary to distinguish the different targets of each potent chalcone analog and verify their safety for non-target organisms. | Poster #: 75 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: anaerobic digestion , hydrogen gas, biogas Project Title: Microbial Populations Shift During Mesophilic and Thermophilic Anaerobic Digestion Author List: Deocampo , Leanne ; Undergraduate, Civil Engineering and Construction Engineering Management , California State University, Long Beach, Presenting Author
Ly , Maggie ; Graduate, Civil Engineering and Construction Engineering Management , California State University, Long Beach, Presenting Author
Asvapathanagul, Pitiporn ; Faculty, Civil Engineering and Construction Engineering Management , California State University, Long Beach
Abstract The goal of this study was to investigate shift of microbial populations using lab-scale batch anaerobic digesters at 35°C (mesophilic condition) and 55°C (thermophilic condition). Two different anaerobic sludge seeds were obtained from two different full-scale anaerobic digesters at a water reclamation plant located in California. Primary and thickened sludge from the same treatment plant were separately employed as substrates. The reactors were incubated at 35°C and 55°C for 14 days. Samples were taken on the day 3, 5, 7, 10 and 14. Alkalinity, chemical oxygen demand (COD), ammonium ions (NH4+-N), volatile fatty acids (VFA) and suspended solids (SS) ranged 3730-7650 mg/L as CaCO3, 152-1198 mg/L, 21.6-96.5 mg/L -N, 290-5243 mg/L and 650-8350 mg/L, respectively. Biogas, a mixture of gases: CH4, CO2, N2, H2S, H2, O2, H2O, was also collected and analyzed along with mixed liquor samples using gas chromatography. Sludge was subjected to DNA extraction and next generation DNA sequencing using iSeq100, Illumina.
| Poster #: 76 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: ceruloplasmin, estrogen, ATP7B, , Project Title: Effects of estrogen on blood plasma ceruloplasmin activity, apo to holo ratio, and mRNA expression for Cp and ATP7B, as determined in ra Author List: Sanqui, Cossette; Undergraduate, Chemistry and Biochemistry , California State University, Fresno, Presenting Author
Garcia, Valerie; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Savage, Cannon; Graduate, Chemistry and Biochemistry, California State University, Fullerton
Kidane, Theodros; Rio Hondo community College, California State University, Fullerton
Linder, Maria; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract Ceruloplasmin (Cp) is a multifunctional Cu binding protein, particularly in the blood plasma, where it accounts for 40-70% of the total Cu[1]. Its functions include oxidation of Fe(II), inactivation of amine signaling agents and NO, neutralization of reactive oxygen species, and delivery of Cu to the surface of cells for uptake, which converts it from the holo to the apo form. It has long been known that levels of plasma Cp increase in response to taking estrogen and progesterone containing oral contraceptive drugs, as well as in pregnancy, when both hormones increase. Plasma Cp is thought to be produced mainly or exclusively by liver hepatocytes, from which it is secreted into the blood by exocytosis, after receiving Cu from the Cu “pump”, ATP7B in the transGolgi network. ATP7B is also expressed in the epithelial cells of the kidney. The objective of the studies to be presented were to determine whether estrogen increases expression of mRNA not only for Cp but also for ATP7B, and whether not just the liver but also the kidney might be responding to the hormone. Since Cp is a major source of copper for the fetus in gestation, we also examined whether estrogen treatment would alter the relative concentrations of holo and apoCp.
For this, female Sprague Dawley rats were injected subcutaneously with 17b-estradiol daily for two weeks, euthanized, and blood (for plasma), liver and kidney were collected for analysis. The enzyme activity of Cp was measured using o-dianisidine oxidation. Cp protein levels and proportions of holo to apoCp were determined by Western blotting of native PAGE gels and densitometry. mRNA levels were determined by Real Time qPCR relative to 18S rRNA.
Estrogen treatment increased circulating Cp oxidase activity and protein levels about 80% over that of controls. It also slightly increased the proportion of holo to apoCp. However, mRNA expression for Cp and ATP7B were unchanged in the hepatocytes, and the same was the case for kidney epithelial cells. We conclude that the increase in Cp circulating in the blood in response to long-term estrogen treatment is not the result of increased expression of ATP7B, and that it occurs either through an increased translation of existing Cp mRNA, and/or through effects of the hormone that decrease its rate of removal from the blood circulation.
[1]Linder, M.C. (2016) Ceruloplasmin and other copper binding components of blood plasma and their functions: An update. Metallomics 8: 887-905. | Poster #: 77 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Aspirin, Unfolded Protein Response, Cancer Project Title: A Mechanistic Insight into the Chemopreventive Action of Aspirin in Colorectal Cancer Author List: Rauth, Nishi; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author, Nagel Award Finalist
Bhandari, Deepali; Faculty, Chemistry and Biochemistry, California State University, Long Beach Abstract Epidemiological studies indicate reduced incidence of cancer in individuals on a daily regimen of low dose of acetylsalicylic acid aka aspirin, but the biochemical mechanism(s) for this protection remains elusive. Aspirin is a non-steroidal anti-inflammatory drug and has been linked to reduced occurrence of many cancers, particularly colorectal cancer. Cancer cells have altered metabolic demands due to the hypoxic environment and rapid proliferation rate that can induce proteotoxic or endoplasmic reticulum (ER) stress. During ER stress, cells activate an evolutionarily conserved signaling pathway known as the Unfolded Protein Response (UPR). The adaptive UPR signaling has been correlated to acquisition of malignancy and chemoresistance in cancer cells. Therefore, we aimed to investigate if aspirin affects the ability of cancer cells to activate the UPR upon induction of ER stress. We treated colorectal cancer cell line DLD1 with sodium salicylate (NaSal), a metabolic derivative of aspirin and induced ER stress with tunicamycin, which halts N-linked glycosylation, an important post-translational modification of proteins in the ER. We then analyzed the expression of various proteins involved in UPR signaling via western blotting. We also used a flow cytometry based assay to detect apoptosis after treatments with NaSal and tunicamycin. Our data indicate that NaSal treatment downregulates UPR signaling. Specifically, we observed significantly reduced expression of glucose-regulated protein of 78kDa (GRP78), a master regulator of UPR whose overexpression has been implicated in cancer progression and chemoresistance. We also observed a greater percentage of early apoptotic cells in NaSal-tunicamycin treated set as compared to the control sets. Together, this work sheds light on a potential mechanistic basis for the chemopreventive action of aspirin. Our current and future experiments are directed toward understanding the molecular mechanism by which NaSal lowers expression of GRP78. | Poster #: 78 Campus: San José State University Poster Category: Biochemistry Keywords: SIRT1, Regulation, Small Molecule Modulator Project Title: Characterizing Novel Small Molecule Regulators of SIRT1 Author List: Wang, Ningkun; Faculty, Chemistry, San José State University
Jumawan, Calvin; Graduate, Chemisty, San José State University, Presenting Author Abstract SIRT1, an NAD+-dependent deacetylase, is involved in a wide variety of cellular processes and regulation, hence the change in activity of SIRT1 may result in a variety of diseases. Resveratrol, a natural polyphenol, has been investigated for its modulatory effects on SIRT1 activity. Recent research indicates that various naturally occurring compounds, such as triterpenoids and alkylresorcinols, also have an effect on SIRT1 activity. To corroborate these findings, we first investigated the kinetic characteristics of SIRT1 in the presence and absence of the selected compounds with a range of peptide substrates. Furthermore, we conducted kinetics experiments through continuous enzyme-coupled assays to determine whether the kcat or KM values are the primary contributor to the change in SIRT1 activity, and compared this kinetic trend to determine whether the chosen small molecule effectively upregulates the kinetic activity of SIRT1. We would examine the EC50 of each compound to determine the optimal concentration to achieve the maximum effect. These studies will help in our understanding of the impact of various small compounds on the activity of SIRT1. | Poster #: 79 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: apolipoprotein, pyrene, self-association Project Title: A Pyrene Conformational Study on the Self-Association of Apolipoprotein A-I Author List: Jauregui, Juliette ; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Monteon, Ramiro; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach
Weers, Paul; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract Cardiovascular disease caused by elevated cholesterol levels is a major cause of death. The correlation between risk of cardiovascular disease and low-density lipoprotein has been well established. High-density lipoproteins (HDL) play a critical role in reverse cholesterol transport, but the protective role of HDL is still not well understood. Apolipoprotein A-I (apoA-I) is the major protein of HDL; the 28 kDa protein is comprised of an ordered N-terminal (NT) domain and a less structured C-terminal (CT) domain. The protein self-associates at concentrations above 0.1 mg/mL, which is mediated by the CT domain. Since this is also the site of lipid binding to form HDL, it is important to understand how the protein forms oligomers. Previous studies showed that key residues in helix 8 and 10 of the CT domain are vital for apoA-I self-association. To determine how apoA-I CT helices align to form an oligomeric protein, a site-specific fluorescent probe, N-(1-pyrene) maleimide (NPM), was utilized. Pyrene is a spatial sensitive probe, producing a strong excimer fluorescence emission peak when in proximity of each other. When NPM is attached to strategic positions in helix 8, 9, and 10, important insight in the orientation of apoA-I CT helices in oligomers can be obtained. The NPM-labeled helices can interact in either a parallel or antiparallel alignment which can be observed via excimer emission peaks. To covalently attach pyrene to apoA-I, site-directed mutagenesis was employed to introduce cysteine at positions S25C (located in the NT domain and served as a control), S201C, Q216C, and S231C (located in helix 8, 9 and 10 of the CT domain). Mutants were expressed in E. coli and purified via Ni-affinity and gel filtration chromatography. The apoA-I S201C mutant was labeled with NPM by preincubation with dithiothreitol (DTT) to ensure cysteine was in a reduced state. NPM was incubated in molar excess with apoA-I S201C for 16 h followed by purification using Ni-affinity chromatography columns to remove excess DTT and NPM. After elution the stoichiometry of labeled protein was calculated by examining the absorbance of protein at 280 nm and NPM absorbance at 345 nm. This showed a labeling efficiency of 68%, which is sufficient for pyrene excimer fluorescence to determine the orientation of CT helices in apoA-I oligomers.
Research is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number GM089564.
| Poster #: 80 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Biochemistry Keywords: biosynthesis, natural products, isotope feeding Project Title: Gene Knockouts and Feeding Studies to Elucidate Biosynthesis in Micromonospora echinospora Author List: Owen, Diana; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, Presenting Author
Ednacot, Eirene ; Undergraduate, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo
Watts, Katharine; Faculty, Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo Abstract TLN-05220 is an aromatic polyketide isolated from the Actinobacteria Micromonspora echinospora, with a unique multi-cyclic angular structure and nitrogen-containing (piperazinone) ring, which is thought to be amino-acid derived. TLN-05220 displays antibiotic activity against resistant pathogens in the same therapeutic range as vancomycin. The long-term goal of this project has been to map out the biosynthetic pathway of TLN-05220 by identifying genes and enzymes responsible for the individual bond-forming reactions that create this molecule. Ultimately, the deep understanding of TLN-05220 biosynthesis gained from this investigation is critical for use of TLN-05220 in future antibiotic development. There have been two main techniques utilized to determine these mechanisms. First, genetic knockout experiments are used to elucidate the enzymes responsible in the pathway. To inactivate a gene encoding an enzyme of interest, we utilize intergeneric conjugation which involves the Actinobacteria genome undergoing homologous recombination with a plasmid carrying apramycin resistance. Apramycin containing media is used to select for successful exconjugants. Polymerase Chain Reaction (PCR) and gel electrophoresis are used to confirm the knockouts by examining for the lack of the gene as well as the insertion of the plasmid. To date, we have analyzed exconjugants from knockout of gernylgernyl diphosphate and ketosynthase genes. The PCR screen determined that genes had not been successfully knocked out, even though they were able grow on the selective media. While more exconjugants will be screened, it is hypothesized that the cells are spontaneously developing a resistance when put under selective pressure. The second technique is an amino acid feeding study, in which M. echinospora colonies are supplemented with stable isotope labeled amino acids in the agar media and products are analyzed via mass spectrometry. This particular method is helpful in determining which amino acids, if any, are incorporated into the piperazinone ring. To prepare samples for mass spectrometry, cells are grown for 5-7 days before being dried and extracted using organic solvent. To date, this technique has confirmed glycine and alanine incorporation, and the investigation of serine and isoleucine inclusion is currently underway. Funding for this project was provided by the Funding: CSUPERB President’s Commission Scholarship. | Poster #: 81 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: RNA binding proteins, RNA binding affinity, post translational modifications Project Title: Characterization of the Linker 2 region of Polypyrimidine Tract Binding Protein 2 Author List: Cuellar, Andres; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Munoz, Gabrial; Undergraduate, California State University, Fullerton
Patel, Milan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Polypyrimidine Tract Binding Proteins, PTBP1 and PTBP2, belong to a family of RNA binding proteins that function to regulate the process of alternative splicing. PTBP1 and PTBP2 can bind to CU rich elements within the premature mRNA to either promote or inhibit splice site selection. Mis regulation of alternative splicing can lead to the production of aberrant proteins and result in neurodegenerative diseases. PTBP1 is expressed in nearly all tissues but is absent in neurons while PTBP2 is expressed almost exclusively in neurons. PTBP1 and PTBP2 share 74% sequence identity and are similar in domain arrangement. These proteins regulate over-lapping and distinct sets of target exons. Notably, differences in expression patterns and splicing activity play a critical role for neuronal differentiation and maturation. How these two proteins can exert different tissue specific splicing outcomes remains unknown.
Recently, we demonstrated that under in vitro splicing conditions, PTBP2 has many more sites of phosphorylation compared to PTBP1 in the less conserved regions including Linker 2. Counterpart PTBP1 residues are non-conservative substitutions that are unable to undergo phosphorylation. Moreover, PTBP2 phosphorylated residues are conserved across lower species signifying an important role for these residues in PTB2 splicing activity. Thus, we hypothesize that phosphorylation in PTBP2 plays a role in its neuronal splicing activity. Previous studies highlight a role for PTBP2 Linker 2 in its neuronal specific splicing activity. We note that Thr298 and Ser308 in Linker 2 is phosphorylated under splicing conditions.
Here, we aimed to determine the role of PTBP2 Linker 2 region phosphorylation in RNA binding affinity. To this end, we generated Thr298Ala and Ser308Ala mutants and recombinant expressed and purified the proteins. We assayed the proteins for RNA binding affinity to the 3’ splice site of the regulated neuronal N1 exon of the c-Src pre mRNA via electrophoretic mobility shift assays. Our results highlight that T298A and S308A have 2.1 and 2.6 lower binding affinity to this substrate compared to wild type PTBP2 suggesting a role for these residues in RNA binding. We have generated phosphomimic mutants T298D and S308D and are currently assaying these mutants for RNA binding activity. Results from this study will address whether PTBP2 Linker 2 region phosphorylation plays a role in its RNA binding activity and in turn in neuronal splicing regulation.
| Poster #: 82 Campus: California State Polytechnic University, Pomona Poster Category: Biochemistry Keywords: BaiK, protein purification, enzymes Project Title: Homology Modeling and Protein Purification of the Enzyme BaiK for Structural and Mechanistic Studies Author List: Mata, Carolina; Undergraduate, Chemistry & Biochemistry, California State Polytechnic University, Pomona, Presenting Author
McCulloch, Kathryn; Faculty, Chemistry & Biochemistry, California State Polytechnic University, Pomona Abstract Primary bile acids play a major role in digestion by assisting in the absorption of fats and oils entering the body. Once in the gastrointestinal tract, naturally occurring gut bacteria such as Clostridium scindens convert primary bile acids into secondary bile acids via a pathway encoded by the bile acid inducible operon, which includes a putative coenzyme A transferase enzyme, BaiK.
Pre-COVID, a recombinant approach using E. coli was chosen to characterize BaiK. First, the E. coli Tuner expression line was transformed using a plasmid containing the baiK gene for overexpression. Large-scale overexpression was used to obtain large quantities of the protein, and the resulting cell pellet was collected for purification via column chromatography. Protein purification strategies have been optimized. Challenges in purifying larger amounts of protein to yield a single peak in chromatograms were circumvented by performing both affinity and gel filtration chromatography on the same day to avoid aggregation. The concentrated protein will next be used in crystallization screenings.
Homology modelling studies were used to obtain a hypothetical BaiK structure, which can help determine function and characteristics. This was done using available web-based tools such as BLAST, SWISS model, EXPASY, and Xtalpred. These servers were used to convert the genomic sequence to a protein sequence, remove the histidine tag using enterokinase, and align the sequence with published structures of PDB proteins. After investigations based on sequence similarity, conserved areas, and proposed function, multiple proteins were selected as templates for BaiK’s homology model. The model was generated using SWISS model, which generated a homo dimer model consisting of a 37% sequence identity. The model had a favorable QMEAN score, with highly conserved areas in the active site. Verification of the model was achieved using the built-in functions from SWISS model.
Dr. McCulloch has received past support from CSUPERB via a New Investigator award and has currently received support from the Cal Poly RSCA program. Funding has also been provided by the Louis Stokes Alliances for Minority Participation fellowship awarded to Carolina Mata. | Poster #: 83 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: DNA Repair, biochemistry , fluorescence microscopy Project Title: Investigating SLX4-Dependent Recruitment of Rad1-Rad10 to DNA Double Strand Breaks During DNA Repair in Dividing Cells Author List: Hakimian, Jonathan; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author, Nagel Award Finalist
Aros, Melissa; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Fischhaber, Paula; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract As DNA damage is introduced into a cell, it triggers checkpoint arrest which inhibits replication initiation. DNA damage also leads to the phosphorylation of Slx4, which facilitates a counteracting pathway called checkpoint signal dampening which promotes homologous recombination, thereby aiding in the repair of some forms of DNA damage including DNA Double-strand Breaks (DSB). Our project focuses on whether the phosphorylation of Slx4 affects recruitment of Rad1-Rad10 at DSB Repair sites in S. cerevisiae when the type of repair employed is either Synthesis-Dependent Strand Annealing (SDSA) or Single-strand Annealing (SSA). To test this, we utilized an Slx4 phosphorylation mutant (slx4-7MUT) that prevents the activation of checkpoint signal dampening and allows us to determine whether fluorescently-labeled Rad10 (Rad10-YFP) is recruited when Slx4 is unable to be phosphorylated. We hypothesize that checkpoint signal dampening results in Slx4-dependent recruitment of Rad1-Rad10 to DSBs during S phase.
Following the construction of a yeast strain containing the slx4-7MUT gene, we combined the slx4-7MUT gene with other genes needed for the assay, including a fluorescently-labeled, inducible DSB site, and Rad10-YFP. With the resulting strains, we conducted fluorescence microscopy experiments with cell cycle-synchronized cultures to visualize Rad10-YFP recruitment to DSBs being repaired either by SSA or SDSA, and as a function of Slx4 checkpoint signal dampening (i.e. containing slx4-7MUT or an Slx4 wild-type control). Preliminary results from a wild-type SSA strain show that 90 minutes following release there is an increase of co-localized foci in S phase cells in the DSB-induced versus uninduced samples. At 120 minutes following release, the percentage of co-localized foci in S phase cells is found to be similar between the induced and uninduced samples. These results show that our assay system is working successfully. Therefore, additional experimental trials comparing SLX4 wild-type and slx4-7MUT strains will reveal whether Slx4 is required to recruit Rad10 to SSA sites in S phase. If these results show diminishment of Rad10 recruitment in slx4-7MUT strains as compared to wild-type controls in S phase, it will support our hypothesis that Slx4 recruits Rad1-Rad10 in S phase. This project could inform our understanding of how the biochemical underpinnings of DSB repair opens possibilities of cancer treatment. | Poster #: 84 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: Cancer, Metformin, Toxicology Project Title: Investigating the ability of metformin to scavenge for methylglyoxal and prevent protein and DNS advanced glycation end-products Author List: Boggess, Ryan; Graduate, Biochemistry, California State University, Northridge, Presenting Author
Tamae, Daniel; Faculty, Biochemistry, California State University, Northridge Abstract Methylglyoxal is a highly reactive ⍺-oxoaldehyde that arises as a byproduct of glycolysis, lipid metabolism, and amino acid metabolism. Recent evidence has established that methylglyoxal reacts with protein, lipids and DNA to form advanced glycation end-products (AGEs) that underlie diabetes pathology. We are particularly interested and well-positioned to quantify DNA-AGEs via a novel, validated liquid tandem mass spectrometry (LC/MS/MS) method. Increased levels of glucose in diabetics leads to increased levels of methylglyoxal which could potentially play a role in the increased rates of certain types of cancer in diabetes patients. Metformin is a cheap and commonly prescribed drug for diabetics and a mounting body of evidence has shown it to lower the relative risk for a variety of cancers in metformin users relative to non-diabetic individuals. The biguanide pharmacophore of metformin and other guanides have been found to react with methylglyoxal forming an imidazolinone ring. My work is focused on investigating metformin’s ability to scavenge methylglyoxal resulting in diminished protein and DNA-AGEs and decreased mutations. I have synthesized methylglyoxal from pyruvic dimethylacetal through an acid-catalyzed reflux reaction. The synthesis was optimized through fractional distillation and two of five fractions contained pure methylglyoxal as determined through 1H-NMR analysis. A known concentration of t-butanol was used to calculate our methylglyoxal concentration via integration of 1H-NMR peaks, indicating 377.7 mM pure methylglyoxal sample was obtained. The reaction between methylglyoxal and metformin has been briefly investigated by incubating both synthesized and commercially purchased methylglyoxal with metformin and semi-quantitative HPLC. In both the synthesized and commercially purchased methylglyoxal reactions, the metformin peak area decreased about 50% in the presence of methylglyoxal. We are in the process of using an enzyme-linked immunosorbent assay (ELISA) to quantify metformin’s effect on the protein AGE, carboxyethyl lysine and will also use LC/MS/MS to quantify the effect of metformin on the DNA-AGE, N2-(1-carboxyethyl)-2’-deoxyguanonsine (CEdG). | Poster #: 85 Campus: California State University, Sacramento Poster Category: Biochemistry Keywords: Drosophila, Aging, behavior Project Title: Principal Component Analysis of aging-related pheromones in D. melanogaster. Author List: Brown, Samuel; Graduate, Chemistry, California State University, Sacramento, Presenting Author
McInnes, Abigail; Undergraduate, Chemistry, California State University, Sacramento
Brenman-Suttner, Dova; York University, Toronto, ON, Canada
Simon, Anne; University of Western Ontario, London, ON, Canada Abstract In insects, social behaviors like aggregation, aggression, attraction, foraging, group behavior and courtship are modulated by pheromones. Molecularly, pheromones are long-chain hydrocarbons that differ in chain length and saturation status. In the fruit fly Drosophila melanogaster, pheromones are produced by specialized cells called oenocytes that are associated with the fly fat body. Following synthesis, those fly pheromones are displayed on the cuticle of the animal, and thus are often referred to as cuticular hydrocarbons (CHC). CHC have recently been proposed to also play a role in in the regulation of aging and longevity. CHC profiles have been shown to change with increasing age, and altering CHC levels in adult flies leads to changes in mating behavior, aging and longevity.
