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Qili Liu, Department of Anatomy, University of California, San Francisco

"Ethanolamine signals protein hunger in Drosophila"

Abstract:

Organisms demonstrate homeostatic regulation of protein intake. Protein hunger, the motivational drive to search for and ingest protein in response to protein depletion, may contribute to overeating and obesity. The mechanisms underlying protein hunger, however, remain largely undiscovered. In the Drosophila model system, prior HPLC data revealed a strong correlation between ethanolamine levels in hemolymph (“fly blood”) and the degree of protein hunger. Building on this observation, we hypothesized that ethanolamine serves as a circulating messenger for protein hunger. In support of this hypothesis, here I provide evidence demonstrating a causal relationship between ethanolamine levels and protein hunger. By supplementing standard Drosophila diets with various concentrations of serine, a biological precursor for ethanolamine biosynthesis, I found that flies subsequently consumed more protein-enriched food in a serine dose-dependent manner compared to those fed on standard diets. Furthermore, my colleagues and I utilized RNA interference to knock down the expression level of different enzymes involved in the biosynthesis and metabolism of ethanolamine. Interestingly, neuronal knockdown of two such enzymes, phosphatidylserine decarboxylase (Pisd) and ceramide phosphoethanolamine synthase (Cpes), significantly reduced the preference for protein when animals were given a choice between protein- and sugar-based food. Our results suggest that the biosynthesis of ethanolamine from serine and the subsequent release of ethanolamine into circulation triggers protein-specific hunger. Future experiments will aim to confirm that circulating ethanolamine levels indeed increase after serine feeding and decrease when Pisd and Cpes are knocked down in the brain. We also plan to identify the receptor for circulating ethanolamine, as well as neuronal circuits responsible for converting this molecular signal into protein hunger. As the molecular pathways described here are largely conserved between flies and mammals, our work may shed light on the homeostatic regulation of protein intake in mammals, and reveal novel targets for addressing obesity in humans.

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

"The Role of Kaposi’s Sarcoma Associated Herpesvirus Open Reading Frame 74 in Spontaneous Reactivation"

Abstract:

Kaposi’s Sarcoma Associated Herpesvirus is the etiological agent of Kaposi’s Sarcoma, Multicentric Castleman’s Disease, and Primary Effusion Lymphoma. One of its many associated oncogenes is Open Reading Frame 74 (ORF74) which encodes the viral G-Protein Coupled Receptor (vGPCR). Several independent observations have classified this gene as a late gene, expressed only after viral DNA replication. Previous reports detail its role in driving angiogenesis. Recent evidence suggests it may respond to alternative mechanisms that activate its expression, and it appears to be involved in many signaling pathway outcomes beyond angiogenesis. In the present study, we generated knock-out and revertant mutants of ORF74 in the BAC-16 using lambda red-mediated recombineering. With infectious virions generated from these mutants, we investigated the role of the vGPCR in spontaneous reactivation of KSHV following de novo infection in iSLK cells. Using RT-qPCR, we demonstrated that knock out of ORF74 results in attenuated transcription of several lytic-associated genes including ORF50, ORF45, and ORF25.

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Matteo Garbelotto, Department of Environmental Science, Policy, and Management, University of California, Berkeley

"Development of degenerate primers used for sequencing and analysis of RPB2 region in Mycena fungi"

Abstract:

Mycena are a large genus of saprotrophic Agaricomycete fungi that have escaped comprehensive genome sequencing to date. As a result, classification of species has largely been dependent upon the morphological characteristics, however, the tiny size, fragile structure, phenotypic plasticity, and ephemeral nature of the fungal sporocarps has called these categorizations into question. Sequences of the ITS region of this group have been achieved in the past, but have not been entirely successful nor the results consistent. In this experiment, multiple variations using degenerate primers targeting the RPB2 region of the genome were used to obtain novel sequences. This has significance from a phylogenetic perspective in that sequencing of the RPB region offers insight into a nuclear region of the DNA to compare to ITS, the traditional ‘barcoding’ sequence, which is the most widely-sequenced DNA region in molecular fungal ecology.

DNA from dried specimens was obtained through NaOH extractions and used at multiple dilutions. The degenerate primers were created using Geneious and Primer3 genomic analysis software to maximize the potential for PCR amplification in this disparate genus. After multiple PCR trials using different degenerate primers and PCR protocols.

