Search Poster #: 1 Campus: California State University, San Marcos Poster Category: Agriculture/Biofuels/Environment Keywords: ectoine, plant, microbe Project Title: Metabolite exchange between plants and soil microorganisms: What is the role of ectoine? Author List: Angelie Arcos Chavelas, Undergraduate, Biological Sciences, Presenting Author; Cesar De La Torre, Undergraduate, Biological Sciences, Presenting Author; Jorge Robles, Undergraduate, BIological Sciences; Robyn Araiza, Chemistry and Biochemistry; Matthew Escobar, Biology, San Marcos Abstract  Ectoine is a cyclic amino acid and compatible solute that is synthesized by many bacteria in response to osmotic or salt stress. Recently we demonstrated that tomato plants (Solanum lycopersicum) can transport microbially-produced ectoine from the soil into their leaves and fruit. To investigate how plants take up ectoine from the soil, Arabidopsis thaliana Col-0 (wild-type) and six mutants lacking root-expressed amino acid transporters were grown in a sterile plant growth medium containing exogenous ectoine. After quantifying shoot ectoine content by HPLC-MS, we found that ectoine levels were significantly reduced in the lysine-histidine-like transporter 1 (LHT1) mutant (56% reduction, P = 3.7E-05), suggesting that this transporter contributes to ectoine uptake. To explore whether ectoine might help alleviate salt stress in plants, tomato seedlings were grown in sterile plant growth medium containing 75 mM sodium chloride, in the presence or absence of ectoine. We found that salt-stressed plants grown in the presence of ectoine produced significantly less proline (55% reduction, P = 7.3E-11) than their counterparts grown without ectoine. Proline is a natural compatible solute produced by tomato, and these results suggest that ectoine may influence the osmoprotective strategies of plants by reducing the need for endogenous proline production. Finally, to complement these targeted approaches, RNA-sequencing was performed to analyze how ectoine affects gene expression in A. thaliana seedlings. Preliminary analyses suggest that plant responses to stress and hypoxia are activated by ectoine. In the long term, we hope to reveal the physiological and ecological significance of ectoine in plant-microbe interactions.
| Poster #: 2 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: Reactive Oxygen Species, Chalcones, Plant Parasitic Nematodes Project Title: Exploring the nematicidal mechanisms of chalcones: ROS production and potential gene dysregulation in Caenorhabditis elegans Author List: Zoie Gavel, Undergraduate, Department of Biology, Presenting Author; Sopanha Peo, Undergraduate, Department of Chemistry, Presenting Author; Alejandro Calderon-Urrea, Biology, Fresno Abstract By 2050, the global population is projected to reach 9 billion, requiring a significant increase in food production. However, plant-parasitic nematodes (PPNs) present a major challenge to crop yields, causing approximately $157 billion in agricultural losses annually. Current PPN control methods are either unsustainable or ineffective. In 2011, Dr. Calderon-Urrea’s lab developed potent nematicidal chalcones—Chalcones 17, 25, and 30—that kill nematodes within three hours at a concentration of 10⁻⁴ M. Nonetheless, the mechanisms underlying their nematicidal action remain poorly understood. A leading hypothesis suggests that these chalcones induce excessive reactive oxygen species (ROS) production, leading to the nematods’s death. To explore this hypothesis, Caenorhabditis elegans nematodes (synchronized at the same life stage) were placed in 96-well plates. M9 buffer served as the negative control, while paraquat (a powerful oxidant) was used as the positive control. The worms were exposed to varying concentrations of chalcones (10⁻⁵ M to 10⁻⁴ M). H2DCFDA, a fluorescent probe, was added to each well to monitor ROS production; the resulting ROSs were detected using a Synergy HTX multimode plate reader. In triplicate experiments, Chalcone 17 significantly increased ROS production in a dose-dependent manner, exceeding the effect of paraquat. Preliminary results for Chalcones 25 and 30 also suggest a positive correlation between dosage and ROS production. Future experiments warrant further investigation into chalcones’ potential involvement in antioxidant gene dysregulation.
| Poster #: 3 Campus: California State University, Monterey Bay Poster Category: Agriculture/Biofuels/Environment Keywords: Bioremediation, Bacteria, Functional Genomics Project Title: Identifying Essential Genes Utilized During Microbial Bioremediation of Insecticides Author List: Mary Snook, Graduate, Department of Applied Environomental Science, Presenting Author; Denisse Emeterio, Undergraduate, Biology and Chemistry; Nathaniel Jue, Biology and Chemistry, Monterey Bay Abstract Insecticides pose a threat to ecological, wildlife and human health when they are carried in runoff water from agricultural fields to surface and ground waters. Microbial bioremediation provides a promising avenue for degrading and removing these pollutants from runoff water before they dissipate into downstream ecosystems. While effective bioremediators for various target pesticides have been identified, we do not yet understand exactly how microbes degrade insecticides in most cases. In this study, we examined bacterial strains with the ability to metabolize imidacloprid and malathion (common insecticides), and used transposon-mediated mutagenesis and RNA sequencing to answer the question of which genes are essential and actively used for this ability. We introduced the tn5 transposon into our study strains, where it randomly inserted into their genomes to create a library of knockout mutants. We subjected these mutants to selective conditions in order to eliminate any that lost the ability to metabolize insecticide. With Illumina DNA sequencing, we sequenced experimental libraries and mapped mutation sites in this selected population, identifying genes lacking in detected mutations as essential for metabolism of insecticide. We also isolated and sequenced RNA from microbes metabolizing insecticide, and compared relative expression of genes in these conditions to expression levels in microbes metabolizing glucose. We expect to find that the same genes identified after transposon mutagenesis and selective growing conditions as lacking mutations will be more highly expressed in microbes actively metabolizing insecticide, indicating that they are both essential, and actively expressed when utilizing insecticide as food source. Identifying these genes will help us understand the metabolic pathways used by these microbes, and predict any potentially harmful byproducts produced. It will also give us a framework for potentially introducing these genes, and the ability to metabolize insecticide, into other microbes. All of this will help design safe and effective systems to remediate insecticide pollution, making agricultural runoff water safer for ecological and public health.
| Poster #: 4 Campus: California State University, Fresno Poster Category: Agriculture/Biofuels/Environment Keywords: Genome modification, Agriculture, CRISPR Project Title: CRISPR/Cas9 mediated knock out of three pigment genes in navel orangeworm, Amyelois transitella (Lepidoptera:pyralidae) Author List: Raavi Riar, Clovis North High School, Presenting Author; Jacob Wenger, Plant Science, Fresno Abstract Navel orangeworm (Amyelois transitella) is a primary pest of tree nuts in the state of California. Larvae of the species bore into nut causing direct damage while also contaminating the kernel with frass, webbing, and aflatoxin producing fungi. As tree nut acreage has expanded and concentrated in California’s Central Valley there has been increased interest in area-wide approaches for the management of this pest, including the use of genetic technologies. Here we describe the application of CRISPR/Cas9 genome editing tools to knockdown three pigment genes (White, Scarlet, and Brown-like) in A. transitella. Knockouts of all three genes were viable through the adult stage and were readily identified phenotypically due to distinct alterations in eye pigmentation. White and Scarlet knockouts also produced visible changes in the larval cuticle pigmentation. Adult Scarlet knockouts produced viable pigmentless larvae, demonstrating a successful germline transformation. Survival and transformation rates of White and Brown-like injected eggs did not produce sufficient adult moths to test viability of progeny. Survival rates for injected eggs were low (2.6-11.4% per sgRNA, averaging 6.5%) as was the transformation rate when calculated across all eggs (0-6.2%), though these rates are in-line with similar insect transformation systems. This work represents the first CRISPR/Cas9 mediated genetic modification of Amyelois transitella, a tool which may be utilized in future research of the pest’s functional genomics, as well as a potential tool for pest management.
| Poster #: 5 Campus: California State Polytechnic University, Pomona Poster Category: Agriculture/Biofuels/Environment Keywords: cattle, biting-fly pests, cortisol Project Title: Effects of Repellent Treatment on Behavioral and Physiological Responses of Cattle to Biting Flies Author List: Jane Rumpak, Graduate, Biological Sciences, Presenting Author; Juanita Jellyman, Biological Sciences, Pomona Abstract Stable flies and horn flies are biting pests of cattle that feed on cattle blood. Fly bites are painful and cattle react with “fly repelling” behaviors including shaking of the head, stamping of legs, flicking of the tail, and twitching of the cutaneous muscle (Mullens et al. 2006), which disrupts feeding, reproductive, and resting behaviors in livestock (Taylor et al. 2012). Biting fly activity increases plasma concentrations of the stress hormone cortisol (Vitela-Mendoza et al. 2016) and reduces cattle weight gain and milk production (Brewer et al. 2021). The economic impact of biting flies on the US cattle industry is estimated at $2.3 billion annually (Kunz et al. 1991). Low toxicity plant-derived fly repellents reduce pests on animals and are safe for animals, non-targets (e.g. farm workers), and the environment. Whether repellent treatment reduces behavioral or physiological responses of cattle to biting flies is not known. We hypothesized that treatment of cattle with repellent would decrease plasma concentration of cortisol in association with lower fly counts and decreased frequency of fly-repelling behaviors.
Ten cattle were used in the study. Data were collected during summer when biting fly activity was at its peak. Animals were restrained in a squeeze chute and treated with Ecovet fly repellent following the label rate to the dorsum (Treatment Group; n=5) or with a water-only sham spray of the same amount (Control Group; n=5). Blood samples were collected from the jugular vein (3 mL) before treatment, 24 and 48 hours later. Blood was centrifuged and plasma was stored at -80℃ until measurement of plasma cortisol concentrations by enzyme linked immunosorbent assay kit (ELISA; Invitrogen). After blood collection a visual count of biting flies on each animal (horn flies and stable flies) was recorded along with a 5-minute count of fly repelling behaviors to estimate biting fly activity. Data collection from cattle was completed in August 2024. The current poster will include analysis of the relationship between the independent variables (number of flies, fly repelling behaviors per animal) and the dependent variable (plasma cortisol) determined using multiple regression analysis. We expect to associate plasma cortisol concentration with fly numbers and the frequency of fly-repelling behavior to evaluate the impact of biting fly activity and repellent treatment on animal behavior and physiology.
Funded by the Agricultural Research Institute.
| Poster #: 6 Campus: California State University, San Bernardino Poster Category: Artificial Intelligence (AI) Keywords: Enhancing Brain Connectivity Analysis with Riemannian Sparse Learning: From Simplex to Sphere Transformation Project Title: Enhancing Brain Connectivity Analysis with Riemannian Sparse Learning: From Simplex to Sphere Transformation Author List: Can Seferoglu , Undergraduate, Presenting Author; Xiangyu Li, Computer Science and Engineering, San Bernardino Abstract “Analyzing brain connectivity poses significant challenges due to the complexity of high-dimensional data, parameter dependencies, and the need for robust and interpretable representations of neural networks. Traditional methods often fall short in these areas, limiting their effectiveness in uncovering meaningful insights into brain function. To address these limitations, we developed Kernelized Spherical Sparse Representation (K-SSR), a novel approach that redefines sparse learning by transitioning from conventional simplex constraints to a Riemannian manifold framework, enhancing data similarity interpretation and clustering in complex datasets. To ensure reliable and efficient optimization, we further introduce the Proximal Riemannian Minimization (PRM) algorithm, an innovative optimization method that guarantees sub-linear convergence rates and offers the potential for global convergence in high-dimensional data environments. This is particularly beneficial for brain connectivity analysis, where the intricate nature of the data and the presence of noise can obscure critical neural connections. PRM’s advanced capabilities allow it to efficiently navigate complex optimization landscapes, leading to more accurate and stable identification of brain network patterns, even under challenging conditions. Applying our method to the Human Connectome Project (HCP) dataset, we successfully uncover significant brain connections, providing new insights into the neural underpinnings of cognitive and emotional processes. Furthermore, this approach holds promise for applications in neurological research, such as identifying altered brain region connections in Alzheimer’s disease. Our work contributes to the field of sparse representation and provides a practical tool for brain connectivity analysis, with relevance to both data science and neuroscience.
“ | Poster #: 7 Campus: California State University, Long Beach Poster Category: Artificial Intelligence (AI) Keywords: Artificial Intelligence, Electromyography, Surgeon skill level Project Title: Enhancing Surgical Training with AI: Predicting Surgeon Skill Levels Using Machine Learning and Explainable Artificial Intelligence Techniques Author List: Areef Hossain, Undergraduate, Computer Science; Rahul Soangra, Chapman University, Physical Therapy; Vennila Krishnan, Physical Therapy, Long Beach Abstract The accurate prediction of surgeon skill levels is critical for evaluating competencies and enhancing surgical training programs. This study harnesses advanced machine learning algorithms and explainable artificial intelligence (AI) techniques to develop robust models capable of determining surgeons' proficiency. Various models including Support Vector Machines (SVM), Random Forest, XGBoost, Logistic Regression, and Naive Bayes were utilized, trained on a dataset comprising twenty-six surgeons categorized by their expertise into 11 novices, 12 intermediates, and 3 experts.
To analyze muscle activations and movement variability, twelve wireless wearable sensors incorporating surface electromyograms (EMGs) and accelerometers were affixed bilaterally to specific muscle groups such as the bicep brachii, tricep brachii, anterior deltoid, flexor carpi ulnaris (FCU), extensor carpi ulnaris (ECU), and thenar eminence (TE). Among these, the Naive Bayes model showcased exceptional effectiveness, achieving an accuracy of 96.4%, precision of 95.7%, recall of 96.2%, and an F1 score of 96.0%. These metrics emphasize the model's capacity to classify surgeon skill levels accurately.
A key aspect of the study was the use of permutation importance for determining feature significance, which illuminated the profound impact of certain features on model predictions. Attributes such as 'decision-making speed' and 'precision of movement' were identified as highly influential. Further, the implementation of SHAP values and permutation importance techniques provided deeper insights into the predictive models, enhancing the interpretability of how different features influence model predictions. This level of transparency is essential for validating AI-driven assessments within surgical training environments.
Our results indicate that precise predictions, combined with a comprehensive understanding of predictive determinants, are imperative for advancing surgical training and improving patient outcomes. This study lays the groundwork for future research into more sophisticated models and larger datasets, aiming to refine prediction accuracy and enhance the interpretability of the models. The goal is to improve surgical training programs and ensure superior patient care.
| Poster #: 8 Campus: California State University, San Bernardino Poster Category: Astrobiology Keywords: Cultivation, Archaea, Thermophile Project Title: Enrichment of the thermophilic archaea Gearchaeales and Calditenuis in laboratory culture Author List: Bianca Garcia, Undergraduate, Biology, Presenting Author; Walter Saldivar, Graduate, Biology; Mylinn Price, Undergraduate, Chemistry and Biochemistry; Jeremy Dodsworth, Biology, San Bernardino Abstract The majority of thermophilic bacteria and archaea have yet to be grown in pure culture and studied in the laboratory. Although genomic data on these lineages are available through cultivation-independent metagenomics studies, obtaining pure cultures can allow detailed analysis of these microbes. This could lead to a better understanding of life and ecology in extreme environments that may be analogous to some found on early Earth, as well as potential biotechnological applications of their thermostable enzymes. Two lineages of yet-uncultivated thermophilic archaea, the genus Calditenuis (formerly Aigarchaeota Group 1) and order Gearchaeales (formerly Geoarchaeota), are often abundant in terrestrial hot springs such as Great Boiling Spring (GBS) in Nevada. Previous work allowed for establishment of laboratory enrichment cultures inoculated from sediments of GBS containing Calditenuis and Gearchaeales, among other microbes, under aerobic conditions at 80 °C using casamino acids as a carbon and energy source. Subsequent experiments found that Gearchaeales were maintained at higher temperatures, and that propionate could serve as an alternative carbon source. Here, we used these two conditions together to attempt to further enrich for these microbes. Cultures were grown at 85 °C with sodium propionate as the sole carbon and energy source for three weeks, and the presence and abundance of Calditenuis and Gearchaeales were assessed using quantitative PCR (qPCR) and 16S rRNA gene tag sequencing. It was found that both lineages were present under these conditions at 4.0*106 and 1.1*107 gene copies/mL for Calditenuis and Gearchaeales, respectively. Dilution-to-extinction (DTE) was then used to enrich for these microbes, by making dilutions of the inoculum of up to 10-10, incubating for up to 8 weeks, and repeating this process with the most diluted culture that showed growth. After three rounds of DTE, with 10-8 being the highest dilution showing growth each time, 16S rRNA gene tag sequencing showed that Calditenuis (65.8% of reads) and Gearchaeales (34.1% of reads) were the only major taxa present. Additional rounds of DTE yielded a culture that was 99.5% Calditenuis, demonstrating high enrichment. Further experiments using shotgun metagenome sequencing and fluorescence in situ hybridization with probes specific for Calditenuis will be used to confirm this level of enrichment.
| Poster #: 9 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: antisense , RNase H, RNase P Project Title: Changing the EGS Technology Paradigm: an Analog that Elicits Degradation Recruiting Endogenous RNase P and RNase H Author List: Angel Magaña, Graduate, Biological Science, Presenting Author; Kimberly Phan, Graduate, Biological Science; Jan Sklenicka, Graduate, Biological Science; David Ngo, Graduate, Biological Science; Maria Soledad Ramirez, Biological Science; Marcelo Tolmasky, Biological Science, Fullerton Abstract Introduction: Antisense technologies use oligonucleotide analogs to inhibit unwanted biological processes by suppressing specific gene expression. Two antisense mechanisms involve inducing target mRNA degradation via endogenous RNase H or RNase P. "EGS technology" employs an antisense molecule (external guide sequence, EGS) to direct RNase P to cleave target mRNA. A major challenge is oligonucleotides' vulnerability to nucleases. Previous studies suggested that nuclease-resistant gapmers containing analogs flanking deoxyribonucleotides are active EGSs. When evaluating the activity of new analog-containing gapmers in mediating RNase P-induced mRNA cleavage, we found some also triggered degradation via RNase H.
Methods: Gapmers consisted of an oligodeoxynucleotide core flanked by bridge nucleic acid residues in a 5-8-4 configuration. The bridge nucleic acid residues evaluated were 2′-O,4′-aminoethylene -β-D-ribofuranosyl (BNANC), 2′-O,4′-methylene (LNA), 2′-O,4′-methoxyethylene (cMOE), and 2′-O,4′-ethylene (cET). Gapmers' ability to induce RNA cleavage was tested by incubation with aac(6')-Ib mRNA and purified RNase P or RNase H. Products were analyzed using 6% denaturing Urea PAGE.
Results: Incubation of aac(6')-Ib mRNA with gapmers consisting of deoxynucleotides flanked by analogs showed that those containing LNA were as efficient as RNA in inducing RNase P-mediated mRNA degradation. Further characterization revealed that LNA-containing gapmers unexpectedly triggered RNase H-mediated mRNA cleavage. This finding allows the degradation of target mRNA by two endogenous RNases that act on different substrates. Additional experiments confirmed the gapmer's efficiency in suppressing the target protein expression.
Conclusions: The discovery that LNA-containing gapmers function as EGSs advanced the promise of EGS technology as a tool to inhibit the expression of unwanted proteins. Furthermore, our breakthrough finding that these gapmers also elicit RNase H-mediated mRNA degradation enhances the potential of these hybrid oligomers that recruit two endogenous RNases that act by fundamentally different mechanisms. We propose that this is an evolution of EGS technology, which we suggest naming EGS-PH technology.
Supported by NIH grants 2R15AI047115, SC3GM125556, and T37MD001368
| Poster #: 10 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: Nitrogen Fixation Regulation, ExoR protein, Protein Crystallography Project Title: Crystallization of ExoR, a Regulatory Signal Protein Controlling Symbiotic Nitrogen Fixation Author List: Katherine Lee, Undergraduate, Chemistry and Biochemistry, Presenting Author; Madeline Rasche, Chemistry and Biochemistry, Fullerton Abstract ExoR from the soil bacterium Sinorhizobium meliloti is a signaling protein that contributes to the regulation of symbiotic nitrogen fixation. Previous research has shown that ExoR is a periplasmic inhibitor of the two-component regulatory system ExoS/ChvI. ExoR activation and binding block the production of the signaling molecule succinoglycan, which is needed for establishing nitrogen fixation with legumes. However, previous attempts to determine the crystal structure of ExoR have been unsuccessful, due to the tendency of the protein to precipitate at greater than 3 mg protein/mL. The goal of the current project is to develop a purification strategy to produce crystallization-quality ExoR protein. ExoR-His6 was produced in Escherichia coli and purified using nickel affinity (NiNTA) chromatography and S200 gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the first elution sample (100 mM imidazole) contained ExoR-His6 at a purity of 46% and a concentration of 1.35 mg/mL. Following gel filtration chromatography of the NiNTA elution sample, the concentration of the targeted protein was 0.21 mg/mL with a purity of about 90%. Crystallization using a 1 M ammonium sulfate buffer produced tiny crystals; Izit crystal dye confirmed that the crystals were indeed protein not salt. Modifying the elution conditions increased the concentration of the targeted protein by more than 2-fold to 0.47 mg/mL; however, the purity was about 60%. Experiments are in progress to increase the purity of the protein to greater than 98% and get a larger crystal in order to determine the three-dimensional structure of the protein.
| Poster #: 11 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: gene regulation, mRNA splicing, protein structure-function Project Title: Determining the role of lysine acetylation in splicing regulation Author List: Christopher Nunez, Graduate, Chemistry and Biochemistry, Presenting Author; Gustavo Salgado, Graduate, Chemistry and Biochemistry, Presenting Author; Niroshika Monerawila Keppetipola, Chemistry and Biochemistry, Fullerton Abstract Alternative pre-mRNA splicing is a process where regulated exons are either included or excluded in the final mRNA transcript and allows one gene transcript to encode for multiple spliced isoforms. RNA binding proteins (RBPs) regulate this process in part by binding to specific cis-regulatory sequences on the pre-mRNA substrates and influence the recruitment of the spliceosome at adjacent splice sites. Neurological diseases and cancers have been associated with aberrant spliced variants underscoring the importance of alternative splicing regulation. Recent studies highlight that lysine side chains of RBPs are modified by acetylation. The ε-amino group of lysine side chains can form hydrogen bonds and stacking interactions with RNA and are frequently found at RNA-protein interfaces. Acetylation neutralizes the positive charge of the lysine side chain, which can disrupt its capacity to interact with the negatively charged RNA. Thus, we hypothesize that lysine acetylation can regulate RBP function and in turn, pre-mRNA splicing patterns, a novel mode of post-transcriptional gene regulation. We are using the Polypyrimidine Tract Binding Protein 1 (PTBP1), a well-characterized RBP to test this hypothesis. PTBP1 contains four RNA binding domains (RBDs) connected via three linker regions and an N-terminal region. It most often functions as a splicing repressor. PTBP1 is acetylated at several lysine side chains in RBDs 3 and 4 and include those that make H-bond and pi-cation interactions with the RNA. Thus, we hypothesize that acetylation/deacetylation of lysine residues within RBD 3 and 4 regulate PTBP1 splicing activity. To test this, we assayed multi-site RBD3, RBD4 and RBD3-4 acetyllyinse mimic (glutamine) and deacetyllysine mimic (arginine) mutants for splicing repression activity on three PTBP1 regulated exons. Wild type PTBP1 served as a control. Our data highlight that lysine acetylation in RBD3 and RBD3-4 significantly decreases PTBP1 splicing activity. We are currently assaying the mutants for RNA binding to determine the role of lysine acetylation in PTBP1 RNA binding affinity. Results from these studies will unravel, for the first time, how RBP acetylation modulates splicing patterns and in turn, cellular gene expression. These studies are important as they will unravel novel enzymes and pathways that can serve as therapeutic targets in rational drug design for aberrant splicing related diseases.
| Poster #: 12 Campus: San José State University Poster Category: Biochemistry Keywords: Molecular chaperones, Protein-protein interactions, Peptide binding assays Project Title: Characterizing a Novel Interaction Between the E. coli Molecular Chaperones DnaK and CbpA Author List: Donna Quach, Undergraduate, Chemistry, Presenting Author; Quynh Nguyen, Undergraduate, Chemistry, Presenting Author; Emiliano Lopez-Ruiz, Undergraduate, Chemistry; Gwen Libozada, Undergraduate, Biological Sciences; Melanie Martinez, Undergraduate, Biomedical Engineering; Andrea Mateo, Undergraduate, Chemistry; Kanika Kolpe, Undergraduate, Justice Studies; Taylor Arhar, Chemistry, San José Abstract DnaK, a bacterial homolog of heat shock protein 70 (Hsp70), is a central molecular chaperone that regulates the folding and triage of the proteome to maintain protein homeostasis. However, DnaK does not function in isolation; instead, it requires assistance from co-chaperones to perform its varied tasks. For example, the J-domain proteins (JDPs) are a class of conserved co-chaperones that target DnaK to interact with specific “client” proteins and allosterically regulate its binding to clients. Recently, a novel interaction was reported between the C-terminus of E. coli DnaK and the JDP CbpA. A similar interaction has been described in eukaryotes, relying on a highly conserved EEVD motif at the C-terminus of eukaryotic Hsp70s. Intriguingly, this EEVD motif is absent in DnaK. The goal of our work is to determine the DnaK residues that are critical this novel interaction. We used fluorescence polarization (FP) and differential scanning fluorimetry (DSF) to assess the binding of CbpA to a peptide corresponding to the DnaK C-terminus, and subsequently evaluated the roles of specific residues by testing mutant DnaK peptides. This preliminary data starts to reveal the relative importance of each residue for binding, and future work will focus on determining the function of this interaction. This work was funded in part by the CSU Biotech New Investigator Grant Program.
| Poster #: 13 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Heart disease, Lipoproteins, Protein biochemistry Project Title: Lipid-binding Activity of Single Cysteine Containing Apolipoprotein AI and Relevance in Cardiovascular Disease Author List: Audrey Tse, Undergraduate, Chemistry and Biochemistry, Presenting Author; Vasanthy Narayanaswami, Chemistry and Biochemistry, Long Beach Abstract Cardiovascular diseases (CVD) are a group of diseases with multifactorial risk factors and are considered the #1 killer in the US. HDL plays a role in promoting cholesterol efflux from macrophages and in reverse cholesterol transport. We examined the major protein component of HDL, apolipoprotein AI (apoAI) basing our studies on apoAIMilano and apoAIParis, two naturally occurring variants whose carriers do not express clinical signs of CVD despite their low levels of HDL, considered a risk factor. We hypothesize that the presence of a single Cys in a segment that is believed to bear an ability to transition between an unstructured loop and a helix, alters the lipid binding and/or antioxidant ability of apoAI. To test this hypothesis, we designed a series of single Cys variants in the segment between 126 and 158 of apoAI (L126C, A130C, L134C, A152C, A154C, A158C) and purified the recombinant protein from E. coli. SDS PAGE analysis under reducing conditions revealed a major band ~26 kDa for all variants; under non-reducing conditions, variants with Cys located towards the edge of the loop (ie, L126C, A130C, A154C & A158C) displayed a mixture of disulfide bonded dimers and monomers, while those with Cys in the middle of the loop segment (L134C & A152C) appear to exist in predominantly non-disulfide bonded monomeric state. Subsequently, the ability of the mutants to bind lipids was determined by the phospholipid vesicle solubilization assay. The assay was carried out under reducing and non-reducing conditions by monitoring the absorbance at 325 nm and expressed as the time required for initial absorbance to decrease by 50% (T½). All mutants displayed a higher lipid binding rate in the reduced state (T½ ranging from 50-125 s) compared to WT apoAI (T½ = 158±13 s). Under non-reducing conditions, all mutants (except L134C) showed a higher rate than WT apoAI (T½ for WT was 178±17 s; L134C=182±18 s; the remaining mutants 54 -136 s). Further studies are in progress to confirm these findings and to separate the monomers from dimers to determine their effect on the lipid binding activities. This will be followed by assessment of the antioxidant activities of these mutants, which will aid in linking with their lipid binding activity. Taken together, these studies will contribute significantly to our understanding of the pleiotropic effect of HDL and its role in mitigating the risks associated with CVD.
