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David Drubin , Department of Molecular and Cell Biology, University of California, Berkeley 

"Cell-cell fusion of genome-edited human cells for rapid screening of novel protein-organelle interactions" 

Abstract: 

Generation of genome-edited cell lines has been critical to the faithful imaging of dynamic cellular processes. Previously, the Drubin-Barnes Lab has used genome-edited mammalian cell lines expressing multiple fluorescently tagged proteins to examine the native dynamics of clathrin-mediated endocytosis (CME). However, generation of these cell lines can require months of effort; the cell line must be genome edited, clonally expanded, validated, and the entire process repeated for each additional tagged protein. Here, I develop a protocol to fuse different genome-edited SKMEL cancer cell lines that each expresses a different fluorescently tagged CME protein to each other to generate a hybrid cell line that expresses multiple fluorescently tagged proteins. This procedure is completed on the time scale of days rather than the months required to genome edit multiple fluorescent tags into a single cell line. I observed that the dynamics of these fluorescently tagged proteins resemble those of cell lines generated through successive rounds of genome-editing, indicating this method allows near-physiological investigation of dynamics of different combinations of proteins by fusion of pre-existing cell lines. Additionally, in collaboration with a lab mate, I found that fused cells could be clonally expanded to generate a new cell line, suggesting usefulness of the hybrid cell lines for long-term investigation of intracellular protein dynamics. Finally, another lab member and I adapted this protocol for induced Pluoripotent Stem Cell (iPSC) cell lines, fusing a cell line that expresses a fluorescently tagged Arp2/3 subunit (nucleator of actin assembly) and AP2 (adaptor protein for CME) with iPSC cell lines from the Allen Cell Collection, comprised of iPSC cells genome-edited to express fluorescently tagged organelle markers. Both AP2 and Arp2/3 signal were found associated with multiple membrane-bound organelles, implicating these proteins in previously unknown functions and demonstrating the utility of this method in discovering novel biology. 

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Craig T. Miller , Department of Molecular & Cell Biology, University of California, Berkeley 

"Identifying and Testing Putative Eda Enhancers in Threespine Stickleback" 

Abstract: 

Ectodysplasin A, or Eda, is a gene involved in trait evolution in humans and fish. Eda is expressed in developing epithelial organs, such as hair, teeth, and scales in fish. Mutations in Eda are known to affect tooth shape and number in humans, mice, and fish. However, little is known about the activation of Eda gene expression during development, specifically via cis-regulatory DNA elements called enhancers. This project aims to identify and test putative Eda enhancers in threespine stickleback fish to gain insight into Eda’s activation. Four putative enhancer regions (3154-3157) were identified in existing ATAC-Seq data from stickleback tooth tissue. We assessed sequence conservation of each enhancer across vertebrates. We found notable sequence conservation suggestive of conserved enhancer function for all except 3157. To test enhancer function, we cloned each region into plasmids upstream of GFP and injected these plasmids into one-cell stage stickleback embryos. Embryos were screened for GFP reporter patterns indicative of enhancer function at 7 days post fertilization. Regions 3154 and 3156 demonstrated expression in the optic tectum, olfactory bulbs, neural tube, and teeth. These repeated patterns of GFP expression indicate that these DNA regions are in fact enhancers. GFP expression in pharyngeal teeth and neuromasts are consistent with Eda expression, suggesting that these are enhancers of Eda. Our results indicate that three out of four regions may be enhancers and that two regions may be enhancers of Eda due to their commonalities with known Eda expression. Identification of Eda enhancers will facilitate mechanistic study of Eda activation during development. 

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David Savage , Innovative Genomics Institute 

"Novel SM-like proteins in archaea Methanoperedens: Implications for gene regulation and protein evolution"

Abstract: 

Candidatus Methanoperedens spp is a methane oxidizing archaea which has been implicated in its potential to target greenhouse gasses. Recently, novel extrachromosomal elements, known Borgs have been identified within Methanoperedens spp, containing multiple genes associated with metabolic capabilities to oxidize methane. As a result, understanding both Borgs and the host Methanoperedens is crucial for elucidating their metabolic pathways. One key component is a novel small nuclear ribonucleoprotein (snRNP)-associated SM-like proteins in the Methanoperedens host genome, which has been implicated in potential nucleic acid binding. SM-like proteins are known to play critical roles in modulating gene expression and could be exploited for bioengineering nucleic acid probes or altering gene expression. In this study, we investigate the phylogenetic placement of this SM-like proteins and hypothesize its function based on protein sequence alignment. Moreover, we seek biochemical validation to determine if this LSm-like protein binds to nucleic acids through purification and subsequent urea gel electrophoresis with RNAse and DNAse treatment. We also initiate in vivo expression experiments of this novel LSm-like protein in E. coli under various stress conditions, including temperature fluctuations and phage introduction, to monitor cellular response. Our findings will contribute to a deeper understanding of the functional role and potential applications of SM-like proteins in Methanoperedens spp. 