Recent reports suggest that aging, as well as metabolic health, such as onset of diabetes or weight, may be transmitted to subsequent generations by epigenetic-inheritance mechanisms. Epigenetic inheritance may affect not just metabolism, but even behavioral traits: young progeny of old D. melanogaster maintain a social spacing behavior that is similar to the behavior of old parents, rather than resembling the behavior of young flies. Together, these data suggest that social behavior may be an age-dependent and epigenetically-inherited trait, with implications for the aging process as well. Thus, CHC are prime candidates to explore the complex relationship between aging, metabolism and epigenetic inheritance.
In order to investigate this relationship, we conducted gas chromatography/mass spectroscopy analysis (GC/MS) of pheromones obtained from young (7-days old) and old flies (50-day old), and their respective offspring. The GC/MS data was then subjected to Principal Component Analysis (PCA) to identify age-dependent patterns. Our data show that the CHC profile of young F1 offspring of young parents is similar to the profile of young parents, while the profile of young F1 offspring to old parents is likewise similar to the old parental CHC profile. Since all offspring were measured when 7-days old, these data demonstrate that age-dependent CHC profile changes are inherited to the offspring, presumably via an epigenetic mechanism. Our data therefore suggest that aging and longevity may be modulated by epigenetically-inherited CHC profiles.
| Poster #: 86 Campus: California State Polytechnic University, Pomona Poster Category: Biochemistry Keywords: Acinetobacter radioresistens 50v1, Minimal metal media, planetary protection Project Title: Spacecraft-associated Acinetobacter radioresistens 50v1 cultivation in minimal metal media Author List: Aldaco, Juan; Graduate, Chemistry and Biochemistry, California State Polytechnic University, Pomona, Presenting Author
Jaramillo, Daniel; Graduate, Chemistry and Biochemistry, California State Polytechnic University, Pomona, Presenting Author Abstract Spacecraft assembly facilities are to be kept clean when assembling spacecraft to prevent forward biological contamination in support of planetary protection. This is performed by applying alcohol wipes such as ethanol on bench tops and spacecraft materials. It was discovered that some microorganisms will survive under these conditions which are later referred to as spacecraft-associated microorganisms. The Acinetobacter species are ones capable of surviving such conditions and under this species is a strain called Acinetobacter radioresistens 50v1 which was isolated from the Mars Odyssey Orbiter. Previous studies have shown that this strain is capable of using ethanol as a carbon source under a minimal nutrient medium containing Fe2+; however, it was also discovered using Fe2+ as a sole trace mineral would not be as effective when comparing metabolic rates of different species. Therefore, the purpose of this research is to conduct an applicable minimal metal media for cultivation. We were able to create two different minimal metal media to compare growth rate, lag time, and cell density. From our results, we were able to conclude that Acinetobacter radioresistens 50v1 grows better in a lower pH media and with the media containing a larger variety of metal ions | Poster #: 87 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Methylotrophic bacteria, Methanopterin Biosynthesis, X-ray crystallography Project Title: Insights into the evolution and mechanism of DmrA, a dihydrofolate reductase homolog Author List: Perez, Ivan; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Potla, Sandesh; Eikon Therapeutics
Axelrod, Herbert; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Rasche, Madeline; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Dihydromethanopterin reductase A (DmrA) catalyzes the reduction of dihydromethanopterin (H2MPT) to H4MPT, which is required for methanol catabolism by some methylotrophic bacteria. The dmrA gene in Methylobacterium extorquens is a homolog of the dihydrofolate reductase gene (dhfr), and it has been hypothesized that DmrA has acquired H2MPT-reducing activity by undergoing alterations in structure and substrate affinity via DHFR gene duplication and mutation. Despite these changes, key enzyme-substrate interactions appear to be conserved, suggesting that the catalytic mechanism from DHFR has been retained in DmrA, albeit with deviations accommodating the larger substrate, H2MPT. To gain insight into the nature of ligand-enzyme interactions, DmrA was purified by nickel chromatography and gel filtration. Two crystal forms were obtained by vapor diffusion crystallography, and structures were superimposed with DHFR using PyMOL. Both structures approximate DHFR in its closed (NADPH-bound) conformation, with NADPH-binding residues (A9, L16, H21, L53, T54, R72, S73, G94, I95, R121) resembling those in DHFR. The enzyme binds H2MPT utilizing W24, D35, and a water molecule. Two alpha-helices likely stabilize the aminobenzene moiety of H2MPT. DHFR-like interactions between an L20 loop and F-G-like loop from DHFR are also seen. Computational modeling indicated that the H2MPT binding pocket is larger than the corresponding pocket in DHFR. The loop interactions point towards DmrA adopting a closed conformation during its catalytic cycle much like DHFR. These findings indicate that DmrA has largely kept the substrate-binding mechanism from DHFR, while a five-residue insertion creates a loop that likely increases the substrate pocket volume to accommodate H2MPT. [Funded by CSUPERB, CSUF, and NSF.] | Poster #: 88 Campus: California State University, East Bay Poster Category: Biochemistry Keywords: Chemical biology, Imaging agent , Aptamers Project Title: Selection of aptamers for a magnetic resonance imaging agent Author List: Reyes, Roelyne; Graduate, Chemistry and Biochemistry, California State University, East Bay, Presenting Author
Abstract Due to the importance of the technique in medicine, millions of magnetic resonance imaging (MRI) scans are performed each year in medical diagnosis. About a third of these scans use gadolinium-based contrast agents (GBCA), a compound that can improve the resolution of the images obtained. Though widely used, GBCAs can cause adverse side effects and along with safety issues, the relaxivity of the current clinical GBCAs are also less than optimal. Relaxivity is one key parameter that measures the effectiveness of an MRI contrast agent. A higher value will allow for the GBCA dosage to be reduced and therefore, can mitigate its side effects. One way to improve relaxivity is to significantly increase the GBCA's molecular size. Our proposal is to couple a GBCA to an aptamer to drastically increase its mass from ~500 Da to ~12 kDa. Aptamers are short sequences of single-stranded DNA or RNA that adopt characteristic three-dimensional conformations. A specific nucleotide sequence in an aptamer can then lead to a unique conformation that displays a selective and strong interaction with a particular compound, also known as the target.
The specific aim of this project is to isolate an aptamer strand that has a high affinity to a GBCA. To achieve this, we chose as our target the compound gadolinium-diethylenetriaminepentacetate, which is the key component of a GBCA currently used in the clinics. To find the high-affinity binding aptamers, we started with a large aptamer library containing ~1015 strands with randomized sequences, which we immobilized on an affinity column. A solution containing the target was then passed through the column, causing the aptamers that bound to the GBCA to be eluted from the column. The aptamers were amplified through polymerase chain reaction, separated into single strands, and subjected to another round of immobilization, elution, and amplification. Through multiple rounds of the selection process, an aptamer pool with high selectivity and affinity for the target was isolated.
20 selection rounds have been completed and will be presented in the poster. The completion and success of each round was monitored via electrophoresis. The amplification reaction product obtained in each round was quantified by UV absorbance. The yield was found to be between 17.5-45 ng/uL. After 20 rounds, our pool of aptamers can bind to a low micromolar (20 uM) concentration of the target.
This project is funded by CSUPERB NIG and CSU East Bay FSG.
| Poster #: 89 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: shuttling proteins, RNA binding proteins, gene mutants Project Title: Identification and Characterization of Nuclear Export Sequences in the RRM2 Region of Polypyrimidine Tract Binding Protein 1 Author List: Horani, Azeem ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author
Cisneros, Greer; Undergraduate, Biological Sciences, California State University, Fullerton
Vu, Kimberly; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Nguyen, Duyen; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Perez, Constanza; Undergraduate, California State University, Fullerton
Miyamoto, Alison; Faculty, Biological Sciences, California State University, Fullerton
Keppetipola, Niroshika; Faculty, Chemistry and Biochemistry, California State University, Fullerton
Abstract Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that shuttles between the nucleus and cytoplasm to provide a variety of cellular functions. In the nucleus, the protein is responsible for regulating pre-mRNA alternative splicing and polyadenylation. In the cytoplasm, it interacts with internal ribosomal entry sites to regulate translation initiation and RNA site-specific localization. PTBP1 is a 557 amino acid polypeptide that is structurally comprised of an N-terminal region and four RNA Recognition Motifs (RRMs) connected via three linker regions. The sequence contains two nuclear localization sequences (NLS) and one nuclear export sequence (NES) at the N-terminal region. However, previous studies highlight that the RRM2 region plays a critical role in PTPB1 nuclear export. Removal of the RRM2 region terminates nuclear export completely, resulting in PTBP1 being nuclear. These results suggests a yet to be discovered role for PTBP1 RRM2 in nuclear export.
Upon scanning the amino acid sequence of RRM2 we identified stretches of sequences similar to the NES consensus Ø-X (2-3) in which Ø is Leu, Val, Ile, Phe or Met and X is any amino acid and the number in parentheses denote the number of repeats. We found five stretches of amino acid sequences in the RRM2 region that correspond to the consensus; LDVLHQIFS (residues197-205), VLKIITFTKN (residues 210-219), LSLDG (residues 239-243), IDFSKL (residues 255-260) and LTSLNV (residues 260-265).We note these regions are conserved in all jawed vertebrates.
Here, we aimed to characterize the five potential nuclear export sequences by creating deletion mutants and assaying the mutants for localization using heterokaryon assays. Deletion mutations were generated by the two step over-lap extension PCR method and cloned into the mammalian expression vector pcDNA3.1(+). We have successfully created four of the five mutants and are currently assaying these mutants for localization in heterokaryon assays. Wild type PTBP1 serves as the control. We are also assaying the mutants in mouse neuro 2A cells for protein expression and splicing activity via Western Blotting and reverse transcription PCR respectively to determine if the mutants have altered splicing activity compared to wild type PTBP1. Overall, results from these studies will highlight whether PTBP1 RRM2 has one or more nuclear export sequences and reveal a novel function for this region of the protein.
| Poster #: 90 Campus: California State University San Marcos Poster Category: Bioengineering Keywords: Cancer Metastasis, Bioengineering, Angiogenesis Project Title: Metastasis of Breast Cancer Cells Through the Chorioallantoic Membrane Vascular Network of the Chick Embryo Author List: Castellanos-Von Borstel, Gilda; Undergraduate, Biotechnology, California State University San Marcos, Presenting Author
Byrne, Patricia; San Diego Zoo Wildlife Alliance
Jensen, Tom ; San Diego Zoo Wildlife Alliance
Luna Lopez, Carlos; Faculty, Biology, California State University San Marcos Abstract Breast cancer is one of the most common types of cancer, affecting 1 in 8 women. Around 170,000 women are affected by triple-negative breast cancer (TNBC) each year. TNBC is highly aggressive, with a 28% chance of survival rate after 5-years if it metastasizes. Many in vitro assays focus on studying cancer migration in 2D or 3D matrices, however they lack a crucial step in tumor metastasis which is the formation of vascularity. While in vivo assays using mouse models can be costly and angiogenesis cannot be easily imaged in real time. In this work we propose to use the chick chorioallantoic membrane (CAM) assay as a method to bridge the gap between the current in vitro and in vivo assays and provide an efficient model to study breast cancer metastasis by imaging cancer cell migration in vascularized 3D cell constructs
The CAM assay is an established method used to study angiogenesis due to its ability to form vessels into the engrafted tissue and provide a native circulatory system. In our case, we engrafted a 3D tumor construct of TNBC cells (MDA-MB-231/GFP) mixed in a collagen matrix. At the same time, we engrafted a secondary 3D construct target made out of an empty collagen matrix. For the CAM components, 3D cell constructs were incubated at 37°C in the CAM for days 10 to 17 of the chick embryonic development. We then imaged the constructs in the CAM using a fluorescence stereo microscope and analyzed the images using ImageJ.
Our study was able to find complete vascularization of the primary tumor, and metastasis happened after just seven days with targets as far as 10 millimeters away. Fluorescence microscopy was used to image the original and metastasized tumor. The area of metastasized tumors was in average three orders of magnitude compared to the original tumor, as measured with ImageJ.
The work helps us build a new accessible model to study cancer metastasis. Other methods have difficulty replicating the natural environment of tumors due to the transitional gap between experimental models and physiological conditions. The CAM provides a dense vascular network that allows for a more physiologically relevant study of cancer metastasis and allows for convenient quantitative assessment of metastasis using standard imaging techniques.
| Poster #: 91 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Bioengineering Keywords: cancer, tumor on a chip, drug development Project Title: 3-D Human Tumor on a Chip for Drug Screening Author List: Nelson, Sabrina; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Reed, Alissa; Undergraduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Breit, Emma; Graduate, Biological Sciences, California Polytechnic State University, San Luis Obispo, Presenting Author
Anastos, Theo; Graduate, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Heylman, Christopher; Faculty, Biomedical Engineering, California Polytechnic State University, San Luis Obispo
Abstract Colorectal cancer (CRC), a solid tumor cancer, is currently ranked as the third deadliest and fourth most common type of cancer in the United States, affecting more than 1.2 million people. Big pharma spends more than 2.5 billion dollars annually on clinical trials for CRC chemotherapy treatments, however, most of these clinical trials fail due to inaccurate preclinical human disease models, such as 2-dimensional (2-D) monolayer human cell cultures or animal models that poorly mimic human physiology. In this study, a systematic approach was taken to develop a 3-dimensionl (3-D) tumor-on-a-chip model that more accurately mimics the in vivo tumor microenvironment and its response to common chemotherapeutics. Specific aspects of the microenvironment that were assessed included extracellular matrix composition and geometry (2-D vs 3-D), CRC tumor density, and static vs dynamic media and chemotherapeutic provision using a microfluidic device. Varying concentrations of SW-620 cells (metastatic human CRC cells) were grown in fibrin and collagen extracellular matrices (ECM) to produce tumors. Fibrin matrices were shown to have significantly larger tumor sizes in comparison to collagen matrices. The optimal plating density for these metastatic cells was determined for varying lengths of experimentation and dimension of culture (2-D vs 3-D). To characterize the ability of the CRC model to mimic the drug response of in vivo tumors, various chemotherapeutic agents with well characterized clinical therapeutic effects, including 5-Flurouracil, Oxaliplatin, and SN-38 were tested in the model. Varying combinations of drug concentrations and cell densities were screened in the model. After optimal model conditions of cell density and drug concentration were established with specificity for each agent, experiments were then conducted within microfluidic devices to add the element of dynamic media and chemotherapeutic provision. Drug efficacy was assessed using metrics of the number of tumors, total area of tumors, and average area of individual tumors using fluorescent microscopy and automated image analysis. Preliminary results in this tumor on a chip model demonstrate a dose-dependent response to the drugs tested that is in line with the response of CRC tumors to these compounds in vivo. Future work will focus on further refining the model for eventual use as a tool in the development and screening of novel drug candidates. | Poster #: 92 Campus: San José State University Poster Category: Bioengineering Keywords: Polyphosphate, Rare earth elements, Genetic Engineering Project Title: The link between rare earth elements and phosphate homeostasis in Methylorubrum extorquens AM1 Author List: Rasouli, Sajede; Graduate, Biological Sciences, San José State University, Presenting Author
Shao, Eric; Undergraduate, Biological Sciences, San José State University
Kaur, Harpreet; Undergraduate, Biological Sciences, San José State University
Skovran, Elizabeth; Faculty, Biological Sciences, San José State University Abstract Methylorubrum extorquens AM1 is a methylotrophic bacterium that can utilize methanol as a carbon source and uses rare earth elements (REEs) as cofactors for three of its alcohol dehydrogenase enzymes. REEs are essential components of many technological devices including smartphones, computers, and optical glasses. We have shown that M. extorquens AM1 can acquire REEs from post-consumer electronics and store these REEs complexed to poly-phosphate (PolyP) in cytoplasmic granules. These granules can be extracted and the REEs recovered. Here we characterize low- and high-Pi conditions, and using a modified Albert’s stain, show that PolyP granules are depleted under Pi starvation. We hypothesize that enhancing Pi uptake results in increased REE storage capacity and REE-PolyP granule size. Using comparative genomics, we identified genes predicted to encode proteins that function in Pi uptake, storage, and use. Transcriptional reporter fusion assays showed that expression of the pstSCAB and pitA Pi transport genes are regulated by Pi as expected. Surprisingly, REEs modulated expression levels resulting in increased Pi transport expression when REEs were in excess. Future studies will determine if increased expression results in increased Pi transport and storage. To enhance Pi uptake, a mutation was constructed in phoU which encodes a regulatory protein that acts as a valve to control Pi intake through the PstSCAB Pi transport system. In other bacteria, phoU is essential or can only be deleted under Pi starvation conditions as deregulated Pi uptake is toxic to cells; negatively charged Pi binds to essential cellular metals like Fe and Mg which inhibits enzyme activities. Growth curves with the phoU mutant in methanol media lacking REEs demonstrated a severe growth defect suggesting that deregulated Pi uptake is also toxic to M. extorquens AM1. However, when REEs were added to the growth medium, growth was rescued, and cells grew at the wild-type rate. We hypothesize that REEs interact with Pi and neutralize the negative charge so that Pi is not available to interfere with cellular metabolism. Further work will determine if REE-Pi storage granules are enhanced in the phoU mutant. Results from these experiments will increase our understanding of the connection between Pi and REE homeostasis, allow us to propose genetic strategies to optimize REE recovery yields, and develop improved bacterial strains and conditions for biometallurgy.
| Poster #: 93 Campus: California State University, Long Beach Poster Category: Clinical Keywords: addiction, reward, animal models Project Title: Blocking serotonin 2A (5-HT2A) receptors attenuates the acquisition of methamphetamine-induced conditioned place preference in adult female rats. Author List: Garcia, Ana; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Verduzco, Gizelle; Undergraduate, Psychology, California State University, Long Beach, Presenting Author
Madden, John; University of New Mexico
Reyna, Nicole; University of New Mexico
Goranson, Emerald; University of New Mexico
Gonzalez, Tiffany; Graduate, Psychology, California State University, Long Beach
Pentkowski, Nathan; University of New Mexico
Zavala, Arturo; Faculty, Psychology, California State University, Long Beach Abstract Background: Methamphetamine withdrawal can induce intense cravings leading to relapse. Contexts/cues paired with chronic methamphetamine use develop incentive motivational properties, promoting future drug-seeking and taking behavior. Research has shown that, in adult male rats, the selective 5-HT2A receptor antagonist M100907 attenuates the acquisition of methamphetamine-induced conditioned place preference (CPP), a measure that examines conditioned associations between the rewarding properties of drugs and contexts. However, these findings have not been extended to adult female rats. Methods: The present study investigated the effects of M100907 on the acquisition of methamphetamine-CPP in adult female rats. During conditioning, rats were administered M100907 (0, 0.025, 0.25 mg/kg, i.p.) 15 minutes before methamphetamine (0 or 1 mg/kg, i.p.) and then were placed into their initially non-preferred chamber for 30 minutes. Following four days of conditioning, the effects of M100907 on the acquisition of methamphetamine-CPP were assessed during a 15-minute drug-free test trial. Results: Pretreatment with M100907 dose-dependently attenuated the acquisition of methamphetamine-induced CPP. Conclusion: Blocking 5-HT2A receptors with a low dose of the selective antagonist M100907 attenuated the rewarding effects of methamphetamine in adult female rats. These data provide further evidence that the 5-HT2A receptor subtype represents a novel target for treating methamphetamine use disorder.
