University Of California Berkeley

NIH Research Projects · FY 2025 · 2016-04

Project Summary: This project is focused on understanding the physical and mechanistic properties of enzymes that underlie their exquisite function. In recent years, protein motions have been implicated as essential to achieve an extremely rapid catalysis of bond cleavage events at enzyme active sites. Methodology for the spatial and temporal resolution of such protein motions has been developed using enzyme prototypes that catalyze hydrogen and methyl transfer reactions. These studies are now being extended to the TIM barrel family of enzymes that represent 10% of known enzyme structures and catalyze 5 out of 7 known EC classes. With this knowledge in hand, new approaches arise for protein redesign, de novo design and drug targeting. A second emerging area in biological catalysis concerns the post-translational modification of peptides that have been synthesized at the ribosome. A combination of structural and biochemical probes is addressing the enigmatic pathway that produces the bacterial cofactor and vitamin, pyrroloquinoline quinone. As the result of a number of recent breakthrough observations, each of the catalysts within the pathway is now amenable for detailed mechanistic study. These enzymes have little or no precedence in humans, making the PQQ pathway a possible new target for antibiotic development.

NIH Research Projects · FY 2025 · 2015-11

ABSTRACT Herpesviruses are endemic within the human population, and cause a wide range of life-threatening diseases. Members of the betaherpesvirus and gammaherpesvirus subfamilies are extremely problematic in immunocompromised individuals, leading to severe congenital disorders and a variety of cancers. This proposal will define how a critical class of herpesviral genes are expressed late in the lifecycle of Kaposi's sarcoma-associated herpesvirus (KSHV) through a mechanism that involves a novel transcription complex, which combines both viral mimicry and co-option of select host machinery. The set of viral proteins required for late gene activation are broadly conserved among the beta- and gammaherpesviruses, indicating that information gained herein for KSHV will likely be applicable for Epstein-Barr virus and human cytomegalovirus as well. The focus of this grant is to define the composition and regulation of this novel gene expression complex, and reveal how its function is linked to viral genome replication. Given that even subtle mutations in this late gene transcription complex effectively `kill' the virus, an understanding of its composition and regulation is anticipated to reveal new targets for antiviral strategies.

NIH Research Projects · FY 2024 · 2015-09

ABSTRACT Light responses are initiated in rod and cone photoreceptors, processed by retinal interneurons, and synaptically transmitted to retinal ganglion cells (RGCs), which send information, in the form of spike trains, to the brain. In degenerative retinal disorders, including Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP), the photoreceptors gradually die off, depriving downstream neurons of light-sensitive input. However, recent evidence suggests that losing photoreceptors is only part of the problem in these disorders. Downstream retinal neurons become hyperactive, with retinal ganglion cells (RGCs) firing spontaneously in darkness at up to 10 times faster than in healthy retina, corrupting the proper encoding of visual information. We recently reported that retinoic acid (RA), a small molecule that activates gene transcription, is the signal that triggers RGC hyperactivity. Blocking the receptor for RA in vivo can reverse hyperactivity, unmasking light responses that would otherwise be obscured by spontaneous RGC firing. Blocking RA receptors in the retina also augments the contrast-sensitivity of learned visual behaviors in a mouse model of RP. Our goal in this project is to assess whether drugs or gene therapies that inhibit RA signaling can improve vision in mouse models of RP, with the hope of extending useful vision for years in humans with degenerative retinal disorders. First, we will ask whether inhibiting RA signaling not only improves light-sensitivity, but actually improves conscious visual function in vision-impaired mice, assessed with behavioral tests of contrast sensitivity and spatial frequency threshold. We will determine how when during the degeneration process RA inhibitors are most effective, revealing the optimal time for beginning treatment. Second, we will investigate retinal neurons that lie upstream of RGCs, namely bipolar cells and amacrine cells. We will ask whether pathophysiological changes in these presynaptic neurons are also induced by elevated RA signaling and whether inhibiting RAR can reverse these changes, providing critical information for effective cellular targeting of gene therapy. Third, we will test whether vision can be improved by inhibiting the enzyme that synthesizes RA, with a re-purposed drug that is already FDA-approved for other indications, paving the way for human clinical studies. Taken together, this project will establish the proof-of-principle behind a new treatment paradigm for augmenting vision in retinal degenerative disorders.