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Yin Shen, University of California, San Francisco 

"Prioritization of Schizophrenia Associated Genomic Variants using Functional Annotation"

Abstract: 

Several studies have identified thousands of single nucleotide polymorphisms (SNPs) associated with various complex diseases or traits including schizophrenia. However, linking variants to their target gene is not trivial since the majority have been found to be enriched in the non-coding genomic regions such as cis-regulatory elements (cCREs). Thus, without functional annotations, it remains unclear which variants have a significant contribution to disease pathogenesis, which genes are regulated by the SNPs, or which cell type is relevant for the disease variant. While prior work characterized cell populations involved in neurogenesis from the mid-gestational human cortex, in this study, we leverage genomic and genetic tools to characterize and prioritize disease-associated variants in neuronal progenitor cells: outer radial glia(oRG) and ventricular radial glia(vRG) which are molecularly distinct subpopulations of radial glia located in separate locations of the neocortex. Although previous studies have implicated glial cell involvement in schizophrenia, the nature of gene regulation of these cell populations to schizophrenia disease pathology remains unclear. Cell type specific data acquired from ATAC-seq, PLAC-seq, and RNA-seq was utilized to identify variants that are located at transcriptionally accessible regions of the genome, participate in 3D-chromatin interactions, and are in contact with highly expressed genes to prioritize 18,263 fine-mapped schizophrenia SNPs to just hundreds of variants. To further prioritize variants, a gapped-kmer SVM classifier model was trained and optimized to predict accessibility for each cell type as accessibility of the genome is associated with enhancer activity. Lastly, the induced change in the gkm-SVM score of each SNP, deltaSVM, quantified the effect of the sequence changes within regulatory elements. This work leverages functional annotation to prioritize SNPs and provide new insights into our understanding of this complex neurodevelopmental disease. In the future, selected variants can be further evaluated by functional validation to confirm their role in disease development.

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Richard Harland , Department of Molecular & Cell Biology 

"Characterization of the function and expression of arhgap8, and arhgap36 in early developing Xenopus laevis embryos. "

Abstract: 

RhoGEFs and RhoGAPs are molecules that regulate the activity of RHO GTPases. These regulatory molecules have been linked to various cellular processes such as cell cycle progression, cell migration, invasion and cytoskeletal rearrangement. In this study, we aim to characterize the function and expression patterns of two of these regulatory molecules in early developmental stages of Xenopus laevis, based on the idea that locally or temporally expressed Rho regulators may control aspects of morphogenesis. Analysis of Bulk RNA sequencing data has revealed expression of arhgap8 and arhgap36 during early developmental stages of Xenopus laevis embryos. We used whole mount in situ hybridization (WISH) to identify the spatio-temporal expression of these genes. Further, we used CRISPR-cas9 gene knockout to understand the function of these genes in early embryonic development. WISH experiments have shown the expression of arhgap8 and arhgap36 during gastrulation, and neurulation. During organogenesis (stage 35), the expression of the two genes was localized in the branchial arches, the otic vesicles and the eyes. arhgap8 was expressed in the somites while arhgap36 is not. WISH combined with antibody staining for 12/101, a skeletal muscle marker suggest in the somite cleft. Functional assessment of these genes using CRISPR-cas9 for knockout experiments have shown defects during gastrulation when either of these genes are absent. Knocking out arhgap8 resulted in a collapse of the dorsal lip during gastrulation. Further, arhgap36 knockout have shown gastrulation delay; however, the embryos proceeded to later stages of development. Absence of arhgap8 is fatal to the embryos while arhgap36 knockout is not. These novel findings show spatio-temporal and functional differences between arhgap8 and arhgap36 in Xenopus laevis embryonic development. 

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Guo Huang , Cardiovascular Research Institute, University of California, San Francisco 

"A novel pacing strategy for analyzing the effects of heart rate and binucleation on cardiomyocyte regeneration." 

