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Ehud Isacoff, Department of Molecular & Cell Biology, University of California, Berkeley

"Characterizing the Functional and Structural Effects of Pasilla Knock Down on the Drosophila Neuromuscular Junction"

Abstract:

Neurons stabilize transmission at synapses by undergoing activity-dependent plasticity in response to factors disrupting their homeostatic balance. In the neuromuscular junction, this synaptic activity is highly regulated by the voltage-gated sodium channels of motor neurons. Pasilla (Ps), a Drosophila mRNA binding protein, is known to regulate alternative splicing of the gene encoding the primary voltage-gated sodium channel in Drosophila neurons, paralytic (para), favoring a more sensitive isoform. However, the effects of pasilla expression on neurotransmission and plasticity have not been well characterized. We investigated this in the Drosophila larval neuromuscular junction (NMJ) by using RNA interference (RNAi) against pasilla and measuring synaptic output in vivo with a postsynaptic activity reporter, SynapGCaMP6f. Structural effects were evaluated through immunohistochemistry using synaptic active zone protein Bruchpilot. Cross-correlation of NMJ ΔF/F and muscle fluorescence, as well as cross correlation of mean NMJ fluorescence and muscle fluorescence were found to be greater in the pasilla knockdown, while the number of contraction bouts was lower. These findings suggest that compensation for pasilla knockdown is postsynaptic.

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Kaoru Sajio, Department of Molecular & Cell Biology, University of California, Berkeley

"Investigating Cell Type Specific Expression of Prostaglandin D2 Receptors in the Fetal Mouse Brain at e15.5"

Abstract:

Prostaglandins (PG) have been found to impact developmental processes such as cell migration in the immune system and dendrite growth in the postnatal brain, but little work has been done relating PGs to fetal brain development. We used quantitative real time PCR (qPCR) to identify cell type specific expression of genes for two prostaglandin D receptors, DP1 and DP2, in the fetal brain. We found that non-myeloid neural cells express both receptors, and that there are no statistically significant sex differences in expression.

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Fenyong Liu, School of Public Health, University of California, Berkeley

"Role of Human Complement Factor H and Its Interaction with Human Cytomegalovirus"

Abstract:

Human cytomegalovirus (HCMV) infection is typically asymptomatic in immunocompetent individuals but can cause severe illness or death in immunocompromised individuals such as HIV/AIDs patients or solid-organ recipients. Drugs currently used to treat HCMV are moderately toxic and can lead to drug resistance, hence the need to identify new viral targets for drug or vaccine development. HCMV has one of the largest genomes of any human pathogen, and while previous studies have elucidated which genes are necessary for survival and replication, the precise functions of many of these genes still remain unknown. HCMV encodes many proteins to inhibit host innate and adaptive immunity, but the role of the complement cascade in conferring immunity towards HCMV has not been studied extensively. Complement is a system of proteins circulating in the blood and on cell surfaces that is part of the innate immune system but can also influence the adaptive immune response. Complement proteins like Factor H (FH) are important for regulating complement to prevent host cell damage but are known to be hijacked by microbes to evade destruction. In this study, I employed a yeast two-hybrid assay to screen for protein-protein interactions between human FH and a library of HCMV genes. Of the 175 HCMV genes tested, 17 (9.7%) high-confidence interactions were identified, seven of which have been previously reported to be involved in immune evasion, but none have previously been reported to interact with Factor H. This study represents the most comprehensive investigation of a single complement protein tested against the HCMV genome for protein-protein interactions to date. These findings have the potential to inform the development of novel antiviral therapeutics and potential vaccine candidates for the prevention of HCMV infections in humans.

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Andreas Stahl, Department of Nutritional Sciences & Toxicology, University of California, Berkeley

"The Effects of NRG4 Ablation in a Synthetic Brown Adipose Tissue on Whole Body Substrate Utilization"

Abstract:

For a long time, brown adipose tissue (BAT) was considered to be important only in human infants and hibernating animals. We now know that it is also present and functional in adult humans as well. Through Uncoupling Protein 1 (UCP-1), the energy of the mitochondrial protein gradient is dissipated as heat as a response to cold temperature. One of BAT’s secreted factors is Neuregulin 4 (NRG4), which reduces lipogenesis in the liver. The goal of this project is to ablate NRG4 in a BAT-specific manner and observe how this affects whole body metabolism. To do this, we used two lentiviral vectors that together use the CRISPR/Cas9 system to knock out the NRG4 gene. The first vector contains a tetracycline-inducible Sniper-Cas9 protein, which is a Cas9 variant with optimized specificity. The second vector contains two sequences encoding their own short guide RNAs that target an early exon of the NRG4 sequence, the td-Tomato gene, and a backbone that expresses the luciferase gene. These gene elements are simultaneously introduced into a backbone vector using the Multiple Lentiviral Expression system (MuLE), which is based on Gateway cloning. These plasmids are delivered into a brown pre-adipocyte cell line and gene ablation is validated through qPCR. The cells are differentiated, implanted in brown adipose tissue organ-like hydrogels, and implanted into mice. The mice are placed on a tetracycline chow diet to induce Cas9 activity and a panel of physiological assays are conducted to assess effects on whole body substrate utilization and thermogenesis.