Funded by CSU BIOTECH Howell Foundation, NIH GM105561 & T34GM149378
| Poster #: 14 Campus: California State University, Los Angeles Poster Category: Biochemistry Keywords: crystal growth, protein inhibitors, kidney stones Project Title: Inhibitory Effects of a Beetle Urine Antifreeze Protein on Human Kidney Stones Formation Author List: Puron Rahman, Graduate, Presenting Author; Alfredo Gonzalez, Graduate; Xin Wen, Chemistry and Biochemistry , Los Angeles Abstract Kidney stones are a common disorder in the human urinary tract. About 80% of all kidney stones are calcium oxalate (CaOx) stones; calcium oxalate monohydrate (COM) is the main constituent. The urine components of human and the beetle Dendroides canadensisis are similar, however, no stones have been found in D. canadensisis. Antifreeze proteins (AFPs) found in many organisms are known to lower the freezing temperature of the body fluids of the organisms and inhibit ice growth. The beetle D. canadensisis consists of 30 AFPs (DAFPs), but only four in hemolymphs, referred to as DAFP-1, DAFP-2, DAFP-4, and DAFP-6. Our previous studies showed that the isoform, DAFP-1, can effectively inhibit the crystallization of a hemolymph sugar, trehalose, in the beetle. Interestingly, DAFP-6 is the only hemolymph DAFP also found in the beetle’s urine. We hypothesize that DAFP-6 can inhibit the formation of COM. Following the established procedures in our laboratory, the two AFPs, DAFP-1 and DAFP-6, were expressed in Escherichia coli (E. coli) cells and then purified using immobilized metal affinity chromatography (IMAC). Bovine serum albumin (BSA) was used as a protein control in this study. The COM stones were prepared in the absence and present of each of the proteinaceous additives at a range of different concentrations up to 5 μM. The COM stones were then collected and analyzed using microscopy and gravimetric methods. Furthermore, the forms of the COM stones were analyzed using Fourier-transform infrared (FT-IR) spectroscopy. The results show that both DAFP-1 and DAFP-6 reduce the sizes and the amounts of formed COM stones, while the effects of DAFP-6 are more significant. In contrast, the presence of BSA promotes the formation of COM stones significantly. The kinetics of COM stone formation is being measured in the absence and presence of the additives. This study suggests a potential use of beetle proteins in inhibiting human kidney stones.
| Poster #: 15 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Cancer, Protein Stability, Differential Scanning Fluorometry Project Title: A possible therapeutic treatment for loss of function of the retinoblastoma protein due to cancer-associated missense mutations using the HPV peptide E7 Author List: Hailey Mims, Undergraduate, Biology, Presenting Author; Vanessa Vasquez Meza, Undergraduate, Biology, Presenting Author; Jason Burke, Chemistry and Biochemistry, San Bernardino Abstract The retinoblastoma protein (Rb) is a tumor suppressor protein that regulates the transition from the G1 to the S phase in the cell cycle. If a missense mutation occurs, the protein structure is altered leading to a loss of function and unregulated cell growth. The peptide E7, introduced by the HPV virus, is found to cause further dysfunction of Rb in its natural state, however an altered version of the peptide has some ability to change Rb’s stability. We hypothesized that when E7 is present, it will bind to Rb and stabilize it to its original state by increasing the melting temperature (Tm) value. To test this hypothesis, we expressed Rb in e. coli cells, lysed the cells, then purified them using affinity and fast protein liquid chromatography (FPLC). We then performed differential scanning fluorimetry by quantitative PCR and found the Tm value for wildtype is 47.74°C, whereas when E7 is present, the Tm increases to 58.34°C. This 10.6°C increase in temperature implies that there is an increase in structural stability to Rb when it binds to E7. In order to further study the effect of E7 on mutations, we expressed, purified and characterized the cancer associated mutations of Rb: M704V, T738I, E539D, and S588F. The Tm values for these mutations are 41.83°C, 41.57°C, 43.02°C, and 46.04°C, respectively, showing that several are significantly destabilized relative to wildtype. From these results, we concluded that several cancer-associated mutations destabilize Rb. Through the presence of E7 and its binding to these mutated proteins, our next step is to attempt to restore their stability.
| Poster #: 16 Campus: California State University, East Bay Poster Category: Biochemistry Keywords: protein, recombinant, HDL Project Title: Expression, Purification, and Characterization of Recombinant, TEV-cleavable human Serum Amyloid A1 Author List: Justine Garcia, Undergraduate, Chemistry & Biochemistry, Presenting Author; Mark Borja, Chemistry & Biochemistry, East Bay Abstract Serum amyloid A-1 (SAA1) is a small acute phase reactant protein expressed during the inflammatory response, which occurs during acute infection, tissue damage, and especially during heart attacks. SAA1 is primarily associated with HDL, and is thought to replace resident apolipoprotein A-I (apoA-I) during acute inflammation, resulting in reduced cholesterol efflux capacity and loss of antioxidant function. Our ultimate goal is to investigate whether SAA1 is able to force apoA-I off of HDL particles in vitro to better understand the mechanisms behind its observed in vivo behavior. To this end, we created a codon-optimized, mature human SAA1 (hSAA1) on a pET-30 expression vector. Our SAA1 construct was engineered with an N-terminal 6His tag followed by a tobacco etch virus (TEV) protease cleavage site. We expressed His-TEV-hSAA1 in E. coli and isolated it using a nickel affinity column. We then performed TEV cleavage and, following passage over a second nickel column to capture TEV and the 6His tag, isolated pure hSAA1 as confirmed by SDS-PAGE.
| Poster #: 17 Campus: California State University, Fullerton Poster Category: Biochemistry Keywords: heat-shock proteins, lipidomics, cancer Project Title: Phosphatidylserine Specifically Regulates HSPA1A Plasma Membrane Localization in Response to Heat Shock Author List: Jensen Low, Graduate, Biological Science, Presenting Author; Azalea Blythe Cuarezma, Graduate, Biological Science, Presenting Author; Rachel Altman, Biological Science; Nikolaos Nikolaidis, Biological Science, Fullerton Abstract HSPA1A is a molecular chaperone crucial in cellular stress response and cancer cell survival. In addition to its cytosolic functions, HSPA1A translocates to the plasma membrane (PM) of heat-shocked and cancer cells, modulating the immune system and regulating cell survival. This translocation depends on HSPA1A’s interaction with phosphatidylserine (PS), a lipid the protein binds with relatively high affinity and specificity. Although PS binding has been demonstrated to regulate HSPA1A’s membrane localization, the specificity of this association remains unclear. We hypothesized that PS is a crucial lipid driving HSPA1A translocation, and the lipid’s heat-induced increase triggers HSPA1A’s PM translocation. We combined pharmacological inhibition and RNA interference (RNAi) targeting PS synthesis alongside confocal microscopy, lipidomics, and western blotting techniques to test this. Heat shock-induced PS increase was confirmed using lipidomics and PS-specific biosensors, showing a peak at 0 hours post-stress. Inhibition of PS synthesis using fendiline and RNAi reduced HSPA1A’s localization to the PM. At the same time, lipid depletion of cholesterol or fatty acids had minimal effects on HSPA1A's PM localization, confirming specificity for PS. Additional experiments showed that PS saturation and elongation changes did not significantly alter HSPA1A’s membrane localization, indicating that the total PS increase, rather than specific PS species, is the critical factor. These findings reshape current models of HSPA1A trafficking, demonstrating that PS is a crucial regulator of HSPA1A’s membrane translocation during the heat shock response. This work offers new insights into lipid-regulated protein trafficking and highlights the importance of PS in controlling cellular responses to stress. Furthermore, our findings have important implications for HSPA1A regulation in cancer, where altered phospholipids and mHSPA1A influence key oncogenic processes.
| Poster #: 18 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: apolipoprotein, lipoprotein, peptide Project Title: Identification of Lipoprotein-Binding Sites Using Cyanogen Bromide Generated Peptides of Apolipophorin III Author List: Danielle Pulido, Graduate, Chemistry and Biochemistry, Presenting Author; Paul Weers, Chemistry and Biochemistry, Long Beach Abstract Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that provides the capability for long-distance flight in insects by utilizing a lipoprotein shuttle mechanism. The protein is made of a bundle of five amphipathic α-helices, which reposition to allow lipoprotein binding. ApoLp-III is an excellent model system for structure function studies of apolipoproteins. In a previous study, apoLp-III was cleaved into two peptides to produce an N-terminal (NT) peptide comprising helix 1 to 3 (NTH1-3) and a C-terminal (CT) peptide of the remaining two helices (CTH4-5). Only the NTH1-3 peptide was able to bind to lipoproteins, similar to the intact protein. This suggests that specific amino acid residues necessary for lipoprotein binding reside within helices 1 to 3. In the current study, two novel peptides of apoLp-III were generated to determine the location of lipoprotein binding sites in the first three helices. By employing site-directed mutagenesis, glutamine-68, positioned at the end of helix 2, was changed to methionine, facilitating cyanogen bromide cleavage. Successful mutation was confirmed through DNA Sanger sequencing. The mutant protein apoLp-III-Q68M was expressed in E. coli, then purified by size-exclusion chromatography and reverse-phase HPLC, yielding 10 mg per L culture. A 1:100 molar ratio of protein to cyanogen bromide was incubated for 24 h to cleave the protein, producing an NT peptide comprising helix 1 and 2 (NTH1-2) with an expected mass of 7438.31 Da and a CT peptide comprising helix 3 to 5 (CTH3-5) with an expected mass of 9980.87 Da. SDS-PAGE revealed a band ~10 kDa, likely representing CTH3-5. The smaller peptide was not visible, which could be due to poor staining or the small size. Since approximately 50% of apoLp-III-Q68M was cleaved, the digestion conditions need to be improved. Reverse-phase HPLC will be used to separate the two peptides and confirmed by SDS-PAGE. The lipoprotein-binding properties of each peptide will then be tested using modified low-density lipoprotein (LDL) and measuring the level of LDL aggregation. If the NTH1-2 peptide provides protection, then the lipoprotein-binding properties reside in the first two helices. If CTH3-5 can provide protection against LDL aggregation, then helix 3 may be most critical. To locate the precise location of the lipoprotein binding site, new peptide synthesis of apoLp-III based on the cyanogen bromide generated peptides may be required.
| Poster #: 19 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: S. cerevisiae, DNA Repair, fluorescence microscopy Project Title: Construction of a Yen1-RFP strain of S. cerevisiae for Investigation of Yen1 in Double Strand Break Repair Author List: Janelli Pineda, Graduate, Chemistry and Biochemistry; Cameel Juman, Undergraduate, Chemistry and Biochemistry, Presenting Author; Paula L Fischhaber, Chemistry and Biochemistry, Northridge Abstract Double strand breaks (DSB) in DNA arise from a range of sources and can stall replication, causing mutations that may lead to biological defects. DSB repair mechanisms differ with respect to the location of the break in the genome, and cell-cycle phase and are facilitated by endonucleases, among numerous other proteins. One of the endonucleases in baker’s yeast (S. cerevisiae) is Yen1 which behaves similarly to Gen1 in humans. Yen1 resolves intertwined chromosomes that otherwise become ultrafine and chromatin bridges during anaphase. However, it is unclear whether Yen1 is recruited to both types of bridges, and perhaps also other anaphase structures. The ultimate goal of this research is to determine whether a red fluorescently-tagged endonuclease (Yen1-RFP) localizes to DSBs found at chromatin and ultrafine bridges following DNA damage, and if it does, to also determine how Yen1 function is coordinated with respect to other steps of bridge management.
The first project toward achieving this was to construct two yeast strains containing multiple fluorescently labeled genes each: Yen1-RFP, Rad10-YFP and Dpb11-CFP in one strain, and Yen1-RFP, Dpb11-CFP, Ina1-CFP, and Ipl1-YFP in the other. To get the Yen1-RFP gene, the GFP gene in our Yen1-GFP plasmid was replaced with the RFP gene from another plasmid using standard cloning techniques. The resulting Yen1-RFP plasmid was transformed into a S. cerevisiae strain containing wild-type YEN1 to create a recombinant Yen1-RFP strain. This strain was screened by PCR, DNA sequencing, and fluorescence microscopy to confirm the presence of Yen1-RFP. The Yen1-RFP strain was then combined with the other fluorescently labeled genes by genetic crosses which were also screened by PCR, DNA sequencing, antibiotic selection, and fluorescence microscopy. All screens showed the presence of the Yen1-RFP gene without any mutations, and bright red nuclear localized fluorescent signal in G1 and anaphase cells. The strains also showed CFP and YFP signals corresponding to Rad10-YFP, Dpb11-CFP, Ina1-CFP and Ipl1-YFP appropriately. Hence, these strains were successfully prepared and can be used to investigate the localization of the Yen1 protein to anaphase bridges following DNA damage, thereby informing our understanding of Yen1 in DSB repair which has implications in cancer and aging.
| Poster #: 20 Campus: California State University, Fresno Poster Category: Biochemistry Keywords: cancer, antibody, mucines Project Title: Investigating the interaction between mucin-16 and binding partners mesothelin, siglec-9, galectin-1 and galectin-3 Author List: Yogeshwari Oka, Graduate, Presenting Author; Cory Brooks, Chemistry and Biochemistry, Fresno Abstract Mucin-16 (MUC16) is a membrane glycoprotein overexpressed in ovarian and pancreatic cancer. The protein is used as a biomarker to track cancer treatment progression. MUC16 lies at the center of the oncogenic signaling pathway and is involved in promoting tumor progression, metastasis, and tumor immune evasion. Four critical binding partners have been known to interact with MUC16 in promoting oncogenic signaling. These proteins are Mesothelin (MSLN), Siglec-9 (Sig-9), Galectin-1 (Gal-1) and Galectin-3 (Gal-3). The interaction between MUC16 and MSLN activates the p38 MAP kinase pathways which leads to increased tumor invasiveness and migration. In ovarian cancer, the interaction between MUC16 and epidermal growth factor receptor (EGFR) is mediated by lectins Gal-1 and Gal-3. This interaction results in cell proliferation and tumor progression. MUC16 suppresses the immune system response through interaction with Sig-9 on the surface of natural killer cells. Due to its role in cancer progression, it has become a target protein for immunotherapy. However, it is critical to understand the interactions between MUC16 and binding partners in order to understand how monoclonal antibodies may inhibit these interactions. The aim of this project was to confirm interaction between native shed MUC16 from a cancer cell line and its binding partners. MSLN, Gal-1, Gal-3 and Sig-9 were purified from E.coli. Shed intact, MUC16 was isolated from ovarian cancer cell line OVCAR-3 using Streptavidin coated magnetic immunoprecipitation (IP) beads bound to a biotinylated antibody. IP was then used to capture MUC16 and binding partners. Elutants of IP were analyzed using Western blot techniques. Through SDS-PAGE and Western blot analysis we were able to confirm successful expression and isolation of MUC16 and its binding partners, as well as confirm interaction between them. This project was made possible through funding by the CSUPERB 2023 Industry Partnership Initiative Grant Program.
| Poster #: 21 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Integrated Stress Response Project Title: Small Molecule Inhibitor b-AP15 Activates Integrated Stress Response in Cancer Cell Lines Author List: Anma Arora, Undergraduate, Presenting Author; Deepali Bhandari, Chemistry and Biochemistry, Long Beach Abstract Cancer cells often utilize an evolutionarily conserved signaling program called the integrated stress response (ISR) to survive adverse conditions such as nutritional limitation, oxidative stress etc. The ISR initially protects the cell by aiding in reestablishment of homeostasis, however, leads to cell death if the stress become chronic. A small molecule inhibitor called b-AP15 (a 2,6-diarylidene cycloalkanone) is a common anti-cancer drug whose primary mechanism of action is thought to be inhibition of dequbiquitinases. However, a recent study from our laboratory suggests that compounds structurally analogous to b-AP15 trigger cell death via ISR. The goal of this project is to investigate whether b-AP15 activates ISR in two cancer cell lines - HeLa, a cervical cancer cell line and DLD1, a colorectal cancer cell line. Cells were treated with increasing concentrations of b-AP15, or vehicle control dimethyl sulfoxide for 3 hours following which they were lysed, and the lysates were analyzed via western blotting to detect ISR specific proteins. Our results indicate that treatment with b-AP15 indeed activates ISR in both cell lines. We found that co-treatment with the antioxidant N-acetyl cysteine (NAC) mitigated ISR in b-AP15 treated cells, suggesting the role of oxidative stress. NAC also restored normal actin filament structure in cells as ascertained via fluorescence microscopy, highlighting the protective role of NAC against b-AP15-induced cellular damage. Our current and future experiments are focused on delineating the exact mechanism underlying b-AP15-mediated ISR activation.
This project is supported by the NIH-NIGMS grant #SC3GM139707 awarded to D.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
| Poster #: 22 Campus: California State University, Long Beach Poster Category: Biochemistry Keywords: Aspirin, Unfolded Protein Response, Protein kinase Akt Project Title: Sodium Salicylate Activates Akt Signaling and the Unfolded Protein Response in a Colorectal Cancer Cell Line Author List: Shayla Tran, Undergraduate, Presenting Author; Amber Peek, Graduate, Presenting Author; Gabriel Tan, Undergraduate; Deepali Bhandari, Chemistry and Biochemistry, Long Beach Abstract Acetylsalicylic acid, commonly known as aspirin, is an over the counter, non-steroidal, anti-inflammatory drug that has been linked to reduced occurrence of colorectal cancer, but the cellular mechanism(s) for its chemoprevention remains elusive. Cancer cells have altered metabolic demands due to the hypoxic environment and rapid proliferation rate that can induce proteotoxic stress. During this stress, cells activate an evolutionarily conserved signaling pathway known as Unfolded Protein Response (UPR) which has been linked to the acquisition of malignancy and chemoresistance in cancer cells. Our group has shown that in addition to UPR, serine/threonine protein kinase Akt is also activated during proteotoxic stress in several cancer cell lines. The goal of this project is to test the activation of UPR effectors and Akt in a colorectal cancer cell line, DLD1. We treated DLD1 cells with sodium salicylate, a metabolic derivative of aspirin, and analyzed the activation status of Akt and the expression of various proteins involved in UPR signaling via western blotting. Our results indicate that Akt gets activated upon treatment with increasing concentration of sodium salicylate. For the UPR, sodium salicylate treatment differentially regulates UPR signaling, activating some effectors and downregulating others. Our current and future experiments are focused on investigating if Akt regulates the sodium salicylate-mediated differential activation of UPR. Together, this study will shed light on the molecular mechanism underlying the aspirin-associated chemoprevention in colorectal cancer.
This project is supported by the NIH-NIGMS grant #SC3GM139707 (DB). ST is supported by the LSAMP program funded through the NSF grant #HRD-1302873 and the CSU Chancellor’s Office. AP is supported by the NIH Bridges to Doctorate program (T32GM138075). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
| Poster #: 23 Campus: San José State University Poster Category: Biochemistry Keywords: proteins, amyloid, oncogenesis Project Title: Discovering chemical determinants of PTEN amyloid formation Author List: Belle Okere, Undergraduate, Chemistry, Presenting Author; Tess Kempner, Undergraduate, Biology; Jay Thompson, Undergraduate, Chemistry; Jennifer Nguyen, Undergraduate, Chemistry; Emma Carroll, Chemistry, San José Abstract Protein aggregation into toxic amyloid fibrils underlies diseases including neurodegeneration and numerous cancers. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a phosphatase and critical tumor suppressor protein that regulates a broad range of pro-cell growth signaling pathways, with tumor suppressor activity occurring principally via dephosphorylation of phospholipid second messengers. Interestingly, PTEN has been observed to accumulate in filamentous amyloid aggregates in cancer cells. PTEN amyloid formation represents a toxic, misfolded state that likely contributes to loss-of-function phenotypes and cytotoxicity observed in cancer; however, the cellular environmental factors driving transformation of PTEN to its amyloid state remain poorly understood. Here, we report a new strategy for recombinant purification of PTEN from E. coli that overcome persistent hurdles in the field to recombinant PTEN purification. These results enable investigation of PTEN conformation, dynamics, and misfolding in vitro. With pure PTEN, we are developing a high-throughput screening platform to test systematically the chemical determinants of PTEN amyloid formation in the cellular environment using metabolite libraries. While it is long established that mutations can alter a protein’s energy landscape, leading to oncogenesis, the influence of cellular metabolic state and PTMs on this landscape is equally significant, yet largely unexplored. Our approach will enable exploration of the potential for components of the cellular environment, including cancer-associated metabolites, to induce amyloid transformation from soluble PTEN. We expect that our studies will enable mechanistic models of PTEN aggregation and inform drug development efforts to prevent PTEN loss of function in oncogenesis.
| Poster #: 24 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Membrane, Protein structure, CRISPR Project Title: Using temperature-sensitive Vps45 in yeast to explore how SM family proteins protect SNARE complexes from premature disassembly during membrane fusion Author List: David Cruz-Rivera, Undergraduate, Biology, Presenting Author; Mayra Silva Barcenas, Graduate, Biology, Presenting Author; Daniel Nickerson, Biology, San Bernardino Abstract Eukaryotic cells use vesicles to transport materials between membrane-bound organelles. Membrane fusion is carefully regulated by proteins, including SNAREs (Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptors) and SM (Sec1/Munc18) proteins, to ensure vesicles fuse only with correct targets. To promote fusion, SNARE proteins anchored in separate vesicle and target membranes form a trans-SNARE complex that "zippers" to brings membranes together. To recycle SNAREs for re-use, α-SNAP (Sec17 in yeast) binds to post-fusion SNARE complexes to aid in their disassembly by chaperone NSF (Sec18 in yeast). However, disassembly machinery can also interfere with pre-fusion trans-SNARE complexes to disrupt fusion. Two of the four evolutionary branches of the SM family (Sly1 and Vps33) have been shown to chaperone SNARE zippering and to protect trans-SNAREs from premature disassembly. Our research aims to determine if the endocytic SM branch (Vps45 in S. cerevisiae) protects zippering SNAREs from premature disassembly in a similar manner.
Temperature-sensitive (ts) alleles produce proteins that can be rendered non-functional when temperature varies from a permissive temperature. Specific mutations in the Vps45 ts allele (vps45-ts) that contribute to destabilization are not yet reported, so we also aim to determine which particular mutation(s) cause a Vps45 ts phenotype.
Sequencing revealed E122G, L355S, L400P, Q518R, and M550V in vps45-ts. Structural analysis predicted that E122G, L400P, and Q518R would be most disruptive. CRISPR plasmids were engineered to individually generate and phenotypically test each high priority mutation. The first mutation introduced in yeast, E122G, was not ts when tested. Construction and phenotypic tests of other mutations are ongoing.
To test whether Vps45 protects trans-SNARE complexes during vesicle fusion, we overexpressed disassembly component Sec17/α-SNAP in cells expressing vps45-ts and examined growth rates at escalating temperatures. At lower temperatures, elevated α-SNAP expression caused no difference in growth rates of yeast expressing vps45-ts, but at 34°C elevated α-SNAP caused a pronounced growth defect not observed in cells with wild type α-SNAP levels. These results indicate that Vps45 function is needed for cells to resist interference by overactive SNARE disassembly.