| Poster #: 94 Campus: San Francisco State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Nociception, Transcriptomics , Insect Project Title: Transcriptomic Analysis after Nociceptive Sensitization in the Insect Model, Manduca sexta Author List: Rodriguez Vega, Ana ; Graduate, Biology, San Francisco State University, Presenting Author
Melton, Rebecca; Graduate, Biology, San Francisco State University
Rice, Alicia; Graduate, Biology, San Francisco State University
Fuse, Megumi; Faculty, Biology, San Francisco State University Abstract The opioid epidemic affects tens of thousands of lives each year, forcing scientists to develop novel and less addicting ways to treat pain. Our laboratory aims to elucidate the complex mechanisms of nociception sensitization, which is often manifested as chronic pain in humans. We have taken advantage of the simple neural circuitry and a measurable behavioral assay our lab has developed to study this phenomenon in the hornworm, Manduca sexta. This invertebrate animal model has the potential of containing homologous pain signaling pathways that may be conserved in vertebrates, making it a possibly ideal system to identify and develop new targets for pain treatment. My current project aims to understand the genetic landscape of the central nervous system and the body wall of Manduca using de novo transcriptomics. We assembled a transcriptome from the central abdominal ganglia of M. sexta in control and sensitized conditions after conducting Next-Generation Sequencing. The transcriptome yielded 211,719 predicted ‘trinity genes’ and 2,768,665 transcripts, with a Bowtie2 alignment of 94.56%. EdgeR, differential expression analysis showed up-regulation of 110 transcripts and 38 down regulated transcripts after injury. Many of these had shared sequences with other well-known learning and memory genes, and at least one insect-specific gene was found, pyrokinin, which inhibited sensitization, suggesting that it may be an endogenous analgesic in M. sexta. These preliminary results show that Manduca shares genetic similarities with other pain model organisms, but may also up-regulate its own endogenous and insect-sepcific analgesic during a sensitized state. | Poster #: 95 Campus: California State University, Los Angeles Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: energy expenditure, optimal linear filter, upper limb wheelchair exercise Project Title: A Cascaded Optimal Linear Filter Estimation of Energy Expenditure Author List: Gonzalez, David; Undergraduate, Electrical and Computer Engineering, California State University, Los Angeles, Presenting Author
Won, Deborah; Faculty, Electrical and Computer Engineering, California State University, Los Angeles Abstract Individuals with lower limb impairment will benefit from monitoring energy expenditure (EE) continuously and accurately using wearable sensors. Our previous work determined that optimal linear filter (OLF) models of EE as a function of heart rate (HR) and muscle activity (EMG) better captured EE dynamics than multiple linear regression models but the model did not generalize well to validation test sets. We hypothesized that a model which cascades OLF estimates of EE based separately on EMG and HR into a third OLF (Cascade OLF) would out-perform the conventional OLF model which combines EMG and HR in a single stage.
Two OLF models were trained to estimate EE during stationary spinning exercises: one as a function of HR, and the other as a function of EMG. A third OLF model, or the Cascade OLF, was trained to model EE as a function of the HR- and EMG-OLF estimates from the first stage of the model. These models were implemented in custom code written in MATLAB (MathWorks, Inc., Natick, MA) according to Equation 1.
EE[n]=∑_i∑_k〖w_i [k] s_i [n-k] 〗 (Eq. 1)
where w_i [k] represents the weight for kth lag of the ith sensor s_i and is found by the cross-covariance multiplied by the inverse of the autocovariance matrix. EMG was acquired from wireless wearable sensors (Delsys, Inc., Natick, MA), placed on four upper limb muscles on each of eight subjects while spinning in wheelchairs. Heart rate and EE were acquired simultaneously from a metabolic cart (COSMED USA, Inc., CA). All OLF models were trained and tested using 15-fold cross-validation with testing blocks of 4 minutes. Goodness of fit was measured by coefficient of determination R^2. The average R^2 for Cascade OLF during training was 0.81 (range of 0.5-0.99), compared to an average R^2 of 0.65 (range of 0.3-0.9) for the single-stage OLF. During validation, the Cascade OLF yielded an improvement in R^2, averaging 0.36 (range of 0.14-0.86) versus 0.29 (range of 0.1-0.6) for the single-stage OLF. Further analysis indicated the two-stage model allows us to better capture different modes of metabolism, as manifested in the way EMG follows EE during higher intensity activity more accurately than HR and vice versa during lower intensity activity. Future work will involve applying this multi-modal model to the other upper limb exercises to see if these promising results generalize.
Acknowledgement(s): This work has been supported by NSF under award No. HRD-1547723. Thanks to IntelliMed Lab and Kin. Dept. | Poster #: 96 Campus: California State University, Northridge Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Molecular Dynamics, 3D-RISM, Machine Learning Project Title: Accelerating Molecular Dynamics Simulations With Machine Learning and 3D-RISM Author List: McMahon, Alexander; Undergraduate, Mathematics, California State University, Northridge, Presenting Author
Donovan, Ezequiel; Undergraduate, Computer Science, California State University, Northridge
Luchko, Tyler; Faculty, Physics, California State University, Northridge
Abstract Molecular dynamics simulation is a fundamental tool for molecular design in biochemistry. However, molecular dynamics simulations are computationally expensive, limiting the size and time scales of the systems that can be modeled. This is particularly true for hydration free energies predictions, an important quantity in drug design. To address these computational limitations, we are training a neural network to emulate hydration free energy output from the 3D reference interaction site model (3D-RISM) theory of molecular solvation. Using supervised learning, we have trained a neural net from a software package, TorchANI, using 6,000,000 3D-RISM data points for 640 small molecules and developed a new feature vector for our software. Preliminary data show a speedup in frame computation time from 30s for standard 3D-RISM to a few microseconds for small molecules with the neural net. The neural net results have an absolute error of 0.5 kcal/mol on hold-out data which is accurate in comparison to the input precision of 0.1 kcal/mol from 3D-RISM. However, errors of 10 kcal/mol are common for larger macromolecules. To improve the accuracy and transferability of the trained neural network, we are implementing an active learning protocol, which will more efficiently explore chemical space. Our preliminary results show a neural network can be trained to rapidly calculate hydration free energies but chemical diversity is key to success. | Poster #: 97 Campus: San José State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Alzheimer’s disease, Glutathione, Single Nucleotide Polymorphisms Project Title: Identify Potential Genetic Variants Associated With Cognitive Impairment and Alzheimer’s Disease Beyond B-Amyloid and Tau Author List: Majmudar, Shamika; Undergraduate, Biology, San José State University, Presenting Author
Newton, Yulia; Faculty, Computer Science, San José State University
Wesley, Leonard; Faculty, Computer Science, San José State University
Abstract Motivation: Alzheimer’s disease(AD) is typically characterized as excessive accumulation, in the brain, of two pathogenic proteins called amyloid beta (Aβ) and tau. However, excessive levels of Aβ and tau are not sufficient to diagnose or explain all instances of AD. Previous work has reported that between 30% and 40% of normal individuals also exhibit high levels of Aβ and tau. Although excess Aβ and tau depositions are required for the pathologic diagnosis of AD, alone they are not sufficient to diagnose mild cognitive impairment (MCI), cognitive dysfunction, or clinical dementia such as AD.
Interference with the expression of the glutathione gene has been demonstrated to play a role in the accumulation of Beta-Amyloid (Aβ) and Tau in the hippocampus region of humans. Previous work has identified 15 genetic mutations that interfere with the glutathione gene and hence increases the likelihood of the onset or progression of AD.
Project Goal: To identify and characterize additional genetic variations that interfere with the glutathione gene and pathway.
Methods Used: The GWAS data for 5,152 AD, MCI, and control subjects from the ADNI DB were analyzed for SNPs that have an epigenetic relationship with the transcription site for the glutathione gene. Chi-square and correlation analysis was conducted to identify candidate chromosomes and genes to investigate. The 3DIV epigenetic DB was used to identify interactions between SNPs and the glutathione gene.
Results: Of the 27 genes investigated to date, the mutation of the NEUROG2 (rs10000007 GWAS SNP ID: rs17589290) gene was identified as interacting with the glutathione gene. Of the 5,152 ADNI subjects analyzed 3,952 were diagnosed as having AD or MCI, and 1,200 were control subjects. Of the 3,952 AD or MCI subjects, 98 (2.47%) exhibited the rs10000007 SNP, and 30 (2.5%) of the control subjects exhibited the same rs10000007 SNP.
Conclusions: Although the rs10000007 SNP alone is not selective between AD/MCI and control subjects, adding the presence of this SNP to the growing list of potential AD/MCI epigenetic biomarkers might collectively serve as indication that additional clinical AD/MCI diagnostic tests might be warranted.
| Poster #: 98 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: spreading depolarization, stroke, automated detection Project Title: Detection of Spreading Depolarization Events for Advancing Stroke Therapy Author List: Abelian, Andrea; Undergraduate, California State University, Los Angeles, Presenting Author
Evans, Cody; Graduate, Neurobiology
Palopoli, Kay; Graduate, Biomedical Engineering
Hoffmann, Ulrike; Faculty, Neurobiology
Won, Deborah; Faculty, Electrical and Computer Engineering, California State University, Los Angeles
Abstract Spreading depolarizations (SD) are large-amplitude changes of local field potentials on the order of minutes typically observed in the brain tissue of patients who have suffered neurological damage such as stroke. SDs appear more frequently in stroke patients suffering greater morbidity, and lead to permanent brain tissue damage, thus resulting in physical disability. The ability to detect SDs in real time is thus expected to enable development of a responsive, targeted therapy to slow or prevent further damage and improve the outcome of the patient’s health. Therefore, we proposed to develop a method to automatically detect SD events based on identified patterns of change in the electrocortiograms (ECoG), or neural field potentials, over time.
We analyzed 16 ECoG recordings which were acquired from seven rats with induced stroke. Sixty-channel multi-electrode arrays (MEA) were implanted in a cortical area covering the infarct and surrounding tissue. Ground truth start and end times of SD events were manually marked by an expert in SDs and ECoG analysis using a custom software application we developed in MATLAB (Mathworks, Inc. Natick, MA). We computed the first-order Euler difference of the ECoG signals and found a consistent triphasic shape in the first derivative at each SD event. The start of the SD events corresponded to the first valley in the triphasic waveform; the end of the SD events corresponded to the return to baseline after the last valley. Based on these findings, we wrote custom software in MATLAB to detect the start and end of SD events and compare them to the ground truth data.
Despite large variability in both DC (baseline) potentials and fluctuations in potential, our algorithm, based on the triphasic waveform in the first derivative of the ECoGs, detected SDs with an average correct detection rate of 91% for event start times and 94% for event end times out of 111 events. Despite such a low missed detection rate, the algorithm produced a high false-positive rate using fixed thresholds. To improve the accuracy, we need to calibrate the parameters used in our detection algorithm to account for inter-subject and inter-channel variability. Future research will include spectral characteristics to create a richer multi-dimensional input space for characterizing, and thus detecting, SD events.
Acknowledgment: The authors would like to acknowledge support for this work by NSF under award No. HRD-1547723. | Poster #: 99 Campus: San Francisco State University Poster Category: Diagnostics/Imaging/Analytical Keywords: Escherichia coli, wild pigeons, Antibiotic resistance Project Title: The Presence of Antibiotic Resistant Escherichia coli in Wild Pigeon Fecal Matter Author List: Velarde, Arianna; Undergraduate, Biology, San Francisco State University, Presenting Author
Chen, Lily; Faculty, Biology, San Francisco State University Abstract Pathogenic strains of gram-negative bacteria have been observed to become progressively resistant to antibiotic treatment, critically affecting human populations at an unprecedented rate. The most frequent gram-negative pathogen that cause disease in humans, Escherichia coli, has been recognized in developing resistance to β-lactams by producing extended-spectrum β-lactamases (ESBLs). The dispersion of antibiotic resistant genes (ARGs) through anthropogenic activities have introduced resistant profiles into the environment and in wildlife. Wild birds may pose as reservoirs for the propagation of antibiotic resistant bacteria and introduce resistant profiles into populated human communities. This study evaluates resistance profiles in the fecal matter of wild Pigeons that associate closely within populated metropolitan areas located in the San Mateo County, California. From two locations in the San Mateo County (Roosevelt Center and Sequoia Station), 31 fecal samples were collected and subjected to phenotypical and biochemical identification. Fecal matter collected from a Psittaciformes species, Budgerigars, served as the negative control. A total of 14 isolates were tentatively identified as E. coli and three strains were selected and tested against three antibiotics, Penicillin, Ampicillin, and Tetracycline, through the Kirby-Baur disk diffusion. The minimum inhibitory concentration was measured of the three E. coli isolates against Penicillin, Ampicillin, and Tetracycline using broth dilution assay. All three strains were susceptible to Tetracycline and displayed moderate resistance to Penicillin and Ampicillin. Strain 1 exhibited higher resistance to Penicillin and Ampicillin compared to Strain 2 and 3. Testing more isolates and verification of E. coli producing extended-spectrum β-lactamases (ESBLs) by PCR and DNA sequencing are in progress. This study will allow the understanding of resistant profiles harbored by wild pigeons in regional community, inform the public about their role as possible vectors, and bring awareness in the associated health risks when encountering them.
| Poster #: 100 Campus: California State University, Long Beach Poster Category: Diagnostics/Imaging/Analytical Keywords: parasite, DNA, nanopore Project Title: Nanopore translocation of topologically linked rings from kinetoplast DNA Author List: Breyer, Sierra; Undergraduate, Physics and Astronomy, California State University, Long Beach, Presenting Author
Klotz, Alexander; Faculty, Physics and Astronomy, California State University, Long Beach Abstract Trypanosome parasites infect millions of people annually. Their mitochondria contain a complex structure called a kinetoplast, which is comprised of thousands of topologically linked DNA rings, like chainmail armor. We are interested in developing nanopore sensing technology to detect these linked ring DNA structures as a means of diagnosis trypanosome parasite infections. Simulations of linked rings in nanopores predict jamming behavior, but experiments have been lacking. Here, we investigated the nanopore translocation of topologically linked rings isolated from the digestion of kinetoplast DNA. We use restriction enzymes to reduce them to simpler topological structures such as double- and triple-linked rings. A solid state nanopore is used to observe their translocation signal, where each topological linkage is identified as a spike in the current blockade. Here, we report on the signatures, statistics, and physics observed in these translocations.