Abstract: 

Understanding the process of cardiac regeneration is essential, as the diminished proliferative capacity of adult cardiomyocytes results in inadequate recovery from heart disease. Phylogeny and ontogeny indicate a remarkable divergence in the regenerative potential of cardiac tissue through the evolutionary shift from ectothermy to endothermy, where ectothermic species retain postnatal regenerative function and particularly mammalian endotherms lose this ability with the acquisition of higher metabolic rates postnatally. Per Kleiber's law, we hypothesize that diploid cardiomyocytes, an indicator of regeneration, decrease through polyploidization and cell cycle exit due to increased cardiac output as compensation for endothermy acquisition: higher standard metabolic rate. We discovered a negative trend between heart rate, a component of cardiac output, and cardiomyocyte ploidy through evolutionary analysis of species along the ectotherm-endotherm axis towards higher standard metabolic rates. To further investigate the role of heart rate in driving cardiomyocyte polyploidization and cell cycle exit, we looked at mammalian embryonic cell cultures, where pacing cells increased binucleation and polyploidization. To improve result validity, we simulated partial in vivo-like conditions by creating a novel neonatal tissue culture protocol that enabled us to visualize the decreased proliferation topographically within the tissue and the diverse interactions. Then, using this tissue model, we investigated various molecular players such as Glycogen Synthase Kinase inhibitor (GSKi) that mitigates the effects of pacing and preserving proliferative activity and regenerative capacity in the mammalian heart or Triiodothyronine (T3)/ Norepinephrine (NE) that may promote binucleation in the myocardial tissue. Further assessment of potential pathway molecules may indicate mechanistic interactions that are interrelated with pacing and regeneration, therefore, assisting our understanding of what contributes to or hinders cell cycle exit and cardiomyocyte polyploidization in the mammalian heart.

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John M. Colford Jr. , Division of Epidemiology and Biostatistics, School of Public Health, University of California Berkeley 

"Micronutrient status during pregnancy is associated with child immune status in rural Bangladesh" 

Abstract: 

Poor immune function increases children’s risk of infection and mortality. Several maternal factors during pregnancy may affect infant immune function during the postnatal period. We conducted observational analyses within the WASH Benefits Bangladesh randomized controlled trial. We measured biomarkers in 575 pregnant women and postnatally in their children. Maternal biomarkers measured during the first and second trimester of pregnancy included: nutrition status via vitamin D (25-hydroxy-D [25(OH)D]), ferritin, soluble transferrin receptor (sTfR), retinol binding protein (RBP); cortisol; estriol. Immune markers were assessed in pregnant women at enrollment and their children at ages 14 and 28 months, including: C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), and thirteen cytokines (including IFN-γ). We generated a standardized sum score of log-transformed cytokines. We analyzed IFN-γ individually because it is a critical immunoregulatory cytokine. All outcomes were pre-specified. We used generalized additive models and reported the mean difference and 95% confidence intervals at the 25th and 75th percentiles of exposure distribution. At child age 14 months, concentrations of maternal RBP were inversely associated with the cytokine sum score in children (-0.34 adjusted difference between the 25th and 75th percentile [95% confidence interval -0.61, -0.07]), and maternal vitamin A deficiency was positively associated with the cytokine sum score in children (1.02 [0.13, 1.92]). At child age 28 months, maternal RBP was positively associated with IFN-γ in children (0.07 [0.01, 0.14]) while maternal vitamin A deficiency was negatively associated with child AGP (-0.07 [-0.13, -0.02]). Maternal iron deficiency was associated with higher AGP levels in children at age 14 months (0.13 [0.04, 0.23]), and maternal sTfR concentrations were positively associated with child CRP levels at age 28 months (0.18 [0, 0.36]). Maternal deficiencies in vitamin A or iron during the first two trimesters of pregnancy may shape the trajectory of child immune status. 