NIH Research Projects · FY 2025 · 2015-07

SUMMARY (OVERALL) The four dengue virus serotypes (DENV1-4) cause the most important mosquito-borne viral disease of humans. Yet, no treatment is available, and the only registered vaccines are problematic. A major challenge is the dual potential of the immune response to protect against or enhance future infection with a different DENV serotype. Thus, it is critical to better understand immune responses to natural DENV infections and vaccines and to identify robust correlates of protection. The overall goal of this P01 is to apply state-of-the-art immunological and virological methods in the context of clinical and epidemiological studies of DENV infections and vaccines to elucidate how adaptive immunity shapes clinical and immunological outcomes. Our current P01 has been very successful, generating ~80 high-level publications since its inception and ~30 publications in the last 3.5 years, producing key findings that are timely and impactful. Further, our P01 consortium has served as a go-to resource for multiple agencies and regulatory bodies regarding dengue medical countermeasures. Here, we build on new tools and hypotheses that emerged from our current P01 to study immunological imprinting and evaluate biomarkers of protection and risk. The overall hypothesis of this P01 is that the magnitude and quality (i.e., repertoire and functionality) of DENV antibody (Ab), B cell, and T cell responses are shaped by distinct structural and antigenic characteristics of the 4 serotypes both in primary (1°) infections and, via immunological imprinting, secondary (2°) infections -- impacting clinical and epidemiological outcomes, with important implications for vaccine design and efficacy. We propose a coordinated P01 with 3 projects: 1) Immunological imprinting and immune correlates of Ab and B cell responses in natural DENV infections; 2) Linkage of vaccine-induced human B cell and Ab response to protective or dengue disease-enhancing immunity; and 3) Immunological imprinting of T cell responses following DENV natural infection and vaccination, supported by 4 cores: 1) Administrative; 2) Viral Assays; 3) Clinical, Data Management, and Statistical Modeling, and 4) Antibodyomics. We leverage unique sample sets from the longest continuous cohort study of dengue (in Nicaragua), currently in its 20th year; a placebo-controlled cohort study of the Dengvaxia® vaccine in the Philippines; and a human vaccine challenge study of a monovalent NIH vaccine. The P01 is highly synergistic in that samples are shared and infection sequences aligned among Projects and Cores. Specific Aims are: 1) Determine how DENV1-4 B cell and Ab responses are shaped by distinct structural and antigenic characteristics in 1° and, via immunological imprinting, in 2° infections; 2) Reveal Ab-mediated mechanisms responsible for vaccine-mediated protection, vaccine failure and vaccine-enhanced dengue disease; 3) Profile the quantity and quality of imprinted DENV-specific memory T cell response in relation to immunophenotype and vaccine outcomes; 4) Compare the breadth of Ab and T cell responses induced by DENV vaccines vs. natural infections; 5) Identify robust correlates of protection or risk enhancement of dengue disease in different contexts.

NIH Research Projects · FY 2024 · 2015-06

PROJECT SUMMARY Nef is an HIV-1 accessory protein whose function is to undermine host defenses. Long term infection with HIV strains bearing defective nef alleles leads to AIDS only over many years, suggesting Nef could be targeted as part of functional cure strategies. This basic research proposal seeks will elucidate the mechanisms of action of Nef. Nef targets include CD3, CD4, CD8, CD28, CXCR4, MHC-I, SERINC3/5, and for HIV-1 group O and SIV, BST2/tetherin. Nef substrates are downmodulated by via the clathrin-coated vesicle (CCV) pathway. Nef does not interact directly with clathrin, but rather with various members of a family of heterotetrameric adaptors known as the adaptor protein (AP) complexes, AP-1 and AP-2. The ability of the human immune system to detect and kill virally infected cells relies on proper presentation of viral antigens on the cell surface by MHC-I complexes. Nef subverts this process by promoting MHC-I complex downregulation by hijacking AP-1 and its associated small GTPase Arf1 at the TGN. MHC-I contains an incomplete version of the normal Tyr-based sorting motif. Nef complements this defective motif and converts MHC-I into a substrate for AP-1 mediated sorting to the lysosome for degradation. Structures of Nef assembled with AP-1 and the MHC-I cytosolic tail in solution suggested that Nef promotes the assembly of hexagonal lattices whose symmetry matches that of clathrin. Now, the previous solution studies will be followed up by reconstitution and structure determination of Nef, MHC-I tail, AP-1 and Arf1 in their functional setting on lipid membranes. Downmodulation of CD28 by Nefs is conserved across SIV and HIV and is mediated by AP-2. CD28 downmodulation phenotypes of Nef mutations follow a distinct pattern from other receptors, and the structural basis for this mode of CD28 is unknown. The structure of the CD28:HIV-1 Nef:AP-2 complex will be determined and leveraged to design mutations that uniquely perturb the CD28 binding site. Of the many host substrates of Nef, the most significant for viral infectivity are the multipass integral membrane proteins SERINC3 and 5. The SERINC binding site appears, on the basis of Nef phenotypes, to overlap with the site used by SIVsmm Nef to downmodulate simian tetherin, but is otherwise distinct from the known locations of CD3, CD4, and MHC-I sites. SERINCs do not share any obvious motifs with other substrates. SERINCs have been purified in monodisperse form suitable for structure determination. The cryo-EM structure of lipid- or detergent embedded SERINC3 or 5 in complex with AP-2 and HIV-1 Nef will be determined, completing a major goal in the field.