This work was funded by CSUSB OSR Research Supply grants to DCR and MSB.
| Poster #: 25 Campus: California State University, San Bernardino Poster Category: Biochemistry Keywords: Mutation, Cancer, Fluorescence polarization Project Title: Missense mutations in Rb affect its ability to bind to ARID4A Author List: Ian Bond, Undergraduate, Biology, Presenting Author; Anthony Castro, Chemistry and Biochemistry; Jason Burke, Chemistry and Biochemistry, San Bernardino Abstract Retinoblastoma (Rb) is a multifunctional tumor suppressor that is crucial in regulating the cell cycle and preventing tumorigenesis. Missense mutations in the Rb1 gene can disrupt its function as a cell cycle regulator, increasing the risk of cell cycle dysregulation and cancer. Missense mutations in Rb can affect its ability to bind these proteins, hindering its binding interactions with other critical peptides. Specifically, Rb suppresses tumorigenesis by binding to E2F transcription factors, halting the cell cycle and controlling cell proliferation. Rb contains a pocket domain that binds an LxCxE binding motif that interacts with cellular proteins including ARID4A. These play a role in repressing genes regulated by E2F. Prior research has shown that specific Rb mutations impair its ability to bind at the LxCxE site, affecting the overall functionality of the Rb protein. Here, we aim to understand how missense mutations in Rb affect its ability to bind to ARID4A. To test this, several cancerous Rb missense mutations found in patients were expressed using E. coli and purified using a series of column chromatography techniques. Fluorescence Polarization (FP) assays were performed to assess the binding affinity (Kd) of wildtype and mutant Rb for ARID4A. Preliminary results show that the Kd of 695MI Rb-ARID4A is 213.3 nM at 25°C and the Kd of WT Rb-ARID4A is 39.65 nM, suggesting that 695MI has a five fold weaker binding affinity compared to the WT. Future work will be done on mutants: 703IV, 751SC and 741RS from prior research resulting in their kD values having a significantly reduced kD in Rb-E7 than WT Rb-E7.
| Poster #: 26 Campus: California State University, Northridge Poster Category: Biochemistry Keywords: GPCR drug discovery, TAS2R38 receptor agonist, diabetes obesity Project Title: Targeting a Bitter Taste Receptor for Diabetes and Obesity Author List: Dylan CapittiFenton, Graduate, Department of Chemistry and Biochemistry, Presenting Author; Ravinder (Ravi) Abrol, Chemistry and Biochemistry, Northridge Abstract Diabetes is a complex metabolic disorder affecting an ever-increasing number of Americans as the adoption of modern diets and a more sedentary lifestyle increases. Molecular mimics of the incretin hormone glucagon-like peptide 1 (GLP-1), which can directly activate the pancreatic GLP-1 receptor (GLP-1R), are commonly prescribed to treat type II diabetes. Activation of the GLP-1R causes an increase in pancreatic insulin secretion. GLP-1R activation has also been linked to weight loss due to its ability to slow gastric emptying, thus extending feelings of satiety after eating. Despite the positive outcomes associated with directly targeting the GLP-1R, this class of drugs has also been linked to many negative side effects. We have identified bitter taste receptor 38 (TAS2R38), an upstream modulator of endogenous GLP-1 release, as a promising drug target for type II diabetes and obesity. This G-coupled protein receptor (GPCR) is expressed extra-orally in the enteroendocrine L-cells of the gut. Activation of TAS2R38 by phenylthiourea, a known agonist of the receptor, correlates to a significant increase in the concentration of GLP-1 in mice (DOI: 10.1016/j.bbrc.2016.04.149). As there is no published molecular structure of TAS2R38, we have built a homology-based model using the active conformation of the closely related bitter taste receptor 46 (TAS2R46) (PDB: 7XP6). We have relaxed this conformation in its native membrane environment via AMBER molecular dynamics (MD) simulations. We have also identified TAS2R38’s agonist binding site using this relaxed model of the receptor’s active conformation. We have performed virtual ligand screening (VLS) using a library of docking actives/inactives to validate our model. The VLS protocol revealed promising, novel TAS2R38 agonists. These ligands are currently being examined with the top-ranking protein-ligand complexes being relaxed via MD simulations to confirm their stability. The most promising TAS2R38 agonists will be validated via biochemical assays quantifying receptor activation and GLP-1 release. This approach of targeting the activation of a bitter taste receptor in the gut, upstream of GLP-1 release, promises to treat type II diabetes and obesity through minimized side-effects.
| Poster #: 27 Campus: San José State University Poster Category: Biochemistry Keywords: Drosophila, intracellular pH, cancer Project Title: Regulation of autophagic cell death by intracellular pH and the proto-oncogene Myc Author List: Alan Wong, Undergraduate, Biological Sciences, Presenting Author; Tiana Tameta-Arenas, Graduate, Biological Sciences, Presenting Author; Antonio Bibiano, Undergraduate, Biological Sciences; Kimberly Nguyen, Undergraduate, Biological Sciences; Israel Palomino, Undergraduate, Biological Sciences; Rachel Ann Soriano, Undergraduate, Biological Sciences; Juan Manuel Reyna Pacheco, Graduate; Bree Grillo-Hill, Biological Sciences, San José Abstract Background: Regulated cell death is essential during development to precisely pattern tissues and avoid developmental errors. Dysregulation of cell death is associated with pathologies including cancer (reduced cell death) and neurodegeneration (increased cell death). Dysregulated intracellular pH (pHi) dynamics are also associated with these diseases, where cancer cells have constitutively higher pHi than normal cells while degenerating neurons have lower pHi. Together, these observations led to the current view that cell death is enhanced at low pHi and inhibited at higher pHi. Our objective is to directly test this prediction in vivo and to determine how pHi regulates cell death.
Methods: We used transgenic Drosophila lines that overexpressed the Na-H exchanger DNhe2 specifically in the eye (DNhe2-OE), which increases pHi and results in a smaller, mispatterned adult eye. We used fly lines with mutations in different cell death pathways. We used another transgenic line that overexpressed the proto-oncogene Myc (Myc-OE), which regulates cellular processes such as proliferation and cell size. Using confocal microscopy, we imaged the eye discs, and then performed cell counts.
Results: In pupal eyes, we found that over-expression of DNhe2 causes a significant decrease in cell number, from an average of 15 cells in control to 12.4 cells. This suggested that cells overexpressing DNhe2 were dying inappropriately. We next tested for genetic interactions between DNhe2-OE and genes that regulated cell death. We found that the pH-dependent cell death is p53-dependent but caspase-independent. When we crossed a fly line with a mutation in a gene that promotes autophagic cell death, Atg1, with Dnhe2, we saw that the eyes were larger and less rough. We also saw altered expression of autophagic markers consistent with increased autophagy at higher pHi. Together, these data suggest that the cells are eliminated through autophagic cell death. Finally, we identified a genetic interaction between the proto-oncogene Myc and DNhe2, where co-expression showed suppression of the rough eye phenotype and rescued the cells that are missing with DNhe2 over-expression. Together, our findings elucidate mechanisms for pH regulation of conserved, critical developmental processes and provide evidence for new paradigms in growth control.
| Poster #: 28 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Biochemistry Keywords: Hydrogel, Responsive Hydrogels , Sequential release Project Title: Synthesis and characterization of a novel dual sequential release hydrogel Author List: Daniel Lopez, Undergraduate, Chemistry and Biochemistry , Presenting Author; Sandra Ward, Chemistry and Biochemistry , San Luis Obispo Abstract The purpose of this study was to produce dual release stimuli hydrogels capable of slow and controlled release of encapsulated payloads that offer various functions in many fields from agriculture to medicine to assist in the the efficacy of the deliverance and reduce the risk of side effects. We designed a vesicle crosslinked hydrogel for controlled stimuli responsive sequential release. Through a dynamic covalent thiol-disulfide exchange a 4-arm thiolated polyethylene glycol (PEG) “crosslinks” vesicles into a hydrogel. The amphiphiles that form the vesicles are sensitive to glutathione (GSH) and a silyl ether core installed in the 4-arm PEG is acid sensitive which creates dual stimuli release capability.The first payload is released by acid hydrolysis of the crosslinker then payload 2 is released via reduction by an antioxidant(GSH). This development of the amphiphile needed to form the vesicles is still ongoing with trials in different techniques of alkyl halides in our william ether synthesis to form a stable high yield amphiphile. However, in the efforts to better understand and characterize the optimal condition of the hydrogel needed once present in the body we used commercially available 4-arm thiolated PEG to conduct studies of swelling and release experiments and rheological and dynamic light scattering(DLS) experiments. Through our rheology and DLS experiments different weight percentages and lengths of the commercial 4-arm thiolated PEG were used to find their corresponding gelation time,when a cross frequency between the storage and loss modulus was represented, from an average of triplicate experiments to be around 2-3 hrs. This gelation time can influence the release rate of the encapsulated drug and the swelling behavior. This understanding led to the swelling and release studies of the same weight percentage and lengths of the 4-arm thiolated PEG. The hydrogel samples were created along with salicylic acid and placed in a tris buffer bath of pH 8.5, where the swelling was measured in intervals of 10 minutes for the first hour then 30 minutes for the remainder. Then aliquots of the release were taken in intervals of 10 minutes for the first hour then 30 minutes for the remainder. This experiment established an ideal condition to help stimulate the dual release kinetics of the hydrogel. Therefore, our characterization studies provide evidence on the ideal conditions for our hydrogel to be placed in when our amphiphile is finally synthesized.
| Poster #: 29 Campus: California State University, Fresno Poster Category: Biochemistry Keywords: Computational Protein Modeling, Virus Like Particles, Protein Arrays Project Title: Integration of Computational Modeling and Experimental Verification to Identify Key Amino Acids of Unexpected Quaternary Structure Formation Author List: Ian Heu, Undergraduate, Presenting Author; Malaika Buendia, Undergraduate, Presenting Author; Masaki Uchida, Department of Chemistry and Biochemistry, Fresno Abstract Decoration proteins (-Dec), are accessory proteins that attach to symmetry-specific sites on the outer surface of the P22 virus capsid. Dec has a homo-trimeric structure and the inherent role of Dec is believed to enhance the stability of the capsid. It is also used in protein-based biotechnology research as a protein linker that can modulate the assembly of P22 virus-like particles (VLPs) into 3-D arrays. To optimize this linker function, computational protein modeling has been employed to design mutants of Dec for modulating the binding affinity between Dec and P22. According to the computational modeling, Dec M53_06, a mutant, which contains three point mutations (D17N, Y49R, AND Q53M) from the wild-type (wt-) Dec, exhibits a greater binding affinity to P22 compared to wt-Dec. However, the purified Dec M53_06 mutant showed an unexpected quaternary structure that differed from the wt-Dec, despite it still binding to P22. To elucidate the cause of this unexpected change in quaternary structure, the three point mutations in Dec M53_06 were systematically reversed. In our study, two of such mutants, Dec M53_06 N17D and Dec M53_06 R49Y have been engineered, expressed, and purified for structural analysis. Purification of the mutants was done using fast protein liquid chromatography (FPLC) with a Histidine-Tag column and examined using SDS-PAGE. Results from the FPLC displayed a strong absorbance peak at 280 nm wavelength (protein) compared to the absorbance peak at 260 nm wavelength (genetic material) during eluent buffer flow indicating successful elution of proteins. Results from the SDS-PAGE displayed bands around 16 kDa-17 kDa matching the expected molecular weight of Dec subunits around the same values indicating that the mutant proteins of interest were successfully purified. In the future, Native-PAGE and size exclusion chromatography (SEC) will be used for the structural analysis of the computationally modeled Dec M53_06 mutants then pull-down assay and SPR will be used to measure binding affinity between the mutants and P22 VLPs.
| Poster #: 30 Campus: California State University, Fullerton Poster Category: Bioengineering Keywords: high-school students research, cross-disciplinary summer internship, undergraduate and graduate student mentorship Project Title: Empowering High-School Students with Cross-Disciplinary Research Skills through Developing Assistive Technologies Author List: Vincent Le, Undergraduate, Presenting Author; Severino Hernandez, Graduate, Presenting Author; Christy Martin; Richard Casillas; Martin Cho; Andrew Herr; Jerry Ku; Ethan Lee; Nirmit Shah; Andy Yang; Nina Robson, Mechanical Engineering, Fullerton Abstract Active participation in research has been shown to play a valuable role in enhancing the educational experience of high-school and undergraduate science and engineering majors. In addition to endorsing learner-centered approaches and participation in discovery-driven research, the National Research Council (NRC) promotes the development of genuinely interdisciplinary courses and curricula, leading to scientists and engineers capable of combining specialized disciplinary knowledge with fluency in complementary disciplines. This vision is consistent with a recent report of the California Life Sciences Institute (CLSI) that workforce-ready biotechnology candidates tend to be equipped with soft skills, as well as experience in using modern data-driven approaches in science, such as robotics, bioengineering, computational modeling.
The authors have combined the NRC with the CLSI calls, by integrating research-based high-impact practices into the Increasing Diversity in Engineering and Labor-force (IDEAL) summer outreach program on Early Experiences in Biomedical Engineering. The four-week program involves high-school students and CSUF undergraduate and graduate mentors from the Colleges of Natural Science and Engineering working collaboratively on projects in the area of Development of Assistive Technologies. The IDEAL program activities are designed to introduce high-school students’ to authentic cross-disciplinary research, as well as strengthen their self-confidence and motivation in pursuing future Biotechnology careers.
Our preliminary direct pre- and post-program self-assessment survey results show that for the short time of one month of summer internship the students enhanced the most their knowledge/skills in reading and understanding research papers, as well as in working collaboratively on research projects. National Science Foundation Middle/High School Student Attitudes Towards STEM (S- STEM) Survey was used to assess the overall impact of the outreach program on the students’ self-confidence and motivation in pursuing future cross-disciplinary Biotech careers. The results showed that the 21st Century skills related to critical-thinking, communication, and collaboration was the section with the most radical improvement.
| Poster #: 31 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: biomedical engineering, brain computer interface, neural engineering Project Title: Laplacian Spatial Filters for Visual Evoked Potential Classification in Brain-Computer Interfaces Author List: Kiran Prasannan Nair, Graduate, Computer Science, Presenting Author; Hubert Cecotti, Computer Science, Fresno Abstract A brain-computer interface (BCI) is a technology that enables direct communication between the brain and an external device, bypassing traditional sensory and motor pathways. It typically involves sensors that detect neural activity and algorithms that translate these signals into commands for controlling computers or prosthetics. A Visual Evoked Potential (VEP) BCI is a type of brain-computer interface that uses visual stimuli to elicit brain responses, which the system then detects and interprets. This allows users to control devices or communicate by focusing on specific visual patterns or images, translating their brain's electrical responses into commands. We consider a code of 63 symbols, with shifts of 0, 8, 16, 24, 32, and 40 (6 classes). Experimental Paradigm was implemented with the MATLAB Psychtoolbox. Participants had to pay attention to a box presenting the visual stimulus based on the sequence of created symbols. In this study, we assess the impact of the Laplacian filter on VEP detection. The Laplacian spatial filter is used to enhance the spatial resolution of electroencephalographic (EEG) signals. It works by subtracting the average of neighboring electrode signals from the signal at each electrode, which helps to emphasize local brain activity and reduce the influence of noise and artifacts. The head is considered a perfect sphere on which the sensors are placed. We consider the distance between two sensors to be the distance between two points on a sphere. The experiments were performed with 8 sensors (O1, O2, Pz, P3, P4, PO7, PO8, and Oz), well-suited for detecting VEP detection. The Laplacian filter increases accuracy, improving signal localization and reducing noise. Different radii (0.5, 1, 1.5, 2) were tested, with radius 1 yielding the best accuracy of 94.8%-97.3%. The methods for calculating Laplacian weights include 1/d, 1/d^2, 1/log(d), and 1/sqrt(d), with the 1/d method providing the highest accuracy. Larger radii like 2 showed a slight drop in accuracy (92.8%-93.03%), suggesting that smaller radii are more effective for signal enhancement. The results support the conclusion that non-invasive BCI based on the detection of code-based visual evoked potential benefits from using a Laplacian spatial filter as a preprocessing method for enhancing the EEG signal. This leads to an improvement of 97% in accuracy compared to the grand average approach, which had an accuracy of 92%.
| Poster #: 32 Campus: California State University, Northridge Poster Category: Bioengineering Keywords: biofilm, laser, Bacillus subtilis Project Title: The effects of different wavelengths of light on biofilm formation Author List: Cindy Quintanilla, Undergraduate, Biology, Presenting Author; Nadia Evans-Lambert, Undergraduate, Biology, Presenting Author; Brooke Walter-Lakes, Physics and Astronomy; Anna Bezryadina, Physics and Astronomy, Northridge Abstract Understanding biofilm development and control strategies is critical because of its impact on human health and the opportunity to produce new biomaterials for future biodegradable technology. Biofilm is a sticky substance formed by microorganisms in response to low-nutrient, hostile environments, serving as a protective mechanism for these organisms. This study investigates the impact of various wavelengths of light on biofilm formation. We concentrate on the biofilm formation of Bacillus subtilis (NCIB 3610 strain) in a minimal salts glycerol glutamate (MSgg) medium. The bio-sample is exposed to different wavelengths of light for several hours to examine changes or consistency in growth patterns. We hypothesize that ultraviolet (UV) and lower visible wavelengths will adversely affect biofilm formation and composition, while certain red and near-infrared wavelengths may have little to no impact or could even promote biofilm growth by enhancing cell proliferation, growth, and repair. Our studies showed that the use of near-infrared laser light with a wavelength range (820 nm - 830 nm) causes minimum harm to bacterial growth; however, exposure to blue light (473 nm) prevents biofilm formation. The results of this work provide optical methods for controlling the growth of biofilm communities.
| Poster #: 33 Campus: California State University, Fresno Poster Category: Bioengineering Keywords: Plasma-enhanced chemical vapor deposition, Oxygen delivery, Collagen Project Title: Encapsulating peroxides on postoperative medical devices via plasma enhanced chemical vapor deposition Author List: Naylene Velasquez, Undergraduate, Chemistry and Biochemistry, Presenting Author; Haylee McFall, Undergraduate, Chemistry and Biochemistry; Morgan Hawker, Chemistry and Biochemistry, Fresno Abstract Periodontal surgeries often leave wounds vulnerable to bacteria, inflammation, and prolonged tissue regeneration, with postoperative hypoxia compounding these challenges. Collagen is widely used in postoperative healing due to its hemostatic advantages, but existing collagen-based materials do not address hypoxia. A promising approach entails plasma enhanced chemical vapor deposition (PECVD) to encapsulate peroxides on polymer surfaces. PECVD occurs when a plasma precursor undergoes gas-phase reactions to generate fragments that form conformal films. PECVD with a 1,7-octadiene precursor was previously used to control oxygen release from polydimethylsiloxane by encapsulating calcium peroxide with a hydrocarbon film. Applying this strategy to collagen has the potential to enhance healing efficacy.
In this work, a collagen patch was designed to sustain in the oral environment, prevent infections, and deliver localized oxygen. PECVD parameters such as exposure time and applied power were optimized to control the plasma-deposited hydrocarbon film thickness, resulting in a device with calcium peroxide encapsulated between two PECVD layers of hydrocarbons. Oxygen release kinetics were evaluated using a dissolved oxygen probe in simulated oral conditions, correlating release rates with diffusion of water into the encapsulation and oxygen being released. Maximum release of oxygen 9.29 mg/L from the encapsulated device occurred after 92 minutes of immersion. Surface hydrophilicity was analyzed using water contact angle goniometry, demonstrating enhanced surface hydrophobicity following PECVD. X-ray photoelectron spectroscopy provided insights into chemical composition. An increase in the C-C/C-H binding environment composition was observed, suggesting a deposition of a hydrocarbon film from the 1,7-octadiene precursor.
We also applied PECVD to clinically-relevant polyglycolic acid-based sutures. This involved modifying the sutures with the same PECVD methods to coat them with hydrocarbon films to encapsulate calcium peroxide, similar to the collagen patches. This approach will enhance suture functionality by providing localized oxygen delivery directly at the suture site to improve the overall healing process.
| Poster #: 34 Campus: San Francisco State University Poster Category: Bioengineering Keywords: beta- lactam antibiotics , enzyme-engineering , pathway-engineering Project Title: Development of an Oxygen and NAD+ Regenerating System for in the Cell-Free Synthesis of Beta-Lactam Antibiotics Author List: Yingyi Huang, Undergraduate, Chemistry and Biochemistry, Presenting Author; Chas Smith, Undergraduate, Chemistry and Biochemistry, Presenting Author; George Gassner, Chemistry and Biochemistry, San Francisco Abstract Beta-lactam antibiotics are the first-line treatment for common bacterial infections, but current industrial methods used for their production are both inefficient and environmentally harmful. Moreover, pharmaceutical companies, driven by more profitable exploits are producing these antibiotics at a rate that fails to meet annual demands. This has resulted globally in recurrent antibiotic shortages that negatively impact vulnerable populations unable to access treatment. To help offset this problem, our laboratory is developing a modular green-chemical approach that joins solvent tolerant bacterial and fungal enzymes in a cell-free enzymatic pathways for the synthesis of beta-lactam antibiotics that begin with alcohol, oxide, aldehyde, or styrenes as synthons. Operation of these cell-free pathways occurs with the net reduction of NAD+ to NADH and molecular oxygen to hydrogen peroxide. This poster will present the development of sensitive stopped-flow luminescence assay for the detection of hydrogen peroxide and its use in establishing the conditions needed to optimize both the enzymatic regeneration of NAD+ and the electrochemical regeneration of oxygen required for sustained transformation of aldehydes to penicillin derivatives.
| Poster #: 35 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Bioengineering Keywords: Microfluidics, CRISPR, Droplets Project Title: Inertial Microfluidic Droplet Encapsulation for Improved Liposomal CRISPR Transfection Author List: Giusseppe Pierotti, Undergraduate, Biomedical Engineering, Presenting Author; Benjamin Hawkins, Biomedical Engineering, San Luis Obispo Abstract CRISPR-Cas9 technology has revolutionized the field of genetic engineering, offering unprecedented precision and versatility in genome editing. Recently, non-viral delivery methods such as liposomes have emerged as a promising vehicle for CRISPR components, offering reduced off-target editing and higher ease of use. However, transfection efficiency with liposomal delivery is low. Microfluidic techniques enable precise control over liposome size and composition, resulting in liposomes tailored for optimal CRISPR plasmid delivery. Non-viral transfection (e.g., electroporation) of suspension cells, such as lymphatic cells, typically occurs at very low percentages (~5%). We aim to demonstrate transfection of hard-to-transfect suspension (e.g., Jurkat Clone E6-1) cells at a high efficiency using inertial microfluidic droplet-encapsulation. Single-cell confinement in droplets with microfluidically controlled liposome synthesis has been shown to improve transfection efficiency to ~50%. Ultimately, we plan to target upregulation of ZNF865, upregulation of which is thought to regulate cell senescence, increasing cell proliferation rates and cytokine release in Jurkat cells. In this study we demonstrate improved liposomal transfection by combining recent advances in liposome-plasmid (lipoplex) synthesis, inertial cell ordering, and droplet confinement in microfluidic systems.
We demonstrate an improved microfluidic device using COMSOL simulation software, integrating high-aspect-ratio microchannel for inertial cell ordering into a droplet-lipoplex encapsulation device. The device was fabricated using soft lithography and tested by encapsulating 5 um dia fluorescent polystyrene particles in aqueous droplets in an Fluorinert FC-40 (oil) continuous phase. The droplets had an average diameter of 53.5±4.3 um and a polydispersity index of 0.006. These results suggest that we can expect to see a high encapsulation efficiency of ~97% as seen in research. With single cell encapsulation efficiencies, we are expecting a high transfection rate of both 3T3 fibroblasts, as well as the Jurkat t-cells using liposome complexes. From previous work with viral CRISPR delivery, upregulation of ZNF865 in acute lymphoblastic leukemia t-cells has been shown to increase the proliferation of the cells as well as increase the production of IL-2 and INF-gamma and can expect the same from non-viral delivery of CRISPR plasmid.
| Poster #: 36 Campus: California State Polytechnic University, Pomona Poster Category: Clinical Keywords: WNV, DEC-205+ DCs, vaccine Project Title: Targeting DEC-205+ Dendritic Cells for Antigen-Specific Immunotherapy Against West Nile Virus Encephalitis Author List: Joshua Herzler, Graduate, Biological Sciences, Presenting Author; Douglas Durrant, Biological Sciences, Pomona Abstract West Nile Virus (WNV), the leading cause of mosquito-borne neuroinvasive disease in the US, remains a major public health challenge due to the lack of an approved therapeutic or human vaccine. Dendritic cells (DCs), crucial for initiating immune responses, play a key role in determining whether T cells are activated to fight infections or are rendered tolerant based on the maturation of DCs. A specific subset of DCs express the endocytic receptor DEC-205, which is responsible for capturing, processing, and presenting antigens to T cells to trigger a robust immune response. Previously, our studies highlighted the role of DEC-205+ DCs in the CNS, where they reactivate antiviral effector T cells, restrict viral replication, and mitigate immunopathology during WNV infection. In this study, we propose an innovative strategy to target DEC-205+ DCs by creating a fusion protein known as a single-chain fragment variable (scFv). An scFv is a genetically engineered antibody fragment that combines the variable regions of an antibody’s heavy and light chains into a single molecule, enabling it to specifically bind to its target—in this case, the DEC-205 receptor on DCs. Our engineered scFv will carry a WNV immunogenic peptide, designed to stimulate a targeted anti-viral immune response. We will purify the aDEC-205-scFv and test its therapeutic efficacy in a mouse model of WNV encephalitis. By co-delivering this fusion protein with a DC maturation signal, poly I:C, we aim to enhance immune activation of anti-viral T cells. We will evaluate the immune response by measuring the upregulation of co-stimulatory molecules (CD40, CD80, and CD86) on DCs and assessing T cell activation through cytokine production and expression of activation markers via flow cytometry. This novel research introduces a targeted antigen delivery approach, focusing on DEC-205+ DCs to create a highly specific and potent immunotherapy against WNV encephalitis. By leveraging the critical role of DCs in modulating immune responses, this study paves the way for potential therapeutic and vaccine strategies not only for WNV but also for other neuroinvasive viral infections.
| Poster #: 37 Campus: California State University, Long Beach Poster Category: Clinical Keywords: Smartphone, activities of daily living, clinical parameters, post-stroke survivors Project Title: Comparison of Smartphone Data Between Laboratory and Home Environments in Post-stroke Survivors Author List: Kevin Tran, Graduate, Physical Therapy; Ethan Le, Graduate, Physical Therapy; Aaron Clark, Graduate, Physical Therapy; Nadav Nankin, Graduate, Physical Therapy; Michael Shiraishi, Chapman University; Rahul Soangra, Physical Therapy; Vennila Krishnan, Physical Therapy, Long Beach Abstract With the rapid advancement of smartphone technology in capturing activities of daily living (ADL) data, it is essential to assess the extent to which data collected in patients' natural environments can mirror that obtained in controlled laboratory settings. This study examined accelerometer and gyroscope data collected via smartphones from four post-stroke survivors performing three ADL tasks: a 10-step walk, a 360-degree left turn, and a 360-degree right turn. Data were obtained in both laboratory conditions (5 trials per participant) and home environments (20 trials per participant over 5 weeks, collected at various times and settings). Independent t-tests showed no significant differences between lab and home environments for time to completion (p = -0.53), peak acceleration (p = 1.19), or peak gyroscope acceleration (p = 0.97) across all tasks. These findings demonstrate that smartphone data collected in home environments, despite being less controlled, are comparable in quality to lab-based data. This suggests that smartphone technology can facilitate meaningful, quantitative ADL data collection in home environments, improving accessibility and cost-effectiveness for clinical and research applications, particularly in underserved populations.
| Poster #: 38 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Modeling simulation, Spectroscopic studies and analysis, Magnetic resonance imaging Project Title: Two-pronged computational and experimental control of magnetic nanoparticle structures for nuclear magnetic resonance imaging Author List: Zulaikha Ali, Undergraduate, Department of Chemistry and Biochemistry, Presenting Author; Yunfei Zhang, Department of Chemistry and Biochemistry; Michael Kaul, Center for Radiology and Endoscopy, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany, Department of Diagnostic and Interventional Radiology and Nuclear Medicine; Billy Truong, Undergraduate, Department of Chemistry and Biochemistry; Deepika Bhanot, Graduate, Department of Chemistry and Biochemistry; , Center for Radiology and Endoscopy, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany; Yuanyuan Li, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, Chemical Sciences Division; He Wei, Department of Chemistry and Biochemistry, Presenting Author; He Wei, Department of Chemistry and Biochemistry, Fresno Abstract In addition to the tens of millions of medical doses consumed annually around the world, a vast number of nuclear magnetic resonance imaging (MRI) contrast agents are being deployed in MRI research and development, offering precise diagnostic information, targeting capabilities, and analyte sensing. Superparamagnetic iron oxide nanoparticles (SPIONs) are notable among these agents, providing effective and versatile MRI applications while also being heavy-metal-free, bioconjugatable, and theranostic. At Fresno State, we designed and implemented a novel two-pronged computational and experimental strategy to meet the demand for the efficient and rigorous development of SPION-based MRI agents. Our MATLAB-based modeling simulation and magnetic characterization revealed that extremely small maghemite SPIONs in the 1–3 nm range possess significantly reduced transversal relaxation rates (R2) and are, therefore, preferred for positive (T1-weighted) MRI. Moreover, X-ray diffraction (XRD) and X-ray absorption fine structure (XAFS) analyses, through our collaboration with Oak Ridge National Laboratory, demonstrated that the diffraction pattern and radial distribution function of our SPIONs matched those of the targeted maghemite crystals. In addition, simulations of the X-ray near-edge structure (XANES) spectra indicated that our synthesized SPIONs, even at 1 nm, maintained a spherical structure. Furthermore, in vitro and in vivo MRI investigations, conducted in partnership with the University Medical Center Hamburg-Eppendorf in Germany, showed that our 1-nm SPIONs effectively highlighted whole-body blood vessels and major organs in mice and could be cleared through the kidney route to minimize potential post-imaging side effects. Overall, our innovative approach enabled a swift discovery of the desired SPION structure, followed by targeted synthesis, synchrotron radiation spectroscopic studies, and MRI evaluations [1]. The efficient and rigorous development of our high-performance SPIONs can set the stage for a computational and experimental platform to develop future MRI agents.
