Biology Room: Session A 125 Baxter microRNA-132 Regulates Hematopoietic Stem Cell Function and B-Cell Development Xiaomi Du Mentors: David Baltimore and Arnav Mehta microRNAs are 22bp non-coding RNA sequences that help regulate a wide variety of immune functions and cell-fate decisions. Each microRNA targets several hundred mRNA sequences and decreases their protein expression by inhibiting translation. We have identified microRNA-132 (miR-132) as a key regulator of hematopoietic stem cell (HSC) function and B-cell development. It modulates these pathways by targeting two transcription factors, FOXO3 and SOX4. We performed quantitative real-time polymerase chain reaction (qPCR), Western blotting, and luciferase assays to confirm that over-expression of miR-132 down-regulates FOXO3 and SOX4. This effect is specific to miR132 because we do not observe the same down-regulation when the miR-132 binding site is mutated. To study the effects of miR-132 in vivo, we compared the phenotypes resulting from over-expressed and knocked out microRNA-132 to those of over-expressed and knocked down FOXO3 and SOX4. miR-132 over-expression leads to rapid HSC proliferation, followed by exhaustion; this is rescued by co-expressing FOXO3., miR-132 over-expression also causes B-cell developmental defects due to the reduced SOX4. These observations establish that miR-132 is significant to maintaining the balance of hematopoiesis. A Study of the Kinetics of mRNA Splicing in the NF-κB Transcriptome Luke Frankiw Mentors: David Baltimore and Devdoot Majumdar The prevailing notion in current literature is that much of mRNA splicing occurs both linearly and cotranscriptionally; however, little work has been done on dynamic systems. My project aims to determine the nature of splicing among the NF-κB transcriptome at different stages of NF-κB induction. I developed and implemented an RNA sense-strand purification technique to “pull-down” specific cDNAs relevant to the NF-kB pathway. This technique has allowed for cleaner Illumina sequencing data. Furthermore, the enrichment offered through the sense strand purification will allow us to use Pacific Biosciences full-transcript sequencing. By sequencing fulllength transcripts, we will be able to determine the exact composition of single mRNA molecules at different stages of induction. Finally, through the summer I have utilized “digital” PCR to analyze the composition of mRNA transcripts for a specific gene in the transcriptome. Preliminary dPCR data shows some non-linearity in the order introns are spliced. A Novel Cell-Specific System for in vivo Studies of Immunological Tolerance Vasant Iyer Mentors: David Baltimore and Mati Mann Immunological tolerance is the process by which immune cells become accustomed to host tissue and to benign foreign matter that the immune system deems harmless; this process is critical in preventing unwarranted inflammations and autoimmune disorders. Although it has been shown that self-recognition and immunoregulatory molecules such as MHCII and IL-10 play a significant role in maintaining systemic tolerance, little is known about how tolerance is conferred and what cells mediate it; investigating these properties of tolerance requires developing a method to disrupt MHCII and IL-10 production in specific cell types in vivo. We developed such a system by creating adeno-associated viruses (AAV), encoding Cre recombinase under the transcriptional control of promoters obtained from cell-specific genes. Similar vectors encoding luciferase were used to optimize the vector transfer process, into the lungs of adult mice. Plans for further work include using this system to silence MHCII or IL-10 expression in cell types of interest and quantifying the subsequent responses of mice to induced inflammations, elucidating the roles of these cell types in maintaining lung immunological tolerance. Measurement of αβ T Cell Receptor Mispairing for Selection of Effective Gene Therapy TCRs Meghana S. Pagadala Mentors: David Baltimore and Michael Thomas Bethune The heterodimeric α/β T cell receptor (TCR) is the sole determinant of T cell specificity. In TCR gene therapy, patient T cells are transduced with tumor-specific α and β TCR genes to impart anti-tumor immunity. However, introduced TCR α and β chains can mispair with the transduced T cell’s endogenous TCR β and α chains, respectively, reducing the number of tumor-specific TCRs on the surface and potentially generating autoreactive TCRs. So far, a TCR’s propensity to mispair has been an undefined property. We are developing a quantitative assay to measure the extent to which mispairing occurs for TCRs of clinical interest. We identified two antibodies that bind the constant portions of the endogenous TCR α and β chains. These epitope sites were mutated in the transduced TCR chains and synthetic epitope tags were added to the N-terminus of these chains to enable their orthogonal recognition by a second set of two antibodies. Using a sandwich ELISA, we capture the transduced α or β TCR chain with a tag-specific antibody and then detect the β or α chain to which each is paired using a second antibody. Signal is observed only when both capture and detection antibody targets are present. Therefore, all possible α/β heterodimers can be distinguished by using different antibody pairs and each heterodimer can be quantified by comparison to a standard curve. The ability to measure mispairing of TCRs will enable selection of clinical candidates that are safer and more effective. A Transcriptional Model for the Feed-Forward Loops