| Poster #: 101 Campus: San José State University Poster Category: Disease (Pathogens) Keywords: sirtuin, insulin, diabetes Project Title: Loss of sirtuin 4 leads to exacerbated age-induced glucose intolerance and insulin resistance in female mice Author List: Penney, Magan; Graduate, Biological Sciences, San José State University, Presenting Author
Lin, Zhihong; Duke Molecular Physiology Institute, Duke University Medical Center
Hirschey, Matthew; Duke Molecular Physiology Institute, Duke University Medical Center
Huynh, Frank; Faculty, Biological Sciences, San José State University Abstract Sirtuins are a family of NAD+ dependent deacylase enzymes that are involved in post-translational modifications of proteins by the removal of acyl groups. These modifications result in alterations in protein function, many of which are considered to be protective against age-related diseases such as diabetes and cancer. There are seven known mammalian sirtuins (SIRT1-7), with sirtuin 3-5 being located in the mitochondria. Of these three, sirtuin 4 (SIRT4), poses a particular interest as it is the least well-studied. Previous work suggests that SIRT4 is involved in the regulation of several metabolic pathways including those related to obesity and diabetes. SIRT4 knockout (SIRT4KO) mice have increased insulin secretion in the presence of both glucose and amino acids. Indeed, we showed that male mice with a loss of SIRT4 had inappropriately elevated glucose- and leucine-stimulated insulin secretion starting at a young age, and this was followed by an early onset of age-related glucose intolerance and insulin resistance. However, similar studies on how a loss of SIRT4 affects female mice during aging have not been completed. Based on preliminary studies, we hypothesized that SIRT4KO female mice may also develop exacerbated age-related insulin resistance compared to wild-type controls. To address this hypothesis, we performed several metabolic studies on 2.5-year-old female wild-type and SIRT4KO mice, including glucose-tolerance tests, insulin-tolerances tests, as well as monitoring fasted blood glucose levels. We also measured glucose-stimulated and leucine-stimulated insulin secretion in these mice. Our findings suggest that similar to male mice, aged female SIRT4KO mice tended to have elevated fasted blood glucose levels. Further, SIRT4KO females had impaired glucose tolerance and insulin resistance compared to wild-type controls. Interestingly, when leucine was administered along with glucose during a glucose tolerance test, glucose tolerance was normalized in the SIRT4KO mice. Taken together, our findings suggest that aged female SIRT4KO mice demonstrate metabolic dysregulation that is consistent with that observed in male SIRT4KO mice. Intriguingly, leucine appears to be able to reverse some of the metabolic abnormalities seen in mice with a loss of SIRT4, and as such, warrants further investigation. | Poster #: 102 Campus: San Diego State University Poster Category: Disease (Pathogens) Keywords: cancer, tumor immune escape, Type I interferon Project Title: VAX014, a novel therapy for bladder cancer, may aid in overcoming tumor immune Author List: Nelson, Kinsey; Graduate, Biology, San Diego State University, Presenting Author
Shingo, Tsuji; External Partner
Reil, Katherine; External Partner
Proclivo, Veronica H.; External Partner
Giacalone, Matthew J.; External Partner
McGuire, Kathleen L.; Faculty, Biology, San Diego State University Abstract Bladder cancer is the 4th most diagnosed cancer today but therapies are limited in efficacy due to
immune evasion by tumors. Two immune escape mechanisms are downregulation of MHC-I
antigen presenting molecules and the overexpression of immune checkpoint molecules, such as
PD-L1, that suppress T-cell activity. Therapies blocking PD-L1 are available for bladder cancer,
but variable PD-L1 expression in patients limits their versatility. Treatments that can overcome
both PD-L1- and MHC-I-facilitated tumor immune escape in broad patient populations are
needed. VAX014, a novel oncolytic therapy, has shown efficacy in two mouse models of bladder
cancer, MB49 and MBT-2, with enhanced survival in treated animals. Previous studies showed
that MBT-2 tumors express high levels of PD-L1 in situ irrespective of VAX014 treatment. In
contrast, MB49 tumors express minimal PD-L1 until treated with VAX014. One goal of this
work is to examine factors contributing to the upregulation of PD-L1 in response to VAX014 in
vitro to gain insight into mechanisms involved. In addition, we aim to evaluate expression of
MHC-I in both models in response to treatment. First, we cultured MB49 cells +/- VAX014 for
20-hours in vitro and evaluated expression of PD-L1 and MHC-I via flow cytometry. We saw
VAX014 facilitated a near 15- and 5-fold increase in PD-L1 and MHC-I, respectively. Next, to
determine if soluble factors were involved, supernatants were isolated from MB49 +/- VAX014
treatment and transferred to naïve MB49 cells for 20-hours. When cultured with treated
supernatant, these cells upregulated PD-L1 and MHC-I 5- and 3- fold, respectively, showing a
soluble factor was present. Similar studies using MBT-2 revealed they didn’t produce this factor
but responded to it after coincubation with VAX014-treated MB49 supernatants by upregulating
PD-L1 (p &lt;0.01) and MHC-I (p &lt;0.05). Given the known role of Type I Interferons (IFNs) in
upregulating MHC-I, we blocked the IFN alpha receptor in naïve cells with an Ab for 1 hour
prior to supernatant treatment and found that the activity was lost, confirming Type I IFNs were
involved in the upregulation of both PD-L1 and MHC-I. These results indicate VAX014 therapy
may provide for a broader use of PD-L1 inhibitors in patients and a more effective way to
overcome tumor immune escape in bladder cancer, highlighting the importance of understanding
these mechanisms. This work was supported by a contract to KMc from Vaxiion Therapeutics. | Poster #: 103 Campus: San Francisco State University Poster Category: Disease (Pathogens) Keywords: Group B Streptococcus , Lactobacillus rhamnosus , Extracellular Vesicles Project Title: Characterize of Group B Streptococcus (GBS) Virulence Factors and Lactobacillus rhamnosus’s Mechanism in Battling GBS Colonization Author List: Quack, Sammy ; Undergraduate, Biology, San Francisco State University, Presenting Author
Cajulao, Jan Mikhale ; Graduate, Biology, San Francisco State University, Presenting Author
Chen, Lily; Faculty, Biology, San Francisco State University
Abstract Group B Streptococcus (GBS), commonly found in the vagina and rectum, is a Gram-positive opportunistic pathogen and a leading cause of preterm birth and neonatal illness. The vaginal tract is also colonized by commensal Lactobacillus species, which are believed to defend the tract from pathogens, including GBS. Previous data from our lab showed that HeLa cells (human immortal cervical cancer cell line), when exposed to either live GBS or culture supernatant, exhibited significant morphology changes and detachment. The data also showed that when Lactobacillus rhamnosus was presented, GBS-infected HeLa cells revealed less cytotoxicity and detachment. However, GBS factors in the supernatant, and the mechanism of L. rhamnosus and GBS on HeLa cell adherence still remain unknown. This study sought to address: (1) The adherence of L. rhamnosus and GBS on Hela cells, and the potential protective role of L. rhamnosus against GBS by partially inhibiting GBS-dependent microvilli and vesicle disruption; (2) The identities of virulence factors presented in GBS supernatant that contribute to the detachment of HeLa cells. Flow cytometry will be utilized to quantify the adherent GBS and L. rhamnosus on HeLa cells. Immunofluorescence microscopy and Scanning Electron Microscopy (SEM) captured images of infected HeLa cells will be analyzed using the image analysis program ImageJ. SDS-PAGE and Western Blotting will be conducted to identify the presence of GBS virulence factors. These results will shed a new light on the virulence factors of GBS and potential mechanisms contributed to a protective role of L. rhamnosus against GBS colonization. | Poster #: 104 Campus: California State University, San Bernardino Poster Category: Disease (Pathogens) Keywords: pandemic, influenza, peptide inhibition Project Title: Competitive Inhibition of Influenza A Replication Using Viral Peptides Author List: Atkins, Cody; Graduate, Biology , California State University, San Bernardino, Presenting Author
Newcomb, Laura; Faculty, Biology , California State University, San Bernardino Abstract Influenza A virus remains a seasonal threat and a source of outbreaks and pandemics. While great progress towards universal influenza vaccines and effective anti-viral treatments are promising, the properties of influenza facilitate rapid change to the viral genetic code, leading to evasion of countermeasures, driving continued antiviral research to prepare for emerging influenzas. The viral ribonucleoprotein (vRNP) presents an ideal target for novel antivirals as it highly conserved among influenzas and responsible for viral gene expression and replication. NP and PB2 are two viral proteins of the vRNP that interact. Previous research identified a region of the NP body domain essential for vRNP function; glycine substitution of 5 amino acids with this domain abolishes vRNP function. Here we show that this body domain mutant of NP (NPbd3) does not co-sediment with PB2 while wild type NP does. These results support that the NP-PB2 interaction within the body domain is essential for vRNP function. To examine the NP-PB2 interaction as a target for antivirals we will express an NP peptide representing this interaction surface and determine if this peptide inhibits influenza virus replication. We have selected residues 276-312 which form a three alpha helix structure and comprise this interaction domain. NP 276-312 will also be expressed with the 5 glycine substitutions present in NPbd3. The DNA coding sequence for these peptides will be inserted into pcDNA3 fused with an encoded nuclear localization signal (NLS) and 13x Myc tag. The NLS ensures the expressed peptides are imported into the nucleus where influenza gene expression and replication occur. The 13X myc tag ensures the peptide is large enough not to diffuse out of the nucleus. The empty pcDNA NLS-13X myc plasmid will express an NLS-13x myc peptide to serve as negative control. Tissue culture cells will be transfected with these plasmids and incubated 24-48 hours to allow expression of the peptides. Cells will then be infected with influenza A Udorn (H3N2) using both high and low multiplicity of infection (MOI). Cytopathic effects (CPE) will be observed, with media samples taken either hours (high MOI) or days (low MOI) post infection. Media samples will be evaluated by hemagglutinin assay to determine virion concentration. We hypothesize that the wild-type NP body domain will inhibit viral replication, but not NPbd3 or the NLS-13X myc control, validating NP-PB2 interaction as a target for antivirals. | Poster #: 105 Campus: California State Polytechnic University, Pomona Poster Category: Disease (Pathogens) Keywords: Candida albicans, Peripheral cannabinoid receptor, Mice Project Title: Role of the Peripheral Cannabinoid Receptor on systemic Candida albicans infection in immunocompromised mice Author List: Little, Michael; Graduate, Biological Sciences, California State Polytechnic University, Pomona
Nadales, Nathalie; Graduate, Biological Sciences, California State Polytechnic University, Pomona, Presenting Author
Buckley, Nancy; Faculty, Biological Sciences, California State Polytechnic University, Pomona
Abstract Candida albicans (C. albicans) is an opportunistic pathogen that can cause systemic candidiasis. Candidiasis is particularly detrimental to immunocompromised individuals, such as those undergoing chemotherapy. The endocannabinoid system (ECS) has immune modulatory effects which may play a role in candidiasis. The ECS includes cannabinoid receptors and endocannabinoid ligands. The best-known cannabinoid receptors are the central cannabinoid receptor (CB1R) and the peripheral cannabinoid receptor (CB2R). CB1R is abundant in the CNS. CB2R is abundant in immune cells. We investigated the role of the CB2R on the severity of systemic C. albicans infection in immune competent and immunosuppressed male and female mice. We used mice lacking CB2R (CB2R-/-) to conduct these studies. Mice were injected intravenously (IV) with either 1XPBS (immune competent (IC)) or 5-fluoruracil, a chemotherapy drug (immunosuppressed (IS)). After three days, mice were infected with 7.5x105 C. albicans cells/mouse, IV. The mice were observed daily for morbidity and survival. Mouse tissues were collected to assessed fungal load (kidneys, brains, liver) and cytokine levels (serum, spleens). The spleen, a major immune organ, expresses high levels of CB2R in wild type mice. Splenocytes were cultured, treated with Lipopolysaccharide (LPS, 1μg/mL) and incubated at 37oC/5%CO2 for 48h after which supernatants were collected and analyzed for secreted cytokines. IC female mice are more resistant to C. albicans infection compared to IC males as assessed by survival and weight loss. IS males died earlier than IS females. Tissue fungal burden was higher in the kidneys and brains of IS mice compared to those of IC mice regardless of sex. Comparing IC mice, serum IL-6 levels were higher in male mice compared to female mice. Serum IL-6 levels were higher in IS mice compared to IC mice, regardless of sex. Comparing splenocytes derived from IC mice, those derived from male mice secreted lower LPS-induced IL-6 when compared to females. Splenocytes derived from IS mice secreted less LPS-induced IL-6 compared to splenocytes derived from IC mice. However, the difference between IC and IS LPS-induced IL-6 in males was less than the difference in females. We suggest that immunocompromised CB2R-/- male mice are more susceptible to C. albicans infection compared to immunosuppressed CB2R-/- females, and that this may be mediated by cytokine production. | Poster #: 106 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: antibody, cancer, molecular biology Project Title: Modular Cloning of Recombinant Antibodies by Assembly of Synthetic Domain Genes Author List: Amaya, Carina; Graduate, Chemistry and Biochemistry, California State University, Fresno
Bedolla, Yazmine; Undergraduate, Chemistry and Biochemistry, California State University, Fresno
Cory, Brooks; Faculty, Chemistry and Biochemsitry, California State University, Fresno
Abstract Cancer is the second leading cause of mortality in the U.S. and a significant health problem worldwide. Antibody immunotherapies have emerged as a constructive way to fight cancer. The antibody isotype IgG (Immunoglobulin gamma) are Y-shaped proteins produced by plasma cells as an immune response. The IgG molecule is sub-divided into an antigen binding region called the Fab, and the effector region called the Fc. For therapeutic development, recombinant antibodies are preferred and, in some cases, it’s desirable to produce intact IgG, or Fab. To facilitate rapid cloning and expression of both IgG and Fab antibody formats, we have developed a modular cloning approach which can rapidly produce these different antibody modalities. Using the MUC1 specific antibody 4H5 as a model, we cloned, expressed, and purified 4H5 as both Fab and IgG. The 4H5 variable domains (VH and VL) were PCR amplified to contain 20 base-pair (bp) overlaps with the pcDNA vector on the 5’ end and a 20 bp overlap to the CH1 and kappa domains, respectively. To produce the Fab, a human CH1 domain was PCR amplified, and a His-tag and a 20 base-pair (bp) overlap to the pcDNA vector were added to the 3’ end of the gene. A human kappa domain was also PCR amplified and a 20 bp overlap with the vector was added to the 3’ end. For IgG production, a human CH1-CH2-CH3 domain was PCR amplified, and the same vector overlap sequence was added to the 3’ end of the gene. The PCR products were purified, and the genes assembled using the HiFi DNA Assembly Cloning Kit. Successful assembly of the genes was confirmed by sequencing, and 4H5 Fab fragments and IgG were produced by transient transfection in Chinese Hamster Ovarian (CHO) cells). Antibody (Fab and IgG) was purified by affinity chromatography, and binding to recombinant MUC1 antigen confirmed by ELISA. The cloning method presented here always simultaneous production of multiple antibody formats. This approach will permit rapid assembly and screening of multiple antibody formats and isotypes for therapeutic antibody discovery and analysis. | Poster #: 107 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: stem cells, Drosophila, genetics Project Title: Functional analysis of Escargot and STAT targets in intestinal stem cells of the Drosophila melanogaster posterior midgut Author List: Khanbabaei, Armen; Graduate, Biology, California State University, Northridge
Petrossian, Cynthia; Graduate, Biology, California State University, Northridge, Presenting Author
Frazier, Courtney; Undergraduate, Biology, California State University, Northridge, Presenting Author
Ca, Donnie; , Biology, California State University, Northridge
Segura, Lina; Graduate, Biology, California State University, Northridge
Loza-Coll, Mariano; Faculty, Biology, California State University, Northridge Abstract Many of our organs contain adult stem cells (ASCs) in charge of replacing cells lost to damage, disease or normal tissue turnover. Like their embryonic counterparts, ASCs can divide asymmetrically, giving rise to a new copy of themselves (i.e. self-renewal) and a sister cell that commits to differentiation into a specific cell type. Decades of research have led to the identification of so-called "master regulators", i.e. pleiotropic genes whose mutations simultaneously affect diverse aspects of normal ASC biology. Furthermore, genome-wide screens around these MR genes have identified hundreds of putative targets that could serve as their downstream effectors. However, in stark contrast to the speed and ease with which bioinformatic predictions can generate lists of putative downstream effectors, their experimental validation through independent approaches is notoriously lacking.
To begin addressing this gap between bioinformatic predictions and experimental validation, we use intestinal stem cells (ISCs) in the posterior midgut of the fruit fly (Drosophila melanogaster). We first integrated genome-wide DNA mapping data for two known MR genes in these cells: the Snail family transcription factor Escargot (Esg) and the signal transducer protein STAT. A series of reverse-transcription qPCR (RT-qPCR) assays allowed us to experimentally identify a subset of these putative targets that showed differential expression in response to the genetic manipulation of Esg, STAT or both. These studies also showed a noticeable false discovery rate (FDR) in bioinformatic predictions of regulation by the MR genes. On the other hand, our results thus far show that the genetic manipulation of confirmed targets often results in noticeable changes to ISC homeostasis, including changes to their number and/or morphology. Therefore, while preliminary, our data would indicate that bioinformatic predictions of regulation by a master regulator are rather noisy. However, once confirmed, the genetic manipulation of these targets suggest that they are critical for ISC homeostasis.
(This study was entirely supported by a NIH Score-2 grant). | Poster #: 108 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: ICER, β-cell, miR-375 Project Title: ICER: A SPECIES-SPECIFIC TARGET OF MIR-375 Author List: Ramirez, Diana; Undergraduate, Biology, California State University, Chico, Presenting Author
Keller, David; Faculty, Biology, California State University, Chico Abstract Type 2 diabetes (T2D) is characterized by defective pancreatic β-cell insulin release. Gene expression is rarely studied in human β-cells, leading to a reliance on rodent models for fundamental answers relevant to human health. However, new human genome analysis has uncovered large numbers of human-specific regulatory DNA domains which are not conserved with other species. The microRNA-375 (miR-375) is a gene that may regulate aspects of the β-cell and may play a role in the pathogenesis of T2D in humans. One possible target gene is the inducible cAMP early repressor (ICER); it can cause diabetes in rats when overexpressed. I want to investigate whether miR-375 binds to the ICER mRNA. A series of co-transfections was performed to test my hypothesis. I transfected human embryonic kidney cells (HEK293T) with the green fluorescence protein (GFP) gene coupled to a miR-375 binding site and either miR-375 or another microRNA control. I found that miR-375 blocked the GFP gene expression eight-fold (n=2). Then, I transfected the GFP gene coupled to a small piece of the human ICER gene, which contains a natural miR-375 binding site, and a rat ICER gene that does not. The results showed a 20 percent down-regulation for human ICER-375 demonstrating the miR-375 binding to the sequence. (n=1). There was no binding of miR-375 to the rat-ICER sequence, proving that the binding of miR-375 was species-specific. The next step is to evaluate the interaction between miR-375 and ICER. To test this, I have identified the ICER mRNAs that are expressed in β-cells. Next, I plan to test the interaction between miR-375 and ICER inside the cells. | Poster #: 109 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: sRNA, in vitor transcription, RT-qPCR Project Title: Quantitative Detection of Short RNA via In vitro Transcription Networks Author List: Stibelman, Aaron; Graduate, Biology, California State University, Northridge, Presenting Author
Takahashi, Melissa; Faculty, Biology, California State University, Northridge Abstract Prokaryotic small RNAs (sRNA) have been identified as powerful regulators of gene expression and some are known to induce a variety of antimicrobial defense mechanisms. As a response, bacterial sRNA have become prime candidates for novel antimicrobial therapies and have surged an interest in the ability to quantitatively identify their production. Currently, there are many technologies for the detection and quantification of RNA that rely on primer design in the initial stages of amplification. Quantitative reverse transcription PCR (RT-qPCR) is one of the most widespread tools in RNA detection for its ability to produce quantitative data with high sensitivity and strand specificity. However, due to the nature of primer design, RT-qPCR is inherently vulnerable to genomic DNA contamination and can generate false positive results. A second caveat to primer-based amplification is in the development of pairs suitable for smaller RNA molecules. The difficulty in designing optimal pairs, with matching properties, increases as target length minimizes. Primer-based detection systems may be avoided through the development of in vitro transcription networks, or genelets. These synthetically designed nucleic acid based biological circuits detect input RNA with high specificity due to predicted Watson-Crick base pairing with the RNA target. We have designed genelet networks with a fluorescence-based output that relies on inducible transcription of an RNA aptamer reporter that results from hybridization of the Escherichia coli sRNA, MicF, within the system. Several network designs were developed using a software algorithm that is able to predict nucleic acid secondary structure and interaction. Each design was tested by recording relative fluorescence every five-minutes for two hours. Our initial tests have revealed a limit of detection in the low nanomolar range but, at minimum, we are aiming to detect femtomolar concentrations. To improve sensitivity, we have incorporated strategies to generate additional copies of the target RNA within the network. Although optimization is required, our preliminary results suggest that improved sensitivity is possible, due to the amplification of target concentration. Overall, genelet systems can be used as diagnostic tools able to detect shorter RNA molecules and can easily be adapted for an array of nucleic acid targets. | Poster #: 110 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Kaposi’s Sarcoma Herpesvirus, G Protein-Coupled Receptors, Cancer Project Title: Exploring the Intracellular movement of the viral G Protein Coupled Receptor of Kaposi’s Sarcoma Herpesvirus Author List: Cajulao, Jan Mikhale; Graduate, Biology, San Francisco State University, Presenting Author
Sanchez, Erica; Faculty, Biology, San Francisco State University Abstract The endothelial and angiogenic cancer known as Kaposi’s Sarcoma is caused by a human herpesvirus: Kaposi’s Sarcoma Herpesvirus (KSHV). KSHV encodes for a viral G Protein-Coupled Receptor (vGPCR) that is homologous to human GPCRs. Much is known about human GPCR signaling mechanisms, including the role of receptor trafficking, subcellular signaling, and host protein interactions. Transcriptional regulation depends on trafficking and localization of human GPCRs. However, vGPCR’s potential trafficking, localization, and host protein-dependent signaling remain unexplored, leaving a gap in understanding KSHV viral-host interactions. We hypothesize that vGPCR’s trafficking, localization, and interaction with human proteins modulate signaling. We examine vGPCR trafficking and localization and determine the role(s) of trafficking and host protein interaction(s) in signal transduction. This study uses immunofluorescence confocal microscopy to monitor the location of vGPCR, quantifying colocalization with various subcellular membranes using the computer program ImageJ. To study potential location-biased signaling, activation of downstream vGPCR targets will be measured after using drugs to restrict vGPCR to specific locations. vGPCR’s interactions with angiogenic human proteins MGAT5 and EMC10 will be interrogated via confocal microscopy and immunoprecipitation to confirm interaction and determine where interactions take place within the cell. Preliminary data show vGPCR localizes in the plasma membrane and the perinuclear region, and co-localizes with EMC10. Detailing the nuances of vGPCR signaling will lay the foundation for future studies that aim to discover much needed therapeutics for Kaposi’s Sarcoma. | Poster #: 111 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: transgene, neuron function, nematodes Project Title: Silencing Neurons to Alter Olfactory Behavior Author List: Vertiz, Johnny; Undergraduate, Biology, California State University, Northridge, Presenting Author
Carstensen, Heather; Graduate, Biology, California State University, Northridge Abstract All organisms rely on their chemosensory abilities to find food, attract mates, and avoid predators. Nematodes can serve as a model system for studying the chemosensory system at a cellular level. In the nematode Caenorhabditis elegans, a histamine-gated chlorine channel system derived from Drosophila can be used to inhibit neuronal function using neuronal type specific promoters and exogenous histamine. Since nematodes do not use endogenous histamine as a neurotransmitter, no changes in behavior is seen when histamine is added exogenously. To test out this knockdown technology in Pristionchus pacificus, we have created a functional construct that has a Ppa-odr-3::HisCl transgene, and hypothesize that the Ppa-odr-3-expressing neurons AM3 and AM4 are responsible for olfactory sensation. Specifically, we are interested in seeing the effects of histamine on adult transgenic nematodes when presented with the attractive odor ZTDO. However, we will be conducting these experiments in the enhancer mutant csu60 background, since wildtype,adults are not attracted to ZTDO. If the His-Cl1 system is successful we would see a decrease in attraction to ZTDO in the transgenic nematodes when histamine is present, but enhanced attraction similar to csu60 when histamine is not present. In theory, this would then reduce the chemoattraction mutant adults have toward ZTDO because of the silencing of AM3 and AM4 neurons caused by the His-Cl1 system. These experiments will help us see if the His-Cl1 system is a viable option to silence neuronal activity in the nematode P. pacificus and would further tell us the role that the AM3 and AM4 sensory neurons play in olfactory sensation. | Poster #: 112 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: cardiomyocyte, hypertrophy, imaging Project Title: Visualizing hormonal effects on cardiac muscle cell hypertrophy using digital holographic microscopy: Does size matter? Author List: Simmons, Jacquelyn; Undergraduate, Biological Sciences, San José State University, Presenting Author