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Allison Xu , Diabetes Center, University of California, San Francisco 

Dynamic Modulation of Pleiotrophin Expression in Young and Old Mice Following Injury 

Abstract: 

Aging results in the deterioration of many physiological processes including the body’s ability to regulate metabolism. The hypothalamus in the brain is one of the main regulators of body weight and metabolism. Interactions between hypothalamic glia and neurons are critical for proper regulation of metabolic processes. One of the mediators of these interactions are tanycytes; tanycytes are specialized glial cells lining the wall of the third ventricle that proliferate to replenish cells, regulate cellular signaling, and secrete paracrine signals. Tanycytes and neurons in the hypothalamus may undergo age-related decline, which contributes to dysregulated energy homeostasis in old age. Our lab has previously shown that the introduction of the neurotoxin monosodium glutamate (MSG) ablates neurons outside of the blood brain barrier in young mice, although they do not exhibit a gross phenotype. MSG treatment has also been observed to induce tanycyte proliferation in young mice, but not in aged mice. We aimed to examine the differences in the ability of tanycytes to produce pleiotrophin (PTN), an important growth factor, in response to hypothalamic damage. Young (3 months) and old (20 months) male, wildtype mice were intraperitoneally injected with MSG to induce damage in neurons exposed to the circulation. After MSG-induced hypothalamic lesioning, young mice demonstrated increased PTN expression in alpha 2 tanycytes. Alpha 2 tanycytes extend their long basal processes into the arcuate parenchyma and are in close vicinity of neurons in the mediobasal arcuate nucleus. The increased expression and release of PTN by tanycytes may promote neuronal repair and enhance neuronal survival. Importantly, our results show that tanycytes in aged mice are unable to upregulate PTN expression in alpha 2 tanycytes, indicative of defective tanycyte functions and hypothalamic repair. This age-related phenotype may contribute to the reduced survival of hypothalamic neurons and metabolic impairment after exposure to circulating toxins. 

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Ian Swinburne , Department of Molecular & Cell Biology, University of California  

Quantifying Iron Uptake in Hematopoietic Stem Cells 

Abstract: 

Understanding the timing of iron internalization during hematopoietic stem and progenitor cell (HSPC) emergence is of high importance, as high or low quantities of iron loading have the potential to make cells sick. Zebrafish are excellent models for studies of blood; therefore, in order to monitor receptor-mediated endocytosis, a process important to iron-loading in red blood cells (RBCs), I engineered genome-edited strains in zebrafish that mark the endogenous localization of known endocytic marker proteins, Clta and Ap2. To determine where and when HSPCs internalize iron during their maturation into RBCs, I designed transferrin delivery experiments to observe iron internalization in vivo for the first time. My results address previous literature controversy regarding the location of the majority of iron absorption, as they suggest that the primary absorption location is in the endothelial cells, not RBCs. Upon imaging the endocytic markers in vivo, I observed the peculiar depletion of Clta, but not Ap2, in RBCs, unveiling an unknown interaction with late stage RBCs. In regards to the timing of iron internalization, I assayed separate life stages with these transferrin injections, predicting that a change in life stage would result in a change in internalization; I then discovered that zebrafish at later stages (~30hp) no longer internalize iron, although the endocytic markers of Clta and Ap2 still prevail. These established baselines of timing and location led me to question if cells would respond to exogenously-elevated iron levels, and I suspect a down-regulation of endocytosis through the detection of iron stores to occur in the subsequent experiments I have designed: delivering iron-dextrans to single-cell embryos, and preparing varying concentrations of hinokitiol and ferric iron soak solutions to deliver iron as an external source. 

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Randy Schekman, Department of Molecular & Cell Biology, University of California, Berkeley   

"Exploring intercellular cargo transfer through open-ended tubular connections"

Abstract: 

Congyan Zhang, Flora Jin, and Randy Schekman

Department of Molecular and Cell Biology

Howard Hughes Medical Institute

University of California, Berkeley

Diverse signaling pathways in cell-to-cell communication systems are required to coordinate the function and behavior of individual cells in multicellular organisms. Differing from the secretion of signaling molecules and signal transmission through gap and synaptic junctions, open-ended membrane tubular connections are membranous transient connections between cells that transfer cargo and facilitate long-range intercellular communication. These tubes show a wide range of functions, but we lack efficient mechanisms to study them. Data from previous experiments showed that the transfer of Cas9 appeared to be mediated by open-end membrane tubular connections, and not all cells were effective donors or acceptor cells in this intercellular transfer of Cas9. We found that HEK293T cells were very effective donors when combined with a breast cancer cell line, MDA-MB231. HEK293T cells were not effective as a receptor in spite of the fact that cultures of these cells form intercellular tubular connections.