of the MET Pathway Albert Ge Mentors: Eshel Ben-Jacob, Bin Huang, Mingyang Lu, and Erik Winfree The MET pathway is a protein network which governs two cell motility phenotypes: amoeboid and mesenchymal. The decisions made by the MET pathway have been linked to metastasis of cancerous cells. Though some of the protein interactions in the pathway have been studied experimentally, little is known about the physical dynamics of the network, and how it changes as a function of the inducing signal. In this project, transcriptional-protein models of the feed-forward loops (self-activating, self-inhibiting, and feedback) in the MET pathway are formalized. Using the models, nullclines were plotted as a function of protein concentration, and analyzed to determine stable and unstable solutions to the system. Bifurcation diagrams were also graphed to observe how each steady-state solution may change with varying input signal. Finally, time dynamics of the systems were examined for peaks in protein concentration. The simulations suggest that feedback systems are relatively independent of the input signal, in comparison to self-activating and self-inhibiting systems. Coupling these loops together may further tie together theoretical with experimental data. Structural and Kinetic Characterizations of Defective Potent VRC01-Like Antibodies Against HIV-1 Courtney Chen Mentors: Pamela J. Bjorkman and Louise Scharf Recently there have been several reports of a “cure” for HIV. Most famous of these are the “Berlin” patient and Mississippi baby. While these cases are promising, they also represent unique instances that are still being studied. Consequently, the discovery of a cure for HIV is still a pressing issue. Current designs of immunogens for vaccines have been spearheaded by expansion in discovery of potent and broadly neutralizing antibodies (bnAbs) against HIV-1. Particularly of interest to the Bjorkman group are antibodies of the proposed class of Potent VRC01-like (PVL) antibodies, which are CD4 binding site bnAbs that share characteristic amino acids. With our collaborators at Rockefeller University, we have an unprecedented set of data on a range of PVL antibodies with in-depth characterization of several PVLs and sequences that span from germline to intermediate, semi-potent forms to mature bnAbs. My project aims, therefore, to study variant PVL antibodies and attempt to identify crucial changes needed to transition from a low affinity form to powerful bnAb. PVL antibodies have been identified from several patients, suggesting a conserved, plausible route of evolutionary attack on HIV, indicating there exists the potential to induce an immune response forming PVL antibodies. Engineering Chimeric Antigen Receptors for the Treatment of HIV Erin M. Isaza Mentors: Pamela J. Bjorkman and Rachel P. Galimidi Chimeric antigen receptors (CARs) are engineered receptors that graft the specificity of a monoclonal antibody onto a T-cell—the cells are removed from a patient, modified to express the desired receptors via retroviral vectors, and reintroduced into the patient. While CAR T-cells have proven effective in treating leukemia, the only attempt in using CAR T-cells to attack HIV proved unsuccessful. However, in the time since this former study, broadly neutralizing antibodies against HIV, which bind to a wide variety of antigens, have been discovered. We propose to engineer CAR T-cells expressing broadly neutralizing antibodies, which bind to and neutralize HIV with a much higher avidity and are safer than the previous generation of anti-HIV CAR T-cells. Assaying Optic Nerve Development in Patient Stem Cell Derived Retina Janani Mandayam Comar Mentors: David Cobrinik, Jennifer Aparicio, Mark Borchert, and Paul Sternberg Optic Nerve Hypoplasia (ONH) is a major cause of neonatal blindness characterized by underdevelopment of the optic nerve—a bundle of retinal ganglion cell (RGC) axons. Leading hypotheses suggest that RGCs either do not develop in sufficient quantities or develop initially, but undergo apoptosis upon failure to project axons properly. This study addresses these hypotheses by comparing RGC production and survival in patient and control-derived induced pluripotent stem cell (iPSC) retinal tissue spheres. Initially, human embryonic stem cell (hESC)-derived retinal spheres were used to develop immunofluorescence (IF) and Western Blot assays to quantify Brn3, an RGC marker, from weeks four through twelve. Due to the challenges in discriminating tightly packed cells in IF, a method comparing the total area of the marker signals was used. Western and IF results indicate RGC production is greatest during weeks five through seven. However, analysis of several spheres, using both methods, shows great variability, even when normalized to total and progenitor cells, indicating that a large sample number could be required to determine if the number of patient and control RGCs differ. Future work will include further establishment of the RGC production curve, quantification of RGC survival with an apoptotic cell marker, and development of an axon outgrowth assay. Characterization of Differentiation Gene Expression During Late Gastrulation of S. purpuratus Eric Qiao Mentors: Eric Davidson and Jonathan Valencia Recently, a near-complete survey of transcription factors expressed in Strongylocentrotus purpuratus during embryogenesis has been performed. However, the expression of terminal differentiation genes (e.g., those including metabolic genes, detoxification genes, and genes coding for digestive enzymes) and their modes