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Gerard Marriott, Department of Bioengineering, University of California, Berkeley

"Mechanoluminescent Probe for Artificial Bones and Joints"

Abstract:

Mechanoluminescence (ML) is light generated by certain materials in response to physical stimuli. ML ions have been, to date, most widely applied to imaging defects in inanimate samples such as girders on bridges. To the best of our knowledge, their application to medicine has been limited to a single study. In my research project, I developed adhesive ML materials that can be used to detect defects and faults in prosthetic devices and bones before they are implanted into patients – importantly, our sensors are unique in showing how stress associated with an external load is distributed throughout the whole device whereas current sensors can only focus on single point contacts. The significance of this work is clear, as we could identify micro-fractures or other defects in an implant that are otherwise invisible before the surgery. In our approach, we apply a thin adhesive film of “ML skin” to the surface of a prosthetic harboring a known defect and then image the ML in response to defined loads. A defect such as a microfracture leads to the channeling of the stress along the fracture, which is reflected in a non-uniform ML intensity that peaks along the lines of increased stress. Specifically, I developed an ML skin in which a biocompatible inorganic crystal (ZnS doped with 1% manganese) is integrated into a thin biopolymer (PLL-PEG) that can tightly bind to the surface of the implant. Finally, I evaluated the effectiveness of this sensor to image stress distribution in the implant during the application of an external load. The usage of ML crystals to detect faults in bone prosthetics prior to the operation could be immensely beneficial in decreasing the number of preventable surgical failures.

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Joshua Woolley, MD, PhD, University of California, San Francisco

"Effects of Oxytocin on Automatic Motor Simulation and Imitation-Inhibition in Patients with Schizophrenia, Schizoaffective Disorder, and Schizophreniform Disorder"

Abstract:

Patients with schizophrenia exhibit social impairments that may be attributed to neurocognitive deficits, such as abnormalities in imitation—the translation of perceptions into executed actions. Oxytocin, a neuropeptide with pro-social effects when administered in humans, has therapeutic potential in improving social cognition. In this randomized, double-blind study, male patients [n=39] with schizophrenia, schizoaffective, or schizophreniform disorder were given oxytocin or placebo on two separate testing days before completing the imitation-inhibition task; this paradigm focuses on the automatic imitation of simple finger movements in response to a cue while observing either congruent (matching) or incongruent (non-matching) movements. Healthy controls [n=51] completed the same task. The results indicate that patients have a higher congruency effect (the difference between incongruent and congruent trials) than healthy controls and therefore exhibit enhanced imitation; previous studies reporting imitation impairment in this clinical population may be confounded by other motoric or cognitive deficits. Oxytocin was also not shown to significantly impact the congruency effect, while anti-dopaminergic medication, attachment styles, and symptom severity were not significant predictors of oxytocin-mediated performance.

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Mark Ansel, University of California, San Francisco

"DeepCLIP: A Deep-Learning Based Approach for Comparative CLIP-Seq Analysis"

Abstract:

DeepCLIP is an algorithm that employs advances in deep learning algorithms to solve the problem of comparative analysis of CLIP-Seq experiments. At the heart of this approach lies the use of deep learning to predict differentially enriched sites in paired CLIP-Seq data. DeepCLIP is enabled by a multi-layer long short-term memory (LSTM) model, a recurrent neural network, trained using published Argonaute HITS-CLIP data from wildtype and miR-155-deficient mouse T cells. DeepCLIP was applied to identify the physically bound targets of miR-23, miR-24 and miR-27 using new comparative HITS-CLIP data generated from wildtype and miR-23/24/27-deficient T helper type 2 (Th2) cells. First, this paper will present an extensive benchmarking of the performance of DeepCLIP and the current prescription for differential CLIP-Seq analysis (that is, dCLIP 1.7). DeepCLIP improves upon dCLIP in terms of the sites called to be differentially enriched, the localization of predictions with greater precision, and in terms of providing a quantitative measure of differential enrichment. Furthermore, DeepCLIP yielded superior insights in the investigation of the RNA secondary structural motifs that enable miRNA-Ago2 targeting of RNA. Lastly, the superior performance of DeepCLIP was shown to hold when predictions are translated to diverse biological milieux.