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Binh Diep, University of California, San Francisco

"Evaluation of the therapeutic potential of two monoclonal antibodies neutralizing alpha-hemolysin and bicomponent leukocidins in a rabbit model of Staphylococcus aureus necrotizing pneumonia"

Abstract:

Antibiotic resistance has become an increasingly urgent public health issue. Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major human pathogen, especially in the context of hospital-acquired infections. S. aureus causes a wide range of infections through secretion of various cytotoxins. In this experiment, we discovered that passive immunization with candidate monoclonal antibody (mAb) that neutralizes Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB led to partial protection, but in combination with MEDI4893, which neutralizes alpha-hemolysin, led to significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 MRSA epidemic clone.

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Guo Huang, University of California, San Francisco

"Investigating Novel Regulators of Cardiac Regenerative Potential"

Abstract:

Unlike adult mammals, neonatal mice are capable of regenerating damaged cardiac tissue during a brief postnatal developmental window. Comparing the expression levels of 1179 transcription factors in adult versus neonatal mice hearts, B-cell lymphoma 6 (Bcl6) is identified as one of the most highly upregulated transcription factors, while Cbp/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich C-terminal domain 1 (Cited1) is one of the most downregulated postnatally. To investigate potential downstream targets of Bcl6, the expression levels of transcription factors in control (Bcl6 f/f) mice versus mice with a cardiac-specific knockout of Bcl6 (Myh6-Cre;Bcl6 f/f) were compared in a preliminary study and Cited1 was the most differentially expressed. Overexpression of Cited1 in neonatal cardiomyocytes (CM) has shown to promote a 4- to 5-fold increase in proliferation rate. Cited1, however, is not a direct target of Bcl6 and the mechanism by which Bcl6 acts on Cited1 is currently unknown. Studies suggest that Cited1 expression is controlled by the PI3K/Akt pathway, which may in turn be controlled by Bcl6 expression as Akt1 is a direct target of Bcl6. I plan to further investigate this mechanism by analyzing the expression levels of Cited1 and cell cycle-related genes in neonatal CMs treated with an Akt inhibitor; I hypothesize that Akt inhibition decreases the expression of Cited1 and these cell cycle-related genes. Moreover, I plan to overexpress Akt1 in neonatal CMs to see whether it promotes CM proliferation, similar to when Cited1 is overexpressed in neonatal CMs.

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

"Role of Serpina3N in the regulation of glucose tolerance and insulin sensitivity in mice"

Abstract:

The worldwide prevalence of obesity is increasing at a rapid pace, bringing along with it higher rates of associated comorbidities such as type II diabetes, hypertension, and cardiovascular disease. Our lab is interested in uncovering the epigenetic mechanisms that link obesity-induced inflammation and insulin resistance to identify new therapeutic targets. A serine protease inhibitor Serpina3n is a protein mainly secreted from adipocytes and is known to have an anti-inflammatory function by targeting pro-inflammatory enzymes such as neutrophil elastase, cathepsin G, and granzyme B. Our initial studies found that adipose Serpina3n expression is highly induced in the state of insulin resistance in vitro and in vivo. Thus, we sought to determine what role Serpina3n plays in adipose tissue in diet-induced inflammation and metabolic dysregulation. To determine the adipose-specific function of Serpina3n, I tested whether this protein has a role in regulating glucose tolerance and insulin sensitivity in mice on chow or high-fat diet (HFD). Our preliminary results showed that Serpina3n KO mice had lower glucose tolerance than wild-type mice on HFD. Additionally, the KO mice had higher levels of plasma insulin than wild-type mice on HFD. This suggests that Serpina3n may play a protective role in resolving adipose tissue inflammation and insulin sensitivity during diet-induced obesity.

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Susana Ortiz, University of California, San Francisco

"Targeting lncRNAs to Discover Novel Kinase Dependencies in NRAS Melanomas"

Abstract:

Current treatment options for malignant melanoma centers around targeted therapies (BRAF and MEK inhibitors) and immunotherapy (anti-PD-1 and anti-CTLA-4 therapies) depending on specific mutation status. While effective for some patients, 25-50% of patients do not respond to existing treatments, including many of those with NRAS mutant melanomas. Through analysis of differential gene expression among normal human melanocytes, melanocytes with induced NRAS mutation, and melanoma cell lines, we identified a pair of novel lncRNAs with potential clinical relevance. We hypothesize that these lncRNAs have key roles in modulating proliferative signaling and interact with canonical oncogenic pathways. Here we show that ASO knockdown of our lncRNA targets result in a durable in vitro decrease in proliferation. Furthermore, using a novel HT-KAM assay, we observe differences in the activity of multiple kinases including AKT, MAPK, and SRC between treatment and control groups. These preliminary results indicate that existing kinase inhibitors approved for other diseases can be effectively co-opted for the management of problematic NRAS mutant melanomas. We are currently testing the in-vitro and in-vivo responses to these kinase inhibitors and performing functional studies to enhance our understanding lncRNAs in oncogenic signaling.