of regulation are less well-known. By relating transcription factor expression to differentiation gene expression, a link can be established between the gut physiology of the sea urchin and the developmental regulatory factors that comprise its gene regulatory network (GRN). In order to do this, we selected sea urchin genes that are homologous to genes encoding human liver- and pancreas-specific proteins and investigated their spatial expression in whole mount in-situ hybridization experiments. Embryos were assayed from 24 to 96 hours post fertilization with 12 hour intervals but with emphasis on later stage embryos. Many of these genes, such as Cpa3, showed specific expression in a ring of cells within the developing gut. The spatial and temporal characterization of these differentiation genes provides a valuable foundation in understanding the regulatory states they occupy and as a result, will provide insight into their mechanism of regulation. Kinetic Characterization of CSN-Mediated Deconjugation of Nedd8 From SCF Ubiquitin Ligase Minsoo Kim Mentor: Raymond J. Deshaies The current model of SCF (Skp1, cullin, and F-box) ubiquitin ligase regulation involves a massive waste of energy. For a target substrate to be ubiquitinated, the corresponding F-box-Skp1 complex has to first bind to a cullin, thereby forming a SCF complex. However, CAND1 could displace the F-box-Skp1 complex from the cullin. When the SCF complex gets neddylated, CAND1 can no longer bind to the cullin and the substrate is more likely to get ubiquitinated. However, CSN that is present nearby could deneddylate the cullin and hence allow CAND1 to bind. As a result, in the current model, it is hard to regulate SCF complexes efficiently. In order to clarify the exact mechanism of how cullin-RING ubiquitin ligases (CRLs) are regulated through neddylation and deneddylation, it is necessary to characterize kinetic properties of CSN. In the following paper, Kd of both neddylated and unneddylated cullins were measured by using a fluorescence binding assay. Exploring Microbial Biofilm Morphology Through Comparative Study and Modeling Zofii Kaczmarek Mentors: Lars Dietrich, Christopher Kempes, and Dianne K. Newman Although many bacterial biofilms form complex patterns, little is known about the mechanisms underlying the formation of these multicellular structures. Pseudomonas aeruginosa colony biofilms that are grown on the surface of an agar plate develop a wrinkled structure to increase oxygen uptake under electron acceptor-limited conditions. Wrinkle width is adjusted to changes in electron acceptor availability, such as oxygen or the endogenous, redoxactive phenazine pigments. We sought to identify genes that play a role in wrinkle formation by screening for mutants with atypical wrinkle morphologies. We screened 27 mutants that were made in the hyperwrinkled phenazine-null background (denoted ∆phz) for deviations in wrinkle thickness. We identified 3 mutants and selected one strain, ∆phz∆ccoN4, was selected for further study. Our findings provide an illustration of the mutability of wrinkle widths. This study can serve as a foundation for more in-depth examinations of genes relevant for the wrinkling process. A Biosynthetic Pathway Using Biochemical Protecting Groups to Dye Cloth With Indigo Aleena Patel Mentors: John Dueber, Zachary Russ, Arthur Fong III, Shyam Bhakta, Nicole Chernavsky, Matthew Chan, Taner Dagdelen, and Jesse Beauchamp The Special Projects in Synthetic Biology (SPSB) team at UC Berkeley has developed a biosynthetic pathway that implements biochemical protecting groups to control the spontaneous oxidation of indoxyl that forms indigo. The enzymatic protection of indoxyl by transferases and deprotection by hydrolases permits controllable release of indoxyl. The indigo-dyeing industry requires a soluble form of indigo to vat-dye large surface areas of cloth. The current industry must dump toxic byproducts into waste-water to produce soluble leuco-indigo before dyeing cloth. Protected indoxyl offers an environmentally friendly alternative that does not require harmful reducing agents to produce a soluble form of indigo. SPSB has identified two sulfotransferases, native to Homo sapiens, and expressed them in E. coli and S. cerevisiae. In vitro experiments confirm that these sulfotransferases are capable of protecting indoxyl with sulfate to prevent spontaneous formation of indigo in aerobic conditions. Sulfatases that hydrolyze indoxyl sulfate to release indoxyl when desired and transporter enzymes that export indoxyl sulfate out of the cell have also been explored to complete the biosynthetic pathway. fMRI Motion Correction for Accurate Implementation of Gaze Estimation Sarthak Sahu Mentors: Ralph Adolphs and Mike Tyszka Gaze estimation in videos is an essential tool in social cognition research because it allows researchers to identify objects and features receiving visual attention in a scene. One of the most common methods of gaze tracking is through the use of infrared illumination and its corresponding reflections from the cornea known as glints. A major drawback of using glints to estimate gaze is that if they are lost in the video then the gaze calibration breaks down and accuracy is lost. Unfortunately, any slight movement of the head, even with the use of restraints, can result in the loss of these glints, which would render the video useless. The goal of this project is to develop a python program that accurately implements a gaze estimation method using a single calibration that would work independently of glints.