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Jay Graham, School of Public Health, GL51 Koshland Hall, University of California, Berkeley, CA

"Comparative Genomic Analysis of Carbapenem Resistant E. coli in Alameda County, California, 2017-2019"

Abstract:

Carbapenem-resistant Enterobacteriaceae (CRE) prevalence has drastically increased and per the CDC, is a serious public health threat. Carbapenems are considered the last line of defense for many severe infections. Escherichia coli (E.coli), a member of the Enterobacteriaceae family, presents a unique health risk and are associated with significant morbidity and mortality in infected individuals, and an increasing number of reports describe extra-intestinal and invasive infections with these organisms. Extra-intestinal pathogenic E.coli (ExPEC) are derived from a small number of phylogenetic lineages, many of which are considered pandemic lineages. Four sequence types (STs, defined using multi-locus sequence typing) are responsible for nearly half of all the E.coli urinary tract infections and blood stream infections in the world. However, few studies have evaluated the genetic make-up of Carbapenem-resistant E.coli, including the antimicrobial resistance and virulence determinants. The aim of this study was to characterize and compare sequence data from carbapenem resistant E. coli isolates provided by Alameda County Public Health Laboratory (ACPHL). Eighty-two CRE E.coli isolates were sequenced using standard unidirectional Illumina sequencing by ACPHL and provided for analysis. The sequencing reads were assembled with Unicycler using Savio (Berkeley cloud computing). Isolates were screened for MLST, antimicrobial resistance genes (ARGs), plasmids, and ExPEC virulence factor genes (VFGs) using Abricate. Molecular analysis revealed a high level of genetic diversity, containing 24 distinct STs, including: ST131, ST69, ST95, ST73. All classes of Carbapenemases were present, with NDM-5 most frequently detected (24.4%). Multi-drug resistant gene mdf(A)-1 was present in all isolates; while, 90 percent of all isolates also contained 3rd generation cephalosporin resistance genes. Genes conveying resistance to aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines were pervasive (>50%) and represented numerous challenges to treatment for these organisms. The number of virulence genes within pandemic STs was significantly higher than non-pandemic lineages (p=3.51839e-2) with virulence genes fimA (92%), trat(71%), kpsM (54%), and iutA(46%) being present in most ST types. Considering the potential public health risk associated with CRE, this data enhances the understanding of clinically important E.coli that are present in Alameda County, California. This study describes the most common types of virulence and resistance genes indicating the need for further collaborative whole genome sequencing-based surveillance.

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David Raulet, Department of Molecular and Cell Biology

"Exploring CD4 Mediated Tumor Rejection in CDN/H9-MSA Combination Therapy"

Abstract:

Many cancer immunotherapies aim to mobilize CD8 T cells. However, numerous tumors evade CD8 T cell responses by losing MHC. It has been shown that intratumoral injections with STING agonists (CDNs) can induce primary tumor regression. Although MHC I-deficient tumors are not susceptible to CD8 T cell-mediated rejection, data from our lab has shown STING activation still leads to tumor regression in up to 100% of animals, mediated by NK cells and CD4 T cells. With the aim of improving CDN therapy, my mentor combined CDNs with an engineered IL-2 “superkine” (H9-MSA) and showed synergistic activity in mobilizing anti-tumor responses by NK cells and CD4 T cells in mice with transplanted MHC I deficient melanoma tumors (B16F10 B2m-/- model). The mechanism by which CD4 cells reject the tumors is largely unknown. I interrogated the CD4 anti-tumor response by addressing whether the tumor cells must be recognized directly by CD4 T cells via MHC II. Further, I investigated the frequency and activation status of tumor-specific CD4 T cells in mice treated with combination therapy. My results showed that NKDTA mice treated with combination therapy showed enhanced tumor rejection and that CDN/H9-MSA therapy induces activated anti-tumor CD4 T cells in vivo. Additionally, the active MHC II gene can be successfully knocked out in B16F10 B2m-/- melanoma cells.