NIH Research Projects · FY 2025 · 2015-05

Genomic data hold the promise of revolutionizing our understanding and treatment of human disease. Multiple barriers stand between the acquisition of the data and realizing these and other benefits. Rapid accumulation of genomic data far exceeds our capacity to reliably interpret genomic variation. New developments in artificial intelligence and machine learning, combined with increased computing power and domain knowledge, provide hope for the deployment of enhanced computational tools in both basic research and clinical practice. Use of these methods critically depends upon reliable characterization of their performance. The Center for Critical Assessment of Genome Interpretation (C-CAGI) will address these needs, through objective evaluation of the state of the art in relating human genetic variation and health. CAGI has had five editions since 2010 with 50 challenges posed to the community taken on by hundreds of predictors, leading to scores of publications about prediction methods and their assessment. We propose for C-CAGI to continue to advance the field of variant interpretation through the following Specific Aims: 1. Develop community experiments to evaluate the quality of computational methods for interpreting genomic variation data. C-CAGI will conduct community experiments in which participants make bona fide blinded predictions of disease related phenotypes on the basis of genomic data. We will engage a diverse predictor community to spur innovation. The CAGI Ethics Forum will vet studies to ensure that privacy and sharing maintain the highest standards and will educate the community. 2. Assess the quality of current computational methods for interpreting genomic variation data; highlight innovations and progress at interactive conferences. Predictions will be evaluated by independent assessors, who will be supported by new assessment approaches from C-CAGI. Results will be presented at CAGI experiment conferences with deep technical engagement, which will be interleaved with reflective CAGIâ meetings that create an environment for a comprehensive evaluation of the field, facilitating identification of major bottlenecks and problems faced by the current genome interpretation approaches. 3. Broadly disseminate the results and conclusions from the CAGI experiments and analysis. C-CAGI will outreach to the broader scientific and clinical community through its publications, and the creation of a calibrated reference integrated into the most common workflows for ready adoption. CAGI will also be represented at international meetings with presentations and workshops. 4. Operate effectively and responsively. C-CAGI will operate efficiently as it closely interacts with hundreds of participants. CAGI will build upon a robust information infrastructure that securely facilitates data dissemination, prediction submission, and assessment.

NIH Research Projects · FY 2025 · 2015-05

PROJECT SUMMARY/ABSTRACT: Following on a very successful launch of this training program at the University of California Berkeley, the purpose of this renewal project is to continue providing high level training workshops for graduate students, postdoctoral fellows and early career faculty interested in acquiring formal demography skills. Formal demography consists of a set of analytic tools that allow for a kind of analysis not possible with standard statistical models and are therefore critical in addressing the kinds of complex population processes occurring in the 21st century. We build on the success of the first 5-year cycle, and introduce new workshop content by having a yearly special emphasis topic chosen to connect formal demography to important and newly emerging areas of demographic research. Pedagogically, we will modernize the workshop format by adding pre-workshop remote learning, on-site team projects, and a new generation of instructors. The proposed program consists of three weeks of guided independent study prior to the workshop, a weeklong workshop in Berkeley – three days of training followed by a two-day research conference; and an annual networking event at Population Association of America meetings. The three days of core training will include instruction on population dynamics and hands-on training in modern demographic computing (in the R statistical modeling language). A third day will include training and methods specific to that year's special emphasis topic. These workshops not only serve as training grounds, but also provide ample opportunities for networking and building relationships and community. During the week, trainees will also work on group projects to give them ample hands-on experience with the data and methodology, and results of these projects are presented on the final day of the workshop. The topics covered in the workshops include the continuation of core topics in the mathematical modeling of fertility and mortality, with the addition of new themes in population models of contagious diseases, impact of natural disasters on demography, and digital demography. These elements – training, research presentations, group projects, networking – work to create a long-lasting community of scholars that can engage in interdisciplinary research for years to come. In this way we expect that the topics of these studies will serve to inform demographic and social science research and have positive impacts in particular on population health policies.