Huang, Herman; Undergraduate, Biological Sciences, San José State University
Payumo, Alexander; Faculty, Biological Sciences, San José State University
Abstract Adult mammals have a limited capacity to regenerate their hearts after injury. In contrast, newborn rodents are capable of cardiac tissue regeneration, which relies on the proliferation of heart muscle cells called cardiomyocytes (CM). Mice and rats lose the ability to regenerate their hearts within the first week after birth when their CMs transition from proliferating mononucleated cells to binucleated cells incapable of cell-cycle completion. After this transition, any increase in heart mass is predominantly due to CM hypertrophy, an increase in cell size. We have recently discovered that combined inhibition of thyroid hormone (TH) and adrenergic receptor (AR) signaling after birth dramatically increases CM proliferation and reduces heart size. However, the link between CM size and cell cycle control are not well understood. We hypothesize that interactions between these pathways directly promote CM hypertrophic growth, which may represent a barrier to CM proliferation and regeneration. The goal of this project is to establish a system that allows us to quantify the effects of TH and AR signaling activation on CM size dynamics. In this study, we innovate the application of digital holographic microscopy to characterize real-time changes in CM cell volume in response to hormonal stimulation in three-dimensions and single-cell resolution. We treated cultured primary neonatal rat ventricular CMs with the sympathetic hormone norepinephrine, which is known to activate AR signaling and stimulate CM hypertrophy in vitro. Norepinephrine treatment for 24 hours results in a 40% increase in CM three-dimensional optical volumes, consistent with previous findings. We then acquired time-lapse videos using our novel digital holographic imaging approach to reveal the kinetics of norepinephrine-induced CM hypertrophic growth. Our preliminary experiments suggest that CM volume increases approximately 10 hours post-treatment. Our results validate the application of digital holographic imaging to quantify dynamic changes in CM size and behavior in response to hormonal stimulation. In upcoming experiments, we will use this technology to define how interactions between TH and AR signaling impact CM size, growth kinetics, and behavior. Understanding the cellular mechanisms downstream of TH and AR signaling that limit the potential for mammalian heart regeneration may one day help inform new strategies in cardiac regenerative medicine. | Poster #: 113 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Tunneling Nanotube, Myosin-X, Antimycin A Project Title: Antimycin A Leads to the Upregulation of Myo10 and Tunneling Nanotube Formation Author List: Putnam, Nicholas; Graduate, Biology, California State University, Fresno, Presenting Author
Gousset, Karine ; Faculty, Biology, California State University, Fresno Abstract Tunneling nanotubes (TNTs) are actin-containing cellular protrusions involved in a myriad of cellular functions. TNTs have been implicated in various forms of pathogenesis. This can be seen through the efflux of chemotherapeutic drugs in cancer cells, as well as the hijacking of TNTs via HIV-1 to promote direct cell to cell spread. Our laboratory has shown myosin-X (myo10) acts as a key regulator of TNT formation. Here, we designed an experiment to test the effects of endogenous reactive oxygen species (ROS) production on Myo10-dependent TNT formation. To establish a relationship between these two factors, HeLa cells were treated with Antimycin A (AMA), a secondary metabolite of Streptomyces. AMA has been linked to mitochondrial electron transport chain (mETC) inhibition via the disruption of complex III. Resulting disruption leads to increased ROS production. To determine the effects of this drug on the expression pattern of Myo10, cells were treated with 50μM AMA for 24 hours. Following treatment with AMA, Myo10 expression patterns were established through western blotting. To determine a correlative relationship between Myo10 expression and TNT formation during AMA treatment, TNTs were quantified via fluorescence microscopy. Our results indicate that after 24 hours of 50μM AMA treatment, cells exhibited elevated levels of Myo10 and TNTs. These results suggest that during the early stages of AMA-induced oxidative stress, Myo10-dependent TNT formation increases. By identifying a potential correlative connection between Myo10-dependent TNT formation and oxidative stress, novel therapies could be developed to slow the proliferation of diseases, such as cancer. | Poster #: 114 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: cardiomyocyte, proliferation, hormones Project Title: A chemically-defined assay to investigate the effects of hormones on heart muscle cell proliferation Author List: Pabla, Nanak; Undergraduate, Biological Sciences, San José State University, Presenting Author
Caampued, Andrew; Undergraduate, Biological Sciences, San José State University
Vanya, Hafsanoor; Undergraduate, Biological Sciences, San José State University
Barragan-Rocha, Berenice; Undergraduate, Biological Sciences, San José State University
Payumo, Alexander; Faculty, Biological Sciences, San José State University
Abstract The heart muscle cells – cardiomyocytes (CMs) – of newborn rodents possess a temporary ability to proliferate and regenerate cardiac muscle after injury. This capacity is lost in the first week of postnatal development when CMs enter the cell cycle but fail to divide and instead binucleate. To enable heart regeneration in adult mammals, it will be critical to define the physiological signals that block CM progression through the cell cycle. We’ve discovered that combined inhibition of thyroid hormone (TH) and adrenergic receptor (AR) signaling after birth promotes CM proliferation and regeneration in juvenile rodents. We hypothesize that interactions between these pathways inhibit CM cell cycle progression. The goal of this project is to develop a neonatal rat primary CM culture assay to directly test the effects of hormones that activate TH and AR signaling on CM cell cycle progression. We developed serum-free culture conditions to isolate the function of hormones otherwise present in unknown quantities in serum. We treated our cultures with 5-ethynyl-2’-deoxyuridine (EdU) to identify CMs entering the cell cycle at S-phase. We then detected EdU incorporation by click chemistry and determined CM-specific EdU labeling by immunostaining against cardiac troponin T. Under serum-free conditions, we observe 0% of CMs labeled with EdU, suggesting low levels of basal CM cell cycle entry. We then innovated the use of glycogen synthase kinase-3 (Gsk3) chemical inhibitors, previously reported to promote CM division, to trigger CM cell cycle entry with temporal control. Treatment with the Gsk3 inhibitor CHIR99021 for 48 hours stimulated EdU incorporation in 40.1% of cultured CMs. Next, we performed time-lapse imaging with digital holographic microscopy and identified events of both complete and incomplete CM division after CHIR99021 stimulation. To quantify how EdU-labeled CMs resolve the cell cycle, we washed out EdU and allowed 24 hours for cell cycle completion. Our preliminary results suggest that 5.8% of EdU-labeled CMs failed to divide and became binucleated after induction with CHIR99021. Establishing these baseline conditions, we will next apply this assay to test if TH and AR signaling activation impacts CM cell division. Unraveling the barriers limiting complete CM cell division will likely help guide the development of new technologies to unlock heart regenerative capacity in the adult human heart. | Poster #: 115 Campus: San Francisco State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila melanogaster, BRCA2, methylparaben Project Title: Determining the LD50 of Methyl and Ethylparaben in BRCA2 Mutants in Drosophila melanogaster Author List: Rizik, Zainab; Undergraduate, Biology, San Francisco State University, Presenting Author
Banuelos, Nicole ; Undergraduate, Biology, San Francisco State University, Presenting Author
Legesse, Lelahiwat; Graduate, Biology, San Francisco State University
Riggs, Blake; Faculty, Biology, San Francisco State University Abstract There have been increases in incidences of breast cancer since 1970 due to increased exposure to chemicals in our daily lives. Parabens are found in our everyday lives and are disproportionately pushed on poor communities via personal products and food. Parabens act like estrogen and attach to the receptors of breast cancer cells and cause them to proliferate. The BRCA2 gene, or the breast cancer gene, plays a vital role in providing instructions for creating a tumor suppressor protein. These proteins prevent cells from excessively multiplying. When there is a mutation in this gene, it could cause breast cancer due to the loss of function of the protein. It is unclear how antibiotics and chemical agents included in our food and grooming products affect those who carry mutations in tumor suppressor genes. This experiment analyzes the effect of the toxins methyl and ethylparaben on organisms with BRCA2 gene mutations using Drosophila melanogaster. They are strategically being used due to being easy to manipulate, store, and have a shorter life cycle. To study this, the lifespan assay of the wild type Drosophila in different concentrations of parabens (300, 700, 1000 mg/l) will be initially determined. There are 4 vials, which include control and three different concentrations of MP + EP or individual paraben with 20 flies placed in each. Dead flies will be counted every day and the remaining living will be transferred into a new vial with an identical diet every 2 days. The mean/maximum lifespan, and 50% survival were calculated based on the efforts observed in the liu, et al. paper. After determining the LD50 of the wild-type Drosophila melanogaster, the same lifespan assay was performed on heterozygous BRCA2 and homozygous BRCA2 mutants. Overall, it was found that there was increased synthetic lethality in flies at increased doses of parabens. | Poster #: 116 Campus: California State University, Los Angeles Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Pseudomonas aeruginosa, biofilm, glycoprotein, , Project Title: System-Wide Approach to Identify Glycoproteins and their Functions in Pseudomonas aeruginosa Author List: Trejos, Aldo; Undergraduate, Chemistry & Biochemistry, California State University, Los Angeles, Presenting Author
Stefan, Schulze; University of Pennsylvania
Pohlschroder, Mechthild; University of Pennsylvania
Abstract Glycosylation is a post-translational modification that adds sugar molecules to proteins affecting their function and properties. Glycosylation in prokaryotes has been detected for a few abundant cell surface proteins like adhesins, pilins, and flagellins. However, system-wide analyses to identify all target proteins of glycosylation are still missing in most prokaryotes. Expansive glycoproteomic analyses of pathogenic bacteria such as Pseudomonas aeruginosa, can reveal glycoproteins and the biological processes in which they are involved, thereby providing the foundation for the development of new drugs and treatments. This study optimized the subcellular fractionation of P. aeruginosa cultures into membrane, cytosol and culture supernatant, which each will be used to conduct future in-depth glycoproteomic analyses. Preliminary Coomassie and glycoprotein staining of these samples showed successful fractionation and potential glycoproteins. Besides the wild-type, we used mutants of three distinct glycosylation pathways to prepare the protein samples, which will serve as crucial negative controls in the glycoproteomic analysis. In addition, we characterized the effects of glycosylation on different biological processes by investigating the impact of these P. aeruginosa glycosylation mutants on colony morphology, swimming, swarming, and biofilm-forming ability. Each glycosylation pathway mutation resulted in a phenotype for at least one of the four assays, highlighting the importance of glycosylation in different cellular functions. This research shows that glycosylation is involved in significant pathogenic processes and may lead to the identification of new target proteins to treat infections. Furthermore, this system-wide analysis can serve as a blueprint to further our understanding of other less understood pathogenic bacteria. | Poster #: 117 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Cervical Cancer, Tumor supression, Anti-proliferation Project Title: Implementation of Novel Drug Compounds and shRNA Constructs to Unravel the Antiproliferative Effects of Hnt1 and p21 on HeLa Cells Author List: Cramer, Amber; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Ahmed, Ramadhan; Undergraduate, Biological Sciences, California State University, Fullerton, Presenting Author
Neil , Ashley; Undergraduate, Biological Sciences, California State University, Fullerton
Walker, Zy'Aan; Undergraduate, Biological Sciences, California State University, Fullerton
Passi, Kevin; Graduate, Biological Sciences, California State University, Fullerton
Patel, Nilay; Faculty, Biological Sciences, California State University, Fullerton Abstract The CDC projects that there will be 1.9 million new cancer cases through 2020, with cervical cancer projecting to make up 12.5% of new upcoming cases. As such, cervical cancer has become increasingly prevalent to public health concerns across diverse populations. In accordance with such need, this study investigates methods to decrease the proliferation of HeLa cells. A promising target for therapeutic treatment is the p21 protein, a tumor suppressor that binds to CDK2/cyclinE, halting the cell cycle at the G1/S phase. Our lab’s previous ICC data has shown robust p21 fluorescence after treatment with the novel compound Camptothecin (CAMP). However, the transfection data has been proven to be inconclusive due the shRNA construct being ineffective at knocking down p21 expression. After several months of troubleshooting, we have synthesized a new p21-shRNA construct that can sufficiently knockdown p21 expression with high fidelity. This study will use novel compounds synthesized by the de Lisjer lab to upregulate p21 expression and utilize shRNA constructs to knock down the expression of both genes. The expression profiles of drug-treated and transfected HeLa cells will be assessed using Western blot, ICC, and qPCR analysis. In conjunction with these assays, Cyquant experiments will be used to identify how modulating p21 expression affects the proliferation and expansion of HeLa cells. By increasing the expression of p21, the proliferation of HeLa cells should decrease due to the inhibition of cell cycle progression at the G1/S phase. | Poster #: 118 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Molecular Identification, Plants, American pika Project Title: Molecular Identification and Reference Genetic Database of American Pika-associated plants Author List: Thang, Stephanie; Undergraduate, Biological Sciences, San José State University, Presenting Author
Moreno, Diana; Undergraduate, Biological Sciences, San José State University, Presenting Author
Guru, Neha; Undergraduate, Biological Sciences, San José State University
Rashid, Muhammad; Undergraduate, Biological Sciences, San José State University
Castillo Vardaro, Jessica; Faculty, Biological Sciences, San José State University
Abstract The American pika (Ochotona princeps) is a generalist herbivore, meaning they eat a wide variety of plants. However, they are known to preferentially consume some plant species over others. In our research lab, we are applying DNA metabarcoding to identify the plants within pika feces to characterize their diets. In order to determine whether pikas select certain plants and avoid others, we are comparing what we find in their feces to what we find within and surrounding their territories. In addition to collecting pika feces, we conducted vegetation surveys and collected plant samples. Many of these plants are challenging to identify in the field, so we are using a molecular identification approach to confirm field identifications and to generate a reference database to compare the diet data to. To accomplish this, we extract DNA from plants collected in northwestern Nevada and the Sierra Nevada mountains, then sequence the internal transcribed spacer (ITS) region of the chloroplast genome. This data will enable us to 1) quantify diet selectivity, 2) characterize diet quality, and 3) compare among these two distinct regions. The reference genetic database generated will also be beneficial for future research on other taxa in the region. | Poster #: 119 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Apoptosis, Brain, Sexual differentiation Project Title: Gene Expression Profiling of Apoptosis-related Genes in the Developing Mouse Cortex and Hippocampus Author List: Weber, Ryan; Undergraduate, Department of Biological Sciences, California State University, Long Beach, Presenting Author, Nagel Award Finalist
Behrend, Anna; Graduate, Department of Biological Sciences, California State University, Long Beach
Tsai, Houng-Wei; Faculty, Department of Biological Sciences, California State University, Long Beach
Abstract Sexual differentiation of the brain is the developmental process by which discrete neural circuits underlying sexually dimorphic behaviors form. Like many other non-gonadal tissues, sexual differentiation of the brain is largely influenced by perinatal rises in testosterone secreted by the developing testes via the activation of androgen receptors and/or estrogen receptors. The cerebral cortex and hippocampus are known to play an important role in cognitive functions and social behaviors, many of which are sexually dimorphic. However, the identity of the molecular elements responsible for the functional and structural differences between the sexes in these two brain regions remains ambiguous. We have previously identified Rbm48 as a sexually dimorphic gene, expressed at higher level in the female mouse cortex/hippocampus than the male at postnatal days (PN) 0 and 7 under the control of testosterone. The neural function of the Rbm48 protein is still unclear, but RBM48 knockdown increases apoptosis in human cancer cell lines. Since sex differences in cell number in several sexually dimorphic brain nuclei is created by apoptosis, we hypothesize that androgen-regulated, differential expression of Rbm48 between the sexes might control brain sexual differentiation via regulation of apoptosis-related genes in a sex-specific manner. To test our hypothesis, we used Mouse Apoptosis RT2 ProfilerTM PCR Array (Qiagen, Germantown, MD) to measure mRNA levels of 84 selected apoptosis-related genes in the neonatal mouse cortex/hippocampus (n= 4-6 per sex). Among the 84 apoptosis genes measured, 82 expressed (with Ct value <35). Using a cutoff of (1) Log 2(fold change) <-1 or >1, or (2) adjusted p-value < 0.10, we identified seven sexually dimorphic candidate genes: two female-biased (Cradd and Naip1) and five male-biased genes (Akt1, Bcl10, Bok, Fadd, and Nfkb1). While Bcl10, Naip1 and Nfkb1 are anti-apoptotic, Akt1 and Bok are pro-apoptotic. In addition, Cradd and Fadd encode two adaptor proteins for caspase activation and recruitment. The expression of these candidates will be validated in the mouse cortex/hippocampus at PN0 and PN7 using RT-qPCR. Our study will not only elucidate the important role of apoptosis in sexual differentiation of the cortex and hippocampus, but also help us better understand the mechanisms underlying the processes regulating sex-specific susceptibility to the neurodevelopmental disorders, such as autism which disproportionately affect men. | Poster #: 120 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: neuroscience, synapse, kinase Project Title: The cGMP-dependent protein kinase egl-4 is necessary for sensory synapse formation in Caenorhabditis elegans Author List: Graves, Josiah; Undergraduate, Biological Sciences, San José State University, Presenting Author
Briseno, Fabiola; Undergraduate, Biological Sciences, San José State University, Presenting Author
Idris, Maryam ; Undergraduate, Biological Sciences, San José State University
Bi, Veronica; Staff, Biological Science, San José State University
Lu, Jiamei; Undergraduate, Biological Sciences, San José State University
Kaur, Sukhdeep; Undergraduate, Biological Sciences, San José State University
Nassif, Cibelle; Graduate, Biological Sciences, San José State University
Nguyen, Christina ; Undergraduate, Biological Sciences, San José State University
Butelet, Julia; Undergraduate, Biological Sciences, San José State University
Varshney, Aruna; Staff, Biological Sciences, San José State University Abstract One of the key elements of a functional nervous system is the ability to regulate synaptogenesis post-embryonically in accordance with the level of neuronal activity. This has been studied in the mammalian visual system, where loss of sensory stimulation to one eye results in the other eye increasing innervation in the visual cortex. However, despite its potential importance in understanding neurological disorders, such as autism and schizophrenia, little is known about the molecular mechanisms that underlie sensory activity-dependent synapse formation. To better understand these mechanisms we are studying the model organism C. elegans, which has a compact and well-characterized nervous system, is amenable to genetic manipulation, and is optically clear, facilitating live imaging. We have focused on synaptic connections between the PHB chemosensory neuron and AVA interneuron pairs. To visualize these specific PHB-AVA synapses, we utilized Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP), a split GFP-based trans-synaptic fluorescent marker. We first conducted a time course experiment to determine if and how synaptic levels change after embryogenesis. Analysis of synchronized populations and single-worm time courses demonstrates that there is a burst of synaptogenesis between the first larval stage and the second and third larval stages. To determine whether the burst of synaptogenesis was regulated by sensory activity, we tested a G-protein coupled receptor mutant in which PHB neurons do not receive key sensory information. Interestingly, the burst of synaptogenesis was reduced, indicating a requirement for sensory activity. In C. elegans, chemosensation leads cGMP production. Therefore, we tested mutations in the C. elegans homolog of the mammalian cGMP-dependent kinase (PKG), egl-4, which is expressed in PHB neurons. We discovered that egl-4/PKG mutants have a striking reduction in synaptogenesis between PHB and AVA neurons, indicating that this gene is required for the sensory-dependent synaptic burst. egl-4/PKG is known to act as a transcription factor and affect synaptic molecules in other neurons. Therefore, egl-4/PKG may convert the sensory signal into a pathway that promotes synaptogenesis. We are currently working to identify additional molecules in this pathway, to better understand the molecular mechanisms that mediate sensory-dependent synapse formation. This work was funded by NIH (R15NS109803 to MV). | Poster #: 121 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Sleep, Synapse, Memory Project Title: Olfactory synapses are modulated by odor training and sleep in Caenorhabditis elegans Author List: Farah, Fatima; Staff, Biology, San José State University
Bokka, Anirudh; Graduate, Biology, San José State University, Presenting Author
Varshney, Aruna; Staff, Biology, San José State University
Jimenez, Vanessa; Staff, Biology, San José State University, Presenting Author
Baradwaj, Anjana; Staff, Biology, San José State University, Presenting Author
Nassif, Cibelle; Graduate, Biology, San José State University, Presenting Author Abstract Sleep is necessary for memory formation in many metazoan systems. However, little is known about the effects of sleep on specific neuronal connections, despite synaptic modulation being hypothesized to underlie learning and memory. Our goal is to better understand how sleep modulates neuronal connections by studying the simple nervous system of the nematode C. elegans. We focus on the AWC olfactory circuit, which mediates attraction to butanone, an attractive odorant secreted by some of their bacterial food sources. We hypothesize that synapses may be altered in animals that acquire a sleep-dependent long-term olfactory memory. C. elegans can learn to ignore butanone when it is paired with removal from food in three successive cycles of training, if they are allowed to sleep in the two hours immediately after training. Using the fluorescent split GFP-based trans-synaptic marker Neuroligin 1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP), we examined whether synapses between the butanone-sensing AWC neurons and their postsynaptic partners, the AIY interneurons, underwent changes after the training. Using a single-worm behavior and imaging assay, we found that between 2 and 16 hours post-training, a greater proportion of butanone-trained animals had large reductions (>50%) in synaptic intensity compared to buffer-trained animals. These results are consistent with our findings from population assays, in which AWC-AIY synapses in butanone-trained animals were significantly reduced 16 hours after training in butanone-trained animals, when compared with buffer-trained animals. Interestingly, loss of sleep disrupted this synaptic modulation in butanone-trained animals. As AWC-AIY connections are thought to govern chemotaxis to butanone, this structural synaptic modulation could explain the diminished response to this odor. We therefore propose that this synaptic change may represent the memory trace. These findings indicate that sleep affects synapses in the simple nervous system of the nematode, and help to define the mechanisms by which sleep promotes learning and memory in metazoans. We hope that these findings may lead to the development of more effective treatments for diseases in which memory is altered, such as Alzheimer’s disease and dementia. This work is supported by the NIH (NIH R01 NS08754401 and NIH R01 DC005991 to MV and NL). | Poster #: 122 Campus: San José State University Poster Category: Other Keywords: protein engineering, fluorescent proteins, cancer Project Title: Engineering Proteins to Measure Acid Levels in Living Cells Author List: Fabillaran, Trisha Marie; Undergraduate, Biological Sciences, San José State University, Presenting Author
Crnjac, Andela; Undergraduate, Biological Sciences, San José State University, Presenting Author
SAndoval, Barbara; Graduate, Biological Sciences, San José State University
Grillo-Hill, Bree; Faculty, Biological Sciences, San José State University Abstract Background: In many cancers, intracellular pH is higher and disrupts many normal cell processes resulting in hyperplasia, dysplasia, and metastasis. In order to study pH changes in cells, scientists have developed different types of tools such as pH sensitive dyes, genetically encoded pH biosensors, nanoprobes in electrodes. However, each of these tools have limitations on how well it can be used in vivo to study tumors in a multicellular organism. Instead, why not engineer a ratiometric pH biosensor that is red and can be used to measure acid levels inside cells? Our project goal is to engineer three mFruits red fluorescent proteins with mutations that we predict will alter the optical properties and confer ratiometric pH-sensitivity. Our approach is based on the protein engineering methods used to create green fluorescent pH sensors. However, we are starting with red fluorescent proteins, as the longer wavelengths are more effective in 3D tissues, and can be combined with many GFP-based imaging tools.