Reference:
1. Ali Z, Zhang YF, Kaul MG, Truong B, Bhanot D, Adam G, Li YY, Wei H*. “Structural control of magnetic nanoparticles for positive nuclear magnetic resonance imaging.” Nuclear Science and Techniques, Accepted and As Soon As Publishable
| Poster #: 39 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Electroencephalogram (EEG), Brain-Computer Interface (BCI), Music Project Title: Quantifying the Neurological Impact of Music on Brain Waves Using EEG and BCI Technology Author List: Zachary O’Neil, Undergraduate, Department of Electrical and Computer Engineering, California State University, Fresno, Presenting Author; Sai Katherapalli, Undergraduate, Department of Electrical and Computer Engineering, California State University, Fresno; Kiran Nair, Graduate, Department of Computer Science, California State University, Fresno; Ethan Phasakda, Undergraduate, Department of Biology, California State University, Fresno; Anthony Westcott, Undergraduate, Department of Biology, California State University, Fresno; Hovannes Kulhandjian, Department of Electrical and Computer Engineering, California State University, Fresno, Presenting Author; Anahit Hovhannisyan, Biology, Fresno Abstract There are five primary brain wave types—alpha, beta, delta, gamma, and theta—each associated with distinct mental states (Figure 1)1. Previous research has shown that music can alter brain wave patterns, significantly influencing both cognitive and physiological processes, particularly in clinical settings2. Bigliassi et al. (2015) found that tranquil music reduces vagal withdrawal by activating the prefrontal cortex. Similarly, Nawaz et al. (2018) observed that stimulating music increases beta waves, while calming music elevates alpha waves in the frontal and parietal brain regions.
Using Brain-Computer Interface (BCI) technology, we established a direct communication pathway between neural activity and external devices (e.g., drones, prosthetic arms) through neural signals. BCIs require the recording of brain activity, which can be accomplished via invasive or non-invasive methods using electrical conductors5. Electroencephalograms (EEGs), which detect neural activity from cortical pyramidal neurons6, are crucial for non-invasive BCI applications.
Although numerous studies have explored the neurological effects of music, there is a lack of quantitative data in the current literature. Our study aims to fill this gap by precisely quantifying the impact of music on brain wave activity. We used BCI technology to collect and analyze EEG data, focusing on how music preferences and personality traits influence brain activity. Our findings indicate that different music genres produce distinct brain wave patterns, and individuals’ preferences for certain genres are reflected in their neural activity.
Figure 1. Brain Waves
| Poster #: 40 Campus: California State University, Monterey Bay Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Non-coding RNAs, Motif Annotation Tool, RNA Visualization Project Title: A Novel Tool for Comparative Analysis and Annotation of RNA Structural Motifs Author List: Shaun Rose, Undergraduate, School of Computing and Design, Presenting Author; Sameer Dingore, Undergraduate, School of Computing and Design, Presenting Author; Judah Silva, Undergraduate, School of Computing and Design; Kalyn Daum, Undergraduate, School of Computing and Design; Shahidul Islam, School of Computing and Design, Monterey Bay Abstract Non-coding RNAs (ncRNAs) play crucial roles in various cellular processes and disease mechanisms. The three-dimensional structure of ncRNAs is a key determinant of their biological function, with many evolutionarily conserved structural motifs identified for their specific roles. However, annotating these RNA structural motifs and understanding their intricate relationships through comparative analysis can be a challenging task. Automated computational tools can annotate highly similar motif instances, but effective analysis of RNA 3D motif variations requires manual intervention, as noted in existing literature. One of the major obstacles in the manual analysis of RNA motifs is the lack of any existing tools dedicated to effective comparative analysis. To address this challenge, we have developed a novel RNA structural motif annotation tool. Unlike existing tools such as JMol or MOL*, which facilitate the analysis of PDB-based 3D objects, our tool offers unique features: 1) it enables objects to be rendered and manipulated independently on a single canvas, and 2) it facilitates motif feature extraction, organization, and classification. Researchers can upload their own motif structures or import ones from our database to conduct in-depth analyses of motif families, such as kink-turn and sarcin-ricin motifs, and build relationships among instances of these families, potentially leading to the recognition of motif subfamilies and modules. Additionally, our tool supports collaboration among researchers, facilitating the sharing and reviewing of annotations to build RNA motif annotations collectively. The data generated from this tool can accelerate the discovery of novel RNA motifs and aid in understanding their biological functions.
| Poster #: 41 Campus: California State University, Monterey Bay Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Non-coding RNAs, Base-pairing Annotation, RNA Base-pair Benchmark Dataset Project Title: Curated Dataset for Benchmarking of RNA Base-Pairing Annotations Tools Author List: Sameer Dingore, Undergraduate, School of Computing and Design, Presenting Author; Shaun Rose, Undergraduate, School of Computing and Design, Presenting Author; Shahidul Islam, School of Computing and Design, Monterey Bay Abstract Non-coding RNAs (ncRNAs) regulate a wide array of cellular processes, with their structural conformation characterized by recurring motifs such as hairpin loops, internal loops, and kink-turns. These motifs are stabilized by base-pairing interactions and have unique biological functions, making accurate base-pairing annotations critical for understanding their roles. However, our evaluation of several widely used base-pairing annotation tools—MCAnnotate, RNAView, ClaRNA, FR3D, and DSSR—revealed significant inconsistencies, such as disagreements or missing annotations, as well as variations in annotation representation. To address these discrepancies, we first standardized the format for base pairing annotations across these tools to enable direct comparison and improve consistency. Utilizing the streamlined annotations, we conducted a comparative analysis with motif cluster data from the RNA Motif Atlas to establish a consensus of base-pairing annotations among different RNA structures within the same motif regions. This approach allowed us to systematically identify inaccuracies in each tool’s annotation, underscoring the need for a standardized approach to evaluating base-pairing annotation tools. To address this need, we developed a curated benchmark dataset based on consensus data from conserved RNA structural motif clusters in the RNA Motif Atlas. This dataset serves as a robust standard for evaluating and refining base pair annotation tools, ultimately providing a valuable resource for enhancing the accuracy of annotations and advancing RNA structural biology research.
| Poster #: 42 Campus: San Diego State University Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: PCL Polymer, 3D Bioprinting, SAW Project Title: 1D Patterning of PCL Polymer via SAW Jetting for 3D Bioprinting Author List: Md Aminul Islam, Graduate, Mechanical Engineering, Presenting Author; Sara Adibi, Mechanical Engineering, San Diego Abstract Three-dimensional (3D) porous scaffolds are critical in tissue engineering as
they provide a framework for cell attachment, proliferation, and differentiation, mimicking
the extracellular matrix in the body. These scaffolds not only support mechanical
stability but also enable the transport of nutrients and waste, which is essential for
tissue regeneration. 3D bioprinting offers a promising approach to precisely fabricate
these scaffolds layer-by-layer, enabling the creation of complex tissue structures
tailored to specific biological needs. In this study, we utilize acoustic jetting technology
to achieve angular modulation of polymer droplet ejection, which is essential for precise
control in scaffold formation. By employing two Interdigital Transducers (IDTs) made
from 128° Y-cut lithium niobate, we control the ejection angle of droplets by adjusting
the burst duration ratio between the two transducers. This setup allows for controlled
angular modulation of jetting from -50° to +50°, using a polymer solution of
Polycaprolactone (PCL) dissolved in DMSO with Sodium Dodecyl Sulfate (SDS) as a
surfactant. Droplets are ejected at varying angles and collected on a Teflon-coated
substrate, forming a 1D array of droplets. These droplets are then solidified to create a
porous scaffold material. By further extending this technology to control jetting in three
directions, 3D bioprinting can be utilized to fabricate complex, customizable tissue
scaffolds for various biomedical applications, with potential for multi-layered structures
that enhance cell interaction and tissue growth.
| Poster #: 43 Campus: California State University, Fresno Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Cell-free DNA, Fragmentomics, Liquid Biopsy Project Title: Saliva Cell-Free DNA as a Biomarker for Early Detection of Gastric Cancer Author List: Hassoon Sarwar, Undergraduate, Bruins in Genomics (B.I.G.) Summer Program, Institute for Quantitative and Computational Biosciences, UCLA, Presenting Author; Louise Oh, Undergraduate, Bruins in Genomics (B.I.G.) Summer Program, Institute for Quantitative and Computational Biosciences, UCLA; Aaron Zander, Undergraduate, Bruins in Genomics (B.I.G.) Summer Program, Institute for Quantitative and Computational Biosciences, UCLA; Dev Trivedi, Undergraduate, Bruins in Genomics (B.I.G.) Summer Program, Institute for Quantitative and Computational Biosciences, UCLA; Irene Choi, Graduate, UCLA School of Dentistry, UCLA; Neeti Swarup, UCLA School of Dentistry, UCLA; Mohammed Aziz, UCLA School of Dentistry, UCLA; David Wong, UCLA School of Dentistry, UCLA; Mario Bañuelos, Mathematics, Fresno Abstract Gastric cancer (GC) is one of the leading causes of cancer death annually and due to its disease heterogeneity, it is difficult to detect during early stages. The tumor microenvironment (TME) of GC includes various molecular components, such as cancer-associated fibroblasts and immune cells, that favor tumor progression. Previous studies have utilized salivary cell-free DNA (cfDNA) as a non-invasive means to guide early cancer detection. This project further explores non-mutational analyses of cfDNA and considers the TME of GC to investigate the underlying genetic differences between non-cancer and gastric cancer patients. We employed a low-coverage single-stranded library NGS pipeline on saliva samples of the two cohorts to study cfDNA characteristics including fragmentomics, G-quadruplex prevalence, and end-motif profiles. Our analysis showed a significant difference between the two cohorts for both saliva cfDNA characteristics and TME-specific biomarkers. These discoveries could potentially improve the application of cfDNA analysis in clinical settings for both early disease detection and for monitoring its progression.
| Poster #: 44 Campus: California State University, Long Beach Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: CAZY, enzyme structure/function, sequence annotation Project Title: From Sequence to Function, Creating A Framework to Predict Carbohydrate Processing Across Environments Author List: Daniel Erdody, Graduate, Presenting Author; Nicholas Griffin, Graduate; Renaud Berlemont, Biological Sciences, Long Beach Abstract While the Carbohydrate-Active enZYme (CAZyme) database is an invaluable resource for studying carbohydrate processing across environments, its limitations for sequence annotation have posed challenges for researchers, especially those attempting to use it for metagenome annotation. Here, we retrieved and reannotated the sequences from characterized CAZymes to include their domain architecture, enzymatic activity, taxonomic origin, amino acid sequence, etc. For each CAZyme family, we further analyzed the domain architectures to identify recombination events leading to multidomain and multifunctional proteins.
By generating a custom database linking individual domains to specific function(s) for characterized CAZYmes, we provide an unprecedented resource to perform precise annotation of newly sequenced datasets (e.g., genomes and metagenomes) and to gain insight into the functioning of environmental microbial communities.
| Poster #: 45 Campus: California State University, Northridge Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Ribosome, Biophysics, Protein-Folding Project Title: Computational Modeling of Magnesium Ion Solvation around Escherichia Coli 70S Using 3D Reference Interaction Site Model of Molecular Solvation Author List: Tiannah York Van Elslande, Graduate, Physics and Astronomy , Presenting Author; Felipe Cavarlho, Physics and Astronomy; Tyler Luchko, Physics and Astronomy ; Tiannah York Van Elslande, Department of Physics and Astronomy, Northridge Abstract The ionic environment around the ribosome affects protein synthesis and
folding and may be critical to understanding deleterious aggregation-based diseases.
However, no existing experimental methods can provide a detailed ionic description of the globular domains lining the ribosomal exit tunnel and the hydrophobic L23 pocket adjacent to it. Therefore, we have computed the density distribution of
ions around the Escherichia Coli 70S ribosome in atomistic detail using the
3D reference interaction site model (3D-RISM) of molecular solvation. 3D-RISM uses the same molecular models as used in simulations but computes the distribution of the solvent using statistical physics, rather than brute-force simulation. To compute the distribution of water and ions, we first parameterized the crystal structure of Escherichia Coli 70S ribosome (PDBID 4YBB) with the Amber force field. Then we used 3D-RISM to calculate the equilibrium distribution of water with 100 mM KCl and 1mM MgCl2. We also computed and observed how the radial distribution functions varied around the RNA versus the protein structures. The density distributions show tight binding of magnesium stabilizing the interior of the structure, as well as diffuse distributions of sodium and magnesium that could impact protein folding.
This material is based upon work supported by the National Science
Foundation under Grants 2102668, 2320846, and 2018427
| Poster #: 46 Campus: California State University, Northridge Poster Category: Computational (Bio, Chem, Math, Eng, etc.) Keywords: Neural Networks, Solvation, 3D-RISM Project Title: Predicting Generalized-Born Molecular Solvation Energies Using An Active-Learning Neural Network Ensemble Author List: Vahe Grigorian, Graduate, Presenting Author; Vahe Grigorian, Physics & Astronomy, Northridge Abstract Simulations are used to predict the real-world behavior of candidate drug molecules interacting with protein binding sites. However, straightforward application of physics-based models often incur exorbitant computational costs when trying to simulate solvation. To address this, we are training neural networks to approximate the 3D reference interaction site model (3D-RISM) and generalized Born (GB) solvent models, which is projected to reduce the computational cost by several orders of magnitude. To efficiently train the neural network, we are using active learning, whereby a neural network iteratively selects only training data that sufficiently differs from the data it has already trained on. This is more efficient than simply iterating through fixed training data, where certain training instances contribute little to the generalizability of the neural network. To implement this, we have trained our active learning network on frames from three different physics-based sampling methods. In each method, we run molecular modeling simulations until the solvation energies differ by a set threshold, before adding the generated frames to the next stage of training. This diverse training set makes the fully trained model able to predict solvation energies for a greater variety of drug molecules. Preliminary results show a root-mean-squared benchmark error on the order of 0.6 to 0.7 kcal/mol on single conformations of small molecules. This material is based upon work supported by the National Science Foundation under Grants 2102668, 2320846, and 2320718.
| Poster #: 47 Campus: California State University, San Bernardino Poster Category: Diagnostics/Imaging/Analytical Keywords: Electroencephalography, Face perception, Evoked related potentials Project Title: Predicting Generalized-Born Molecular Solvation Energies Using An Active-Learning Neural Network Ensemble Author List: Alexandra Ackerman, Graduate, Presenting Author; Nicolas Brunet, Psychology, San Bernardino Abstract “Facial recognition processes are closely associated with specific electrophysiological markers, particularly the N170 event-related potential (ERP), which reflects activity in the Fusiform Face Area (FFA), a key brain region involved in processing facial information. Damage to this region can result in prosopagnosia or face blindness. An ongoing debate in the field of neural face processing concerns whether FFA activity is modulated by facial expressions, with some studies reporting modulation and others finding no effect. A frequently cited 2007 study by Blau et al.* supported the former view, suggesting that fearful expressions elicit larger N170 amplitudes compared to neutral expressions. However, upon closer examination, we identified a potential confounding variable in their stimuli: in the “”fearful”” condition, many faces displayed exposed teeth, which could independently influence N170 responses.
To test this hypothesis, we replicated Blau et al.’s study using a factorial design, systematically controlling for mouth position (teeth exposed vs. not exposed) across emotional expressions. Preliminary data revealed no significant effect of emotional expression on N170 amplitude. However, faces with exposed teeth, regardless of expression, elicited a small but statistically significant increase in N170 amplitude. These findings suggest that the effect reported by Blau et al. may have been driven by the presence of exposed teeth, rather than emotional expression.
Blau, Vera C., et al. “”The face-specific N170 component is modulated by emotional facial expression.”” Behavioral and Brain Functions 3 (2007): 1-13.” | Poster #: 48 Campus: California State University, Los Angeles Poster Category: Diagnostics/Imaging/Analytical Keywords: alpha synuclein, nanoplastics, Parkinson’s disease Project Title: Evaluating the effect of polystyrene nanoplastics on the abnormal aggregation of alpha synuclein Author List: Isabel Garcia, Graduate, Presenting Author; Yixian Wang, Chemistry and Biochemistry, Los Angeles Abstract Alpha-synuclein, a protein linked to Parkinson’s Disease, is typically found in its monomeric state but can aggregate into toxic oligomeric or fibril forms that contribute to neuronal cell death. Research indicates that various factors, including oxidative stress and environmental toxins, can trigger alpha-synuclein aggregation. Very limited studies have investigated the possible contribution of nanoplastic pollution to this. Understanding this mechanism is crucial for revealing how nanoplastics might influence cellular health and potentially accelerate the development and progression of Parkinson’s Disease. This project aims to investigate the impact of nanoplastics on the abnormal aggregation of alpha-synuclein. Specifically, the aggregation of monomeric alpha-synuclein, both in the presence and absence of nanoplastics 0.03 μm carboxylate-modified polystyrene nanoplastics, was characterized using Atomic Force Microscopy (AFM) and Circular Dichroism (CD) spectroscopy. The results showed that, over a six-day period, the observed results were contradictory to those reported in the literature. While fibrils and aggregates are shown in the protein samples, those with nanoplastics observed little to no fibrils or aggregates present. This project is supported by the CSUBIOTECH 2024 Faculty-Graduate Student Research Collaboration Grant Program, the MORE Bridges to the PhD Program, and NIH (R15NS120157).
| Poster #: 49 Campus: California State Polytechnic University, Pomona Poster Category: Disease (Pathogens) Keywords: West Nile Virus, DEC-205 DCs, Neuroinvasive disease Project Title: DEC-205+ Dendritic Cells as Key Regulators of Neuroprotection in West Nile Neuroinvasive Disease Author List: Nadeem Halasah, Graduate, Biological Sciences, Presenting Author; Douglas Durrant, Biological Sciences, Pomona Abstract West Nile virus (WNV), a mosquito-borne virus endemic to the United States, can cause severe West Nile neuroinvasive disease (WNND), often resulting in meningitis, encephalitis, and substantial neuronal damage. Both virus-mediated and immune-mediated pathologies have been implicated as major contributors to neuronal loss during WNND, highlighting the necessity of a finely tuned immune response to clear the virus without exacerbating damage in the central nervous system (CNS). Dendritic cells (DCs), particularly those expressing the endocytic receptor DEC-205, have emerged as critical players in this balance. Our previous work established that DEC-205+ DCs localize to the CNS during WNND and remain at the blood-brain barrier (BBB) throughout the infection. While their presence has been associated with improved disease outcomes, the precise neuroprotective mechanisms remain elusive. To further investigate these mechanisms, we infected wild-type (WT) and DEC-205-deficient (DEC-205-/-) mice with WNV. Compared to WT, DEC-205-/- mice exhibited a striking increase in viral neuroinvasiveness and replication, particularly on days 6 and 9 post-infection (p.i.), as measured by standard plaque assay. Immune profiling via flow cytometry at these critical time points revealed heightened immune cell activation and increased trafficking into the CNS in DEC-205-/- mice, potentially driving immunopathology. Moreover, using a cytometric bead array (CBA) multiplex assay, we found elevated granzyme B (GZMB) and IFNg levels in the DEC-205-/- mice, indicating increased cytotoxic activity and inflammatory responses in the CNS. These findings strongly suggest that DEC-205 plays a pivotal role in restricting both viral entry and the infiltration of activated immune cells into the brain, thus mitigating damage. Our results establish DEC-205+ DCs as essential gatekeepers at the BBB, revealing a previously uncharacterized neuroprotective mechanism. These discoveries have broader implications for understanding neuroinvasive viral infections and could guide the development of targeted therapies to enhance neuroprotection during viral encephalitis.
| Poster #: 50 Campus: California State University, San Bernardino Poster Category: Disease (Pathogens) Keywords: Influenza virus, antiviral, peptide inhibition Project Title: Influenza NP peptide serves as antiviral in human lung cells. Author List: Caroline Vanegas, Undergraduate, Biology, Presenting Author; Cindy Ramirez , Graduate, Biology; Megan Hudson, Graduate, Biology, Presenting Author; Laura Newcomb, Biology, San Bernardino Abstract BACKGROUND: Despite yearly vaccination efforts, seasonal influenza remains a health concern. More alarming, spillover of highly pathogenic avian influenza (HPAI) H5N1 in mammals threatens emergence of novel H5N1 with pandemic potential. Antivirals are important to slow spread of novel viruses, but antiviral use selects resistance, demanding identification of targets to facilitate development of new antivirals. Influenza nucleoprotein (NP) is a conserved and attractive target. We previously identified amino acids within the body domain of NP essential for influenza RNA synthesis and interaction with polymerase basic 2 subunit (PB2). HYPOTHESIS: Disruption of NP-PB2 interaction is a viable target for development of new antivirals against influenza. METHODS: Here we engineered single-integration lentiviruses and transduced human embryonic lung cells (A549) to integrate DNA encoding NP peptide encompassing amino acids essential for PB2 interaction or control NPbd3 peptide, with 5 amino acid substitutions that eliminate PB2 interaction. Clonal populations were isolated, passaged, cryopreserved, and resuscitated as distinct cell lines. Cells were infected with influenza A virus (H3N2) at both low or high MOI and cytopathic effect (CPE) was observed post infection, comparing to uninfected control. RESULTS: Transduced cells express NP or NPbd3 peptides and remain healthy and viable through multiple passages. Infected cells expressing NP peptide exhibit less CPE than control, demonstrating NP peptide antiviral function. RNA was isolated and will be analyzed to quantitate viral mRNA and host antiviral mRNA expression. CONCLUSION: This research establishes disruption of NP-PB2 interaction as a viable target for development of needed new antivirals to protect against influenza.