Therefore, we designed a novel strategy to study cargo transfer through membrane nanotubes. Using NanoLuc Binary Technology (NanoBiT) and the NanoBRET Nano-Glo Detection System, we constructed a split nanoluciferase assay with HEK293 cells and MDA-MB-231 cells. We tagged one cell line with HiBiT, a small 11 amino acid peptide, and another with LgBiT, another larger subunit, and enhanced them with SBP–streptavidin complex to optimize complementation. The bound complex shows luciferase activity, allowing us to measure cargo transfer through membrane tubes alone, as the HiBiT and LgBiT complexes complement through the tubes to form the luminescent nanoluciferase. The observation that cargo transfer through open-ended membrane tubular connections only occurs between different cell lines allows us to further confirm the signal’s source. Controlling for cell confluence with tagged firefly luciferase, co-culture assays with both 293-HiBiT + 231-LgBiT-Fluc and 293-LgBiT-Fluc + 231-HiBiT coambinations showed ~90-fold increase in luminescence intensity. These results confirm that the split-nanoluciferase assay can be applied to the study of intercellular cargo transfer through membrane nanotubes. Next, we used the strategy to screen potential fusogens and receptors, identifying a number of candidate proteins including retroviral protein syncytin, dynamin, and multiple GPCR-associated proteins. Further investigation through forskolin and H89-treated co-culture experiments supports GPCR involvement in tube formation, and actin inhibitor assays with latrunculins A and B support dynamin involvement. I will further examine these protein candidates through CRISPR knockouts and examine the localization of these proteins through fluorescence microscopy.

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Bruce Wang , University of California, San Francisco 

"Longitudinal Transcriptomic Profiling of Rodent Liver Fibrogenesis at the Single Cell Level" 

Abstract: 

Fibrogenesis is a process of excessive wound healing response that results in the deposition of scar tissues. In the liver, fibrosis is the most common pathology observed in chronic liver diseases, and is often initiated with injury to epithelial cells, such as hepatocytes. Therefore, hepatocytes are likely to play an important role, not only in the initiation, but also progression of liver fibrosis. As a result, characterizing the network of downstream molecular events upon liver injury may present attractive targets for therapeutic intervention. Based on a previously established liver single-cell RNA sequencing (scRNASeq) and single molecule fluorescent in situ hybridization (smFISH) workflows, analysis of the longitudinal hepatocyte spatial and transcriptomic changes can be performed in a well-established liver fibrosis rodent model where carbon tetrachloride (CCl4 ), a hepatotoxin, leads to pericentral hepatocyte death. To investigate gene expression changes, we have generated an experimental setup with acute CCl4 injury to male mice at timepoints of day 1, 2, 3, 5, and 14, chronic CCl4 injury at timepoints of week 2, 4, 6, and 8, as well as control male mice. We have successfully captured various cell populations representative of the rodent liver via scRNASeq, and are analyzing cell population proportions and gene expression changes across experimental groups. Current analysis has exhibited the conserved zonation of well-characterized liver cell populations across injury experimental groups. Namely, within hepatocytes, typical hepatocyte subpopulations are largely maintained, with an expanded proliferating hepatocyte population present in both acute and chronic experimental groups, that is otherwise absent in the control groups. Further analysis will involve deeper investigation into other major liver cell populations, such as endothelial cells and macrophages, and eventually an effort to delineate the regulation of different signaling pathways between the injury groups. 

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Sona Kang , Department of Nutritional Sciences and Toxicology, University of California Berkeley 

"Investigating the Functional Role of CILP in Adipogenesis" 

Abstract: 

DNA demethylation has been shown to be critical for adipogenesis, which significantly influences energy homeostasis and metabolic health. The Kang Lab previously found that mice with adipocyte precursor cells (APCs) deficient in TET3, a DNA demethylase, showed reduced adipose mass. Furthermore, genome-wide RNA-seq and DNA methylation profiling studies revealed target genes differentially regulated in the tissues of Tet3 knockout mice. The functional roles of these differentially regulated genes in adipose development remained unknown. Among the downregulated candidates, this study narrowed down to the cartilage intermediate layer protein (CILP). CILP is known to be involved in extracellular matrix remodeling in hearts and muscles. The protein is also enriched in particular APC sub-populations. However, the effect of CILP on adipogenesis has not been previously investigated. The purpose of this study is to figure out the functional role of CILP and how it affects the capacity of preadipocytes to differentiate. We demonstrated that Cilp expression significantly increased during adipogenesis in vitro. Additionally, we observed a higher expression of Cilp in the white adipose tissues of obese mice. We further investigated the functional role of CILP in TET3-mediated adipogenesis using gain-of-function studies. The results from Oil Red O staining and gene expression analysis showed that recombinant CILP treatment enhanced the adipogenic capacity of 3T3-L1 cells. Overall, the data suggest that CILP may be an important molecular target of TET3 to promote adipogenesis. 