NIH Research Projects · FY 2026 · 2015-01

Y chromosomes of many organisms contain a large number of transposable elements (TEs), which are transcriptionally constrained by repressive chromatin marks. When relieved of these epigenetic modifications, many TEs can readily move from one genomic location to another (toxic Y chromosomes). Yet evolutionarily young Y chromosomes still contain a large number of essential genes that are actively transcribed, and competition between the opposing mechanisms of heterochromatin formation and active transcription can result in incomplete silencing of TEs on evolving Y chromosomes. Our proposal aims to characterize epigenetic conflicts between host-specific genes and selfish genetic elements on evolving Y chromosomes at various stages of degeneration, whose resolution may select for adaptive degeneration of the Y. The accumulation of repetitive elements on the Y chromosome appears to be universal during sex chromosome evolution. We will take advantage of evolving neo-Y chromosomes in Drosophila with varying levels of degeneration, to catalog toxicity of Y chromosomes using expression profiling and chromatin analysis. We will link epigenetic and expression profiles across neo-Y chromosomes that differ in gene and repeat density, to identify whether active transcription of genes on neo-Y chromosomes results in incomplete silencing of TEs. We will establish whether toxic Y chromosomes incur a fitness cost on males, by forming a mutational burden and reducing male longevity, as suggested by our preliminary work. Integrating our results across aims will provide a full picture of how the toxicity of the Y chromosome changes over time, and how epigenetic conflicts between host genes and selfish elements may be resolved.

NIH Research Projects · FY 2025 · 2014-12

PROJECT SUMMARY / ABSTRACT Cells have evolved intricate quality control systems to prevent the accumulation of aberrant or damaged macromolecules, such as proteins and DNA. Lipids are chemically diverse macromolecules that have important functions in membrane biology, energy homeostasis, and signaling. Similar to proteins and DNA, lipids can also be damaged (e.g., oxidized). However, our understanding of the mechanisms that regulate lipid quality control remains at an early stage, representing a key gap in knowledge. The accumulation of oxidatively damaged lipids is a hallmark of ferroptosis, an iron-dependent regulated form of cell death that is an emerging target for the treatment of drug-resistant cancers. Understanding the regulation of lipid peroxidation and ferroptosis provides an exceptional system to uncover the mechanisms that govern lipid quality control and to identify novel therapeutic targets for the treatment of drug-resistant cancers. The prevailing paradigm has focused on the glutathione-dependent peroxidase GPX4, which suppresses ferroptosis by converting lipid peroxides into non- toxic lipid alcohols. Employing CRISPR-based genetic screens, we recently discovered FSP1 as a new ferroptosis resistance factor that acts parallel to GPX4. Mechanistically, FSP1 reduces non-mitochondrial coenzyme Q10, which functions as a lipophilic antioxidant to suppress the propagation of lipid peroxides. Genetic disruption or chemical inhibition of FSP1 sensitizes cancer cells of diverse tissue origins to ferroptosis. FSP1 is present on lipid droplets, the primary lipid storage organelle in cells, but its role on lipid droplets is unknown. The current proposed research aims to exploit lipidomics, imaging, and biochemically reconstituted and cell-based assays of FSP1 function to address several outstanding questions regarding the cellular role of FSP1 and its regulation. In Aim 1, we will test the hypothesis that lipid droplet-localized FSP1 reduces coenzyme Q10 to prevent oxidation to stored neutral lipids such as triacylglycerol. In Aim 2, we will validate and characterize a series of regulators that we identified through genetic screens that govern FSP1 post-translational stability. The completion of these aims will advance our understanding of the mechanisms that mediate cellular lipid quality control, including determining how neutral lipids stored in LDs are protected from oxidative damage and how FSP1 levels are regulated in cancer.

NIH Research Projects · FY 2025 · 2014-09

We study how neural activity in the hippocampus and connected areas mediates their roles in learning and memory. We are interested in circuit mechanisms responsible for activation of hippocampal units in precise sequences that depict past and future behavioral trajectories. These sequences, called "replays", are attracting increasing attention because of the unique way they allow a subject to re-experience events from another time and place. Despite these intriguing features, several questions remain. We do not know how replays impact other brain activity and what role they play in behavior. We also do not know how other circuits outside of the hippocampus are involved in generating replays. Here, we will find answers to these questions, by recording and manipulating neural activity, in hippocampus and in a closely connected area called entorhinal cortex, in awake and freely behaving rats. (Aim 1) Previous attempts to disrupt replay have only revealed relatively subtle effects on behavior. For example, disruption during a post-training consolidation period has relatively weak effects on a spatial memory task. Here we present preliminary evidence that disrupting replay while a rat learns a new goal location in a spatial memory task dramatically affects performance during a probe test performed immediately afterward. We will use this effect to determine which parts of replays are important. For example, it could be that replays must join up the goal location and more distant locations in the environment to enable later navigation to the goal from those distant locations. These and other hypotheses will be tested systematically to reveal how replay contributes to spatial learning. (Aim 2) The medial entorhinal cortex (MEC) has been implicated in the representation of spatial goals, and in supporting longer hippocampal replays. However, this latter result was found with only a partial suppressive effect on MEC activity, and in mice, where replay is difficult to measure. We use an innovative new optogenetic technique using more penetrative wavelengths of light, and an innovative form of optical fiber geometry, to shut down activity along the entire length of the MEC in the rat. We will use this to look for stronger effects on hippocampal replay, and for effects that are specific to certain types of replay, such as those that travel toward the goal. Further, we can test whether MEC is necessary for replay-dependent spatial learning as shown in Aim 1. (Aim 3) We also use advanced silicon probes to measure activity from hundreds of units along the length of the MEC. Therefore we will look for replay within MEC itself, and how it relates to hippocampal replay. This has been controversial in the literature, but with our increased cell yield we will be able to resolve this, and also examine sub-types of MEC cell such as grid cells, border cells, head direction cells etc. Taken together, our results will provide insight into fundamental mechanisms of learning and memory, that are affected in diseases such as Alzheimer's disease, epilepsy, stroke and normal aging.