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David Agard, University of California, San Francisco

"DNA Binding Activity of a Protein Forming the Nucleus-like Structure in Jumbo Bacteriophage ɸPA3"

Abstract:

The ɸKZ-like bacteriophage ɸPA3 creates a novel nucleus-like compartment, isolating viral DNA from the cytoplasm of its host. This “phage nucleus” assembles after DNA injection and grows up to half the host cell size or about 0.5 um in diameter as DNA replicates within. One of the primary proteins thought to comprise this compartment is gene product 53, or gp53. Gp53 is the most abundant non-virion particle protein but has no previously characterized homologous proteins, making its structure and function unknown. In unpublished work, our lab has found that purified gp53 assembles into nanometer-scale structures, perhaps indicative of its role in forming a large structure in vivo. Utilizing fluorescence anisotropy, this work further characterizes gp53 reporting previously unknown DNA and ATP binding activities. Notably, we report that gp53 binds DNA in a concentration dependent manner and prefers AT-rich nucleotide sequences. Ongoing work aims to determine whether nucleotides and oligonucleotides compete for the same site or whether gp53 possesses two separate binding sites for these substrates.

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Charles Chiu, Department of Laboratory Medicine, University of California, San Francisco

"Novel Methodology for Rapid Detection of Pathogens with CRISPR-Cas12a and Sequencing"

Abstract:

As of April 2021, over 141 million people were infected with SARS-CoV-2, a virus which rapidly and dramatically changed (and continues to change) people’s lives during the COVID-19 pandemic. Separately, in 2016, nearly 60,000 people were diagnosed with a tickborne infection; however, due to the nature of tickborne diseases, diagnosing and rapidly treating a tickborne illness is immensely difficult. Due to recent advances in CRISPR, sequencing, and primer design technology, we can now detect pathogens, especially emerging ones, through universal amplification of a conserved region, and then selective fluorescence with CRISPR to help figure identify pathogens within large quantities of samples. From there, this expedient test can directly be inputted into the Oxford Nanopore real time sequencing machines to directly sequence, thus verifying the pathogen. Through the development and testing of this assay, new variants of the SARS-CoV-2 virus was identified, leading to the reinforcement of the immense power of these novel technologies.

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Iswar Hariharan, Department of Molecular and Cell Biology, University of California, Berkeley

"Characterizing the Role of Fos Isoforms in Growth Regulation Pathways in Drosophila"

Abstract:

Fos is a transcription factor involved in various genetic pathways, including through a heterodimeric interaction with Jun, forming the transcriptional activator protein complex AP-1. AP-1 is an important downstream target in JNK signaling, a pathway that has been shown to trigger a wide array of downstream consequences from apoptosis to tumorigenesis when AP-1 is activated. AP-1 has been shown to be important for the activation of many different transcriptional targets. However, recent work in the Hariharan lab suggests that Fos may also as a transcriptional repressor in some contexts. Sumabat et al. found that the transcriptional co-repressor, C-terminal Binding Protein (CtBP) is a negative regulator of growth that represses a number of AP-1 target genes including the microRNA bantum (ban). The repression of ban expression occurs through the brc12 enhancer, which is known to contain AP-1 binding motifs. In searching for possible binding partners between CtBP and other transcription factors, it was observed that two out of five isoforms of the Drosophila Fos protein (also referred to as Kayak) contain the recognized CtBP binding motif (PxDLS). Some preliminary evidence also suggested that these Fos isoforms could be forming a transcriptional repressing complex with CtBP and acting to repress the expression of ban.

To further characterize the interaction between Fos and CtBP, we investigated the role that Fos protein isoforms play in growth regulation in Drosophila. CRISPR/Cas9 was used to create mutations in the genome that altered only the Fos isoforms that contain the PxDLS binding motif (isoforms A and G). These new isoform-specific mutants allow us to explore three main questions: (1) Are these isoforms of Fos required for normal development and growth? (2) Do these isoforms, Fos.A and Fos.G, negatively regulate the expression of JNK/AP-1 target genes (such as ban)? and (3) Finally, does negative regulation by these isoforms of Fos depend on interactions with CtBP? We addressed these questions in Drosophila by comparing mutant and wild-type cells within a developing tissue, the wing imaginal disc. Using genetic tools, we created tissues that contain both wildtype and Fos.A mutant cells to specifically compare the level of target gene expression via transcriptional reporters and immunofluorescent imaging techniques. In addition, co-overexpression experiments of different Fos isoforms together with CtBP were conducted to determine if the predicted complex can act to repress gene expression targets. Preliminary analysis of these experiments suggested that Fos.A plays a role in negatively regulating gene expression of AP-1 target genes, and that Fos.A could be acting together with CtBP to repress gene expression.