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Matthew Springer , Division of Cardiology, University of California San Francisco, 

Evaluation of NIDA Research Marijuana through Rat Serum Analysis in the Study of Effects on Cardiovascular Health 

Abstract: 

Summary: With progressive widespread use of marijuana, there is an urgency to understand the potential detrimental health effects of active use and secondhand exposure. Much of the current known use of marijuana comes from studies using DEA-approved research marijuana from the University of Mississippi, administered by National Institute on Drug Abuse (NIDA). However, the significantly lowered THC levels in NIDA-supplied marijuana, among other differences between NIDA and real-world material, have raised concerns about the validity and accuracy of these studies in relation to human health. The goal of this study is to validate or identify limitations in the use of NIDA-research marijuana, in conjunction with a study of cardiovascular and physiological effects of cannabis.

Methods: We chemically analyzed cannabinoids and terpenes in plant material, smoke, and vaporizer aerosol to characterize the products and determine how chemical composition is changed by the drying regimen. We also determined if the drying regimens and/or cannabinoid profiles influence the known impairment of vascular function in exposed rats, and examine effects on blood pressure and platelet aggregation. Finally, we examined the serum from exposed rats to determine functional and pro-inflammatory effects, and potential changes in serum that impair function of cultured endothelial cells.

Current results: While acute exposure to marijuana smoke or aerosol does not significantly vary inflammatory properties, there is a significant difference in permeability between testing and control groups (but not among groups). 

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Matt Springer  , University of San Francisco Parnassus Campus 

Coiled vs Coil-less Vapes: Differences in Cardiac Fibrosis and Heart Rate Variability 

Abstract: 

Vaping devices have gained an immense amount of attention over the past decade as a “healthier” alternative to cigarettes and have become the most popular way for teenagers and young adults to smoke nicotine. Since the early 2000s there have been different types of vaping devices released into the market including coiled vaping devices such as the Juul and other coil-less vaping devices such as the Surge that utilize ultrasonic technology to heat the vapor instead of directly through nichrome coils that Juuls use. Our study examines if the Surge coil-less vaping device is less toxic to the body and destructive to the function of the cardiovascular system compared to the Juul Coiled vaping device through measurement of cardiac fibrosis, ultrasound of the heart, and heart rate variability.  

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Jay Keasling  , Department of Energy: Joint BioEnergy Institute 

Taking off on a Journey Towards Renewable Jet Fuel Using Directed Evolution

Abstract: 

Polyketide Synthases (PKS) are enzyme complexes that generate polyketides in antibiotics/plastics. My project tests the NADPH turnover of both a polyene PKS (SgcE) versus a Fatty Acid Synthase (FAS1A) within MX203 E. Coli. SgcE has a very low product turnover because its accumulated NADPH requires a heterologous reductase for metabolism. Additionally, FAS1A undergoes two essential reduction steps with NADPH that I hoped to engineer a more efficient PKS out of. In both cases of metabolism, the Pentose Phosphate Pathway (PPP) requires a reduction of NADPH per glucose. My aim was to couple the MX203 growth to its reductase activity, and to have the more efficient of SgcE or FAS1A to convert its substrates (Malonyl-CoA and acetyl-CoA respectively) into a sustainable/alternative jet fuel product called Pentadecaheptaene.

After growing SgcE and FAS1A in 3 different E.Coli strains, I found that SgcE was no longer growth coupled (lower NADPH turnover) compared to the FAS1A which exhibited significantly higher growth with a functional Acyl Carrier Protein (ACP) compared to its inhibited ACP form (measured by OD). I discerned that FAS1A reduces more NADPH generated from the PPP (glucose→ malonate generates 6 NADPH per 3 glucose and 5 Malonyl-CoAs), while SgcE only has one reductive step that cannot reduce all NADPH made from MCoA. 

Focusing on FAS1A’s redox cell rescue, I hypothesized that FAS1A’s metabolism is induced by IPTG and malonate transporters in the cell. I tested FAS1A growth +/- malonate in the media for several malonate transporters, and witnessed cells growing at 3x the rate (Flaviolin as a colored indicator for MCoA) faster with increasing IPTG induction relative to the absence of exogenous malonate. With MCoA in the cells, my PhD mentor Aidan and I have successfully improved selection by uncoupling malonate reduction and glucose oxidation to generate MCoA without oxidizing glucose. 