NIH Research Projects · FY 2025 · 2014-09

PROJECT SUMMARY/ABSTRACT Pathogenic Rickettsiae are obligate intracellular bacteria that cause diseases such as spotted fever and typhus. We study the spotted fever group (SFG) species Rickettsia parkeri, which causes an eschar-associated human rickettsiosis and is experimentally tractable, making it an ideal model for revealing molecular mechanisms of SFG Rickettsia infection and virulence. Following invasion of host cells, SFG Rickettsia escape from the phagosome into the cytosol, replicate while avoiding ubiquitylation and autophagy, and polymerize host actin to promote intracellular motility and cell-cell spread. However, there are fundamental gaps in our knowledge of the molecular mechanisms by which SFG Rickettsia exploit or disrupt host cell structures and pathways to promote their infection cycle. Towards bridging these gaps, in the current granting period we discovered that the patatin- like phospholipase enzyme Pat1 is important for virulence and for escaping from host membranes including phagosomes and autophagosomes. We further showed that outer membrane protein OmpB and lysine methylation are crucial for virulence and for shielding bacteria from ubiquitylation and autophagy. We also observed that two actin-based motility proteins, RickA and Sca2, function independently in cell-cell spread and virulence. Finally, we developed an interferon receptor-deficient mouse model of eschar-associated rickettsiosis that can be used to evaluate the role of bacterial factors in virulence. These findings support the overall scientific premise that rickettsial proteins manipulate host cell components to enable bacterial escape from host membranes, avoidance of host ubiquitylation pathways, and mobilization of the host cytoskeleton for movement. However, key outstanding questions remain. How does bacterial phospholipase activity contribute to infection? How does bacterial surface architecture prevent ubiquitylation by host machinery? How and why do Rickettsia use two mechanisms for intracellular movement? We will address these outstanding questions, testing the overall hypothesis that the structure, function, and regulation of bacterial secreted and surface proteins is critical for manipulating or avoiding host cell molecules and structures, enabling infection of cells and virulence in animals. This general hypothesis will be tested in three Aims focused on uncovering the roles of Pat1, OmpB, RickA, and Sca2 in escape from host membranes, intracellular survival, and motility. The Aims are to: (1) determine how Rickettsia phospholipase activity contributes to infection; (2) reveal how Rickettsia surface architecture enables avoidance of ubiquitylation; and (3) establish how and why Rickettsia use two mechanisms for intracellular movement. The impact will be to reveal crucial molecular mechanisms used by Rickettsia and other pathogens to manipulate host cells and the importance of these mechanisms to infectivity. Our studies may also lead to improved diagnostics and treatments for rickettsial and other infections.

NIH Research Projects · FY 2025 · 2014-04

PROJECT SUMMARY / ABSTRACT Visual information is transmitted from the primate eye to the brain through at least 20 distinct retinal ganglion cell (RGC) types, each of which is thought to extract specific features from the visual scene. Although the most numerically abundant primate RGC types have been well characterized, the physiological functions of a dozen or more sparse types remain unknown. These uncharacterized RGC types are likely involved in crucial visual reflexes and image-forming vision, but their sparsity has made them difficult to study. To address this knowledge gap, we recently developed a robust experimental approach to link genetically defined RGC types with their specific morphologies and physiological functions. Our approach led to the discovery of On-type direction-selective ganglion cells (On-DSGCs) in the macaque retina, a sparse but highly conserved RGC type that plays a critical role in gaze stabilization across vertebrates. Here, we will build on this discovery and leverage recent insights from comparative genomics to test our central hypothesis that sparse primate RGCs serve the same critical visual functions as their orthologous cell types in lower vertebrates. In Aim 1, we will test the hypothesis that the preferred directions. In Aim 2, we will test the hypothesis that the macaque retina contains object-motion sensitive RGCs that, like their orthologs in mouse retina, selectively express the transcription factor, RUNX1. To address these aims, we will utilize our proven multimodal approach, which combines ex vivo two photon calcium imaging to measure functional responses, with post-hoc molecular identification to link each recorded RGC type to its transcriptomic identity. Our integrated approach will reveal novel primate RGC types with complex receptive field properties that have hitherto escaped detection due to their sparsity. Moreover, understanding the correspondence between RGC types in primates and other species will permit rational application of findings from lower vertebrates to advance understanding of the primate, and by extension, the human visual system. These outcomes will be crucial to develop more realistic models of human vision and ultimately to improve diagnostics and therapeutics for retinal disease.