| Poster #: 51 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Disease (Pathogens) Keywords: Microparticles, coagulation , neutrophils Project Title: Investigating the production of pro-coagulant extracellular vesicles following phagocytosis of Staphylococcus aureus by neutrophils Author List: Justin Grapentine, Undergraduate, Biological Sciences, Presenting Author; Benjamin Schiff, Undergraduate, Biological Sciences, Presenting Author; Mallary Greenlee-Wacker, Biological Sciences, San Luis Obispo Abstract Extracellular vesicles (EVs) are nano-sized particles that are released from the plasma membrane of cells and may play a role in sepsis. Sepsis is defined as a dysregulated immune response to infection, and increased coagulation is one of the major molecular hallmarks. We studied the relationship between EVs and coagulation using differential centrifugation to isolate EVs and thrombin generation (TG) to measure coagulation. Selecting S. aureus for its propensity to cause sepsis, our group demonstrated that EVs produced from human neutrophils following phagocytosis of S. aureus (SA-EVs) are pro-coagulant. We extended these findings to neutrophil-like, differentiated HL-60 cells, and showed that activity came from human EVs and not bacteria because depletion of human EVs with anti-CD66b-Dynabeads abolished TG. TG was dependent on coagulation Factor XII, suggesting the involvement of a negatively charged molecule on EVs as an initiator of the pathway. To investigate the involvement of negatively charged proteins, SA-EVs were incubated at 60˚C for 15 minutes. Compared to mock-treated SA-EVs, heat treatment partially diminished TG. Given the relationship between SA-EVs and coagulation, our second objective was to decrease EV production by interfering with biogenesis. Although other groups showed that neutral sphingomyelinase (nSmase) inhibitor GW4869 inhibits production of some EV subsets, we found that treatment of neutrophils and HL-60 cells with GW4869 failed to diminish EV production. As a means to gain insights into the factor initiating pro-coagulant activity and the pathway of EV biogenesis, we performed proteomics on spontaneously released EVs and SA-EVs. Compared to spontaneously released EVs, we found that 238 proteins were enriched in SA-EVs. Although no obvious candidates for pro-coagulant activity were found, several Annexin proteins were enriched. Since Annexins play an important role in the membrane-repair process, our future goal will be to investigate whether membrane repair processes contribute to EV biogenesis. This research was generously supported by start-up funds and the William and Linda Frost Fund in the Cal Poly Bailey College of Science and Mathematics.
| Poster #: 52 Campus: California State University, Fullerton Poster Category: Disease (Pathogens) Keywords: parasites, kinesin, flagellum Project Title: Establishing the function and localization of kinesin in the human parasite Trypanosoma cruzi Author List: Angelica Lopez, Graduate, Biological Science, Presenting Author; Akshara Kannan, Graduate, Biological Science; Abigail Kamal, Undergraduate, Biological Science; Veronica Jimenez , Biological Science, Fullerton Abstract The contractile vacuole complex (CVC) of Trypanosoma cruzi is a bipartite structure formed by the spongiome, a network of vesicles budding out of the Golgi complex, and a central bladder that regulates the volume of the cells. Water and osmolytes are collected in the bladder and expelled to the flagellar pocket by contact with the membrane via a transient pore. The filling state of the CVC is sensed by a mechanosensitive channel (TcMscS) required for normal CVC function. Beyond its osmoregulatory function, the CVC has newly revealed roles related to the trafficking of flagellar and surface proteins, but the mechanisms regulating protein distribution has not been characterized. We have found that mechano-deficient parasites lacking TcMcsS, have important defects in cytokinesis and flagellar formation. In these parasites, a novel kinesin (TcKLIF) is significantly downregulated. We sought to establish the expression and localization of this protein in the three life stages of T. cruzi and found a robust expression in the flagellar attachment zone, where the flagellum adheres to the body of the parasite. TcKLIF ablation by CRISPR-Cas9 targeting results in growth defects, abnormal kinetoplast/nuclei ratio and cell cycle dysregulation. TcKLIF-KO epimastigotes show “twirling” motility with decrease in progressive directional movement and bent flagellar structures that could explain the abnormal motility pattern. The KO parasites also show an important decrease in differentiation as well as infectivity. Our results indicate that TcKLIF plays an important role in maintaining flagellar structures and repositioning of organelles required for parasite division and life stage transition. Unraveling the role of kinesins in T. cruzi will provide valuable insights into how the parasite transitions into its infective form, ultimately contributing to our understanding of Chagas disease transmission.
| Poster #: 53 Campus: California State University, Long Beach Poster Category: Disease (Pathogens) Keywords: SARS-CoV-2, Antivirals, Viral Attachment Project Title: A SARS-CoV-2 Virus Like Particle (SC2-VLP)-Assay to Evaluate Heparan Sulfate-Like Compounds for Use as Broad-Spectrum Antivirals Author List: Julian Lopez, Graduate, Biological Sciences, Presenting Author; Peter Ramirez, Biological Sciences, Long Beach Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a zoonotic, enveloped, betacoronavirusthat was the causative agent of the worldwide COVID-19 pandemic. SARS-CoV-2 contains a positive sense single stranded RNA (+ssRNA) genome and shares approximately 80% genetic similarity to SARS-CoV. SARS-CoV-2 primarily infects epithelial cells of the upper and lower respiratory tract. While effective SARS-CoV-2 antivirals exist (Paxlovid, Remdesivir), none target the early stages of infection to limit SARS-CoV-2 transmission. Heparan Sulfate Proteoglycans (HSPGs) are widely expressed cell surface proteins that contain unbranched, negatively charged heparan sulfate (HS) polysaccharides. Diverse viruses including HIV-1 and SARS-CoV-2 use HS to attach to the cell surface, aiding viral entry into host cells. Sulfoglycodendrimers (SGDs) act as synthetic mimics of HSPGs that inhibit HIV-1 binding and entry. Whether SGDs also act to inhibit SARS-CoV-2 entry is unknown. Here, we adapted a SARS-CoV-2 Virus Like Particle (SC2-VLP) system to determine the effects of an SGD-like synthetic polysaccharide (dextran sulfate) on SARS-CoV-2 infectivity. SC2-VLPs are non-infectious and contain all the SARS-CoV-2 structural proteins (Spike, Envelope, Membrane, and Nucleocapsid) along with a luciferase transcript that is packaged within the virus particle. Thus, SC2-VLPs represent authentic aspects of SARS-CoV-2 entry, assembly, and release. We hypothesized that addition of dextran sulfate would reduce SC2-VLP infectivity. To test this, target cells expressing the SARS-CoV-2 receptors (ACE2/TMPRSS2) were or were not pre-incubated with varying concentrations of DS prior to infection with SC2-VLPs. We then measured luciferase activity as a readout for infectivity. Our results showed a significant decrease in infectivity with over 80% inhibition at sub-micromolar concentrations with a half-maximal inhibitory concentration (IC50) around 34nM. These results indicate that our SC2-VLP assay serves as a reliable model to study inhibition of SARS-CoV-2 attachment and entry and for our future directions of testing distinct SGDs as potential antiviral compounds.
This project is supported by the CSULB-CIRM Stem Cell Biotechnology Training Program and National Institute of Allergy and Infectious Disease award R16AI184450.
| Poster #: 54 Campus: California State University, San Marcos Poster Category: Disease (Pathogens) Keywords: virus, viral replication, gene function Project Title: Characterization of the Ranavirus ambystoma1 89R gene. Author List: Matthew Leslie, Undergraduate, Biological Sciences, Presenting Author; James Jancovich, Biological Sciences, San Marcos Abstract Ranaviruses (family Iridoviridae, genus Ranavirus) are large double stranded DNA viruses known to infect a diverse range of ecologically and economically important cold-blooded vertebrate organisms of freshwater environments, including various species of salamanders, fish, and amphibians. The ranavirus Ambystoma tigrinum virus (ATV), now referred to as Ranavirus ambystoma1, originally isolated from the tiger salamander (Ambystoma tigrinum), has a genome of 106,332 base pairs and contains approximately 92 open reading frames (ORF) each encoding for a protein. The function of approximately half of the ATV encoded ORFs have been determined through experimentation or inferred by sequence homology to other proteins with a known function. The goal of this project is to characterize the ATV 89R ORF, a gene that does not have any identifiable functional domains but is highly conserved across the genus Ranavirus. The first step in understanding ATV 89R’s role in viral replication is to determine if the gene is essential or non-essential. If the gene is essential, it cannot be deleted from the virus. However, if the gene is non-essential the gene can be deleted and a virus, ATVΔ89R, can be successfully generated. Therefore, a linear recombination cassette was constructed containing a screenable (green fluorescent protein – GFP) and a selectable (neomycin resistant gene – NR) marker that is flanked by 89R upstream and downstream homologous sequences. This linear recombination cassette was then transfected into fathead minnow cells infected with wtATV and the viral particles were harvested after 48-72 hr of incubation. The resulting preparation containing virus was used in a plaque assay with neomycin and any resulting plaques were screened for GFP expression and neomycin resistance. After multiple rounds of plaque purification our data suggest the 89R ORF is non-essential for viral replication in cells in culture and can be removed or deleted from the viral genome. To further characterize the function of the ATV 89R protein, a pipeline of in vitro assays designed to determine the gene expression profile, cellular localization, and impact on growth kinetics as well as in vivo assays to examine the role of 89R in viral pathogenesis will be performed. The results of this work will provide insight into how ATV 89R contributes to the viral replication cycle, pathogenesis and/or immune evasion that may result in developing methods of disease intervention for these important pathogens.
| Poster #: 55 Campus: California State University, Dominguez Hills Poster Category: Microbiology Keywords: Fungal Natural Products, Antibiotics, Drug-lead Discovery Project Title: Identifying Novel Fungal Metabolites with Antibiotic Activity Author List: Luis Lopez, Graduate, Biology, Presenting Author; Erin McCauley, Chemistry & Biochemistry, Dominguez Hills Abstract A major challenge facing the future of human health is the rise of antibiotic-resistant pathogenic bacteria. As modern-day antibiotics are becoming ineffective against nosocomial multi-drug resistant pathogens, the global mortality rate has escalated to critical levels and solving this problem is an urgent issue. Therefore, researchers are focusing on finding novel chemical scaffolds that can be used to develop new classes of antibiotics that can circumvent current antimicrobial resistance genes. This research seeks to identify natural products with novel chemical scaffolds that can be used to drive forward antibiotic drug-discovery research. Natural products are secondary metabolites from living organisms, they have played a role in traditional medicine for thousands of years and are an essential part of the current therapeutic arsenal for modern medicine. The objective of this research will be to identify novel chemical scaffolds from marine-derived fungi that exhibit antibiotic activity. To achieve this, we will explore the chemical diversity from taxonomically unique marine-derived fungal strains cultivated from Indonesian marine sponges and screen them in an antibiotic assay against a BSL1 strain of bacteria that is taxonomically similar to the pathogen P. aeruginosa. This research was initiated by growing 50 taxonomically distinct fungal strains on rice media and extracting the metabolites they produced. These complex extracts were simplified into “Peak Library Fractions” (PLFs) using a reverse phase C18 flash chromatography system. The PLFs were screened for antibiotic activity against P. putida and analyzed using a liquid chromatography - high accuracy mass spectrometer (HAMS). PLFs that exhibit activity against the P. putida and that did not correspond to any known antibiotic natural products based on HAMS analysis were prioritized for further study. The PLFs of interest were purified to 99% purity using preparative high-performance liquid chromatography, and their structures were determined using 1D & 2D nuclear magnetic resonance along with HAMS. This research will aid in the field of antibiotic drug-lead research by adding possible leads to combating the P. putida’s taxonomical relative P. aeruginosa, a World Health Bacterial Priority Pathogen.
This research is supported by NIH (SC2GM144172).
| Poster #: 56 Campus: San Francisco State University Poster Category: Microbiology Keywords: antibiotics resistance, transporter, bacteria Project Title: A conserved but atypical ABC transporter in the alpha-proteobacterium Sinorhizobium meliloti confers resistance to a range of antimicrobials Author List: Klara Christensen, Undergraduate, Biology, Presenting Author; Malea Tuimavave, Undergraduate, Biology, Presenting Author; Joseph Chen, Biology, San Francisco Abstract Sinorhizobium meliloti is a Gram-negative bacterium known for its ability to form endosymbiosis with compatible legume plants: it induces the formation of root nodules, colonizes them, and fixes nitrogen for the host in exchange for carbon compounds. This mutualistic relationship serves as a model for investigating molecular mechanisms that underpin microbe-host interactions, including how related pathogens bypass host defenses. We recently discovered that loss-of-function mutations in an S. meliloti gene encoding an ABC transporter increased sensitivity to a range of antimicrobials, including beta-lactams, chloramphenicol, and sodium dodecyl sulfate (SDS). Resistance can be restored in these mutants by introducing an ortholog from S. medicae. This gene encodes both the nucleotide binding (NBD) and transmembrane domains (TMD) associated with an ABC transporter, as well as a third unknown domain. To test if the transporter activity encoded by the gene is necessary for antimicrobial resistance, we introduced three different point mutations into the S. medicae ortholog, made in specific conserved residues of the NBD necessary for function in other ABC transporters. Surprisingly, these mutant orthologs were still able to complement the deletion in S. meliloti. We also assessed if three other genes near the ABC transporter are involved in resistance to the same antimicrobials. Deletions of these genes, which encode proteins involved in polysaccharide synthesis, led to the same phenotypes as the deletion of the ABC transporter gene. The mechanism by which these genes confer resistance to antimicrobials is under active investigation. Elucidating this mechanism can further our understanding of antibiotic resistance in alpha-proteobacteria, as well as how soil microbes compete against one another via chemical assaults.
| Poster #: 57 Campus: California State University, Northridge Poster Category: Microbiology Keywords: LPS biosynthesis, multidrug efflux pumps, gene regulation Project Title: Role of lipopolysaccharide biosynthesis on regulation of the multidrug efflux pump AcrAB-TolC of Escherichia coli Author List: Rafael Estrada, Undergraduate, Biology, Presenting Author; Cristian Ruiz Rueda, Biology, Northridge Abstract Gram-negative bacteria are resistant to antibiotics are a major threat to public health. Multidrug efflux pumps are among the major mechanisms for antibiotic resistance in these bacteria. This project focuses on AcrAB-TolC of Escherichia coli, which is the main multidrug efflux pump of this bacterium. The AcrAB-TolC pump expels toxic substances from the cell such as a broad range of antibiotics, dyes, bile salts, and cellular metabolites. Previous studies have shown that several transcriptional regulators control the expression of the AcrAB-TolC pump in response to toxic chemicals and several physiological functions. Regarding the known regulators, we recently discovered that AcrR (a transcriptional repressor of acrAB operon) regulates physiological functions such as motility and metabolism. Our group has hypothesized that AcrR can respond to metabolic and stress signals to modulate the expression of efflux, motility, and other mechanisms associated with antibiotic resistance, stress responses and metabolic balance. Specifically, our prior findings have shown that three cellular metabolites (polyamines) act as ligands that control the function of AcrR and the expression of the acrAB operon as a result. However, the regulation of AcrR in response to other metabolites and cellular functions remains unknown. To close this gap in knowledge, our goal is to identify novel genes, pathways, and functions that control acrAB expression, mainly via AcrR, by using random transposon mutagenesis. Mutagenesis experiments were performed using an E. coli strain carrying an acrAB promoter-lacZ fusion to identify functions and metabolic pathways whose inactivation up-regulates the expression of the AcrAB-TolC pump and thus produce blue colonies in the presence of X-gal. Using this approach, we have identified 40 mutants that caused an up-regulation of acrAB expression from a screen of 27,000 mutants. Among these 40 mutants, we identified eleven novel genes and three known genes whose inactivation activates the acrAB promoter. Three of these mutants were involved in the Lipopolysaccharides (LPS) biosynthesis pathway and were studied in further detail to confirm their role in controlling acrAB expression. Overall, these findings suggest a co-regulation between LPS/outer membrane biosynthesis and multidrug efflux in Gram-negative bacteria that may enhance their survival in response to antibiotics and other stresses.
| Poster #: 58 Campus: California State University, Los Angeles Poster Category: Microbiology Keywords: circadian rhythms, fluorescence microscopy, RNA binding proteins Project Title: Investigating the role for Rbp2 in the regulation of the circadian clock in cyanobacteria Author List: Parker Saikley, Undergraduate, Biological Sciences, Presenting Author; Nidhi Alle, Undergraduate, Chemistry and Biochemistry, Presenting Author; Susan Cohen, Biological Sciences, Los Angeles Abstract Circadian rhythms are biological processes that follow an approximately 24-hour cycle, governing a wide range of physiological functions. In cyanobacteria, where Synechococcus elongatus is the primary model system, these rhythms are regulated by a core oscillator comprised of KaiA, KaiB, and KaiC proteins. This oscillator drives global gene expression patterns, regulates cell division, and facilitates natural transformation. Using functional fluorescent proteins it was found that during the day, KaiA and KaiC proteins are diffused throughout the cell, while at night, KaiC localizes to the cell pole. Recent studies have revealed that the RNA-binding protein Rbp2 plays a critical role in the regulation of this system by associating with KaiC. Rbp2's RNA-binding activity is essential for proper clock function. Deletion of rbp2 leads to a longer circadian period and disrupts the nighttime localization of KaiC at the cell pole, highlighting its importance in maintaining proper circadian rhythms. KaiC and Rbp2 have been shown to associate maximally via co-immunoprecipitation (Co-IP) at dusk, whereas Rbp2 co-localizes with KaiC at the poles of cells later in the night. Here we investigate the KaiC-Rbp2 association using Co-IP and fluorescence microscopy. Specifically, how RNA(s) influence the interaction between Rbp2 and KaiC and what other factors might be involved in complex formation. Using mutant variants of Rbp2 that disrupt its RNA binding activity, Rbp2-Y4A, and Rbp2-R42AF44AF46A, we find that Rbp2-Y4A and KaiC still associate at dusk; however, Rbp2-R42AF44AF46A localization to the cell poles is severely reduced. Moreover, treating our samples with varying concentrations of RNaseA found that the removal of RNA during the Co-IP process improved association. Rpb2 and KaiC do not interact in vitro, suggesting other components are required to mediate their interaction in vivo. We have determined that circadian input protein CikA is not required to mediate the association between KaiC and Rbp2 at dusk and that KaiC and Rbp2 each influence each other's localization to the cell poles at night. Taken together our data have demonstrated that RNA is not needed to mediate the initial association between KaiC and Rbp2 at dusk but is required for complex formation at the poles of cells later in the nighttime.
This project is funded by NSF CAREER award (MCB 1845953).
| Poster #: 59 Campus: California State University, San Marcos Poster Category: Microbiology Keywords: Adopt-a-genome, JGI, microbial Project Title: The JGI-CSU “Adopt a Genome” Project: Microbial Genome Exploration and Publication for Undergraduates Author List: Keely Berner, Undergraduate, Biological Sciences, Presenting Author; Michelle Zoza Veloz, Undergraduate, Biological Sciences; Rekha Seshadri, Department of Energy Joint Genome Institute; Matthew Escobar, Biological Sciences, San Marcos Abstract In 2023, the Department of Energy’s Joint Genome Institute (JGI) and several CSUBIOTECH faculty established an informal collaborative network called the “Adopt a Genome” project. The project builds upon the thousands of microbial genomes that have been sequenced by JGI, many of which have never been formally analyzed or published. The Adopt a Genome project allows CSU faculty to “claim” JGI’s unpublished microbial genomes, with the expectation that faculty will work with CSU undergraduates in the analysis and publication of the claimed genome sequences. There are currently ~1900 microbial genomes available to the project, and ~120 have been claimed by nine CSU faculty. To date, three peer-reviewed publications in Microbiology Resource Announcements have been produced (with publication fees paid by JGI), involving three faculty authors and seven undergraduate student authors. Access to the microbial genomes and associated curricular resources/tutorials is freely available, and any interested CSU faculty are encouraged to join the project by contacting Dr. Matthew Escobar at CSU San Marcos (mescobar@csusm.edu) and Dr. Rekha Seshadri at JGI (rseshadri@lbl.gov). | Poster #: 60 Campus: San José State University Poster Category: Microbiology Keywords: CRISPR/Cas9, Oral Microbiome, 16S rDNA gene Project Title: Optimizing CRISPR/Cas9 to Enrich for Rare and Novel Bacteria in The Human Oral Microbiome Author List: Adrian Ordonez, Undergraduate, Biological Sciences, Presenting Author; Jimmy Cruz, Undergraduate, Biological Sciences, Presenting Author; Marianna Velasquez, Undergraduate, Justice Studies; Cleber Ouverney, Biological Sciences, San José Abstract With over 700 species of bacteria, the human oral microbiome serves as the portal entrance for many microbes into the human digestive tract and it plays a crucial role in both health and disease. Despite advances in high-throughput sequencing, challenges remain to fully characterize the complexity of this microbial community, in part due to the overwhelming abundance of six prevalent bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria, Fusobacteria, Spirochaetes, Bacteroidetes) which account for nearly 96% of the total sequences and hinder the detectability of rare DNA templates. Recently, we proposed a modification of the standard metagenomics approach which relies on the CRISPR/Cas9 system to enrich rare bacteria, by digesting the DNA of the most common lineages. That protocol, however, faced challenges, including the presence of the remaining small pieces of DNA in the sequencing reaction, and here we propose an improved method. We hypothesize that by diminishing the most abundant 16S rRNA genes in a sample before PCR amplification, we can enrich for rare bacteria bringing them to detectable levels. Our novel approach was initially validated using pure cultures of known bacteria digested using CRISPR rRNA guides (sgRNA) to target those templates respectively. We then treated genomic DNA purified from human oral plaque with our Cas9-sgRNA cocktail. The remaining DNA was PCR amplified using barcoded primers targeting the full length of the 16S gene. The PCR amplicon was cleaned using AMPure XP magnetic beads at a ratio that filtered for the full-length gene while discarding the smaller fragments. The final DNA was quantified using a Bioanalyzer before pooling samples and sequencing using Oxford Nanopore. Preliminary results showed a decrease in the abundant bacterial phyla as compared to the untreated control. The proportion of target sequences that were eliminated from the sample ranged from 99% for Actinobacteria to 9.8% for Bacteroidetes. The experiment was replicated with similar outcomes. Furthermore, lesser-known bacterial phyla were revealed only in Cas9-treated samples including Campylobacter, Agrobacterium, and Parvimonas associated with immune deficiencies and chronic renal insufficiency. CRISPR-Cas9 enrichment of the human oral microbiome may provide an alternative technological advancement to increase the detectability of bacterial phyla that were previously unaccounted for.
| Poster #: 61 Campus: San José State University Poster Category: Microbiology Keywords: E. coli bacteriophage, Host range specificity, Genomic sequencing Project Title: Characterization and sequencing of the novel bacteriophage “Halophage” to determine therapeutic potential Author List: Azriel Montalvo, Graduate, Biological Sciences, Presenting Author; Marina Fiaz, Undergraduate, Biological Sciences, Presenting Author; Akiko Balitactac, Graduate, Biological Sciences; Vashaki Lohadas, Undergraduate, Biological Sciences; Ervin Bose, Undergraduate, Biological Sciences; Edward Hayek, Undergraduate, Biological Sciences; Karen Cao, Undergraduate, Biological Sciences; Wendy Lee, Computer Science; Robert Fowler, Biological Sciences; Steven White, Biological Sciences; Sonia Singhal, Biological Sciences, San José Abstract As the number of bacteria developing antibiotic resistance increases, it is becoming difficult to treat what were once routine infections. Bacteriophages, or phages, viruses that only infect bacteria, can provide an alternative treatment solution. It is currently estimated that there are 1031 bacteriophage particles in the world; however, relatively few have been studied. We isolated a novel phage, named “Halophage,” from the Santa Clara Valley Waste Treatment plant. Our research objective was to characterize the phage’s host range, infection dynamics, and genome sequence to determine its viability for phage therapy.
We assayed the host range of the phage to determine what bacteria it infects. It was found to have a narrow host range for the bacteria tested, only infecting Escherichia coli B. The phage causes lysis from without on E. coli K (high concentrations of phages burst the bacterial cell without infecting). We also characterized the phage’s infection dynamics on E. coli B, including the time it takes to attach to the host (attachment time), replicate within and burst the host (burst time), and the number of new phages produced (burst size). Halophage had a short attachment time of 40-120 seconds, a burst time of 24 minutes and 35 seconds, and a burst size of approximately 134.7 viral particles per infection. Finally, the phage was sequenced with Nanopore and Illumina technology. The Halophage genome contained 39,721 base pairs and had a GC content of 48.47%. The genome had 62 open reading frames, and a 93.80% similarity to the laboratory phage T7.
The rapid attachment, replication, and narrow host range of Halophage suggest that this phage could be effective in treatment of diseases caused by E. coli B. We plan to extend our study to determine whether Halophage can infect other enteric bacteria of clinical relevance, such as Yersinia pestis and Salmonella species, and to identify the host receptors the phage uses for attachment.
| Poster #: 62 Campus: California State University, Fresno Poster Category: Microbiology Keywords: IS elements, antibiotic resistance evolution, Acinetobacter baumannii Project Title: Genomic analysis of IS elements in Acinetobacter baumannii antibiotic resistance Author List: Ramon Lomeli, Graduate, Biology, Presenting Author; Francine Arroyo, Biology, Fresno Abstract Multidrug resistant bacterial infections pose a significant challenge to human health, affecting millions globally and compromising the efficacy of once-reliable antimicrobial therapies. The emergence of resistant Acinetobacter spp. strains necessitates the further understanding of the underlying genetic mechanisms that confer antibiotic resistance. Insertion sequence elements (IS elements) are transposable segments of DNA that possess the ability to move within and between genomes. IS elements play a crucial role in shaping the genomic landscape, increasing genetic diversity, as well as influencing the evolutionary trajectory of various organisms. Despite this, the contribution of IS elements on A. baumannii resistance evolution are understudied.
The purpose of this study was to characterize how IS elements impacted antibiotic resistance in A. baumannii under different bacterial lifestyles. A. baumannii is a hospital-acquired pathogen that often attaches to medical devices as a biofilm. We evolved A. baumannii ATCC17978 against ciprofloxacin and ceftazidime for 15 days under biofilm and planktonic conditions. We sequenced whole-populations and identified mutations that resulted in antibiotic resistance. We found IS element insertions in all treatment groups. Two major pathways for resistance emerged and both related to a multidrug efflux pump AdeIJK: efflux pump negative regulator AdeN and phospholipid-related PgpB. Next, we characterized IS element diversity and sequence target motifs.