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Rodrigo Almeida, Department of Environmental Science, Policy, and Management, University of California, Berkeley

"Evaluation of Temperature-Sensitive Genes in Xylella Fastidiosa"

Abstract:

Xylella fastidiosa is a pathogenic bacterium that infects many plant species including grape and olive crops, causing diseases that can wipe out large plant populations and hinder the industries associated with these crop species. Previous studies have affirmed X. fastidiosa’s natural competence and the effects of temperature on its growth and spread, and identified genes that may be associated with its response to temperature. Using a method to knock out genes published in a recent study, I have created DNA fragments containing kanamycin resistance cassettes between the regions upstream and downstream of the gene to be knocked out, and am taking advantage of X. fastidiosa’s natural competence to transform the bacteria using these DNA fragments. Once these mutant strains have been cultured on plates containing kanamycin to ensure only the recombined bacteria persist, I will grow the bacteria at different temperatures to determine if any of these genes have a causal effect on X. fastidiosa’s temperature-dependent growth. This could help determine causes of X. fastidiosa’s pathogenicity and potentially identify better ways to treat or prevent the diseases it causes.

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Robert Tijan and Xavier Darzazq, Department of Molecular and Cell Biology, University of California, Berkeley

"Differential Transcriptional Activation by p53 in Human Cancer Cells"

Abstract:

The most frequently mutated gene in human cancers is TP53, which encodes the tumor suppressor protein p53. While p53’s role in the DNA damage response pathway has been well-characterized, its tissue-specific functions remain poorly understood. In particular, p53’s role in 3D chromatin organization has been virtually unexplored. To further investigate these unaddressed issues, I induced p53 in 10 human cancer cell lines from different tissue types and am mapping genome-wide p53 DNA binding sites by chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq). I will then correlate p53 DNA binding profiles with gene expression and chromatin organization patterns using RNA-seq data and 3D interaction maps obtained by Micro-C (a high resolution version of Hi-C), which were generated by other members of the Tjian-Darzacq Lab. We anticipate that by using this multi-omics approach we will uncover the role of p53 in remodeling the functional genome in a cell-type specific manner.

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

"Testing Natural Killer cell response using NKG2D-Ligand knock out mice"

Abstract:

Natural Killer (NK) cells are well characterized for their role in killing virally infected and cancerous cells. NK cells perform immunosurveillance and are differentially activated by stress-induced ligands binding to their activating receptors. These ligands have been shown to be highly expressed on infected or cancerous cells. Recently, however, ligands for the NKG2D receptor were found on the surface of normal cells at steady state. It has since been proposed that certain NKG2D ligands (NKG2D-L), such as RΑΕ-1δ and RAE-1ε, are involved in inducing NK cell hyporesponsiveness by persistently stimulating the cells. However, whether other NKG2D ligands are involved in inducing NK cell desensitization is unknown. In this study we utilize different knockout (KO) mice to investigate novel NKG2D-L involvement in inducing NK cell hyporesponsiveness in steady state. If more ligands other than the RAE-1 proteins were to be involved in NK cell desensitization, we hypothesized that, upon stimulation, RAE-1 MULT-1 KO and RAE-1 H60b KO mice would have increased responsiveness compared to WT, and that mice lacking all ligands (All Ligand KO, ALKO) would react most strongly when stimulated. We found that ALKO mice had the highest degree of responsiveness. However, RAE-1 H60b double KO and RAE-1 MULT-1 double KO NK cells had varied and predominantly unaltered responsiveness compared to RAE-1 KO, suggesting that tH60b and MULT-1 might not be involved in NK cell desensitization. Our findings provide important progress for the identification of ligands that tune NK cells responses and the development of novel immunotherapies against cancer.