Methods: We identified three mFruit proteins (mNectarine, pHorange, and pHuji), and performed a protein sequence alignment and 3D structural alignment to identify conserved amino acids that confer pH-sensing in green fluorescent proteins. We used site-directed mutagenesis to mutate three amino acids (A145E, S146H, L204H), including two to histidine, which has a pKa near neutral and thus will become protonated and deprotonated at physiological pH. We made the a single mutation (L204H), a double mutation (A145E, S146H) and triple mutation. These genes were next cloned into a plasmid for expression and purification in E. coli. Following expression in E. coli, we made crude lysates and protein fluorescence was characterized in a fluorescent plate reader.
Results: Sequencing results show that we successfully generated one set of mutations in all three mFruits proteins. Preliminary protein fluorescence data shows that L204H does not significantly change the optical properties of pHorange, but shows an overall decreased fluorescence intensity. Next, we will characterize protein fluorescence in the other two proteins, and will proceed with making the next sets of mutations. This new pH biosensor will be an important tool to understand how increased intracellular pH promotes cancer.
| Poster #: 123 Campus: Sonoma State University Poster Category: Other Keywords: curriculum, inclusivity, pedagogy Project Title: Moving Towards Inclusivity in Chemistry Using Group-Worthy Learning Tasks and Class Support Activities to Decrease Equity Gaps Author List: Lillig, Jennifer; Staff, Academic Programs, Sonoma State University, Presenting Author
Works, Carmen; Faculty, Chemistry, Sonoma State University, Presenting Author
Daubenmire, Paul; College of Marin
Antonakos, Cory; Diablo Valley College
Palmer, Erin; Diablo Valley College
Stacy, Angy; University of California Berkeley
Beaulieu, Ellen; Diablo Valley College
Nguyen, Hien; College of Marin
Claesgans, Jennifer; College of Marin
Clark, Chase; Staff, Chemistry, Sonoma State University Abstract The overall goal of our work is to disrupt pervasive narratives and misplaced assessments of what defines scientific brilliance, specifically in the introductory chemistry classroom. We have developed a collection of 45 chemistry activities grounded in data and critical thinking. Activities are grouped under the umbrella topics of matter, measurement, and change and are free through the CanvasCommons learning management system. In these activities, students use various types of data to investigate a question and develop an understanding of the key chemical concept for that lesson. Over three semesters, activities were gradually incorporated into an Introductory Chemistry course taught by the same instructor. ~40% of students in the course identified as an underserved minority (USM). During this pilot, the USM GPA increased by 0.88 GPA units and the USM DFW rate decreased by 13 percentage points. In contrast, the non-USM GPA remained relatively unchanged over the course of the pilot and change in DFW rate. Additional data from a partner college showed a 10% increase in successful responses to an embedded test question for the course section that utilized these activities as the primary pedagogy compared to their section not using the materials. Retention and success rates in introductory chemistry at this institution increased by 7.5% and 13.4%, respectively, for students that utilized these materials (N = 49) vs. those that did not (N=581). These data are promising and we are now working to complete a more rigorous comparison. This work is supported by a California Learning Lab award. | Poster #: 124 Campus: San José State University Poster Category: Other Keywords: muscle spindle, electrophysiology, sympathetic nervous system Project Title: Effect of Sympathetic Neurotransmitters on Muscle Spindle Afferent Sensitivity to Muscle Stretch in Adult Mice Author List: Gomez, Teodomiro; Graduate, Biological Sciences, San José State University, Presenting Author
Ortiz, Serena; Undergraduate, Biological Sciences, San José State University, Presenting Author
Harnisch, Arthur; Graduate, Biological Sciences, San José State University
Snyder, Erika; Undergraduate, Biological Sciences, San José State University
Andrade, Tim; Undergraduate, Biological Sciences, San José State University
Wilkinson, Katherine; Faculty, Biological Sciences, San José State University Abstract Proprioception is the sense of body position and movement in space. Proprioception is necessary for complex motor movements, maintenance of posture, and also helps to protect the body against injurious movements. The primary sensory input for proprioception comes from muscle spindle afferents (MSAs), which are mechanoreceptors present in muscle spindles that relay information about changes in muscle length to the Central Nervous System. The muscle spindle is also innervated by sympathetic neurons, but the role of this sympathetic innervation on muscle spindle function is not well understood. Here we test the hypothesis that MSAs will exhibit decreased firing in response to muscle stretch following exposure to the sympathetic neurotransmitters norepinephrine and epinephrine. To test this hypothesis we used an ex vivo mouse muscle-nerve preparation. We recorded MSA firing activity during ramp-and-hold stretch and sinusoidal vibration before and after exposure to norepinephrine, epinephrine, or adrenergic receptor agonists. We observed significantly decreased firing during the end of stretch after exposure to both norepinephrine (100 µm , n=6 ) and epinephrine (30 µm , n=6 ). To identify the adrenergic receptor(s) involved, we also tested two ɑ2 adrenergic receptor agonists, Clonidine and Dexmedetomidine, and the ɑ1 receptor agonist phenylephrine. Both ɑ2 receptor agonists caused a decrease in MSA firing (1 mM clonidine, 5 of 8 decreased firing; 100 µm dexmedetomidine, 2 of 3 decreased firing). Phenylephrine had no significant effect on MSA firing (100 µm n=2; 30 µm n=6). These results show direct effects of sympathetic neurotransmitters on MSA stretch sensitivity and further support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating MSA tone and therefore motor control. Future studies will utilize optogenetic techniques to specifically stimulate sympathetic neurons innervating the EDL muscle to further probe the importance of this mechanism in the regulation of MSA sensitivity. | Poster #: 125 Campus: California State University, East Bay Poster Category: Other Keywords: acute pain, cannabinoid, behavior Project Title: Behavioral effects of the Cannabis-related terpene ß-caryophyllene in male rats Author List: Sanchez, Stephanie; Undergraduate, Psychology, California State University, East Bay, Presenting Author
Quintana, Taylor; Undergraduate, Psychology, California State University, East Bay, Presenting Author
Kandasamy, Ram; Faculty, Psychology, California State University, East Bay
Abstract Despite decades of research examining the biological mechanisms of pain, few new treatments have been developed. Understanding the behavioral effects of novel pain-relieving agents (i.e., analgesics) requires the use of tests of pain and locomotor activity in laboratory animals. Combining these two measures allows for the identification and understanding of whether a drug can inhibit pain-related behaviors and whether the drug affects general movement. A good analgesic should inhibit pain without affecting general movement in healthy animals. To date, few studies have combined tests of pain and locomotor activity to identify effective doses of novel analgesics. The objective of our experiments was to combine acute pain tests (i.e. tests that do not inflict long-term pain) and home cage wheel running to evaluate the analgesic and behavioral effects of a novel class of analgesics, Cannabis-related terpenes. ß-caryophyllene (BCP) is a terpene found in many strains of Cannabis, and some animal studies indicate that they may provide pain relief without dangerous or unpleasant side effects. We hypothesized that BCP would alleviate acute pain and not affect wheel running activity in healthy male rats. We used male Sprague-Dawley rats to evaluate the analgesic effects of BCP in acute pain tests (hot plate and tail withdrawal tests). Home cage wheel running was used to measure general locomotor activity and changes in behavior not related to pain. After BCP administration via intraperitoneal injection, both pain tests revealed that low doses (3 and 10 mg/kg) of BCP did not produce antinociception on the hot plate and tail withdrawal tests; however, a 30 mg/kg dose produced antinociception in the 15 min following intraperitoneal (i.p.) administration. Further, doses of 30 mg/kg and 100 mg/kg BCP did not affect wheel running, suggesting that these doses may not produce behaviorally disruptive side effects. These results indicate that low to medium doses of BCP may be effective at alleviating acute pain without side effects. Future studies can use a combination of tests of acute pain and wheel running to evaluate behavior changes for novel analgesics without inducing long-term injury to animals. Funded by the College of Science at Cal State East Bay and the CSUPERB President’s Commission Scholars Program. | Poster #: 126 Campus: California State University, Sacramento Poster Category: Other Keywords: Drosophila melanogaster, neurodevelopment, bisphenol A Project Title: Bisphenol A exposure impacts neurodevelopmental gene expression, learning, and synaptic morphology in Drosophila melanogaster Author List: Anderson, Judith ; Graduate, Biological Sciences, California State University, Sacramento, Presenting Author
Raghulan, Rashi; Graduate, Computer Science, San José State University, Presenting Author
Johnson, Eden; Graduate, Computer Science, San José State University
Welch, Chloe; Graduate, Biological Sciences, California State University, Sacramento
Tupikova, Angelina; Graduate, Biological Sciences, California State University, Sacramento
Lee, Wendy; Faculty, Computer Science, San José State University Abstract INTRODUCTION: Bisphenol A (BPA) is an environmentally prevalent endocrine disrupting chemical that can impact human health. Emergent data indicate BPA may be an environmental risk factor for neurodevelopmental disorders—BPA has been associated with behavioral impairment in children and it causes a variety of neurodevelopmental phenotypes in model organisms. Further analysis is needed to comprehensively delineate the cellular and molecular impacts BPA has on developing nervous systems. In this study, we used Drosophila melanogaster as model to explore the consequences of developmental BPA exposure on gene expression, cognitive function, and synapse development. METHODS & RESULTS: Following RNA-sequencing, we performed Gene Set Enrichment Analysis (GSEA) and found neurodevelopmentally relevant pathways were significantly impacted by BPA (0.2mg/ml). Among the top misregulated genes were those associated with learning, synapse development, neural stem cell development, and axon guidance. We had previously found that BPA (0.2mg/ml) affects neural stem cell development and axon guidance, but we had not examined learning or synapse development. Here, we used an established behavioral paradigm called conditioned courtship to evaluate associative learning. In this paradigm, male flies are exposed to an unreceptive/pre-mated female (an “aversive stimulus”) for an hour. Courtship activity is assessed for the first and final ten minutes of the hour. Flies with unimpaired learning exhibit a significant decrease in their courtship activity in the final ten minutes compared to the initial ten minutes. We found that BPA-treated flies did not reduce their courtship activity in the final ten-minute interval, indicating that BPA impairs learning. We also used immunofluorescence and confocal microscopy to examine synapse morphology within the larval neuromuscular junction and found BPA significantly increased the number of axonal branches. Our findings align with studies of BPA in mammalian model organisms, suggesting that BPA impairs functionally conserved neurodevelopmental pathways. Further, because Drosophila do not possess classic estrogen receptors or estrogen, this research indicates that BPA can impact neurodevelopment by molecular mechanisms distinct from its role as an estrogen mimic. FUTURE DIRECTIONS: We are currently investigating how structural analogs to BPA affect neurodevelopmental phenotypes. FUNDING: NIH SCORE SC2 Award, 1SC2GM132005. | Poster #: 127 Campus: California State University, East Bay Poster Category: Other Keywords: chronic pain, cannabinoid, behavior Project Title: The Effects of ß-caryophyllene on Mechanical and Thermal Hypersensitivity in Rats with Chronic Inflammatory Pain Author List: Chin, Christopher; Undergraduate, Psychology, California State University, East Bay, Presenting Author
Ron, Katiana; Undergraduate, Psychology, California State University, East Bay, Presenting Author
Kandasamy, Ram; Faculty, Psychology, California State University, East Bay
Abstract Cannabis has been used to treat pain for centuries; however, our understanding of the compounds in the plant that produce pain relief is limited. The antinociceptive (i.e., pain-relieving) effects of major cannabinoids such as ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have been extensively studied in rodents. These studies have led to formulations of THC and CBD for human use; however, humans use different strains of Cannabis that contain several hundred different compounds. The contribution of these compounds to pain relief produced by Cannabis is unclear. ß-caryophyllene (BCP) is one compound found in the essential oils of Cannabis. Despite some early studies, the extent to which these compounds produce pain relief against chronic inflammatory pain in living biological systems remains unclear. We hypothesized that BCP would reverse mechanical and thermal hypersensitivity in male Sprague-Dawley rats with inflammatory pain. Three different doses of BCP (10, 30, and 100 mg/kg) were administered to rats via an intraperitoneal injection after hindpaw inflammation. We also used tests of mechanical and thermal hypersensitivity (von Frey and Hargreaves tests) to evaluate thresholds in rats with an inflamed hind paw. Briefly, the von Frey test requires the application of a blunt probe to the plantar surface of the hindpaw to detect the amount of mechanical force required to elicit paw withdrawal. The Hargreaves test requires the application of an infrared heat source to detect the amount of time needed to elicit paw withdrawal from the heat. Neither the low dose (10 mg/kg) nor the medium dose (30 mg/kg) of BCP reversed hypersensitivity in injured rats. However, a high dose of BCP, 100 mg/kg, was necessary to reverse mechanical hypersensitivity; however, this dose did not reverse thermal hypersensitivity. Therefore, BCP produces pain relief although it only does so against certain types of pain. Future studies of the pain-relieving effects of Cannabis constituents must include tests of many pain-related behaviors to understand dose-response relationships and their therapeutic potential. Funding provided by the College of Science at Cal State East Bay.
| Poster #: 128 Campus: San Francisco State University Poster Category: Other Keywords: Vision, Retina, Connectome Project Title: Connectomic Reconstructions of Retinal Cell Circuitry in the Leucoraja erinacea All-Rod Retina Author List: Magaña Hernandez, Laura; Graduate, Biology, San Francisco State University, Presenting Author
Perry, Priscilla ; Undergraduate, Biology, San Francisco State University
Hamada, Elizabeth; Undergraduate, Biology, San Francisco State University
Dibaj , Parsa; Undergraduate, Biology, San Francisco State University
Anastassov, Ivan ; Faculty, Biology, San Francisco State University
Abstract Lining the back of the eye is a thin neural tissue called the retina which is responsible for converting light rays into neural signals. In order to process visual information coming from the outside world, our photoreceptors; the rods and cones, detect photons of light and convert that into an electrical signal. These light sensitive neurons are the first cells in the visual pathway and are essential for proper vision. Most vertebrate retinas are known to contain both photoreceptors and task each population with handling a particular range of light intensities. Our research is aimed at understanding how the pure-rod retina of the Little skate (Leucoraja erinacea) can perform rod and cone functions with a monotypic population of photoreceptors. My objective is to describe the anatomical properties of rod circuitry and the morphology of individual synaptic connections within the context of the evolutionarily optimized visual system of the little skate retina. Serial block-face scanning electron microscopy (SB-3DEM) was performed on individual retinal samples. The datasets analyzed are from a region of interest (ROI) in the outer plexiform layer, as well as from a full cross-section of the skate retina. I used serial section electron microscopy data to obtain 3-D reconstructions of circuit and cell-level ultrastructural features in the little skate retina. Preliminary data shows at least ~12-14 (and as many as 25) anatomically distinct processes that invaginate into the rod terminal and can be traced to one or several different synaptic ribbons. Extensions of the rod terminal, referred to as telodendria, make contacts with the telodendria of other rods. The skate rod does not have an axonated terminal and ~6-8 telodendria (~2-18 μm in length) can be traced to either neighboring rods, or yet unidentified cellular processes. Therefore, I hypothesize that skate rods in the outer plexiform layer make significantly more contacts to postsynaptic cells when compared to rods found in duplex retinas, i.e. retinas with both rods and cones. These preliminary results suggest that the rods in the little skate retina could have a hybrid rod-cone anatomy. Results from this research could provide new insight about the evolutionary modifications that a duplex vertebrate retina can make. Addressing these issues may also lead to the developmental efforts to preserve and rescue the function of the rods for patients with degenerative diseases or injury. | Poster #: 129 Campus: California State University, San Bernardino Poster Category: Other Keywords: Retinoblastoma Protein, Cancer Biochemistry , Differential Scanning Fluorimetry Project Title: Biochemical Characterization of Cancer-Associated Missense Mutations in the Retinoblastoma Protein Author List: Castro, Anthony ; Undergraduate, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Moorman, Chad; Staff, Chemistry and Biochemistry, California State University, San Bernardino, Presenting Author
Wolf-Saxon, Emma; Staff, Chemistry and Biochemistry, California State University, San Bernardino
Burke, Jason; Faculty, Chemistry and Biochemistry, California State University, San Bernardino Abstract The retinoblastoma protein (Rb) was first discovered in a malignant childhood eye tumor, retinoblastoma. Since its discovery, Rb has been observed as dysfunctional in various human cancers. Rb is a tumor suppressor that regulates a critical checkpoint in the cell cycle (G1 to S-phase) and represses cell growth through stable interactions with the E2F transcription factors. Genome sequencing data have revealed several significant recurrent missense mutation variants across many cancer types. The hypothesis is that these mutations destabilize the structure of Rb and disrupt the natural interactions between Rb and its protein binding partners. The methods used to test this began with a search through cancer genome sequence data to identify missense mutations in Rb. We transformed E. coli cells to overexpress the Rb protein wild-type or mutation variants. The protein underwent purification through affinity chromatography and fast protein liquid chromatography (FPLC). Bradford assays measured the protein expression levels relative to wild type and SDS-PAGE analysis demonstrated the purity of the mutation variants. Using differential scanning fluorimetry (DSF), thermal shift and binding affinity assays measured the changes in protein stability and protein-ligand binding interactions. Our results from the Bradford assay and SDS-PAGE analysis revealed that missense mutations tend to reduce Rb expression compared to the wild type, sometimes nearly halting protein expression altogether. The DSF data showed that mutations disrupted the overall stability and binding interactions of Rb variants. The conclusions drawn from the variants studied are that missense mutations adversely alter expression levels, structure stability, and binding interactions of Rb. These mutations introduce structural modifications to Rb that may contribute to the dysregulation of the cell cycle and uncontrolled cell proliferation observed in tumorous cancers. This study was made possible by the National Institute of Health and CSUPERB. | Poster #: 130 Campus: California State University, Fullerton Poster Category: Proteins (Include Proteomics) Keywords: cell signaling, TGF-beta, extracellular matrix Project Title: Investigating MAGP2’s potential regulation of the TGF-β signaling pathway, and its role in heritable FTAAD Author List: Glaab, Michael; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Miyamoto, Alison; Faculty, Biological Science, California State University, Fullerton Abstract Aortic abnormalities exist in several human genetic disorders, including thoracic aortic aneurysms and dissections (TAAD), Loeys-Dietz, and Marfan syndrome. In many of these cases, mutations in genes related to the TGF-β signaling pathway have been identified. Such mutations can result in excessive TGF-β signaling pathway activity, which is implicated as the causal factor for many diseases featuring TAAD. In some cases of familial (heritable) TAAD (FTAAD), mutations have been found in microfibril associated glycoprotein 2 (MAGP2). MAGP2 has been shown to interact with TGF-β; however, the potential regulatory effect of MAGP2 on TGF-β is still unknown. To determine if MAGP2 binding with TGF-β inhibits TGF-β pathway activity, TGF-β and MAGP2 were added to cells in vitro, then activity of the TGF-β pathway was checked. TGF-β pathway activity causes the phosphorylation of the downstream proteins SMAD2 and SMAD3 that can serve as indicators of pathway activity and are detectable via Western blotting. Preliminary results show a decrease in downstream SMAD2/3 phosphorylation when MAGP2 is pre-incubated with TGF-β, suggesting that MAGP2 has a direct inhibitory effect on the TGF-β pathway. Experiments will also be performed where TGF-β and the either the wild-type MAGP2 or the FTAAD mutant form of MAGP2 are pre-incubated and added to cells in vitro. This will test my hypothesis that the FTAAD mutant form of MAGP2 does not bind to TGF-β properly, thereby reducing the inhibition of TGF-β compared to the wild-type form. Elucidating the role of MAGP2 may reveal novel functionality of MAGP2 and could help develop therapies for TAAD where mutations in MAGP2 cause aortic abnormalities.
I would like to thank CSUPERB for Graduate Student Research Restart funding for this project.
| Poster #: 131 Campus: California State University, Northridge Poster Category: Proteins (Include Proteomics) Keywords: chaperone protein, NMR spectroscopy, structure-function studies Project Title: Learning about protein stability and allostery through a conservative mutation Author List: Duguil, Debra; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Martinez, Amanda; Undergraduate, Chemistry and Biochemistry, California State University, Northridge, Presenting Author
Crowhurst, Karin; Faculty, Chemistry and Biochemistry, California State University, Northridge
Abstract HdeA is an acid-stress chaperone protein that protects the survival of pathogenic gram-negative bacteria in the stomach and intestine, thereby aiding the spread of dysentery. At neutral pH, HdeA is an inactive folded dimer, while at pH 2 it is active and partially unfolded; exposed hydrophobic portions of HdeA bind other unfolded client proteins at the low pH of the stomach and protect them from irreversible aggregation and bacterial death.