NIH Research Projects · FY 2026 · 2014-02

PROJECT SUMMARY (See instructions): The assembly and release of HIV-1 from infected cells are essential steps in the viral replication cycle. HIV assembly is driven by the virally encoded Gag polyprotein. Bending of the plasma membrane into spherical buds, packing of the RNA genome (gRNA), and incorporation of the envelope glycoprotein (Env) are among the key events of assembly and budding. Release depends on the the host-encoded ESCRT proteins, which are recruited by Gag to the neck. Building on considerable progress in understanding structural and cellular mechanisms of HIV assembly, we are now in a position to answer the major open questions about how HIV-1 orchestrates its own release by hijacking ESCRT complexes. This process is especially timely in the wake of the discovery of the endogenous retroCHMP3 factor that restricts ESCRT-mediated release of retroviruses and other enveloped viruses in some new world monkeys, without unduly compromising host ESCRT functions needed for normal cell function. We will take a three-pronged approach to explaining how HIV-1 assembly and release are coupled through the ESCRT system. In the first aim, structural studies of biochemically tractable subassemblies of ESCRTs from humans and other species will be used to build up experimentally validated models of those aspects of the system that are still missing. These include the linkages downstream of ESCRT-1. Single particle cryo-EM structures will be determined and integrated models will be build in collaboration with Greg Voth. In the second aim, the entire system consisting of HIV-1 Gag, membranes, and human ESCRTs will be reconstituted in vitro. Scission function will be assessed biophysically using membrane nanotubes pulled by optical tweezers. Structures of the entire reconstituted system will be determined by cryo-electron tomography (cryo-ET) and subtomogram averaging {STA), with modeling guided by atomistic structures, either pre-existing or obtained in the first aim. In the third aim, a structural movie of release will be obtained by stepwise in situ cryo-ET imaging of HIV-1 as it escapes infected cells. ESCRT-mediated HIV-1 release will be trapped at various stages through the use of targeted inhibition of each step in the assembly. We will begin with targeted inhibition of the AAA+ ATPase responsible for the final stage release, VPS4A/B. RetroCHMP3 and other targeted dominant negative factors will be used to inhibit earlier steps in the pathway. Cryo-ET imaging will be carried out for budding events at the cell periphery,

NIH Research Projects · FY 2025 · 2013-12

OVERALL CORE: PROJECT SUMMARY The Berkeley Population Center (BPC), now in its 18th year of operations, requests a five-year P2C renewal to continue its unique contributions to population dynamics research. Taking advantage of both faculty affiliate expertise as well as institutional initiatives, the five Primary Research Areas (PRAs) to be supported in the renewal period continue with the five existing, well-established, thematic areas of: (1) Formal Demography, (2) Data Science and Demography, (3) Population Health, (4) Reproductive Health and HIV, and (5) Family Policy. Collectively these are methodological and substantive areas in which Berkeley holds academic prowess, and to which BPC contributes essential knowledge to population science. BPC affiliates are drawn from Demography, Public Health, Economics, Sociology, Public Policy, Social Welfare and other disciplines. Early- stage investigators are warmly welcomed to the Center and offered extensive mentoring and financial assistance to advance their intellectual development and research activity, fostering the development of new cohorts of researchers, especially those from underrepresented backgrounds. The BPC offers staff support for logistics on all aspects of research administration, real and virtual meeting space, and shares information in various ways. BPC expands population research through expert consultation on every phase of scientific projects, plus easy access to newly expanded top-notch physical facilities and other resources such as a data and computing lab as well as high performance computing. These combined activities and initiatives converge into an interlocking hub of support for interdisciplinary collaboration, intellectual interaction, and new and novel research initiatives. The BPC is poised to continue to be a world leader in its contribution to innovative and interdisciplinary population research that will transform how contemporary population challenges are addressed and translated.