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

"The Effects of GLUT1 Ablation in a Synthetic Brown Adipose Tissue Organ on Whole Body Substrate Utilization"

Abstract:

Brown adipose tissue (BAT), since its relatively recent discovery in humans, has been characterized by its functions as a chemical thermoregulator of the body. Through Uncoupling Protein 1, BAT is able to dissipate the mitochondrial proton gradient as heat which increases core body temperature during acute cold exposure. Glucose Transporter 1 (GLUT1), coded by SLC2A1 gene, is a crucial glucose uptake transporter in all cell types including brown adipocytes. The goal of this project is to ablate SLC2A1 in a BAT-specific manner and measure the resulting whole body physiological changes. Rather than using a traditional Cre/LoxP mouse strain to study this, we have developed a system to inducibly knockout any gene within a BAT cell line that has been embedded in a hydrogel, creating a synthetic BAT. We used two lentiviral vectors to achieve the ablation of GLUT1. First, a plasmid containing a tetracycline-inducible CRISPR Sniper Cas-9 protein, an optimized Cas-9 protein with improved gene editing efficiency is added. Second, with the Multiple Lentiviral Expression system, a flexible Gateway cloning-based lentiviral system, we generated a plasmid containing two short guides targeting one early exon of a specified gene, Td-tomato for FACS sorting, and a backbone that expresses luciferase. Once the plasmids are delivered, I then used qPCR and Western Blot to ensure that the target gene is no longer being expressed and functional proteins are no longer being produced. Once validated, cells are differentiated ex vivo, embedded in hyaluronic acid hydrogels, and implanted into mice. A week after implantation, we conducted a panel of physiological measurements to establish before and after metabolic parameters of implanted mice. Additionally, various serum tests as well as indirect calorimetry was used to infer changes in overall metabolic tone by measuring metabolite levels and thermogenesis, respectively.

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Ari Molofsky, University of California, San Francisco

"Negative regulation of ILC2s by IFNg in the context of mixed inflammation"

Abstract:

Group 2 innate lymphoid cells (ILC2s) play a major role in type 2 inflammation, tissue remodeling, and allergic pathophysiology. However, the negative regulation of ILC2s in the context of mixed inflammation is not well described. Here, we will use flow cytometry and quantitative confocal microscopy to demonstrate that after IL33-mediated activation, ILC2 are robustly enriched in the adventitial layer of large vessels as well as in the tissue parenchyma. Type 1 cytokine, Interferon-gamma (IFNγ), which is released during bacterial or viral infection, directly restricts IL-33 mediated activation of ILC2s and distribution into the parenchyma. The failure of ILC2 restriction by IFNγ during Listeria monocytogenes infection, a well studied type 1 inflammation model, results in increased mortality, bacterial burden, and morphological differences in the infectious foci known as granulomas. The sphingosine 1-phosphate (S1P) pathway, which is crucial for T cell trafficking from lymphoid organs through endothelial barriers has recently been shown to be a key player in ILC2 mobilization after activation. By using an antagonist for the S1P signaling pathway, we can block the IL-33 mediated accumulation of ILC2s in the liver, resulting in lower mortality, bacterial burden, and granuloma morphology favoring a healing response post type 1 inflammation.

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

"NimB4, Hemocyte Activity, and Tumor-Host Interaction"

Abstract:

The mechanism of how the immune system of hosts recognizes and interacts with tumors has long been an intriguing aspect in cancer research. In particular, the role of innate immune cells in survival of tumor-bearing hosts remains relatively unclear. To address this puzzle, we used Drosophila melanogaster, which has only innate immunity, as the model system to investigate functions of hemocytes, a type of innate immune cells, on the survival of tumor-bearing hosts. Interestingly, depletion of hemocytes in tumor-bearing flies reduced their lifespan, suggesting that hemocytes are essential for hosts’ defense against tumor. Furthermore, we searched for tumor-secreted factors, which are abnormally elevated and involved in regulating hemocyte functions. By performing RNAi screen, we identified NimB4, which belongs to the Nimrod protein family known to associate with hemocyte activities such as phagocytosis and pathogen recognition, and we predicted that NimB4 is likely to share functional commonalities with other Nimrod family proteins based on its protein structure. Knocking down NimB4 in Drosophila ovarian carcinoma showed significant reduction in tumor growth and extended host lifespan. To further investigate the connection between NimB4 and hemocyte activities, we transplanted tumors lacking NimB4 into flies having fluorescence-labeled hemocytes and visualized recruitment of hemocytes to tumors using confocal microscopy. Flies bearing NimB4-knowndown tumor exhibited both reduced growth of tumor and more hemocyte recruitment around their tumor. In conclusion, our research demonstrates that knocking down tumor-secreted factor NimB4 reduces tumor growth and promotes host survival in both genetic ovarian carcinoma model and transplant. As we continue to investigate the correlation between tumor-secreted factor and innate immune cells, the findings may shed light to innovative cancer therapeutics that makes use of players in the innate immune systems.