We previously found preliminary evidence that an isoleucine to leucine mutation (HdeA-I62L) is more disruptive to the folded protein than expected for a conservative single-point mutation. For this project we therefore wanted a deeper study of this mutant at pH 6, investigating the allosteric impact of the mutation, its overall stability, and chaperone activity. Our main tool is NMR spectroscopy, a technique that allows for atomic-level analysis. An overlay comparing the 1H-15N NMR spectra (which probe mainly backbone amides) for wild type and mutant showed evidence for widespread chemical shift changes, indicating disturbance to the protein. To analyze the location of the largest chemical shift changes of HdeA-I62L, we recorded a set of NMR spectra that enabled us to assign backbone amide chemical shifts and to pinpoint which residues were impacted by the mutation. Chemical shift assignment is not quite complete, but preliminary analysis illustrates that residues around the single-point mutation are more prone to a shift. Although local effects may be expected, calculated chemical shift perturbation (CSP) values indicate that these residues differ by at least two standard deviations above the average chemical shift change, which is much larger than anticipated, considering that the I to L mutation involves only the transfer of a methyl group from the beta to the delta carbon. Additionally, numerous residues not adjacent to the mutation also have large CSPs, indicating that an allosteric impact is observed in HdeA-I62L. Future experiments include additional NMR experiments to observe changes in backbone flexibility, and chaperone function tests of HdeA-I62L with a client protein. From this work, we hope to better understand HdeA’s sensitivity to small changes and its effects on structure and function.
We are grateful to the CSUPERB Presidents’ Commission Scholars Program for research support to D.D, as well as the NIH for research funds (SC3-GM116745) and the NSF for our NMR spectrometer (CHE-1040134). | Poster #: 132 Campus: California State University, Long Beach Poster Category: Proteins (Include Proteomics) Keywords: Enzyme replacement therapy, Protein engineering, Nanomedicine Project Title: Development of Nanodiscs with Apolipoprotein E3 Based Chimera for the Treatment of Lysosomal Storage Diseases Author List: Nguyen, Christy; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Metkar, Vidya; Graduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Narayanaswami, Vasanthy; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract Lysosomal storage diseases (LSDs) are a group of inherited metabolic diseases that are characterized by enzyme deficiencies that affect the function of the lysosome. Niemann-Pick disease types A and B are LSDs that are caused by the deficient enzyme, acid sphingomyelinase (ASM), resulting in the toxic buildup of sphingomyelin in the lysosome. Despite the promising outcomes of potential treatments such as enzyme replacement therapy (ERT), there are also limitations to be considered; ERT lacks the ability to undergo the biodistribution of recombinant enzymes into the central nervous system and to penetrate the cell membrane for eventual delivery to the lysosomes. Alternatively, developing a reconstituted high-density lipoprotein (rHDL) nanodisc surrounded by apolipoprotein E3 (apoE3) and recombinant ASM may prove effective in targeting the lysosome. rHDL is an ideal platform for the delivery of enzymes since apoE3 is known to bind low-density lipoprotein (LDL) receptors and can facilitate receptor-mediated endocytosis where the lipoprotein components are delivered to the lysosomes. We hypothesize that recombinant ASM can be delivered as a chimeric protein with apoE3 in a nanodisc. Our overall objective is to determine whether the fusion of a receptor-binding segment of apoE3 with functional ASM (ASM-apoE3NT) can bind to LDL receptors and be targeted to the lysosomes. We designed a plasmid construct consisting of the N-terminal (NT) domain of apoE3 and the C terminal domain of ASM, linked by a flexible linker, with a hexa-His-tag attached at the NT end. We subcloned the chimeric insert into a pET20b(+) expression vector. In preliminary studies, we carried out an expression of ASM-apoE3NT using E. coli and purified the protein nickel affinity chromatography but obtained poor yields. In the next step, we will optimize expression, carry out the structural characterization of the chimera through fluorescence and circular dichroism spectroscopy, assess the enzyme activity of ASM, and generate rHDL nanodiscs. Subsequently, the goal is to determine cellular uptake and lysosomal localization through fluorescence microscopy. Successful findings of this study may provide a non-invasive and targeted method for the delivery of large molecule therapeutic agents to treat LSDs. | Poster #: 133 Campus: California State University, Long Beach Poster Category: Proteins (Include Proteomics) Keywords: Targeted drug delivery, Nanotechnology, Lipids and Proteins Project Title: Targeting Nanodiscs to Breast Cancer Cells Expressing Matrix Metalloproteinase 14 Author List: Abdulhasan, Zahraa; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Kulkarnia, Daanish; Graduate, Chemistry and Biochemistry, California State University, Long Beach
Narayanaswami, Vasanthy; Faculty, Chemistry and Biochemistry, California State University, Long Beach
Abstract Triple-Negative Breast Cancer (TNBC) counts for 15-20% of breast cancer cases. It lacks common biomarkers and is highly aggressive and metastatic. It is shown that TNBC cells overexpress matrix metalloproteinase 14 (MMP14) on their surface, a protein that degrades the extracellular matrix to allow the cells to metastasize and spread to distal areas of the body, worsening disease prognosis. It is also shown that MMP14 protein is present on the surface of MDA-MB-231 cells, an in vitro model for late-stage TNBC. Our overall goal is to target MMP14 using a peptide ligand (called AF7p) attached to apolipoproteinA1(apoAI)/lipid nanodiscs as bait. An important aspect of targeting is to demonstrate that the bait is specific for MMP14. We hypothesize that the peptide ligand attached to nanodiscs will bind to cancer cells expressing MMP14. We used flow cytometry to test our hypothesis by comparing nanodiscs binding to MDA-MB-231 cells, an in vitro model for late-stage TNBC that have been reported to express MMP14 and A172 human glioblastoma (GBM) cells, which have been reported to lack MMP14. In initial studies, MDA-MB-231 and A172 GBM cells were grown to confluency, isolated, probed with anti-MMP14 primary antibody followed by treatment with goat-anti-mouse Alexa-555 labeled secondary antibody and suspended in flow cytometry buffer. In control experiments, cells were incubated as such with no added antibodies, or treated with secondary antibody only or with isotype and secondary antibody. Flow cytometric analysis revealed the presence of MMP14 on MDA-MB-231 cells and negligible presence on A172 GBM cells. Our next step is to conduct targeting studies with nanodiscs prepared with AF7p and independently validate the results by immunofluorescence. Results from this study are significant since they have potential to selectively target cancer cells using nanodiscs loaded with a cocktail of chemotherapeutic drugs for TNBC treatment. | Poster #: 134 Campus: California State University, Chico Poster Category: Synthetic Chemistry Keywords: Synthesis, Small Molecule, Medicinal Chemistry Project Title: Development of Potent Isoflavone Carbamate Derivatives to Target Alzheimer’s Disease Author List: Chatha, Baljot; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Waite, Cody; Undergraduate, Chemistry and Biochemistry, California State University, Chico, Presenting Author
Arpin, Carolynn; Faculty, Chemistry and Biochemistry, California State University, Chico
Abstract Alzheimer’s disease (AD) is a progressive neurodegenerative disorder commonly found among the elderly. In 2021 a total of 6.2 million in the U.S. were living with Alzheimer’s, which represents 1 in 9 people over the age of 65. It is projected to affect nearly 14 million Americans by 2060. There is no definitive cure, and little is known about the formation of the hallmark trait(s) of the disease. On a biochemical level, the disease is associated with decreased levels of the neurotransmitter acetylcholine (ACh) in the brain. Acetylcholinesterase (AChE) is the enzyme responsible for the hydrolyzing of ACh. Current pharmaceuticals used to treat AD are reversible inhibitors of AChE. Previous research has shown that AChE inhibitors with isoflavone scaffolds and others with carbamate moieties have all been exhibiting great potential to be potent inhibitors of AChE. For example, the carbamate moiety is present in rivastigmine, a pharmaceutical currently used to treat AD, and many non-approved FDA AChE inhibitors like neostigmine, physostigmine, and pyridostigmine. Carbamates are a key structural motif in many other FDA-approved drugs. Thus, we have designed and are currently preparing a library of novel AChE inhibitors that combine both the isoflavone and carbamate functionalities within the same molecule. Up to this point, our isoflavone diol synthesis has been successfully completed in a 35% overall yield. Next, we attempted to install carbamates onto one or both alcohols of our isoflavone diol to no avail. However, we did successfully prepare diester compounds that were then assessed for inhibitory activity against AChE using Ellman’s assay. Thankfully, several newly prepared and tested compounds showed some inhibitory activity, which was also consistent with our findings from computational docking studies carried out using PyRx and Chimera. Currently we are troubleshooting other reaction conditions to yield carbamates on the isoflavone diol, and we are preparing other isoflavone derivatives and testing all new compounds for their inhibitory activity. Inhibitor design, synthetic efforts, computational docking studies, and preliminary biological activity will all be presented. | Poster #: 135 Campus: California State University San Marcos Poster Category: Synthetic Chemistry Keywords: gold catalysis, intramolecular, etherification Project Title: Microwave-assisted, gold-catalyzed intramolecular etherification of diols to afford cyclic ethers Author List: Wagner, Madison; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Iafe, Robert; Faculty, Chemistry & Biochemistry, California State University San Marcos Abstract As a relative newcomer to the field of homogeneous catalysis in organic methods development, gold catalysts have demonstrated high catalytic activity in numerous organic transformations. The structural motifs attainable by these methods are a common building block for the synthesis of several natural and synthetic products ubiquitous in biologically active compounds. For instance, several potent anticancer, antidepressant, antifungal, anti-inflammatory, antimicrobial, antitumor, antiviral (including antiretroviral), and cytotoxic agents can be synthesized from gold catalysis. The methodology presented herein builds off prior studies using gold(I) salts to catalyze etherification reactions. A novel one-pot methodology utilizing gold(I) catalysts has been developed to yield five- and six-membered benzylic cyclic ethers, a common structural motif found in antibiotic and antidepressant treatment drugs, from readily available diols. To our knowledge, an intramolecular gold(I)-catalyzed etherification reaction is currently unprecedented in the literature. Results will be presented on reaction optimization and substrate scope. 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 intramolecular gold-catalyzed etherification reaction has been optimized using 1,4-diols and 1,5-diols as the nucleophile, 5 mol % of the gold(I) and silver(I) salts, polar solvent, and slightly elevated temperatures to afford 80% of the target cyclic ethers. Using these conditions, the substrate scope for this reaction will be presented for electron rich and electron deficient substrates. The 1,4- and 1,5-membered rings are also synthesized from commercially available starting materials. All starting materials and reaction targets were fully characterized by 1H-NMR and 13C-NMR spectroscopy.
| Poster #: 136 Campus: California State Polytechnic University, Pomona Poster Category: Synthetic Chemistry Keywords: Anti-cancer drugs , Thiohydroxamic acid, Histone deacetylase 6 (HDAC6) Project Title: Investigation of Thiohydroxamic Acid Analogs as Potential Aniti-Cancer Drugs Author List:
Abstract “Our research focuses on addressing the key problems associated with hydroxamic acid anti-cancer drugs. Hydroxamic acid functionality is toxic and non-selective. Our long-term goal is to develop thiohydroxamic acid compounds as histone deacetylase 6 (HDAC6) selective inhibitors. HDAC6 selective inhibitors, HPB and HPOB, were chosen as the lead compounds in our research because they have shown excellent selectivity and bio-activity. To better understand the metal-chelating properties of the thiohydroxamic acid functional group, we have designed and synthesized a series of benzothiohydroxamic acids. The products were characterized with 1HNMR, 13CNMR, IR, and GC/MS. Currently, we are exploring the binding nature of these compounds to metal ions (Zn, Co, Mn, Ni, Fe, and Cu) through UV-Vis spectroscopy. Here we present our key findings of thiohydroxomaic acid model compounds. | Poster #: 137 Campus: California State University San Marcos Poster Category: Synthetic Chemistry Keywords: bioactivity, benzotriazoles, synthesis Project Title: Synthesis of Benzotriazole Derivatives and Evaluation of Antifungal Activity on Candida albicans Author List: Stockton, Leeluleilani; Undergraduate, Chemistry & Biochemistry, California State University San Marcos, Presenting Author
Vargas, Sidney; Undergraduate, Chemistry & Biochemistry, California State University San Marcos
Iafe, Robert; Faculty, Chemistry & Biochemistry, California State University San Marcos
Abstract Background: Candida albicans is an opportunistic pathogenic yeast and one of the leading causes of mycoses in the United States. The most prescribed antifungal used for C. albicans infections is fluconazole, a member of the azole class of antifungals. Extensive documentation in clinical settings indicates C. albicans develop drug resistance quickly to azole treatments. AIDS patients and other immunocompromised individuals contract fungal infections through the course of their disease, and these patients are particularly at risk because C. albicans are the most common opportunistic pathogenic fungi. As a result, there is a critical need for novel antifungal agents to treat drug-resistant fungi associated with immunosuppressed conditions. The goal of this research project is to evaluate fungicidal and fungistatic properties of novel pharmaceutically relevant scaffolds containing the 1,2,3-benzotriazole moiety, considered to be a bioisostere of the azole functional group. This was accomplished by performing minimum inhibitory concentration (MIC) tests on C. albicans propagated in YPD broth with novel benzotriazole derivatives prepared by gold methodologies developed in our lab.
Methods: Minimum Inhibitory Concentration (MIC) assays were used to determine the lowest concentration of benzotriazole derivatives needed to inhibit growth of C. albicans. An overnight culture and subculture of C. albicans were propagated in Yeast Peptone Dextrose (YPD) broth. The subculture was done to maintain the culture in its active form. Fluconazole was used as the positive control against the tested benzotriazole derivatives. Optical density at 600 nm (OD600) using a UV-Vis Spectrometer was performed to determine the cell number of C. albicans after the assay.
Results: The current evaluation of twenty novel benzotriazole derivatives by MIC testing shows minimal antifungal activity of para-substituted benzotriazoles. Studies are currently underway to investigate if the activity is fungistatic or fungicidal, as this data will be presented also. Further substrate scope expansion using a gold-catalyzed substitution protocol developed in our lab for the substitution of benzylic alcohols using gold(I) salts as catalysts is currently being performed.
Acknowledgements: We thank CSUPERB (Presidents’ Commission Program) for support of this work. | Poster #: 1 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: isotopes, nitrogen, nitrous oxide Project Title: Assessing Nitrous Oxide Cycling in the Coastal Waters of the Eastern Tropical South Pacific Using Stable Isotopes Author List: Chen, Jacquelin; Undergraduate, Chemistry and Biochemistry, California State University, Long Beach, Presenting Author
Gluschankoff, Noah; Graduate, Earth System Science, Stanford University
Casciotti, Karen; Faculty, Earth System Science, Stanford University
Abstract Nitrous oxide (N2O) is a climatically relevant greenhouse gas that can be produced in the ocean and emitted to the atmosphere, where it can later lead to ozone depletion. Previous studies show high accumulations of N2O above the oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP), but the relative roles of the microbial pathways that dictate N2O cycling vary regionally. The purpose of this study is to identify the primary pathways controlling N2O cycling in the coastal waters of the ETSP. In this study, N2O concentrations and its isotopes, as well as O2 concentrations and the isotopic compositions of nitrate and nitrite, were analyzed at three stations between the shallow coastal shelf and the shelf slope along the GEOTRACES GP16 Zonal Transect. Our results demonstrated a rapid decline in O2 concentrations coinciding with accumulation of N2O. This N2O accumulation is estimated to support significant fluxes of N2O to the atmosphere at our stations. N2O isotopocule results illustrate intense N2O cycling, driven by a combination of incomplete denitrification and hybrid N2O production in the oxycline. In the anoxic waters of the ODZ N2O isotopes indicate concurrent N2O production and consumption through changes in 𝛿15N⍺, 𝛿18O, and 𝛿15Nβ. Overall, N2O cycling in the coastal ETSP waters appears to be mainly driven by denitrification, with a smaller role from hybrid formation. | Poster #: 2 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: Cellulase, Biofuels, Bioprospecting Project Title: Novel Carbohydrate-active Enzymes from the Cow Rumen Microbiome as Tools for Biofuel Production Author List: Ponce, Melanie; Graduate, Biological Sciences, California State University San Marcos, Presenting Author
Escobar, Matthew; Faculty, Biological Sciences, California State University San Marcos Abstract The global impacts of climate change have highlighted the importance of carbon neutral energy sources such as biofuels. Cellulose is one of the most abundant biopolymers on the planet, and cellulose-rich agricultural and forestry waste products are very promising resources for biofuel production. However, a primary limitation in the commercial production of cellulosic biofuels is the inefficiency and high cost of degrading cellulose-rich plant biomass into glucose. Thus, the focus of this study was to characterize novel carbohydrate-active enzymes (CAZYmes) that can efficiently break down plant cell wall carbohydrates for the purpose of biofuel production. The cow rumen is a rich reservoir of cellulolytic microorganisms that produce novel, uncharacterized CAZYmes. Using a previously published cow rumen metagenome dataset, we identified 2,232 sequences containing glycoside hydrolase 5 (GH5), GH8, GH9, or GH 48 protein domains, which are characteristic of cellulase enzymes. We then used NCBI Batch CD Search to characterize the domain structure of these sequences. 125 genes contained multiple carbohydrate-relevant protein domains, suggesting that they may encode multi-functional cellulases, which have been shown to be highly efficient in cellulose degradation. PCR primers were designed to amplify 36 of these multi-domain sequences, and we were able to successfully amplify 11 novel CAZYme sequences from cow rumen DNA. We are currently cloning these sequences into the pET100-TOPO vector for use in recombinant protein production in Escherichia coli. We will characterize the enzyme kinetics and substrate specificity of these putative multi-functional cellulase enzymes, and compare their performance to commercially available cellulase enzyme cocktails that are currently used for biofuel production. | Poster #: 3 Campus: California State University, Fullerton Poster Category: Agriculture/Biofuels/Environment Keywords: Aquaculture, Chlamydomonas reinhardtii, Classical Strain Improvement Project Title: Algae for Aquaculture: Increased Protein and Lipid Production in Chlamydomonas reinhardtii Using Classical Strain Improvement Techniques Author List: Salman, Tanya; Graduate, Biological Science, California State University, Fullerton, Presenting Author
Xia, Angela; Undergraduate, Biological Science, California State University, Fullerton, Presenting Author
Cohen, Amybeth; Faculty, Biological Science, California State University, Fullerton
Abstract Over the past twenty years, global farmed-fish production has increased by more than 500%, necessitating a parallel increase in fish food production. Historically, fish meal and fish oil (FMFO) supplied farm-raised fish with the needed essential proteins and omega-3 fatty acids. However, using ocean resources for FMFO is unsustainable. This project aims to develop high-fat and high-protein strains of the single-celled, green microalga Chlamydomonas reinhardtii through non-GMO practices, which could be used to supplement FMFO. We employed the classical strain improvement (CSI) techniques of UV mutagenesis with nitrogen-deficient induced gametogenesis and mating, as they had been successfully used to produce strains of C. reinhardtii with an increased chloroplast-protein content (Fields et al., 2019) and survivability in high salinity conditions (Takouridis et al., 2012). In our study, five pairs of wild-type (WT) C. reinhardtii strains were subjected to CSI techniques, establishing fifteen genetically diverse offspring populations: five from sexual recombination during mating and 10 from UV mutagenesis. Ten isolated colonies from each population were surveyed for crude protein and lipid content, using a crude protein extraction and Bradford assay and BODIPY 493/503 lipid staining and fluorescence assay, respectively, to isolate the first set of top-producers. The five selected strains from mating underwent UV mutagenesis, while the 10 strains that were first exposed to UV light underwent mating in order to develop the second round of genetically recombinant populations. Crude protein and lipid assays of the newly isolated colonies revealed eight high protein and 18 high lipid strains. The two top-producing strains have either a 57.6% increase in total protein or a 150.2% increase in total lipids compared to the WT parents. The next steps will involve fluorescent-activated cell sorting of desired populations to screen for strains with an even more significant increase in lipid production than the WT parents, followed by confocal microscopy of BODIPY 493/503 lipid-stained cells, SDS-PAGE of extracted proteins, and determination of the amino acid and lipid profiles in the selected strains.