A total of 17 different IS elements were identified. Fifteen IS elements were associated with adeN and 2 with pgpB. The most frequent IS element family mutation type was ISAba1 and was found in both gene targets (adeN and pgpB) and both lifestyles. However, the biofilm lifestyle resulted in a larger diversity of IS family types including ISAba11, IS701, and ISAba12. To determine if target location was due to a common motif indicative of a hot spot or random, a 30-nucleotide window flanking the IS element insertion site was compared for all IS types. No clear pattern was evident of a hotspot between pgpB and adeN. All 15 adeN sequences were rich in Adenine and Thymine suggesting a preference for AT rich sites for insertion. Overall, we were able to show that IS elements are a significant mutation type affecting antibiotic resistance. The impact of biofilm lifestyles on selecting for a diverse range of IS family types is worth further study.
| Poster #: 63 Campus: California State Polytechnic University, Pomona Poster Category: Microbiology Keywords: Project Title: The Role of Sex and Mouse Strain in a Systemic C. albicans Infection Author List: Edgar Perez Valdes, Graduate, Biological Sciences, Presenting Author; Alexander Royas, Graduate, Biological Sciences; Nancy Buckley, Biological Sciences, Pomona Abstract The role of sex in immunological defenses against bacterial and viral infections are better understood than the defense against yeast infections such as those caused by Candida albicans (C. albicans). C. albicans is an opportunistic pathogen which poses varying risks depending on the individual's immune status. Our lab has found that, in mice from a C57BL/6 background, males with a systemic C. albicans infection are more susceptible to the infection than infected females, as evidenced by survival rates and weight loss. The objective of this project was to determine whether sex played a role on the susceptibility to the infection in a different mouse strain. Thus, Swiss Webster (SW) male and female mice, along with mice on a C57BL/6 background were infected with C. albicans (7.5x105 yeast cells/20g mouse). Survival rates, weight changes, tissue fungal load, and serum IL-6 levels were assessed. We found that SW mice were more susceptible to the yeast infection compared to mice with a C57BL/6 background as assessed by greater disease symptoms. In both mouse strains, males were more susceptible to the infection compared to female mice as assessed by survival, weight loss, disease scores and blood IL-6 levels, but not tissue fungal load. These data suggest that SW mice have a higher susceptibility to systemic C. albicans infections than mice on a C57BL/6 background. The link between IL-6 levels and bone marrow neutrophil egression and neutrophil maturation stages is forthcoming. In addition, our data supports our previous findings where males are more susceptible to females to a C. albicans infection regardless of strain.
| Poster #: 64 Campus: California State University, East Bay Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, Decision making, Neurons and circuits Project Title: Mommy Knows Best: Genetic and Circuit basis of female Drosophila decision making Author List: Yi Shen Lee, Graduate, Biology, Presenting Author; Namrata Dhungana, Graduate, Biology, Presenting Author; Divya Sitaraman, Psychology, East Bay Abstract Decision making processes dictate the lives of every living organism. The core of decision making is to select the best choice considering multiple factors including preference and consequences. Even the simplest organisms make decisions in their natural environment and studying these in animals with reduced complexity and increased accessibility can provide critical insights into how the nervous system underlies this process. Here, we propose to use the fruit fly Drosophila melanogaster, as a system to understand the neural circuit mechanisms underlying decision making by studying the effects of neuronal and genetic manipulation on egg laying site selection or oviposition. Preliminary experiments show that female flies prefer to lay eggs on protein-rich non-fermenting yeast and avoid laying eggs on pure sugar substrates that attract predators. However, in their natural environment, flies do not encounter pure sugar or yeast and must sample combinations.
As a first step in characterizing the decision making process in egg laying preference behavior, we presented female flies with different substrates with various concentrations of yeast protein and sugar. We found that although yeast and sugar are favored nutritional sources, the female flies avoid laying eggs on sugar-yeast combinations which shows their aversion towards sugar over preference towards yeast.
To investigate the circuit basis of decision making we systematically inhibited input, outputs and core processing regions of the mushroom body, a structure implicated in associative learning and other decision making tasks and assayed oviposition preferences. We also screened the role of sugar and amino acid taste receptors in this choice behavior and found that Gr6f, 6e/d play a critical role in oviposition preference. Further, several Mushroom body output neurons (MBON1, MBON4 and MBON 9) are involved in decision making and relay these signals to oviposition command neurons (specifically pCd and OviN). Taken together, we have developed a novel decision making task and mapped sensory, central processes and premotor circuits underlying these processes. We will present these data and additional hypotheses related to how sensory processes change as a function of mating and influence egg laying decision making.
| Poster #: 65 Campus: San Diego State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: germ cell, developmental biology, toxicology Project Title: Preservers of reproductive integrity: ABC transporters and their potential roles in the human germline Author List: Timothaus Haddad, Graduate, Biology, Presenting Author; Priscilla Plascencia, Graduate, Biology; Courtney Anderson, Graduate, Biology; Catherine Schrankel, Biology, San Diego Abstract Cellular defense systems are fundamental to life and dictate survival in an increasingly polluted world. The germline of an organism is the most important cell type to protect, as it creates the next generation. The germline is first defined at the primordial germ cell (PGC) stage: the embryonic stem cells to future eggs and sperm. In humans, PGC specification occurs weeks before the formation of a protective placenta. However, little is known about the protection of PGCs from outside toxicants during their inception and early development. Pivotal to the front lines of well-known cellular defenses are transporters of the ATP-binding cassette (ABC) transporter superfamily. The primary conserved function of the ABCB, -C, and -G subfamily transporters is to export xenobiotics, metabolic byproducts, and other small molecules out of the cell. In addition, the C-subfamily of ABCs export signaling molecules that are essential for the growth and proliferation of stem cells. Given the duality of transporter functions, we sought to identify which ABCs were utilized in human PGCs, using a cell culturing approach for inducing human PGC-like cells (hPGCLCs) from embryonic stem cells (hESCs) in-vitro. We first identified novel localization of ABCG2, -C4, and -C1 proteins in hPGCLC membranes, with ABCC1 being the most enriched compared to hESCs. Next, pharmacological inhibition of ABCC1 during PGC derivation resulted in 50% less hPGCLC induction compared to DMSO vehicle controls. Furthermore, the addition of high concentrations of the toxicant DMSO during the hPGCLC derivation protocol resulted in less than 10% induction of PGCs compared to control, but without significant cell death. Together these findings suggest that ABCC1 plays a developmental role in PGCs, likely for proliferation or growth, and that PGC induction is not efficient under a certain threshold of stress or toxicity. Future work will improve our understanding of how organisms use ABC transporters to initiate and/or protect their reproductive capability at the earliest developmental stages possible. This could help us mitigate the impact of early life exposures to toxicants and pollutants on human fertility. *This work was in part funded by a 2024 CSUBIOTECH Faculty-Graduate Student Research Collaboration Grant.
| Poster #: 66 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Escherichia coli, antibiotic resistance, riboregulator Project Title: Comparing the Sensitivity of RNA Detection Tools Author List: Bao Ngoc Nguyen, Undergraduate, Biology, Presenting Author; Mary Geuvjehizian, Undergraduate, Biology, Presenting Author; Melissa K Takahashi, Biology, Northridge Abstract The rising issue of antibiotic resistance is prevalent within clinical settings. Some antibiotics infiltrate cells by entering through outer membrane proteins, which facilitate the uptake of nutrients and small molecules. To mitigate the danger of antibiotics, bacteria utilize small RNAs (sRNAs) to repress outer membrane proteins and block the entry of antibiotics into the cell. The focus of this study is examining Escherichia coli and two of its sRNAs, RybB and MicF. As a first-response mechanism to stress conditions, they play a crucial role in antibiotic resistance. Consequently, detecting when and how these sRNAs are made can enhance our understanding of resistance mechanisms, and how they are influenced. This project aims to develop sensitive and specific RNA detection tools that can identify low concentrations of sRNAs. We have examined two methods of RNA detection: RNA toehold switches and three way junction repressors (3WJ). Both toehold switches and 3WJs are riboregulators that use specific RNA structures to control protein expression upon binding of a specific RNA sequence. When the sRNA binds to the toehold switch or 3WJ, green fluorescent protein (GFP) expression is changed, which provides a method of detecting the sRNA. We set out to design both types of riboregulators for RybB and MicF to compare the sensitivity of the tools. Thus far, we have designed 3WJs for MicF and RybB, as well as a toehold switch for MicF. We performed a quantitative analysis of the detection of sRNAs by testing the two riboregulators with varying concentrations of RybB and MicF to determine their sensitivity. Plasmids encoding the sRNAs and riboregulators are then inserted and expressed in E. coli. The performance of each riboregulator is assessed by measuring optical density and GFP fluorescence of the cells. Identifying the most sensitive riboregulators will bring us closer to the overall goal of developing tools to monitor sRNA production. This will then allow us to begin investigating the environmental triggers of RybB and MicF and their impact on the mechanisms of antibiotic resistance.
| Poster #: 67 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Quantitative Polyermase Chain Reaction, non-model species, gene expression Project Title: Optimized method for differential gene expression analysis in the non-model plant species Leptosiphon parviflorus Author List: Weston Gonor, Graduate, Biological Sciences, Presenting Author; Ed Himelblau, Biological Sciences, San Luis Obispo Abstract Next-generation sequencing methods allow for the efficient and accurate analysis of differential gene expression, however are often limited to organisms with high-quality reference genome sequences available. These methods are often not plausible for pilot studies of non-model organisms with limited resources and no available genome. The aim of this study was to develop a bioinformatic and molecular biology pipeline for analysis of differential gene expression in non-model plants. In the context of this research, the analysis was completed on the California native plant Leptosiphon parviflorus (common name: Variable Linanthus), in which field observations have shown a difference in floral pigmentation across various environmental stressors. Gene expression analysis was achieved through the utilization of various genomic databases such as PLAZA, and NCBI BLAST to identify genomic and amino acid candidate gene sequences within closely and distantly related species. Sequence alignment tools were then used to locate highly conserved exonic regions of candidate genes and the primer3 program allowed for the efficient design of Polymerase Chain Reaction (PCR) primers specific to the regions with the highest conservation between both genomic and amino acid sequences. Subsequently, quantitative PCR was performed using high quality cDNA and amplification of candidate genes was confirmed. Results currently show that housekeeping genes ACT1 and CAB1 are expressed at the same levels throughout leaf and flower tissues of all Leptosiphon samples. The success of this pilot has allowed for the continued analysis of gene expression and molecular ecology within Leptosiphon parviflorus, and provides an easy to follow guideline for future studies of organisms with a similar lack of genomic resources.
| Poster #: 68 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Gibson Assembly, Course-Based Undergraduate Research Experience, Molecular Cloning Project Title: Improving Student Outcomes with an Adaptable Molecular Cloning Course-Based Undergraduate Research Experience Author List: Samuel Catania, Undergraduate, Presenting Author; Christopher Cummings , Undergraduate; Eirene Ednacot, Undergraduate; A.J. Kinsella-Johnson, Undergraduate; Claire Meeds, Undergraduate; Jack Reynolds, Undergraduate; Ava Sanderson, Undergraduate; Rachel Johnson; Katharine Watts, Chemistry and Biochemistry, San Luis Obispo Abstract The continuous advancement of molecular biology techniques requires that molecular biology curricula are regularly refined to effectively prepare students to enter the workforce with modern competencies. In particular, the use of Gibson Assembly has become more predominant in the scientific community due to being highly customizable as well as its ability to easily ligate multiple linear DNA fragments into one recombinant plasmid. Thus, we created a Gibson Assembly cloning module for deployment in a molecular biology laboratory course at California Polytechnic State University, San Luis Obispo, and evaluated student learning outcomes. This module was completed over three weeks, spanning six three-hour lab periods and two fifty-minute lecture periods. In addition to Gibson Assembly, the independent project gave students experience with other common molecular biology techniques including polymerase chain reaction (PCR), DpnI restriction digest, DNA transformation, DNA isolation, agarose gel electrophoresis, and PCR screening. Over three iterations of the course, students participated in an experiment-based independent project that involved cloning three unique plasmid libraries to support research projects in natural product biosynthesis. Students were given pre and post questionnaires to evaluate their understanding of molecular cloning and their confidence in molecular biology terms and techniques. Two tailed paired t-tests revealed that students’ responses showed a significant increase in both learning molecular cloning concepts and in self-reported confidence with molecular cloning terms and techniques. Cohen’s d test indicated that specialized Gibson Assembly questions showed significant increases with the largest effect sizes. After participation in the independent project, students reported being significantly more likely to pursue a career using these molecular cloning techniques. This module framework can be generalized to teach Gibson Assembly for various applications, providing instructors with a toolkit for teaching an adaptable and emergent cloning technology while advancing their research projects.
| Poster #: 69 Campus: California State University, Channel Islands Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: behavior, genetics, mechanisms Project Title: Deciphering the molecular mechanisms and sensory cues that mediate multisensory behavior Author List: Lily Yered, Undergraduate, Biology, Presenting Author; Julia Moffa, Undergraduate, Health Sciences, Presenting Author; Gareth Harris, Biology, Channel Islands Abstract An environment is often represented by numerous sensory cues. In order to better survive, an animal often needs to detect and process simultaneously present sensory cues to make a behavioral decision. Integrating multiple sensory cues generates a more accurate evaluation of the environment and provides important adaptive values. Intriguingly, humans have several neurological diseases that are linked to sensory processing or decision-making including, autism spectrum disorder, Parkinson’s disease, bipolar disorder, depression and schizophrenia. These diseases can involve differences in encountering multiple sensory stimuli that evoke certain behavioral choices under normal conditions. These studies reveal multi-sensory integration as a common neuronal and behavioral process. The importance of these studies will help understand the underlying mechanisms of disease in humans. Using genetic mutants, microscopy and behavior, we investigate the behavioral differences across, 1) spontaneous food leaving, and, 2) an escape behavior known as 2-nonanone-dependent food leaving. Specifically, using genetic molecular mutants and various strains of nematodes that have originated from distinct geographical locations. We have also begun to dissect the components that make up food signals, and ask how this factors into our multisensory behavior. We use a behavioral paradigm to address these questions to determine how these different worm strains behave. We have found a number of molecules expressed in the nervous system, neurons that are involved and that control these behaviors and identified differences in behaviors across distinct nematodes. We hope to provide insight into how different organisms may behave in different conditions during exposure to conflicting attractive food sources and adverse environmental conditions.
| Poster #: 70 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Lipotoxicity, Cell Stress, Gene expression Project Title: SUGAR, FAT AND THE IMPLICATIONS OF GLUCOLIPOTOXICITY IN PANCREATIC BETA CELLS Author List: Alejandro Marquez , Graduate, Biology, Presenting Author; David Keller, Biology, Chico Abstract Glucolipotoxicity in pancreatic beta cells is a leading theory for type 2 diabetes. This overnutrition causes cascading effects from fatty acids and glucose. Eventually these cells exhibit increased metabolic stress, radical oxygen species production, endoplasmic reticulum (ER) stress, and eventually apoptosis. Beta cells manage their fatty acids by balancing the rates of storage and breakdown in subcellular structures called lipid droplets (LDs) which are derived from the ER. Recently, studies on LDs in beta cells have yielded conflicting results with regard to their contribution to pathology. We believe LDs can mitigate the effects of glucolipotoxicity by storing excess lipids, however, under severe conditions we predict that ER stress will increase and discontinue the production of LDs. Do cells increase the number and size of LDs in response to elevated fatty acids? Would ER stress from glucolipotoxicity cause lipid droplet accumulation and size to increase? Do LDs have protective or negative effects when glucolipotoxicity is involved? The cell line I will be conducting my research with is INS-1 and I have shown through an MTS assay that prolonged exposure and increasing amounts of glucolipotoxicity via elevated levels of glucose and palmitate cause cell death. We visualized fatty acids and triglycerides within treated cells at varying degrees of glucolipotoxicity and exposure using Nile Red staining and confocal microscopy. Utilizing qPCR we found that cells exposed to glucolipotoxicity in greater degrees for 48 hours saw a decrease in CHOP, a gene that is expressed during ER stress, when compared to the control. Observing expression of PLIN2, a gene associated with LD function, in the same conditions showed that PLIN2 increased compared to the control for all glucolipotoxicity conditions. Our preliminary conclusion is that glucolipotoxicity does not cause ER stress mediated apoptosis and that glucolipotoxicity is associated with an increase in LD accumulation. To continue we will be visualizing LDs with Nile Red and PLIN2 antibody to confirm the presence of LDs. This will be used to test varying degrees of glucolipotoxicity and exposure to quantify the characteristics of LDs. qPCR will be utilized to measure genes associated with LD triglyceride storage and ER stress while under the same conditions. My research will shed a light on pancreatic beta cell physiology within glucolipotoxicity and build a greater understanding towards type 2 diabetes.
| Poster #: 71 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: muscle spindle afferent, proprioception, sensory physiology Project Title: Muscle Spindle Afferent Responses Become More Slowly Adapting During Early Postnatal Development Author List: Thao N. Pham, Graduate, Biological Sciences, Presenting Author; Mayra Cardenas Rojo, Graduate, Biological Sciences, Presenting Author; Katherine Wilkinson, Biological Sciences, San José Abstract Proprioception is the body’s ability to sense its position in space and is necessary for completing complex motor tasks. Muscle spindle afferents (MSAs) report muscle length and movement information and are the most important sensory input for proprioception. Prior studies have found that the anatomy and gene expression of MSAs change during early postnatal development. We hypothesized that the MSA response to stretch also changes by becoming more slowly adapting over development due to increased glutamate neurotransmission. We measured MSA stretch response in two groups of mice: 7-11 days old and 17-21 days old. The extensor digitorum longus muscle was isolated with the sciatic nerve and placed in an ex vivo bath with oxygenated synthetic interstitial fluid. An extracellular electrode was used to record stretch sensitive neural signals. The muscle was stretched to three physiological lengths and firing rate during rest and at the end of stretch was calculated. Then Xanthurenic acid (XA), a vesicular glutamate transport 1 (VGLUT1) inhibitor, was introduced into the bath to block glutamate packaging into vesicles and therefore release. Stretch responses were recorded for another 60 minutes. A hallmark of a more rapidly adapting sensory neuron is an inability to maintain firing at resting length (resting discharge, or RD). Our results show that 16% of afferents from the p7-11 age group (n=6) exhibited RD, while 44% in the p17-21 age group (n=9) had RD. Previous work in adults shows 50-75% of afferents exhibit RD. XA addition resulted in 60% of p7-11 days old mice (n=5) and 75% of p17-21 days old mice (n=4) decreasing firing especially near the end of stretch. This is similar to what we previously saw in our adult animals and suggests that glutamate neurotransmission is similarly important earlier in development for the maintenance of MSA firing during stretch. Our preliminary sample supports the hypothesis that MSAs are more rapidly adapting during the early postnatal days (p7-11). However, the switch to more slowly adapting responses is unlikely due solely to changes in glutamate neurotransmission. Future studies will look at how other proteins and neurotransmitters change during development to identify potential mechanisms for the maturation of the MSA stretch response.
| Poster #: 72 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: small RNA, antibiotic resistance, MicF Project Title: The Application of a Synthetic Gene Circuit to Study the Role of sRNA MicF in Antibiotic Resistance Author List: Christian Romero, Graduate, Biology, Presenting Author; Jezriel Punzalan, Undergraduate, Biology, Presenting Author; Melissa K Takahashi, Biology, Northridge Abstract Antibiotic resistance is a public health concern as it limits available treatment options for bacterial infection. Bacteria such as Escherichia coli utilize outer membrane proteins to transport small molecules across the outer membrane, which antibiotics use to enter the cell. OmpF, one of these proteins, is downregulated by the non-coding small RNA (sRNA) MicF and accomplishes this by antisense binding. Restricting the production of this transport protein prevents the entry of antibiotics into the cell. sRNAs like MicF are expressed only under the presence of a cognate afflictor (e.g. toxic compounds and nutrient availability). These triggers and the degree to which they elicit expression eludes us. Measuring the concentration of sRNAs will help us ascertain their effect on antibiotic resistance when exposed to these various triggers. However, methods for quantitative detection of sRNAs are limited. Quantification of RNA is usually done with quantitative reverse transcription polymerase chain reaction (RT-qPCR), which requires cell lysis and purification of the sRNA, resulting in a loss of yield that will vary depending on the method used. To overcome this, we aim to construct a synthetic gene circuit to quantitatively detect MicF activity, wherein the presence of MicF triggers a cascade of interactions that lead to the expression of the reporter gene, green fluorescence protein. A gene expression assay is used to determine circuit efficacy through optical density and fluorescence readings of cells containing the circuit. After various optimizations, we selected the best performing circuits and tested their ability to detect endogenous levels of MicF. Further optimizations of the circuit will provide a precise method for quantifying sRNA activity, allowing future studies into the intricate triggers that prompt sRNA activation. Research that will further elucidate how sRNAs like MicF regulates bacterial resistance mechanisms may inform us of ways to combat antibiotic resistance.
| Poster #: 73 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Papillary, Thyroid , Carcinoma Project Title: From Cuboidal to Columnar: The Transition of the Thyroid Follicular Cell in Tall Cell Papillary Thyroid Carcinoma Author List: Terone Dunbar, Graduate, Biology, Presenting Author; Terone Dunbar, Jason Bush, Fresno Abstract Abstract
The lack of comprehensive molecular studies and absence of reliable biomarkers for distinguishing variants of Papillary Thyroid Carcinoma (PTC) has led to uncertainty in their classification and treatment. Our research focuses on Tall Cell Papillary Thyroid Carcinoma (TCPTC), recognized as one of the most aggressive and controversial PTC subtypes. This ambiguity results in inconsistent treatment strategies for both malignant and non-malignant forms of PTC. Current diagnostic approaches rely on morphological features, often leading to disagreement among pathologists regarding variant identification. To address this, we aim to investigate molecular markers using RT-qPCR to analyze gene expression changes in TENS1, CBY1, CELSR1, CTNNB1, and CTNNBIP1. We utilize the K1 thyroid cancer cell line, housekeeping genes, and human adult normal thyroid tissue RNA for comparison controls. Preliminary data indicates that these low-abundant transcripts require greater than 30ng RNA/sample to yield sufficient Ct slope curves. Through ∆∆Ct analysis, we are quantifying gene expression alterations, to identify potential biomarkers to improve clinical classification. This could lead to the development of refined diagnostic protocols and personalized treatments, particularly for patients with TCPTC. Ultimately, this research has the potential to uncover biomarkers for other PTC variants and enhance evidence-based treatment strategies, reducing diagnostic uncertainty.
| Poster #: 74 Campus: California State Polytechnic University, Pomona Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Project Title: Investigating the Lysogenic Cycle of STIV viruses Author List: Kimberly Aguilar, Graduate, Presenting Author; Chloe Grant, Undergraduate, Presenting Author; Kimberly Aguilar, Biological Sciences, Pomona Abstract Viruses are the most abundant biological entities found in all domains of life. Archaeal viruses display similar characteristics to viruses found in the eukaryotic and bacterial domains. However, our knowledge of archaeal viruses is limited compared to our knowledge of viruses infecting the other two domains of life. Recent work on a particular virus group has resulted in the emergence of a model system for studying archaeal viruses. The Sulfolobus turreted icosahedral virus (STIV) family infect Sulfolobus species that inhabit acidic (pH 1- 4) hot springs (70-80°C) within Yellowstone National Park. STIV1 was the first member of this growing virus family. It was the first lytic virus isolated from Sulfolobus. Viruses can undergo two replication strategies: a lytic and a lysogenic cycle. In the lytic cycle, a virus will attach to the host cell, release its genetic material, hijack the host’s machinery to replicate, assemble virions, and egress the host cell. In the lysogenic cycle, also known as latency, the virus integrates into the host’s genome and persists in the host until the conditions are no longer favorable. Subsequently, as the cell replicates, the viral genome replicates as well; the viral DNA is passed onto progeny cells. Despite the abundance of archaeal viruses, little is known about viral integration. Integration of viral DNA is a form of site-specific recombination, but integrases can serve other functions. STIV1 engages in a lytic replication cycle but encodes an integrase-like protein (A510). However, there is no evidence demonstrating STIV1 integration into its host. With the recent discovery of an STIV variant that integrates, we decided to investigate the potential integrase protein in both STIV1 and STIV3. The overall goal of this project is to learn more about the lysogenic cycle in STIV viruses. In order to learn more about integration, viral mutants will be created in both STIV1 and STIV3. Virus replication and integration will be assayed using qPCR, western blots, and Southern blots. Two viral mutants have been generated and have yet to be transfected into the host. Understanding the role of A510 will provide further insight into virus evolution and replication.
| Poster #: 75 Campus: Sonoma State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Retinal Pigmented Epithelial Cell, interchromosomal linkage, Haploid Set Project Title: A new model of chromosome organization: Interchromosomal linkages Author List: Jason Romero, Graduate, Biology, Presenting Author; Amada Castro, Undergraduate, Biology, Presenting Author; Lisa Hua, Biology; Jason Romero, Biology, Sonoma Abstract It was discovered that human cells impede homologous chromosome pairing by keeping two haploid chromosome sets apart along the centrosome axis throughout mitosis. Using live cell imaging of Retinal Pigmented Epithelial (RPE1) cells, recent data showed the movement of each chromosome was restricted along a subcellular, centrosome axis. The spatial restriction of chromosomes was present prior to mitotic spindle assembly, and after its disassembly. Using 3D high resolution confocal imaging of Human Umbilical Vein Endothelial Cells (HUVECs), a region of low centromeric DNA satellite and protein components were found along the centrosome axis in metaphase cells. Taken together, these data suggest the haploid set segregation pattern may be present throughout the cell cycle, and may be microtubule independent. However, how the haploid sets associate together along the centrosome axis, yet remain separate with little chromosome mixing remains elusive.
We hypothesize that chromosomes within a haploid set are tethered via interchromosomal linkages. To test whether there is a single main linkage between chromosomes within a haploid set, we use two drugs, Paclitaxel (Taxol) and Reversine, to stabilize the mitotic spindle and override the spindle checkpoint for progression of metaphase/anaphase, respectively, to observe for micronuclei formation. Micronuclei are visualized by Immunofluorescence to LaminB1, a nuclear envelope protein, and a DNA DAPI counterstain. Formation of more than two large micronuclei may rule out a single main interchromosomal linkage that holds individual chromosomes within a haploid set together.
Our preliminary data of drug-treated RPE1 cells show the presence of multiple micronuclei varying in size. We show that 93% of treated cells (n=57 cells) have developed two or more micronuclei.Our data suggests it is unlikely that there is a single, or main,interchromosomal linkage within each haploid set. Findings from our project will expand our understanding of the mechanism of haploid set segregation, and provide further insight into our knowledge of cellular biology.