NIH Research Projects · FY 2025 · 2013-09

Project Summary Neural circuits of the primary visual cortex (V1) are critical for generating perceptions of our external world. In V1, most neurons exhibit potent modulation by stimuli that are outside their receptive fields, a process termed `surround modulation'. Importantly, the magnitude and sign of surround modulation depend on the orientation of the center and surround – leading to the notion that flexible surround modulation contributes to scene segmentation, salience detection, contour integration, and figure/ground segregation. The specific neural circuits in the visual cortex that explain the orientation dependence of surround modulation are largely unknown. By combining two photon calcium imaging, two photon holographic optogenetics, in vivo patch clamp electrophysiology, and the first use of a two photon holographic mesoscope, we aim to reveal the precise synaptic and circuit architecture in the mouse visual cortex that mediates the earliest stages of image segmentation. First, we will measure the visually evoked synaptic conductances in cortical interneurons that explain their highly differentiated tuning properties to contextual visual stimuli. Next, we will precisely map the local and long-rate connectivity onto specific subtypes of cortical interneurons in vivo using two photon photo- stimulation. Finally, we will probe how two photon holographic co-activation of surround co-tuned ensembles in V1 or higher visual areas give rise to figure/ground modulation in V1 principal neurons. Together, our aims will establish a highly detailed mechanistic understanding for a visual computation responsible for object recognition.

NIH Research Projects · FY 2024 · 2013-07

Project Summary During this time of rapid evolution and uncertain future in the US healthcare system, it is critical that the US prepare a new generation of scientific leaders with the interdisciplinary training, flexible critical thinking skills and real-world grounding to provide the necessary leadership to make sound decisions for our future. The University of California, Berkeley (UC Berkeley) and University of California, San Francisco (UCSF), widely regarded as two of the top public universities in the world, both have long-standing health services research (HSR) training programs. UC Berkeley and UCSF propose a five-year renewal of the joint HSR T32 training program for four predoctoral and two postdoctoral trainees. The overall objective of the fellowship program is to harness collaboration, complementary skills, and training capacity of the two institutions to provide an outstanding training experience that emphasizes the application of interdisciplinary advances in social, behavioral, and data sciences to real world challenges of clinicians, healthcare delivery systems, and health policy makers. The predoctoral program provides rigorous training in research methods central to HSR, including data science, quasi-experimental methods, econometrics, and implementation science, as well as opportunities to partner with health care organizations and systems to conduct research that is responsive to their priorities. Predoctoral trainees are required to have previously completed coursework in epidemiology, economics, and statistics, and during our program they complete courses in research design, advanced statistics, health economics, public policy, and organization behavior. The four predoctoral traineeships are selected on an annual basis and each can be reappointed for an additional year, depending on coursework and research performance. The postdoctoral program, administered by the UCSF Philip R. Lee Institute for Health Policy Studies, is a two-year fellowship program that provides intensive experiential HSR training to doctorally-trained social and behavioral scientists, as well as doctoral-level health professionals with the equivalent of an MPH degree. Throughout their training, trainees participate in biweekly colloquia at UCSF and UC Berkeley to expose them to cutting edge research in progress by nationally and internationally recognized health services researchers.

NIH Research Projects · FY 2025 · 2013-05

Project Abstract The Environmental Health Sciences Division of the School of Public Health at the University of California, Berkeley proposes to continue its summer research internship for undergraduate students, to introduce them to the field of environmental health science with the long-term aim of increasing the number of talented students who pursue graduate degrees and careers in this field. Students will be recruited from the University of California, Berkeley, San Francisco Bay Area colleges, and targeted universities across the state and country that meet diversity criteria. Students admitted to the program will be matched to faculty conducting environmental health research of interest to the intern. Student interns will conduct research with the designated faculty member and, when appropriate and in accordance with the learning objectives, members of the research team, including staff scientists, graduate students and postdoctoral scholars. The interns will attend twice weekly seminars including topics on environmental health sciences and the responsible conduct of science, as well as participate in research discussion groups and group field trips. They will meet at least weekly with their faculty mentor. The faculty mentors will define projects suitable for their intern, monitor the intern's progress on a weekly basis, and provide written feedback on the performance of the intern and the structure and administration of the program. Likewise, the interns will provide written and verbal feedback about their mentor and the program as a whole. Research projects may include direct data collection, literature reviews, laboratory experiments, computer-based modeling, data analysis, and research to practice initiatives, among others. The grant management team will maintain its Advisory Committee made up of project management staff, faculty, and students to ensure that the summer internship program benefits from experience and services available on the UC Berkeley campus. It will improve its recruitment process to better reach minority and disadvantaged students and maintain an evaluation process to ensure that the program continuously improves over the five- year period of the grant.