This project is supported by a CSUPERB Faculty-Graduate Student Collaborative Research Grant, the COAST Undergraduate Student Research Support Program, and funding from the Department of Biological Science at CSU Fullerton.
| Poster #: 4 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: Arsenic, Halobacterium , Bioremediation Project Title: Cloning and characterization of arsenite efflux transporter metallochaperone arsD- related protein from Halobacterium salinarum Author List: Patel, Lipi; Graduate, Biology, California State University, Northridge, Presenting Author
LNU, Shaurya; California State University, Northridge, Presenting Author
Perl, Scott; NASA JPL
Basu, Chhandak; Faculty, Biology, California State University, Northridge Abstract Halobacterium salinarum is a model organism to study microbial stress physiology because of its ability to survive in extremes of environmental conditions, including high salinity, UV light, and heavy metals like arsenic. Arsenic is one of the most toxic metals known to have devastating effects on humans, such as cancer, low mortality rate, and inhibit plant growth and photosynthesis. It also is a significant contaminant in many food commodities such as seafood, poultry, and rice. Halobacterium salinarum comprises genes responsible for heavy metal resistance and enzymes for detoxifying heavy metals. Therefore, this trait of Halobacterium could be used for promising bioremediation of environments contaminated with highly toxic metals due to urbanization and industrialization. An effective way of performing bioremediation is phytoremediation, which treats pollutants or wastes by using plants to remove, degrade, or stabilize unwanted substances such as toxic metals by sequestering them from the soil. It is very important to remediation practitioners because they can put plants to work and save money on excavation costs. The soil structure remains preserved as well. This method could be used by people in developing countries where comparatively, such problems are more prevalent due to a lack of resources. The project aims to clone and characterize the arsenite efflux transporter metallochaperone arsD- related protein (GenBank accession number NC_001869.1) from halobacterium and express it in hyperaccumulator plants like tobacco. Once this gene is expressed, these plants are expected to sequester the arsenic compounds and ameliorate soil health. We have successfully established the required conditions for the growth of Halobacterium salinarum and transformed it using pBI121 by the osmotic shock method. We are currently in the process of cloning the arsenite efflux transporter gene from Halobacterium and inserting it into the pBI121 plasmid vector by replacing the GUS reporter gene. The recombinant plasmid will be transferred to Arabidopsis thaliana by the ‘floral dip’ method. Arabidopsis plants will be used as a ‘proof of concept’ to evaluate the plants’ abilities to remove arsenic from soil. The project’s long-term goal is to evaluate a transgenic tobacco plant expressing the arsenite efflux transporter gene for its ability to remove arsenic from soil. | Poster #: 5 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: Algae, Dunaliella salina, Biofuel Project Title: Production of cold-tolerant strains of Dunaliella salina through genetic transformation with Agrobacterium tumefaciens Author List: Hein, MacKenzie; Graduate, Biology, California State University, Northridge, Presenting Author
BASU, CHHANDAK; Faculty, Biology, California State University, Northridge Abstract Genetically modified microalgae have considerable potential as bio-factories for the production of value-added compounds such as biofuels, glycerol, and carotenoids. However, production may be disrupted by the sensitivity of microalgae to conditions that inhibit their growth, such as high salinity and low temperatures. Many microalgae also possess a cell wall, which must be removed before the cells can be transformed. Dunaliella salina is a eukaryotic microalgae species with several traits that make it attractive as a platform for genetic modification. This microalga is also halotolerant,i.e., able to survive across a wide range of salinities. This means that the salinity of water used for the aquaculture of this algae does not need to be precisely maintained, and the cells can be grown in seawater or hypersaline brine. However, observations in our lab indicate that this algae’s growth can be severely inhibited by low temperatures, meaning that cultivation in cold or temperate climates may prove difficult. A possible solution may involve the genetic transformation of Dunaliella salina with genes from psychrophilic microalgae species, which confer the ability to grow at low temperatures. We have already succeeded in the cultivation of Dunaliella salina in the lab, and we have compared the growth of the organism at both room temperature and 4oC. We have used electroporation to transform Agrobacterium tumefaciens with PBI121 plasmids containing a GUS reporter gene to test the efficacy of the transformation protocol. We are currently in the process of genetically transforming the algae via co-cultivation with Agrobacterium tumefaciens harboring a potentially cold-tolerant ferredoxin gene from Chlamydomonas sp. UWO241. If the transformation is successful, it will allow us to study the potential benefits cells resistant to multiple types of extreme conditions may have for biomanufacturing. Microalgae grown for the production of valuable compounds are cultivated in open-air pools. Engineering microalgae to grow at low temperatures would allow them to be grown continuously year-round in temperate climates, without interruption by decreases in temperature during fall and winter. | Poster #: 6 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: arbuscular mycorrhizal fungi, agriculture, amf Project Title: Evaluating the impacts of varying healthy soil practices on arbuscular mycorrhizal fungi spore presence Author List: Martinez, Katherina; Undergraduate, Biology and Chemistry, California State University, Monterey Bay, Presenting Author
Hynes, Meagan; Faculty, Biology and Chemistry, California State University, Monterey Bay
Haffa, Arlene; Faculty, Biology and Chemistry, California State University, Monterey Bay
Kortman, Stefanie; Staff, Science & Environmental Policy, California State University, Monterey Bay Abstract Arbuscular mycorrhizal fungi (AMF) are soil microorganisms that can form mutualistic symbiosis with plants. The AMF acts as an extension of the plants’ root system to aid in nutrient exchange and increased resilience against stressors. Utilization of commercial AMF inoculum in soil health practices may prove beneficial to combat climate change effects, sustain agricultural production, and maintain soil aggregate stability. This research aims to see if applications of mycorrhizal inoculum to the soil would yield an increased presence of AMF spores in a commercial organic cropping system of romaine lettuce and celery. Treatments of AMF inoculum, AMF inoculum plus compost, as well as a control were used in determining changes to the overall AMF spore count in the soil. From 2020 to 2022, soil core samples were collected, extracted, and are currently being counted for spore presence. Currently, there are no significant findings regarding the different treatments. However, at this time, an overall decrease in spore counts was found from preplant to harvest for the fall core samples in 2020 and 2021 suggesting utilization of mycorrhizal spores by the plants over the growing season. AMF inoculation was confirmed for Fall 2020, 2021, and Spring 2021 by analyzing root samples from plants in each treatment. The spring samples need to be completely evaluated before we can reach a definite conclusion regarding the findings on the different treatments and sampling time. | Poster #: 7 Campus: California State University, Northridge Poster Category: Agriculture/Biofuels/Environment Keywords: climate change, Permafrost Project Title: Isolation of Polarmonas spp. from ancient thawing permafrost Author List: Sarkisyan, Eleonora; Undergraduate, Biology, California State University, Northridge, Presenting Author
Mackelprang, Rachel; Faculty, Biology, California State University, Northridge Abstract Permafrost is soil that remains permanently frozen throughout the year and is located in polar regions of the Earth. Climate change is causing permafrost to thaw on a large scale. Permafrost contains large amounts of organic carbon, and microbes are able to convert the carbon into greenhouse gasses when it is thawed. In this study permafrost samples, frozen for 5,000 and 40,000 years, were collected from central Alaska and transplanted into the active layer (soil overlying permafrost that freezes and thaws annually) in order to stimulate thaw. After the permafrost samples were collected and transported to our laboratory. Initial studies from our samples show that Polarmonas spp. (phylum Proteobacteria, family Comamonadaceae) are highly abundant in thawed permafrost. Previously cultivated members of this genus are known to be psychrotolerant, which means they are capable of growth during low temperatures.
To better understand the physiology of these potentially important bacteria, we are attempting to culture Polarmonas spp. from thawed permafrost in the laboratory. After the species are cultured and the genome sequenced, we will better understand survivability and growth characteristics.These data will yield insight into the activity and carbon degradation processes in a species that may be crucial in producing greenhouse gasses during permafrost thaw.
| Poster #: 8 Campus: California State University San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: marine science, plankton, microscopy Project Title: Investigating the relationship between nutrient levels and autotrophic and heterotrophic plankton biomass off the coast of southern California Author List: Gutierrez, Luis A.; Undergraduate, Biology, California State University San Marcos, Presenting Author
Garcia, Anissa M.; Graduate, Biology, California State University San Marcos, Presenting Author
Taniguchi, Darcy A.A.; Faculty, Biology, California State University San Marcos
Abstract Marine drifters made of one cell, known as single-celled plankton, are the base of the oceanic food web and play a major role in the movement of elements and compounds around the ocean. These single-celled plankton can obtain their energy through photosynthesis, known as autotrophs, or through consumption, known as heterotrophs. Autotrophic plankton rely on nutrients such as phosphate, nitrogen, and silicate for growth and cell maintenance. In turn, heterotrophic plankton consume autotrophic and other heterotrophic plankton. Since nutrient concentrations can have a direct impact on planktonic abundances, we aimed to study the relationship between these two factors in a gradient from near the coast to the open ocean. Samples of plankton and nutrient concentrations were taken off the coast of southern California during 2020 and 2021 in locations ranging from nearshore to offshore. Two different forms of microscopy, brightfield and epifluorescence, were used to image, identify, and categorize cells into two groups, diatoms (autotrophic plankton) and non-diatoms (a mixture of autotrophic and heterotrophic plankton). We used the image analysis software ImageJ to obtain cell size measurements, which we then used to calculate the total carbon biomass of both groups. Our results indicated that as nutrient levels increased, so did the total carbon biomass of diatoms. Additionally, results showed no correlation between the total carbon biomass of non-diatoms and nutrient levels. Future analysis of additional microscopy samples will allow us to see the strength of these relationships over a greater amount of time and space. | Poster #: 9 Campus: California State University, Long Beach Poster Category: Agriculture/Biofuels/Environment Keywords: Geometric Morphometrics, Justicia, Reproductive Biology Project Title: Morphological Variation in Justicia (Acanthaceae) Corolla and Pollen Author List: Wang, Yuting; Graduate, Biological Sciences, California State University, Long Beach, Presenting Author
Fisher, Amanda; Faculty, Biological Sciences, California State University, Long Beach Abstract Justicia (Acanthaceae) species have a wide variety of flower forms and have adapted to many types of pollinators in the western hemisphere including bats, bees, butterflies, hummingbirds, and moths. Justicia species pollinated by the same animals tend to share similar corolla shapes and colors. Before molecular science, botanists used to classify species based on morphology. For instance, some moth-pollinated Justicia were formerly classified in the genus Siphonoglossa based on their long and narrow corolla tubes. However, a molecular phylogeny of Justicia found that Siphonoglossa is not monophyletic and there have been many transitions between pollinator types among closely related Justicia species. This means that similarities in Justicia corolla morphology evolved through convergence. Other flower characteristics such as pollen size and shape may have also co-evolved with animal pollinators. My objectives are to compare: (1) corolla shape and size and (2) pollen shape and size among Justicia species in the ‘sessile-axillary inflorescence’ and the Dianthera/Sarotheca/Plagiacanthus clade. The species in these clades are known to have bee, hummingbird, and lepidopteran pollination. One-way ANOVA and Tukey’s tests detected variation in corolla and pollen length, width, and length-to-width ratio among Justicia species. Supporting my prediction, lepidopteran pollinated Justicia species have a significantly higher corolla length-to-width ratio when comparing to Justicia relatives that are pollinated by bee, hummingbird, or insect generalists. Results from geometric morphometric analyses show differences in corolla shape of Justicia species with different pollinators. Lepidopteran pollinated Justicia species have a narrower corolla tube and a higher corolla tube opening while generalist pollinated species has a larger corolla pouch. I will compare pollen shape in Justicia species pollinated by different animals using outline analyses. I expect that lepidopteran-pollinated Justicia species will have pollen that is more similar in size, shape, and texture compared to their close Justicia relatives that are bird or bee pollinated. This study will help to advance our knowledge of flower-animal co-evolution and transitions between different animal pollinators in western hemisphere Justicia. This project is funded by National Science Foundation Award #1754792 and a CSUPERB Faculty-Student Collaboration Grant. | Poster #: 10 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Hot spring, Thermophile, Exobiology Project Title: Growth of Caldatribacterium (OP9) in pure culture and in co-culture with sulfate-reducing bacteria Author List: Torres, Aries; Graduate, Biology, California State University, San Bernardino, Presenting Author
Alvarado, Toshio; Graduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Faculty, Biology, California State University, San Bernardino
Abstract The Atribacterota (formerly called Atribacteria or OP9) is a phylum of bacteria whose members are found in many anaerobic environments such as hot springs, non-thermal ocean sediments, and petroleum reservoirs. Currently there is only one cultivated representative in this entire phylum, and little is known about the physiology and ecology of these microbes. Members of our lab recently isolated another member of this phylum, Caldatribacterium, from Great Boiling Spring, NV. During initial enrichment, Caldatribacterium appeared to be growing in co-culture with a member of the genus Thermodesulfobacterium, a sulfate-reducing thermophile, in media with fucose as a sole carbon source. Caldatribacterium was eventually isolated by including yeast extract in addition to fucose, but the isolate could not grow on fucose alone. This led to the hypothesis that the sulfate-reducing bacterium may be supporting growth of Caldatribacterium. To test this, co-cultures were made of Caldatribacterium with three different species of Thermodesulfobacterium and a species of Thermodesulfovibrio, which is also a thermophilic sulfate reducer. All four of these sulfate reducing bacteria were able to support growth of Caldatribacterium (2.1-6.8*10^7 cells/mL) in co-culture on fucose alone, whereas none of these microbes can grow on their own under these conditions (<10^6 cells/mL). This raised the question of what benefit the sulfate-reducer might provide to Caldatribacterium. The vitamin solution that is routinely used for the Caldatribacterium and co-cultures is typically filter-sterilized, but we noticed that some of the vitamins may not have been fully soluble and may have been retained on the filter, not allowing for growth. To test this, the vitamin solution was autoclaved rather than filter sterilized. The autoclaved vitamin solution allowed for growth of Caldatribacterium on fucose alone (0.3-1.2*10^8 cells/mL) to similar levels obtained when yeast extract is included (0.3-2.9*10^8 cells/mL), indicating that the autoclaved vitamins are providing or transforming a compound required by Caldatribacterium in the vitamin solution. These data suggest that the partner organism might be producing a vitamin needed by Caldatribacterium to grow in co-culture. Learning more about the nutritional requirements of Caldatribacterium may help inform cultivation of other members of the Atribacterota present in different environments.
Supported by NASA Exobiology grant 80NNSC17KO548.
| Poster #: 11 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Archaea, Astrobiology, Thermophile Project Title: Laboratory Cultivation of and Substrate Utilization by the Novel Thermophilic Lineage Gearchaeales Author List: Saldivar, Walter; Graduate, Biology, California State University, San Bernardino, Presenting Author
Moya, Jennifer; Undergraduate, Biology, California State University, San Bernardino
Dodsworth, Jeremy; Faculty, Biology, California State University, San Bernardino
Abstract The majority of microbes on Earth have yet to be grown in pure culture and studied in the laboratory. Cultivation-independent genomic methods like metagenomics can predict what these microbes can do, but cultivation is still valuable in assessing these predictions. Uncultivated microbes are truly diverse, especially within hot springs. Great Boiling Spring (GBS) in Nevada is home to many uncultivated microbes, including the archaeal order Gearchaeales, which is found in other hot springs around the world. Previous students established laboratory cultures from GBS that contained Gearchaeales at ~2% abundance based on 16S rRNA gene tags, along with other novel microbes. These cultures have 0.02% casamino acids as a carbon source, ~10% oxygen, are incubated at 80 ℃, and are transferred every 3 weeks. This mixed culture has been maintained since 2016 and we were unsure if Gearchaeales was still present in the culture. The goals for this project were to confirm the persistence of Gearchaeales in the cultures and to find conditions that will further enrich for Gearchaeales. Quantitative polymerase chain reaction (qPCR) was used to determine Gearchaeales abundance on DNA extracted from cultures grown under different conditions in triplicate, including incubation temperature and time, oxygen levels, and growth substrates. The qPCR confirmed that Gearchaeales was present at ~1.2-2.8*10^7 16S rRNA gene copies/mL culture and that 3-4 weeks of incubation was required to maintain stable populations. The abundance of Gearchaeales increased as culture temperature was increased from 75-85 °C, but were not detected at 90 °C. Tests with different oxygen concentrations confirmed that 10% oxygen yielded the best growth (1.2-4.0*10^7 copies/mL), with 20% oxygen (fully aerobic) showing reduced abundance (6.5-9.9*10^6 copies/mL); microaerobic (2.5-5%) or anaerobic conditions did not support growth (<10^5 gene copies/mL). Starch (5.6-7.4*10^6 copies/mL) and a mixture of butyrate, propionate, and acetate (BPA; 0.6-3.6*10^6 copies/mL) as alternative carbon and energy sources supported growth, but at lower levels compared to casamino acids (1.5-3.7*10^7 copies/mL), although overall cell abundance was lower with BPA, suggesting enrichment. Further experiments will combine some of these conditions to help further enrich for Gearchaeales, which will help us to better understand this lineage and possibly lead to a pure culture.
Supported by NASA Exobiology grant 80NNSC17KO548.
| Poster #: 12 Campus: San Francisco State University Poster Category: Astrobiology Keywords: hot springs, microdiversity, metagenomics Project Title: Metagenomics-Based Analysis of Population-Level Genomic Variation in Hot Spring Microbial Communities Author List: Hogan, Daniel R.; Graduate, Biology, San Francisco State University, Presenting Author
Williams, Andre's W.; Graduate, Biology, San Francisco State University, Presenting Author
Salcedo, Rebecca S. R.; Stanford University
de la Torre, José R.; Faculty, Biology, San Francisco State University Abstract Metagenomics, sequencing genomic DNA from entire microbial communities, has altered our understanding of the microbial world, revealing unsuspected levels of biodiversity. Most metagenomic analyses have focused on obtaining metagenome-assembled genomes, which represent the consensus genome sequence of organisms in the community. However, these datasets also contain information about the genetic variation present within each species in the community. To date, the extent of this microdiversity, along with the environmental and evolutionary forces driving it, have largely been ignored. Prior work in our lab examined the microdiversity of natural populations of thermophilic ammonia-oxidizing archaea (ThAOA) using metagenomic datasets sequenced with Illumina short-read technologies (reads of 100-150 bp). We found great variability in the amount of genetic diversity of ThAOA populations in hot springs across three continents, as indicated by the density of single nucleotide polymorphisms (SNPs). Some populations (Spain) appeared almost clonal, with little or no genetic variation across the entire genome. In contrast, we found that populations in the United States (Nevada) and China (Yunnan) showed significant diversity across all loci. We have now examined whether other dominant microorganisms in the samples show similar patterns of diversity. In the Spanish community, the second most abundant organism was a close relative of Thermoflexus hugenholtzii. Like the ThAOA in this sample, we found remarkably low numbers of SNPs for T. hugenholtzii, suggesting that the microbial community in this engineered hydrothermal system may be extremely new. In springs with genetically diverse populations of ThAOA, we likewise found significant SNP densities across the genomes of other abundant microorganisms. More recently, we have looked at genomic variability at larger scales within populations. Flexible genomic islands, often containing multiple genes or operons, result from the acquisition by individual cells of large genomic fragments by horizontal gene transfer. Genes in flexible islands have higher proportions of non-synonymous to synonymous changes when compared to genes in the core genome. These results suggest different selective pressures may be acting on genes found in the core or flexible regions of the genome. In the future, we will explore the use of long-read sequencing technologies to improve our assessments of genomic variability in these communities. | Poster #: 13 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: inflammation, blood plasma ceruloplasmin, rates of turnover Project Title: The role of the copper-containing blood plasma protein, ceruloplasmin, in inflammation Author List: Savage, Cannon; Graduate, Chemistry and Biochemistry, California State University, Fullerton, Presenting Author, Eden Award Nominee
Sanqui, Cossette ; Undergraduate, Chemistry and Biochemistry, California State University, Fullerton
Perez, Madelynn; Fullerton College
Linder, Maria C; Faculty, Chemistry and Biochemistry, California State University, Fullerton Abstract Ceruloplasmin (Cp) is a 132 kDa Cu binding glycoprotein in blood plasma involved in numerous processes including delivery of copper to cells and neutralization of reactive oxygen species. It is largely produced by liver hepatocytes, with initial synthesized as copper-less “apo” Cp in the rough endoplasmic reticulum, then glycosylated, with Cu added in the trans-Golgi network before secreted into the circulation as “holo” Cp. Upon donating its Cu to cells, Cp reverts to apoCp and is removed from the circulation by the liver upon desialylation and endocytosis. In addition to its Cu transport duties, there is a clear role for Cp in the acute phase response of inflammation. We have been studying the time-course of the Cp response to inflammation using the turpentine inject model in rats, following changes in Cp enzyme activity, mRNA, and proportions of holo to apo Cp. After a single turpentine injection, Cp mRNA levels increased 3.5 fold, peaking at 16 h and returning to normal after 24 h. a similar pattern was observed for kidney Cp mRNA. This spike in expression was followed by a tripling of Cp enzyme activity in the blood, which reaches a maximum by 48 h, implying an increase in transcription and translation. Interestingly, Cp activity in the blood remained elevated for upwards of 7 days, and not returning to control levels until 14 days. While the normal half-life of Cp is not clearly established, but some evidence suggesting ~10 h, our findings imply that the inflammatory response while transiently increasing the rate of Cp synthesis reduces the rate at which Cp is removed from the blood. Our next objective is to test this idea by measuring the rates of synthesis and degradation of Cp in control and turpentine-treated rats using incorporation and retention of 14C-leucine and 67Cu in circulating Cp over time, and for which we have developed a new Cp isolation procedure. If correct, this will add a whole new dimension to our knowledge of Cu metabolism and immunity. Samples from the time-course study described were also analyzed for effects of inflammation on the ratio of holo to apo Cp, after developing a new and reproducible native PAGE immunoblotting procedure. Ratios of holo to apo were markedly enhanced, which is consistent with the idea that the active Cu-dependent form of Cp is required in inflammation.
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