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Randy Schekman  , Department of Molecular & Cell Biology

Characterizing the Extracellular RNA Profile of Fission Yeast and Human Cells

Abstract: 

RNAs of various sizes and classes are present outside of the cell membrane in all three domains of life. These extracellular RNAs (ExcRNAs) are proposed to mediate intercellular, interorganismal, and even interkingdom communication. We show here that the apoplastic space of Schizosaccharomyces pombe provides a powerful model system for investigation of ExcRNAs. As shown through protease, detergent, and nuclease treatments, the ExcRNA profile of fission yeast closely resembles that of human cell culture. This opens the door for fission yeast to be used as a model for probing ExcRNA pathways and functions.

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Babak Javid  , University of California, San Francisco

Generating a High Mistranslating Strain of BCG as a Novel Vaccine Candidate 

Abstract: 

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the world's deadliest bacterial infection, killing approximately 1.5 million people annually. The only licensed vaccine for the prevention of TB is the century-old live attenuated strain M. bovis-BCG (BCG). Although BCG can mitigate serious forms of childhood TB, it has limited efficacy in preventing the transmissible form of pulmonary tuberculosis. Preliminary data shows that Mtb has a high rate of specific errors in protein synthesis (mistranslation). Furthermore, Mtb requires mistranslation to adapt to the host environment. These data suggest that mistranslated proteins and peptides may be required for Mtb to evade host innate and/or adaptive immunity. We hypothesize that by constructing a strain of BCG with high mistranslation rates, this may provoke immunity that would specifically target these mistranslated proteins, and hence mistranslating BCG may be a superior vaccine candidate. In this thesis I propose to isolate and characterize strains of BCG with high, specific rates of mistranslation. 

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David Weisblat  , Department of Molecular & Cell Biology

Leech Neurons Differentiate in Size Similarly Across Ganglia After Reaching Juvenile State 

Abstract: 

Mapping the structure and cellular inventory of nervous systems allows for precise manipulation and observation such as knockouts targeting specific neurons. For example, in C. elegans, each of the 302 neurons has been identified and characterized by cell lineage and connections to other neurons. The anatomically simple C. elegans system appears to be invariant in terms of neuron number and anatomy, in contrast to vertebrate nervous systems. At what point in the spectrum of neural complexity does variability emerge? This study contributes to the investigation of this question by studying the nervous system of the leech Helobdella austinensis. Leech central nervous systems comprise 32 segmental ganglia, each of which contains about 400 neurons, many of which are uniquely identifiable on the basis of the size and position of their cell bodies. Preliminary segmentation analysis results reveal that ganglia nuclei volumes increase and become more variable as leeches develop to adulthood. Neuron count and nuclei volume are similar between midbody ganglia and between adult individuals.

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Nina Dronkers  , Department of Psychology, University of California, Berkeley

The Cognitive Effects of Acute High-intensity Interval Training on Healthy Young and Older Adults 

Abstract: 

About one-third of stroke patients develop aphasia, a debilitating language deficit caused by damage to the language areas of the brain. The standard treatment for aphasia is Speech-Language Therapy, which involves working on targeted language tasks to improve communication. However, recovery with Speech-Language Therapy is often partial and slow. Aerobic exercise has been shown to enhance cognition in stroke survivors in functions commonly impaired in people with aphasia. This study seeks to pilot a new high-intensity interval training (HIIT) routine that could boost cognitive functions impaired by stroke, such as executive control, with the ultimate goal of validating this routine as an adjuvant treatment for people recovering from stroke. This new Aphasia Physical EXercise (APEX) program was designed with stroke survivors in mind to provide the same cognitive boost as traditional continuous aerobic exercise regimens while still being safe and feasible for individuals who may have limited mobility. The APEX program was piloted in two populations: healthy young adults and elderly adults age-matched to the target stroke population. The young adults underwent an acute bout of exercise and subsequently reacted faster in tasks that tested executive control (Stroop Task and Visual Search Task) compared to pre-exercise reaction times. The older adults had greater working memory capacities following exercise. These results demonstrated the feasibility and safety of the APEX program, and future studies will test the effectiveness of APEX in the stroke population.