This work was supported in part by SSU MESA Summer Scholars to A.C., the National Science Foundation (NSF RUI Award#2027746 to L. L.H.), and National Institutes of Health (NIHAward R16GM153517-01 to L. L.H).
| Poster #: 76 Campus: San José State University Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Myogenesis, Limb-Girdle Muscular Dystrophy , AMPs Project Title: TRIM 32 regulates myogenesis in Drosophila Author List: Benjamin Marsh, Graduate, Biological Sciences, Presenting Author; Isabel Renteria, Undergraduate, Biological Sceinces, Presenting Author; Maximus Riad, Undergraduate, Biological Sciences; Kumar Vishal, Biological Sciences, San José Abstract Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) is an inherited disease of skeletal muscle characterized by progressive muscle weakness and loss of muscle strength resulting from mutations in the E3 ubiquitin ligase TRIM32. However, the mechanisms by which TRIM32 regulate muscle differentiation and regeneration or the pathological conditions that lead to loss of muscle function are not understood. A comprehensive genetic analysis in vertebrate models is complicated by the ubiquitous expression of TRIM32 and neurogenic defects in TRIM32 mutant mice that are independent of the muscle pathology associated with LGMD2H. The model organism Drosophila melanogaster possesses a Trim32 (dTrim32) homolog whose expression is muscle-specific has been long been used as a model to understand evolutionarily conserved aspects of muscle development and maintenance. In this study we have used Indirect flight muscles (IFMs) of Drosophila to investigate the mechanism through which TRIM32 regulates muscle formation and maintenance. We hypothesize that dTrim32 controls the number of adult muscle progenitors (AMPs), which in turn regulates muscle formation and maintenance to prevent the pathological changes associated with LGMD2H. To test this hypothesis, we blocked dTrim32 function (using RNA interference) in the AMPs and muscles using Gal4/UAS expression system. We monitored the effects of dTrim32 loss on AMP number (by counting the total number of AMPs using anti-Twist antibody), on MEF2 (a prominent myogenic transcription factor) expression (using anti-MEF2) and progression of muscle formation (using anti-22C10 and anti-Ewg). We utilized confocal microscopy to image the samples and ImageJ and Graphpad Prism for the image analysis. We find that disrupting dTrim32 in AMPs reduces the AMP numbers and decreases MEF2 expression. Furthermore, our results show that disrupting dTrim32 function blocks the initiation of muscle formation and results in impaired muscle patterning. These defects are due to reduced AMP fusion and lack of muscle differentiation. Finally, we find that blocking dTrim32 after the muscle formation does not affect muscle function. Our next step is to determine how loss of dTrim32 during myogenesis impacts muscle maintenance during aging. This study will help us better understand the mechanism through which Trim32 regulates LGMD2H. This study was funded by SJSU startup fund.
| Poster #: 77 Campus: California State University, Fresno Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, oxidative stress, neurobiology Project Title: Blue light promotes sleep independently of photoreceptors through oxidative stress Author List: Ivan Soto, Undergraduate, Biology, Presenting Author; Gauri Paul, Undergraduate, Clovis Community College, Biology, Presenting Author; Cynthia Hsu, Biology, Fresno Abstract With the advent of electronics and the increasing prevalence of blue light from screens, a better understanding of its effects on sleep and oxidative damage will also help the fields understanding of other related physiological disruptions such as mood disorders, cancer, and neurodegeneration. In this study, we are using the Drosophila model to understand the mechanism through which blue light drives sleep. To determine if light drives sleep through oxidative stress, we overexpressed the antioxidant genes superoxide dismutase 1 (Sod1), superoxide dismutase 2 (Sod2), and catalase-A in neurons and found that this upregulation prevents the sleep increase in response to blue light. We also silenced cells that express ninaE, a visual photopigment found in the compound eye, through expression of the inward rectifying potassium channel KIR2.1 and found that signaling through the compound eyes is dispensable for the sleep promoting effects of blue light. In contrast, flies with the null allele cryptochrome, a cytosolic photopigment implicated in circadian phase resetting, do not show an increase in sleep in response to light. Long term, understanding the mechanisms through which light affects sleep will enhance our understanding of how sleep need and ability changes in the presence of different environmental conditions and the possible impact light has on long-term neurological function.
| Poster #: 78 Campus: California State University, San Bernardino Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Transcriptome, Feline Coronavirus, Macrophages Project Title: Transcriptome analysis of novel feline macrophage cell line in response to feline coronavirus. Author List: MariaCeleste Padilla, Graduate, Biology, Presenting Author; Novalee Erickson, Undergraduate, Biology, Presenting Author; Adam Espinoza, Graduate, Biology; Laura Newcomb, Biology, San Bernardino Abstract BACKGROUND: Infection of domestic cats with feline coronavirus (FCoV) typically results in symptomless or mild enteric disease and considered feline enteric coronavirus (FECV). However, in ~5-10% of domestic cat infections, virus tropism, or cell type infected, changes and macrophages become infected. Macrophages release virus throughout the body, resulting in systemic infection and organ failure and considered feline infectious peritonitis virus (FIPV). To study transition of FCoV at the molecular level, our laboratory generated a novel domestic feline macrophage cell line (FMAC) susceptible to both FECV and FIPV infection. HYPOTHESIS: We speculate genetic differences in host factors influence transition of FCoV from FECV to FIPV. METHODS: RNA transcriptome sequencing, often referred to as RNA-seq, is a powerful method used to study the transcriptome and ascertain differential mRNA expression. To characterize host mRNA response to FCoV, we infected FMAC and Crandell-Rees Feline Kidney (CRFK) with FECV and FIPV isolates, including uninfected controls for both cell lines. At various times post infection cells were visualized by microscopy to confirm expected presence or absence of cytopathic effect (CPE), and collected for isolation of total RNA using Trizol. RESULTS: RNA concentration and purity were assessed by optical density and integrity confirmed by resolution on 1% bleach agarose gel to visualize rRNA. RNA was then sent to UCI Genomics for RNA-seq. We received annotated raw read transcriptome data that will be normalized and investigated to reveal mRNA expression differences between uninfected and infected cells, FECV and FIPV infections, and FMAC and CRFK. CONCLUSIONS: We expect our initial analysis will reveal differential expression of select host mRNAs in all comparisons and identify host factors that may influence the transition from FECV to FIPV. We aim to generate more feline macrophage cell lines from genetically diverse domestic cats for similar analysis and comparison to understand how genetic diversity in host factors may influence FECV to FIPV transition.
This project is funded by CSU-Biotech Developmental Grant to LLN, CSUSB Office of Student Research grants awarded to MCP and NE, and NIH URISE fellowship awarded to NE.
| Poster #: 79 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Wnt signaling, developmental biology, qPCR Project Title: Expression of Wnt and Fzd Genes in a Marine Invertebrate Capable of Whole-Body Regeneration Author List: Pejalyn Balanon, Undergraduate, Biological Sciences, Presenting Author; Lauren Emigh, Undergraduate, Biological Sciences, Presenting Author; Zane Sieger, Undergraduate, Biological Sciences; Elena Keeling, Biological Sciences, San Luis Obispo Abstract An ongoing question in developmental biology is the extent to which different members of a gene family exhibit specialized functions versus overlapping roles in various biological processes. An example is the Wnt family of signaling proteins and corresponding Fzd family of receptors, known for pivotal roles in cell communication during development and regeneration. In the marine invertebrate Botrylloides violaceus, the Wnt genes are implicated in regulating its whole-body regeneration (WBR). B. violaceus is a valuable organism for studying the roles of Wnts, as this species undergoes both sexual and asexual reproduction in addition to WBR, allowing for the exploration of these proteins across different developmental processes. Previous students obtained a draft genome for B. violaceus; this allowed us to investigate gene expression in the Wnt signaling pathway. Using the draft genome, we found thirteen Wnt genes and five Fzd genes. We have optimized PCR conditions for all Wnt primers and most Fzds. We also refined DNA and RNA extraction methods, cDNA synthesis, and quantitative PCR (qPCR) assays. Currently, we are quantifying relative gene expression via qPCR. We have determined that colonies undergoing asexual budding express all thirteen Wnt genes with expression detectable within 32 cycles. This confirms that the putative genes identified through bioinformatics are active, establishing a basis for examining their expression in different tissues and developmental stages. After completing quantification of Wnt and Fzd expression in colony tissue, we will initiate studies on expression patterns in different tissues and developmental stages. Overall, these gene expression patterns can provide insight into the possible individualized roles of different Wnts in the development, function, and regeneration of B. violaceus.
Funding provided by the Frost Fund, California Polytechnic State University, San Luis Obispo
| Poster #: 80 Campus: California State University, San Marcos Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Saccharomyces cerevisiae, Mismatch repair, Repeat expansion diseases Project Title: Investigate the Role of Mismatch Repair Protein Msh3 on Myotonic Dystrophy Type 2-causing CCTG DNA Repeat Instability Author List: Emma Segovia, Undergraduate, Biological Sciences, Presenting Author; Lorena Ibarra-Quinonez, Undergraduate, Biological Sciences, Presenting Author; Jane Kim, Biological Sciences, San Marcos Abstract There are over 40 genetic diseases caused by expanded simple DNA repeat sequences. Myotonic Dystrophy type 2 is one such disease, and affected individuals can have between 75-11,000 CCTG DNA repeat sequences in the human CNBP gene. Contraction and expansion mechanisms of these repeat sequences are not well understood. However, previous studies in the Kim lab demonstrated that knocking out MSH3, a gene involved in mismatch repair, reduced the rate of large-scale CCTG contractions in budding yeast Saccharomyces cerevisiae. Because inducing large contractions could be beneficial therapeutically, we sought to make small deletions and specific point mutations in MSH3 to investigate how specific protein domains and amino acids might be involved in CCTG repeat instability. We used a plasmid encoding Cas9 and guide RNA specific to MSH3 along with DNA repair templates that would introduce either a 30 amino acid deletion (269-298) or mutate threonine 275 in the MSH3 gene, using yeast transformations to carry out CRISPR gene editing. The equivalent point mutation was shown previously to affect Msh3 protein stability in mice. Using Polymerase Chain Reaction (PCR) and agarose gel electrophoresis, we observed PCR products consistent in size with the designed deletion. Using PCR, restriction digest, and agarose gel electrophoresis, we observed yeast clones consistent with T275I mutation, as the DNA repair template was designed to introduce a new EcoRV restriction site. Overall, these results demonstrate the ability to mutate the MSH3 gene. Western blot analysis will be used to determine protein levels in these yeast strains since Msh3 is tagged with the Myc epitope. Characterizing the role of Msh3 in CCTG repeat instability in yeast may help inform how single nucleotide polymorphisms in the MSH3 gene may correlate to DM2 patient outcomes.
| Poster #: 81 Campus: California State University, Northridge Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Transcription, Promoter, Luciferase Project Title: Quantifying Gene Expression of the Human UVRAG Promoter Author List: Angel Cermeno, Undergraduate, Biology, Presenting Author; Lesslie Jocol-Perez, Biology; Cindy Malone, Department of Biology, Northridge Abstract In the gene expression pathway, DNA sequences are transcribed into RNA as RNA polymerase II is bound to the promoter site which is responsible for transcription initiation at the transcription start site. Transcription factors operate as regulatory elements by aiding in the binding of RNA polymerase II and serving as activators or repressors of promoter activity when bound to transcription factor binding sites. UV Radiation Resistance Gene (UVRAG) functions as a regulator gene involved in autophagosome maturation for macroautophagy, a process which degrades impaired cytoplasmic substances upon autophagosomal transport. Inhibiting UVRAG expression prevents its interaction necessary to degrade Epidermal Growth Factor Receptor protein to combat cancer cell proliferation. Characterizing the UVRAG promoter by localizing transcription factor binding sites will assess its functionality and the region responsible for the highest transcriptional activity will be found. The human putative promoter was identified through NCBI and amplified through PCR creating a 1489 base pair promoter product. The product was subcloned into a promoterless pGL3 Basic vector for formation of a recombinant plasmid that will assess the luciferase gene found within the vector determinant on the promoter's functionality. Human embryonic kidney cells were transfected with the complete recombinant plasmid to quantify luciferase expression through a dual luciferase assay which revealed a 57-fold increase in luciferase compared to lone pGL3 Basic indicative of an active promoter. Multiple bioinformatic databases were used to discern and map consensus transcription factor binding sites on the recombinant plasmid. Subsequent deletion constructs within the promoter were successfully derived using site-directed mutagenesis to create restriction enzyme sites surrounding the recognized transcription factor binding sites at positions -1148, -935, -725, -478, -177, and -89 relative to a preliminary transcription start site. Deletion constructs will be transfected and assayed to evaluate the transcription factors and region responsible for maximum transcriptional expression. Modulation of the UVRAG promoter may be conducted to achieve desired levels of expression for maintaining macroautophagy regulation.
| Poster #: 82 Campus: California State University, Monterey Bay Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: C. elegans, genomics, bioinformatics Project Title: Skih-daddle: Microsatellites, Genomic Instability, and Possible Correlations to skih-2 Deletion Using Nanopore Sequencing Author List: Vanessa Phan, Undergraduate, Department of Biology and Chemistry, Presenting Author; Matthew Modena, Graduate, University of California Santa Cruz, Department of Molecular, Cellular, and Developmental Biology; Chloe Wohlenberg, Graduate, University of California Santa Cruz, Department of Molecular, Cellular, and Developmental Biology; Josh Arribere, University of California Santa Cruz; Vanessa Phan, Department of Biology and Chemistry, Monterey Bay Abstract Various studies have established that an increase in microsatellite instability (MSI) – accumulated unstable repeat regions within DNA – play a role in cancer development. Normal DNA mismatch repair corrects errors associated with MSI, however, when incapacitated mutations accumulate. The aim of our current research is to understand the potential role of skih-2 in the regulation of microsatellites. The exonuclease involved in mRNA decay and part of the exonuclease complex, skih-2, has been found downregulated in MSI-related cancers. Although the function of skih-2 is not fully understood, we hypothesize that it is involved in maintaining genomic stability through curtailing excessive MSI expansion. To test this hypothesis, we investigated whether skih-2 deletion led to intensified MSI. This was carried out via collection of wildtype and skih-2(Δ) C. elegans in the initial generation and after 30 generations, allowing plenty of time for possible MSI to occur. High molecular weight DNA was subsequently extracted and sequencing using nanopore to create individual libraries. The resulting sequences were then aligned, with our initial generation 0 acting as the reference genome to track developments in microsatellite expansion. From these libraries we’ve been able to find areas of insertion and possible microsatellite instability, however further genomic analysis is needed. Interactions between skih-2 and MSI could lead to further understanding in how genomic stability is maintained.This in turn will illuminate the mechanisms by which genetic diseases occur, in conjunction with greater insights on the impact of epigenomic alterations within cells. Future investigations aim to fully characterize the role of skih-2 in relation to mismatch repair and microsatellite regulation, defining its roles in preserving important biological pathways and cellular health.
| Poster #: 83 Campus: California State University, San Marcos Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: breast cancer, metastasis, adipocytes Project Title: Metastatic and metabolic gene expression changes in breast cancer cells exposed to white or brown adipocyte conditioned media Author List: Casandra Nguyen, Undergraduate, Presenting Author; Valeria Castellanos Rodriguez, Undergraduate, Presenting Author; Carlos Luna, Biology, San Marcos Abstract Approximately 2.3 million women are diagnosed with breast cancer each year. Research has shown that paracrine signaling from adipocytes can lead to the progression of breast cancer. However, there is a lack of understanding of how different types of adipose tissue in the breast contribute to tumor progression. While most research has been done on white adipocytes, progress in imaging techniques has found that brown adipocytes are present in the adult human body. There is contradictory research on whether brown adipocytes have an affect on breast cancer prognosis in patients. The limited understanding of the role of different adipocyte types in cancer highlights the importance of further research.
MDA-MB-231 and immortalized human brown and white preadipocytes were cultured. RT-qPCR, immunofluorescence, and imaging were used to observe the changes in the metastatic potential of human breast cancer cells exposed to conditioned media from differentiated human brown and white adipocytes.
qPCR results showed that SNAI1 had no change in MDA-MB-231 exposed to white adipocyte conditioned media and was upregulated in brown adipocyte conditioned media compared to control. SNAI2 was upregulated in MDA-MB-231 when exposed to both brown and white adipocyte conditioned media. Metabolic-related genes, FASN and SREBF1, were upregulated in MDA-MB-231 when exposed to both white and brown adipocyte conditioned media.
Immunofluorescence with Ki-67 and cell count analysis showed enhanced proliferation in brown and white conditions compared to control. Morphological analysis and vimentin staining showed that cells exposed to white, and brown had a more elongated shape with increased vimentin expression. Image analysis revealed statistical differences with control conditions, but not between white and brown adipocyte conditioned media.
Exposure of MDA-MB-231 cells to adipocyte conditioned media caused an increase in metastatic (SNAI1, SNAI2, vimentin, Ki-67) and metabolic (FASN, SREBF1) markers. Interestingly, there was no difference between white and brown adipocyte conditioned media, only in the case of SNAI1 expression. Further research needs to be conducted on the types of secreted factors common and specific to brown and white adipocytes to find potential therapeutic targets.
| Poster #: 84 Campus: California State Polytechnic University, Pomona Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Leukemia , Gene Fusion, Cell Signaling Project Title: Utilizing Kinase Inhibitors to Target Daple-RTK Gene Fusions in Leukemia Author List: Arnel Ibarra, Graduate, Biological Sciences, Presenting Author; Jason Ear, Biological Sciences, Pomona Abstract Gene fusions frequently occur in many cancers including leukemia, resulting in unregulated cell proliferation, survival, and migration. Specifically, the Daple (CCDC88C) protein fused with receptor tyrosine kinases (RTKs) like FLT3 and PDGFRB, leading to hyperactive signaling pathways that exhibit oncogenic properties. All Daple-RTK gene fusions retain the coiled-coil domain (CCD) of Daple, and it is hypothesized that this region is responsible for the dimerization and activation of the tyrosine kinase domains. Daple-FLT3 gene fusions, identified in chronic myeloid leukemia patients, include the CCD of Daple and the tyrosine kinase domains of FLT3. The CCD is hypothesized to trigger auto-phosphorylation of the FLT3 tyrosine kinase domain at tyrosine 842, activating pathways such as STAT5, AKT, and MAPK independent of extracellular ligands. Similarly, Daple-PDGFRB gene fusions, found in various leukemia types, use the Daple CCD for activation of the PDGFRB tyrosine kinase domain at tyrosine 857. These activated fusion proteins promote cancer through hyperactive Ras/Raf/MAPK, AKT, and Jak/STAT signaling pathways which affect cell proliferation, survival, and migration. Tyrosine kinase inhibitors (TKIs) like Imatinib and Sorafenib can block these pathways by inhibiting ATP binding and preventing phosphorylation. Our study demonstrates the potential of TKIs in directly blocking tyrosine kinase domain activation, thereby preventing hyperactivation of downstream signaling and offering a promising therapeutic strategy for leukemia patients with these gene fusions.
| Poster #: 85 Campus: California State University, Channel Islands Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: desiccation, stress, proteome Project Title: Proteome Dynamics in Desiccation and Rehydration: Unveiling Adaptive Mechanisms for Biotechnology and Agriculture Author List: Sheila Ferer, Presenting Author; Hugo Tapia, Biology, Channel Islands Abstract The growing impact of climate change has heightened the urgency to understand how organisms adapt to environmental stresses, such as fluctuating water availability. Anhydrobiotes—organisms capable of surviving extreme desiccation—enter a state of stasis during water loss and resume normal metabolic functions upon rehydration. In recent years, the molecular mechanisms underlying this survival strategy have started to be revealed. However, this study represents the first effort to investigate how the majority of an organism’s proteome is restructured in response to desiccation and rehydration. By leveraging the Yeast GFP-Fusion Collection, which covers 75% of the Saccharomyces cerevisiae proteome, we can monitor the localization and expression of 4,156 proteins during these stress conditions. A pilot study involving 20 GFP-tagged strains has already identified three distinct patterns of protein behavior following desiccation and rehydration: 1) proteins with unchanged localization, 2) delocalized or degraded proteins, and 3) proteins forming puncta. Expanding on this initial finding, this summer, we have employed automated microscopy and custom machine-learning algorithms to analyze thousands of yeast strains, clustering protein localization patterns based on their similarities under varying stress conditions. This large-scale screening will enable us to create a comprehensive map of proteome dynamics, highlighting key proteins and families that undergo significant shifts during desiccation and rehydration. By identifying proteins crucial for survival in extreme desiccation, we can open new avenues for developing drought-resistant crops, which is essential for future agricultural resilience. Additionally, understanding the mechanisms behind desiccation tolerance could revolutionize industries dependent on cold storage by reducing the need for refrigeration, potentially breaking the cold-chain in sectors like food preservation and pharmaceuticals.
| Poster #: 86 Campus: California State University, Chico Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: scoliosis, hormone, biomedical Project Title: Testosterone as a protective agent in idiopathic-type scoliosis Author List: Karen Contreras, Undergraduate, Chemistry, Presenting Author; Kristen Gorman, Biological Sciences, Chico Abstract Idiopathic scoliosis (IS) is a prevalent genetic pediatric syndrome defined by abnormal spinal curvatures of unknown cause. Although boys and girls tend to demonstrate similar rates of curve onset, as children approach adolescence females are up to 7 times more at risk for severe curve magnitudes that require corrective surgery. Estrogen has been extensively explored as a risk factor, but studies using human cohorts have failed to explain the female bias. A female bias for severe curve magnitudes has been noted in the guppy and medaka fish models for IS, and conservation of endocrine factors among humans and fish is well established. We hypothesized that testosterone is a protective agent against progression of curve magnitude during growth. Using an inbred lineage of medaka true-breeding for idiopathic-type spinal curvatures, embryos were dosed with 7α-Methyldihydrotestosterone from pre-gastrula stage until hatching. Age matched control groups received no testosterone treatment. Fish were raised to sexual maturity individually and the magnitude of spinal curvatures of the treated and untreated fish was compared using an established scale applied to guppy scoliosis. We conducted 3 cohort experiments to assess a total of 83 treated and 31 control fish. A portion of the control cohort died due to unrelated issues. The results showed a broader range of phenotypes in the treated cohorts, with skewing towards lower magnitude curves. We compared cohort curve magnitudes using a two-tailed t-test (assuming unequal variances) to demonstrate a significantly protective effect from testosterone (p=1.1 x 10exp-8 ). This is the first study to investigate testosterone as a protective agent in progressive scoliosis. We will continue to collect control data to balance our statistical comparisons. Although all treated fish matured into phenotypic males, we would like to genetically test the XX/XY status of fish to see if this explains variation in the dosed cohort. Further investigation into the mechanisms by which testosterone protects could lead to new treatment approaches for IS.
| Poster #: 87 Campus: California State University, San Bernardino Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Agrobacterium , Genomics, CURE Project Title: Development of a new research project to study functional microbial genomics of amino acid biosynthesis in Agrobacterium as a Course-based Undergraduate Research Experience (CURE) Author List: Isabel Zaragoza, Undergraduate, CIRM-COMPASS, Biology, Presenting Author; Manuel Robledo, Undergraduate, Biology; David Rhoads, Biology, San Bernardino Abstract Course-based undergraduate experiences (CUREs) are high impact practices for high school, college & university curricula. The application of projects involving functional microbial genomics of amino acid biosynthesis in CUREs has been done at CSUSB for several years and here we report the development of a new project in this area. Agrobacterium is a soil bacterium that creates transgenic plants naturally and is a major system for creating transgenic plants for biotechnology and agricultural applications. Employment of a genomics approach to this important organism provides a better understanding of Agrobacterium and contributes to the field of agricultural biotechnology. Specifically, we focused on the argG gene encoding the enzyme argininosuccinate synthetase (A.S.S.), which is involved in the biosynthetic pathway of arginine (Arg) in bacteria. A putative insertional E. coli knockout (KO) mutant of the argG gene (JW3140-5, CGSC8306) was obtained and confirmed to require addition of Arg to the growth media for cell growth. The E. coli argG gene was then cloned and shown to genetically complement the E. coli argG mutation in our expression-vector-based system. We also performed genomic studies to identify several putative argG genes in three strains of Agrobacterium. This establishes the cloning and functional testing by complementation of the E. coli argG KO mutant using cloned putative Agrobacterium argG genes as a new research project to be used in future CURE courses and traditional undergraduate research projects.
| Poster #: 88 Campus: California State University, Fullerton Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Human induced pluripotent stem cells, Organoids, Neurons Project Title: Effects of Heparin Concentration and Wnt/β-catenin Signaling on Differentiation of Human-Induced Pluripotent Stem Cells into Neuro-cortical Organoids Author List: David Sanchez, Undergraduate, Biological Science, Presenting Author; Suraj Singh, Undergraduate, Biological Science, Presenting Author; Ashley Neil, Sanford Burnham Prebys Medical Discovery Institute, Conrad Prebys Center for Chemical Genomics; Kevin Passi, Graduate, Biological Science; Nilay Patel, Biological Science, Fullerton Abstract The directed differentiation of human-induced pluripotent stem cells (hiPSCs) into neuro-cortical organoids (NCO) provides insights into how different brain cortical layers are formed during neurodevelopment. The formation and maturation of these organoids are influenced by various factors, including timing and signaling pathways. The Wnt/β-catenin signal transduction pathway is an evolutionarily conserved pathway that is integral in directing stem cells to a neuronal fate and rostrocaudal patterning. This project aims to investigate the function of heparin, specifically its role in modulating neuronal differentiation through the Wnt/β-catenin pathway. Most sources of heparin include animal products, which could limit the possible use of these stem cell-derived neurons for clinical application. Thus, it is essential to identify the role of heparin in NCO formation and the replacement of heparin with xeno-free small molecules. We have tested three levels of heparin (none, 0.5μg/μL, and 1μg/μL), a Wnt/β-catenin agonist SB216763, and Wnt/β-catenin antagonist XAV939. SB216763, a GSK-3 inhibitor, was used as a heparin substitute as it increases β-catenin levels. These compounds were added during the neuronal induction medium stages of the Lancaster lab protocol (DOI:10.1038/nprot.2014.158). We evaluated the effects of systematically manipulating heparin levels in combination with the Wnt pathway modulators XAV939 and SB216763 on NCO formation and maturation. The results of this experiment were characterized by the use of immunofluorescence assay and qPCR at key points along the development of the NCOs. Staining was performed for neurons, progenitors, and forebrain development (Tuj1, Sox2, and FoxG1). Expression of these markers at various stages confirmed the successful formation of NCOs. Our results suggest that activation of the Wnt/β-catenin pathway by heparin is essential for the formation and maturation of NCOs.