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Lin He, Department of Molecular & Cell Biology, University of California, Berkeley

"Characterizing the ultrastructure of a new type of choroid plexus cilia that regulate cerebrospinal fluid production"

Abstract:

Cilia are traditionally classified by their function and structure as either motile or non-motile (primary). There is a new type of cilia found on choroid plexus epithelial cells (CPECs) that are important to mammalian homeostasis as they are responsible for regulating cerebrospinal fluid (CSF) production in the brain. Because little is known about CPEC cilia and their corresponding basal bodies, this project characterizes their ultrastructure using images taken from Focused Ion Beam milling combined with Scanning Electron Microscopy (FIB-SEM). We used 3-D rendering software such as Amira and ITK-SNAP to analyze raw FIB-SEM images from choroid plexus samples of wild type CPEC mice from 3 different life stages: E12.5, P0, P14, and segmented cilia and basal bodies to provide an overview of the structure. We quantified cilia length and subdistal appendage (SDA) numbers on each basal body and discovered that CPEC cilia have key novel characteristics not present in any other type of cilia. We found that each CPEC had multiple cilia, which displayed axoneme shortening during development, and that each cilium originated from heterogenous basal bodies with differing SDA numbers. Although very little is understood of CPEC cilia, our project was able to characterize their ultrastructure during development, and our observations suggest CPEC cilia have many novel characteristics not found in either motile or primary cilia. This new finding can translate to better understanding the homeostasis of CSF production and have many applications in medicine, namely, treating and preventing hydrocephalus.

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David Raulet, Department of Molecular & Cell Biology, University of California, Berkeley

"Engineering Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells for Treatment of Glioblastoma"

Abstract:

Immunotherapy has quickly evolved to become a robust form of cancer treatment. A cellular immunotherapy, chimeric antigen receptor (CAR) engineered immune cells such as CAR-T cells and CAR-NK cells are providing platforms to treat various malignancies. Solid tumors such as Glioblastoma (GB), or Glioblastoma multiforme, have tumor microenvironments that are highly immunosuppressive. GB is the most common and aggressive brain tumor in adults, which successfully suppresses the self-defense functions of the T and NK cells from the immune system, which allows for invasion into healthy tissue. This proposal is aimed at exploring the use of CAR-Natural Killer (NK) cells to treat cancer. NK cells are natural cytotoxic cells of the innate immune system in the body, which can be isolated from healthy donors, and then engineered with chimeric antigen receptors (CAR). The characteristics of the NK cell highlight it’s potential as a form of adoptive cell therapy. My project aims to develop an EGFRvIII-targeting CAR-NK to treat Glioblastoma through two components – optimizing transduction efficiency of the EGFRvIII-targeting lentiviral vectors and propagating the CAR plasmid construct. NK cells are notoriously difficult to transduce, so developing an efficient protocol is essential for success. My results showed that culture conditions using mixtures of IL-12, IL-15, and IL-18 were very promising. Furthermore, the use of dextran, a glucan polysaccharide, resulted in improved lentiviral transduction of NK cells, in comparison to the cationic polymer, polybrene. Taken together, these data provide means to carry out studies that will examine the therapeutic usefulness of CAR-NK cells in treating patients with glioblastoma multiforme.

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Matthew Krummel, Department of Pathology, University of California San Francisco

"Characterization of Antigen Uptake and Presentation via Different Fluorescent Proteins"

Abstract:

Despite recent advances in cancer immunotherapy, many cancer patients still have non-favorable clinical outcomes and do not regress tumors. Tumor regression and effective long-lasting immune response towards cancer relies on antigen uptake and presentation by antigen presenting cells (APCs) and is thus studied intensively. In these studies the flow of antigens is commonly visualized through the expression of fluorescent protein (FP) ZsGreen, GFP, Venus, and mCherry. So far ZsGreen is the only stable FP that can be tracked efficiently towards the lymph node. We aim to analyze and compare different FPs in their ability to be tracked in antigen uptake and transfer models.

Based on sequence homology and/or species of origin, we chose to examine ZsYellow, DsRed, AsRed, mCherry, and mRFP. Expression of fluorescence in five respective cell lines, along with ZsGreen, was assessed in vitro via a stability assay across different pH environments. Fluorescence stability was then assessed in vivo through analyzing FP antigen uptake and transfer in immune cells isolated from subcutaneous tumors and draining lymph nodes. Strikingly, we observed that expression of mRFP and mCherry in tumor cells altered antigen uptake patterns when compared to ZsGreen expression. As such mRFP and mCherry signal was present predominantly in conventional dendritic cells (cDC) 1 and not in cDC2s or tumor associated macrophages (TAMs). The conventional pattern of antigen uptake should follow that of ZsGreen, where highest uptake is observed in cDC2s and TAMs. Future experiments need to consider that uptake and transfer might be impacted by protein expression, stability, or other unknown factors. Thus our data sheds light on potential under-appreciated processes regulating antigen transfer.