| Poster #: 89 Campus: California Polytechnic State University, San Luis Obispo Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Mastitis, Metabolites, Dairy cattle Project Title: Biomarker Detection for Mastitis Development in Dairy Cattle Author List: Amara Kramer, Undergraduate, Animal Science, Presenting Author; Kevin Chen, Graduate, Animal Science; Desiree Seto, Graduate, Animal Science; Julie Huzzey, Animal Science; Fernando Campos-Chillon, Animal Science; Daniel Peterson, Animal Science; Siroj Pokharel, Animal Science; Kim Sprayberry, Animal Science; Paul Anderson, Computer Science & Software Engineering; Joy Altermatt, Animal Science; Mohammed Abo-Ismail, Animal Science, San Luis Obispo Abstract Mastitis is the prevailing disease affecting dairy cattle and is characterized by inflammation of the mammary gland due to infection, causing significant economic loss for the dairy industry. The goal of this project was to utilize metabolomics to identify the dynamic variations in metabolite molecules, elucidate metabolic changes during clinical mastitis development, and detect biomarkers for mastitis in blood serum. We collected 148 blood serum samples from 25 cows from the Cal Poly dairy herd, over 5 time points during the transition period. A case-control design was employed on periparturient Holstein and Jersey cattle, at five time points (pre-calving, week 1, 2, 3, and 4). The statistical analyses were performed using MetaboAnalyst software. We fit a linear model that included group (Healthy/Mastitis), breed, and age at calving on 759 identified as known metabolites. A total of 9 metabolites were statistically significant at 5% false discovery rate in all the stages. Some of the metabolites were linked to biological pathways involved in inflammatory response. Biological pathways such as the Cyclooxygenase (COX) pathway, lipid biochemistry that interacts with the Cytochrome P450 pathway, and the N-Ethylmaleimide Sensitive Factor pathway all rely on the significant metabolites identified to properly generate the inflammatory response, thus, linking rising concentrations of said metabolites with the development of mastitis inflammation. This study proposed potential metabolites as biomarkers for management and early predictors of mastitis during the transition period in dairy cattle.
| Poster #: 90 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Arabidopsis, ANAC046, qPCR Project Title: The Regulation of Bolting-Induced Leaf Senescence by the ANAC046 Transcription Factor in Arabidopsis thaliana Author List: Amber McNicol, Undergraduate, Biological Sciences, Presenting Author; Alejandro Duran, Undergraduate, Presenting Author; Judy Brusslan, Biological Sciences, Long Beach Abstract Leaf senescence is the natural aging process that recycles nutrients from older leaves to newly developing regions of the plant. Bolting is the transition from the vegetative to reproductive state of a plant. ANAC046 is a NAC-family transcription factor that is reported to be a positive regulator of leaf senescence in the model plant species, Arabidopsis thaliana and we want to test whether it plays a similar role in bolting-induced leaf senescence. We suggest that ANAC046 loss-of-function mutant plants (anac046 mutants) will show delayed bolting-induced leaf senescence, as measured by a delay in NIT2 gene expression. These mutants carry a T-DNA insertion that disrupts the ANAC046 gene. The Gene Regulatory Network (GRN) shows the genes that may be positively regulated by ANAC046: AtATG18a, ATG8C, and MT1C. Our hypothesis is these genes will show a smaller increase in gene expression in anac046 mutants when compared to WT. The first experimental replicate was sown to compare WT and anac046 through a timepoint analysis, where specific leaves were harvested at the time of bolting (T0) and 12 days after (T12). These plants were harvested for their leaves 4 & 5 for NIT2 gene expression, which increases during leaf senescence. For each trial, there were 10 WT T0, WT T12, anac046 T0, and anac046 T12 samples. RNA was isolated from harvested samples and used as a template for cDNA synthesis, which was then used to perform real-time qPCR to quantify NIT2 mRNA levels, normalized to ACT2 as well as levels for the target genes. Gene expression of NIT2 showed a small but significant difference between T0 and T12 but did not show reduced expression in the anac046 mutants. The gene expression of ATG8C in the anac046 samples is significantly less at T12, showing that ATG8C may be regulated by ANAC046. MT1C displayed similar inductions of gene expression between WT and anac046, whereas AtATG18a showed a decrease in expression at T12 in anac046, opposite of that observed in WT. These initial data suggest that bolting-induced LS is not affected in anac046 and is similar to WT. This differs from high light (HL) induced LS as seen in previous work. The data also suggests that the ANAC046 TF may regulate the autophagy-related genes AtATG18a and ATG8C. While there was no strong significant difference in NIT2 gene expression under lower light intensity, the findings for AtATG18a show support for the predicted GRN. Two other trials are underway to strengthen these findings.
| Poster #: 91 Campus: California State University, Long Beach Poster Category: Molecular Biology (Include Regulation and Genomics) Keywords: Drosophila, Tumor cell dissemination, G-coupled protein receptors Project Title: Identifying the upstream G-protein receptor mediating Calcium influx in RasV12 tumor cell dissemination Author List: Vincente Abatay, Undergraduate, Biological Sciences, Presenting Author; Alan Vu, Undergraduate, Biological Sciences, Presenting Author; Jiae Lee, Biological Sciences, Long Beach Abstract Dissemination is the initial process for tumor cells to metastasize throughout the body. To understand the dissemination mechanism, we express the fly ortholog of the oncogene RasV12, a common mutation in human cancers, in Drosophila midgut to describe cellular events of the disseminating tumor cells. During dissemination, RasV12-expressing tumor cells undergo morphological changes and form invasive protrusions that breach the extracellular matrix (ECM) and the visceral muscle (VM). Our previous results have suggested that E-cadherin/beta-catenin dissociation which is essential for the dissemination is controlled by intracellular calcium signaling pathways where Ca2+ is released from the endoplasmic reticulum (ER) via the inositol triphosphate receptor (IP3R). E-cad/beta-catenin assembles invasive protrusions at the actin-and cortactin-rich sites that breach the extracellular matrix for dissemination of RasV12-tumor cells. We have performed candidate-based RNAi screening to determine the upstream G-coupled protein receptors (GCPR) responsible for the influx of Ca2+ signaling. The genes of interest are moody and mthl-14, GCPRs enriched in the digestive system. Especially, moody is known to be expressed in the plasma membrane and the septate junction (SJs) that preserves cell adhesion and epithelial polarity, which could be the possible mechanism of decreasing dissemination in the midgut upon RNAi knockdown. Our study provides a unique opportunity to investigate the molecular pathway controlling tumor cell dissemination using an in vivo model with a native tissue context, giving insight into how calcium signaling affects tumor cell dissemination
| Poster #: 92 Campus: California State University, Northridge Poster Category: Product-focused Innovation Keywords: Haptic feedback, Gesture recognition, Prostheses control Project Title: An age-adaptable below-the-shoulder prosthetic arm with a touch notification system and a gesture-recognizing foot controller Author List: George Matthews, Undergraduate, Mechanical Engineering, Presenting Author; Logan DeHay, Undergraduate, Mechanical Engineering, Presenting Author; Joseph Sunbaty, Undergraduate, Mechanical Engineering; Daniel Sanchez, Undergraduate, Mechanical Engineering; Oren Gravenhorst, Undergraduate, Mechanical Engineering; Jocelyn Camarena, Undergraduate, Mechanical Engineering; Nicholas Fanderlik, Undergraduate, Mechanical Engineering; Daniel Haley, Undergraduate, Mechanical Engineering; Romel Angelo Melgar, Undergraduate, Mechanical Engineering; Mary Kesablian, Undergraduate, Mechanical Engineering; Peter L Bishay, Mechanical Engineering, Northridge Abstract Prostheses play a vital role in improving the quality of life for individuals who have lost their limb due to injury or illness. Unfortunately, powered below-the-shoulder prosthetic options are very limited due to the complexity of the human arm’s range of motion and its corresponding degrees of freedom in a prosthetic arm. The market is also limited in terms of adjustable sizing options. Currently, children who use prosthetic arms need to purchase entirely new devices as they grow and adapt to such devices. Prosthetic arms are also dominated by myoelectric controls, which have many limitations and face high rejection rates. The main focus in designing current prosthetic devices has been the input (control) aspect, and very little effort has been put into the output (feedback) aspect. Haptic feedback systems have been dominated by a very complex and costly design, resulting in usage strictly in robotic arms, which in return leaves no feedback systems for prostheses users. In response to these challenges and using the advantages of 3D-printing, this work presents a scalable below-the-shoulder prosthetic arm with an advanced tendon-driven finger design. Leveraging the power of parametric design tools in computer-aided design (CAD) software, the arm can be customized in size and shape based on some dimensions taken from the user’s body. This enables a user to continue using the same prosthetic arm design they got used to as they grow. The arm is controlled by an innovative foot controller that uses a combination of buttons and sensors to control the grips and articulation of the arm. The controller contains an inertial measurement unit (IMU) that tracks the user's gestures, which are used to intuitively control the arm. Haptic feedback is sensed through a glove that processes vibrations and forces into real-time feedback to the adjustable feedback band. The feedback given simulates reaction forces of different grips and textures of different surfaces.
| Poster #: 93 Campus: California State University, East Bay Poster Category: Product-focused Innovation Keywords: Virtual Reality, Patient Safety, STEM Education Project Title: Advancing Nursing Education through Immersive Virtual Reality Training Author List: Sai Gayam, Graduate, Computer Science, Presenting Author; Fay Zhong, Computer Science, East Bay Abstract Patient safety is a critical aspect of healthcare that demands continuous attention and improvement. In the field of nursing education, ensuring that students are equipped to identify and address unsafe practices is essential for enhancing patient outcomes and reducing medical errors. Traditional classroom and hospital-based training methods have inherent limitations in providing students with extensive opportunities to practice recognizing safety issues in realistic healthcare settings. As such, there is a pressing need for innovative approaches that can bridge this training gap and better prepare nursing students for the complexities of clinical practice.
In response to the challenges posed by traditional training methods, we propose the development of an immersive virtual reality (VR) simulation as a revolutionary approach to advancing nursing education. By leveraging VR technology, nursing students can immerse themselves in realistic hospital room environments, point out unsafe parts of image and interact with 3D objects representing safe and unsafe medical items, and engage in deliberate practice to enhance their ability to identify safety issues. This innovative training tool offers a unique opportunity for students to build expertise in recognizing and addressing unsafe practices in a controlled and risk-free virtual space, ultimately empowering them to translate their knowledge to real-world clinical settings.
The integration of immersive VR training in nursing education not only addresses the current limitations of traditional training methods but also paves the way for future breakthroughs in healthcare training methodologies. By providing students with remote accessibility, realistic simulation experiences, this innovative approach aims to redefine the training landscape and prepare nursing students for the challenges they will encounter in their professional careers.
| Poster #: 94 Campus: California State University, Long Beach Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM) Keywords: Keck Undergraduate Research Experiences (KURE) Incubator, KURE Bridge program, environmental toxins Project Title: Keck Undergraduate Research Experiences (KURE) Incubator: environmental toxins are sneaking into your life Author List: ; Yuan Yu Lee, Biological Sciences, Long Beach Abstract The KURE Incubator project launched a research bridge program in summer 2024 that was designed for any undergraduate/high school students who are interested in Science, Technology, Engineering and Mathematics (STEM) but have not yet had an opportunity to participate in research. This program provides an overview of environmental toxicology, including an examination of the major classes of pollutants, their fate in the environment, their disposition in organisms, and their mechanisms of toxicity. An emphasis of the program are the principles and methods of biological testing for toxicity and health effects, risk assessment, and the impact of pollutants on daily life items and ecosystems. Bisphenol A (BPA) and triclosan (TCS) were chosen toxins of interest, because they are pervasive environmental toxins with human impacts that raise concerns. BPA, commonly found in plastics, epoxy resins, and thermal paper, leaches into food and water, exposing humans through ingestion and skin contact. Triclosan, an antimicrobial agent, is common in personal care products like soaps, toothpaste, and cosmetics, along with household items like plastics and textiles. Both chemicals disrupt endocrine function by mimicking or blocking hormones. Endocrine disruption can lead to developmental abnormalities, reproductive issues, and increased risk of certain cancers. Throughout this program with the goal of not only introducing the early authentic research experiences but also educating students about overlooked dangers in household items and how we use those items safely”.
| Poster #: 95 Campus: California State University, Northridge Poster Category: Programmatic (Core, Stem Cell, Bridges, PSM) Keywords: placenta, trophoblast stem cell lines , EVOM machine Project Title: Modeling a Placenta In Vitro Author List: Jazmine Vasquez, Undergraduate, Biology, Presenting Author; Mark Sharpley, Cedar Sinai Medical Center, Pediatrics; Ophir Klein, Cedar Sinai Medical Center, Pediatrics; Cindy Klein, Biology, Northridge Abstract Over the years, the scientific community has been working to develop human patient-specific placenta models. These models can be very useful for testing the effects of drugs and pathogens, as well as to characterize patient-specific pregnancy complications. To develop an in vitro placental model, we used trophoblast stem cell lines that were established by taking tissues from patients undergoing chorionic villus sampling who are currently in an ongoing pregnancy. After generating more than 50 trophoblast stem cell lines, we tested these lines for those that would be advantageous for modeling placental transport. An effective barrier is necessary to model nutrient transport and the ability to block the passage of drugs or pathogens. We established conditions to create a proper monolayer by seeding epithelial cells from the trophoblast stem cell lines on both sides of a transwell plate. We then differentiated these cells into two types of cells, cytotrophoblast cells and syncytiotrophoblast cells. To confirm their differentiation, RNA was isolated and purified using the Qiagen RNeasy kit and quantified by Nanodrop. We confirmed that the rophoblast stem cell lines differentiated by measuring their gene expression of
known corresponding genes for their specific types. We tested the plates that had cytotrophoblast cells and syncytiotrophoblast cells confirmed by RNA expression and confirmed placenta models for the effectiveness as a barrier using an EVOM machine. This machine was used to take Trans-Epithelial
Electrical Resistance readings used to quantify the resistance,strength and durability of each line. We were able to test the durability of the placenta
models by taking these Trans-Epithelial Electrical Resistance readings over several days. After analyzing the data for all of the placenta models we created, we found that one line, CSTB002E, stood out from the rest in both effectiveness and durability. For the next steps, we will characterize the cells of this placenta model to determine why they make such a great barrier to guide future experiments.
| Poster #: 96 Campus: California State University, Sacramento Poster Category: Proteins (Include Proteomics) Keywords: amyloid, apolipoprotein, protein folding Project Title: Self-Association of Human and Canine Apolipoprotein A-I using SEC-MALS and Chemical Cross-linking Author List: Isaias Iniguez-Sandoval, Undergraduate, Chemistry, Presenting Author; Khaled Jami, Graduate, UC Davis, Chemistry; Linda Roberts, Chemistry, Sacramento Abstract Apolipoprotein A-I (apoA-I) is the major protein component of HDL (high density lipoprotein) whose primary role is to remove cholesterol from the bloodstream. Previous studies suggest methionine-oxidized apoA-I readily leads to the formation of amyloid, a deleterious insoluble aggregate that has been linked to cardiovascular disease and other neurodegenerative ailments. Additional research points to the loss of self-association in methionine-oxidized protein, implicating the loss of naturally occurring oligomers as a possible precursor to amyloid formation. Human and canine apoA-I, while having about 85% sequence similarity, differ in their methionine content with the latter having only one methionine versus three in human protein. Despite fewer methionine residues, and therefore fewer sites of oxidation, amyloid apoA-I has been found in the vasculature of aged dogs, whose life span is much shorter than that of humans. This suggests canine apoA-I may form amyloid at a faster rate than its human analogue. As a first step in exploring this issue, we sought to determine whether self-association in canine apoA-I differs from that of human apoA-I. The molecular weight and quantity of the various species present in solution were analyzed using SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering) and the bifunctional chemical cross-linker, BS3 [bis(sulfosuccinimidylsuberate)]. The cross-linked species were visualized by coomassie staining of SDS-PAGE in the absence of beta-mercaptoethanol. Results suggest that human apoA-I exists mostly in monomeric form, whereas canine apoA-I under the same conditions exists primarily as higher order oligomers. These results demonstrate that there is a difference in self-association between the species, which may influence amyloid formation.
| Poster #: 97 Campus: California State University, San Bernardino Poster Category: Proteins (Include Proteomics) Keywords: Nucleoprotein, Influenza A Virus, Protein-protein interactions Project Title: Analysis and alteration of influenza Nucleoprotein (NP) to identify and define new antiviral targets. Author List: Valentin Acosta, Graduate, Biology, Presenting Author; Alyssa Salgado, Graduate, Biology, Presenting Author; Laura Newcomb, Biology, San Bernardino Abstract BACKGROUND: Influenza virus remains a health threat. Yearly vaccination protects against severe disease, but efficacy varies as viral genetic changes may render the vaccine less protective. Furthermore, highly pathogenic avian influenza (HPAI) H5N1 has jumped to mammals with outbreaks among cattle and cats at many dairy farms in the US, raising pandemic concerns. Seasonal vaccines are not formulated to protect against H5N1. Antivirals are an important first line of therapeutic defense to slow an emerging influenza, but antiviral use selects resistant viruses, exemplified by the fact that first generation influenza antivirals are no longer effective or in use. HYPOTHESIS: Conserved influenza nucleoprotein interactions provide multiple targets for development of innovative antivirals effective against seasonal and emerging influenza viruses. METHODS: NP protein sequences isolated from human, avian, swine, and cattle were analyzed for polymorphism and conservation using the National Center for Biotechnology Information (NCBI) Virus database. NP crystal structure was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and scrutinized to identify conserved amino acids on the surface of NP accessible to participate in weak interactions with other molecules. Published literature on NP interactions with influenza PB1, PB2, and NS1, was considered and NP amino acids were selected for alteration aiming to disrupt these NP interactions. Standard molecular techniques including polymerase chain reaction (PCR), restriction enzyme digestion, recombination and ligation, were used to construct recombinant DNA plasmid designed to encode NP with select amino acid alterations. Mammalian cells will be transfected to assess altered NP expression, function, and interactions using Western Blot, reconstituted viral ribonucleoprotein assays, and gradient fractionation, respectively. DNA plasmids are also suitable for in vitro transcription and translation which will be used with co-immunoprecipitation to assess altered NP interactions in vitro. RESULTS: Seven recombinant DNA plasmids encoding different NP alterations were constructed and sequence confirmed. We are evaluating the consequence of NP alterations on NP expression, function, and interactions. CONCLUSIONS: Defining NP amino acids involved in interactions will inform molecular details of essential domains and facilitate development of innovative antivirals.
| Poster #: 98 Campus: California State University, Fullerton Poster Category: Proteins (Include Proteomics) Keywords: PTBP1, heterokaryon, export Project Title: Determining the Nuclear Export Sequence In RNA Recognition Motif 2 of Polypyrimidine Tract Binding Protein 1 Author List: Riley Scanlon, Undergraduate, Biological Science, Presenting Author; Christian Hernandez, Undergraduate, Biological Science, Presenting Author; Madelynn Perez, Undergraduate, Chemistry and Biochemistry; Niroshika Keppetipola, Chemistry and Biochemistry; Alison Miyamoto, Biological Science, Fullerton Abstract Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that regulates fundamental cellular processes including alternative splicing in the nucleus and messenger RNA (mRNA) transport from the nucleus to the cytoplasm for protein translation. PTBP1 contains four RNA Recognition Motifs (RRMs), as well as a classic nuclear localization sequence and a classic nuclear export sequence (NES). Additionally, there is evidence that the second RRM domain has nuclear export activity, although which amino acids in RRM2 are required for this activity is still unknown. The Keppetipola lab identified potential NES sequences in RRM2, deleted each separately, and this project is testing the mutants in a nuclear export assay. The assay for nuclear export requires the fusion of human osteosarcoma cells (MG63) transfected with FLAG-tagged PTBP1 to untransfected mouse NIH 3T3 cells; fused cells (known as heterokaryons) share a cytoplasm in which export from a human nucleus to a mouse nucleus can be visualized using immunofluorescence. Initially the assay was not optimal; heterokaryons were rare, and when they were found a PTBP1 signal under fluorescence was often absent. We optimized the following parameters and achieved the following: 1) Cell fusion: using polyethylene glycol with a molecular weight of 8000 daltons for 2 minutes yielded fused cells that could contain more than 10 nuclei, 2) MG63 cell transfection: using the Avalanche ® Omni Reagent and 1 microgram of plasmid increased the percentage of transfected MG63 cells from 30% to 40%,3) Mouse cell line selection: a mouse myoblast C2C12 cell line was initially used, but when directly transfected with wild-type PTBP1, the protein was aberrantly found in the cytoplasm instead of the nucleus, therefore a second mouse cell line, NIH 3T3 fibroblasts, was tested and found to correctly localize PTBP1 to the nuclei, allowing us to utilize this cell line, and 4) Cell mixing: a 2:1 ratio (MG63: NIH 3T3) generated the highest number of fusions containing both human and mouse cells. The optimized protocol has now been successfully performed independently by three research students. We are currently using this assay to analyze two mutant PTBP1 constructs, each of which contain deletion mutations in the RRM2 domain of PTBP1. Based on the results from a related project, we believe that these are the most likely candidates for a novel nuclear export sequence in RRM2 of PTBP1.
| Poster #: 99 Campus: California State University, Fullerton Poster Category: Proteins (Include Proteomics) Keywords: stem cell, iPSC, in vitro differentiation Project Title: Optimizing Stem Cell Differentiation into Neurons with Synthetic Proteins Author List: Danica Banzon, Undergraduate, Biological Science, Presenting Author; James Pierpoint, ThermoFisher Scientific, Cell Biology Research and Development Services ; Christian Stadler, ThermoFisher Scientific, Cell Biology Research and Development Services ; Roxanne Strahan, ThermoFisher Scientific , Cell Biology Research and Development Services ; Alyssa Webb, ThermoFisher Scientific, Cell Biology Research and Development Services ; Alison Miyamoto, Biological Science, Fullerton Abstract To advance clientele research and projects, the Cell Biology Research and Development Services team at ThermoFisher strives to provide and perform innovative experiments. Such methods, like generating mRNA and transforming stem cells into specialized cell types, are a main focus so that optimized protocols can become available services to clients. This project's objective was to create and introduce mRNA into human and stem cells, including stem cells, and then influence the stem cells to become neurons. Like the natural cellular process of creating proteins from DNA, mRNA was artificially created using ThermoFisher kits and introduced to two human cell lines to produce proteins. The fluorescent proteins produced by the introduced mRNA were detected in individual cells using flow cytometry. After 24 hours, an average of 58.55% of the cells showed successful RNA uptake and protein production in two human cell lines. This method was also applied to induced pluripotent stem cells (iPSCs), resulting in an average success of 78.8%. Using growth factors, these stem cells were then influenced to transition into matured neurons, by day 10, the influenced stem cells resembled neural cells by microscopy. As part of this team, I independently maintained human and stem cell lines, and worked alongside scientists to perform mRNA synthesis, transfection, flow cytometry, and differentiation on these cells to obtain data for an ongoing project. This project, including the first-time performance of iPSC differentiation with synthesized mRNA in our group, provides the opportunity to build neurological models to study neurogenesis and disorders. The applied techniques across various cell types demonstrate the potential expansion of iPSC studies.
| Poster #: 100 Campus: California State University, Fullerton Poster Category: Proteins (Include Proteomics) Keywords: cardiotoxicity, sarcomere, cancer Project Title: Myosin Activation as a Strategy to Rescue Targeted Therapy Induced Cardiotoxicity Author List: Sebastian Ochoa Vazquez, Undergraduate, Biological Science, Presenting Author; Kai Zhang, City of Hope, Diabetes Complications & Metabolism; June-Wha Rhee, City of Hope, Division of Cardiology, Department of Medicine; Zhao Wang, City of Hope, Diabetes Complications & Metabolism; Alison Miyamoto, Biological Science, Fullerton Abstract Cancer therapies are usually constructed in a way that allows for specific pathways of a cell to be targeted. However, some of these therapies like tyrosine kinase inhibitors and proteasome inhibitors, can also immobilize the pathways in off-target healthy cells. One important example of this is the targeting of healthy heart cells, which induces cardiotoxicity. To determine which type of cancer therapies specifically are attributed to heart dysfunction, an in vitro approach was constructed implementing heart muscle cells called cardiomyocytes. Cardiomyocytes function as contractile units, whose basic structure consists of a sarcomere which encompasses both an actin and a myosin filament that bind together to form a contraction when myosin is activated through phosphorylation to bind to actin. Our hypothesis is that myosin could play a role in this off target cardiac dysfunction. An assay was designed around Myosin light chain 2 protein (MYL2); it can be used as a marker for sarcomeric cardiotoxicity since it promotes downstream pathways of contraction. The drugs Carfilzomib and varying tyrosine inhibitors were then tested in this assay. As part of this team I performed the following experiments. Drug treatment of cardiomyocytes with Carfilzomib and varying tyrosine inhibitors, notably when comparing Carfilzomib to the control group I saw a reduction in MYL2 based on western blot data and a reduction and loss of contractile function based on contractility data. Moreover, cardiomyocyte contractility seemed to have been rescued when the cells were also treated with Omecamtiv, a drug that is a myosin activator that can reverse myosin inactivation. Based on the data, Carfilzomib could potentially be targeting MYL2, leading to cardiac dysfunction, but was able to be rescued by Omecamtiv.
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