NIH Research Projects · FY 2025 · 2012-08

Project Summary/Abstract Youth who exhibit an evening circadian chronotype (“night-owls”) follow a delayed sleep schedule, increasing activity later in the day and both going to sleep and getting up later, compared to morning types (“larks”). Eveningness arises from a confluence of psychosocial, behavioral and biological factors and is an important contributor to vicious cycles that escalate vulnerability and risk among youth. While the basic biological shift toward eveningness—initially triggered around the onset of puberty—may be difficult to modify, the psychosocial and behavioral contributors are modifiable. Supported by R01HD071065, we have conducted a “treatment experiment” in which we delivered the Transdiagnostic Sleep and Circadian Intervention for Youth (TranS-C) to reduce eveningness among 10-18 year olds. We randomly allocated youth with an evening chronotype, and who were “at risk” in at least one of five health-relevant domains (emotional, cognitive, behavioral, social, physical), to either: (a) TranS-C (n = 89) or (b) Psychoeducation (n = 87). While the results were promising, some drop off in treatment gains were observed. This is consistent with prior research documenting that a drop-off in the years following treatment is too commonly observed. Indeed, there have been calls to study if and how behavioral interventions are maintained (NOT-OD-19-040). Hence, in this revised renewal application, we propose to study the maintenance of behavior change by conducting a 6-year follow-up of the unique cohort of youth recruited for R01HD071065. The youth will be 16 to 26 years old. They will be assessed for sleep and circadian functioning and functioning in five health-relevant domains (emotional, cognitive, behavioral, social, physical) (SA1) and for their utilization of sleep health behavior (SA2). As a next step in this research program, we propose to evaluate if a Habit-based Sleep Health Intervention (“HABITs”)—a novel low-cost approach derived by leveraging the science of habit formation—improves the utilization of sleep health behavior and improves sleep and circadian outcomes and functioning in the five health-relevant domain outcomes in the short and longer term (SA3). An independent sample of youth who exhibit a high level of eveningness and are “at risk” in at least one of the five health-relevant domains will be randomly allocated to HABITs alone or HABITs plus Text Messaging (“HABITs+Texts”) (n = 160). The text messaging portion is derived from learning theory, the Behavior Change Wheel and focus groups. We will also examine if sleep health behavior that has become habitual mediates the effects of treatment on improvement in sleep, circadian and health outcomes. Moderation analyses will examine if intervention effectiveness is related to age/developmental stage, sex, SES, racial/ethnic minority group and season of participation. This research will advance knowledge on longer-term outcomes, the role of eveningness as a mechanism contributing to poorer youth outcome and the value of leveraging of learning theory and the science of habit formation in health promotion.

NIH Research Projects · FY 2025 · 2012-07

This competing application for a training program in Immunology and Molecular Medicine (IMM) supports the training activities of 18 field-leading scientists whose research centers on immunobiology. The training faculty are all members of the Department of Molecular and Cell Biology (MCB), and each has ample research support and a strong commitment to mentoring. The training program is centered in the MCB Division of Immunology and Molecular Medicine, in which most training faculty are members. The IMM Training program focuses on basic immunological mechanisms in the context of infections, cancer, and immunopathology. We are motivated by the shared belief that achieving a thorough, basic understanding of recognition, activation, regulation, differentiation, and interactions of cells of the immune system will lead to therapies for infectious disease, cancer and other ailments, and that training young investigators in this approach, while providing broad training in basic molecular and cell biology, offers a most productive avenue for enhancing success in this endeavor. The program is strongly committed to recruiting and training students and postdocs from diverse personal backgrounds. The IMM training program will provide a home for trainees in Immunology and Molecular Medicine and support for training related activities. We propose support for 5 graduate students and 3 postdoctoral fellows. Graduate students will generally be supported starting in their second year to ensure commitment to the research area and to provide additional information to evaluate their capability of providing exceptional contributions to the program. Postdoctoral trainees will generally be supported starting in their first or second year. In general trainees will be supported for 2 years on the training grant, with additional years supported from other sources. While this training grant recently completed its 10th year of funding, the grant is the successor of an NCI- supported training grant (Molecular Immunology and Tumor Biology Training Program, T32 CA009179), which supported immunology research at Berkeley for 35 years.

NIH Research Projects · FY 2025 · 2012-05

PROJECT SUMMARY/ABSTRACT Biophysical cues encoded in the structure, mechanics, and dimensionality of the stem cell microenvironment are now appreciated as important regulators of self-renewal and differentiation. For the past 15+ years, including two periods of R01 support, we have been exploring mechanistic and translational aspects of this regulation in hippocampal neural stem cells (NSCs), which generate new neurons into adulthood and contribute to neurological disease and repair. We have made several important contributions to the field’s understanding of stem cell mechanobiology, including the discovery that NSC lineage decisions are maximally sensitive to extracellular matrix (ECM) mechanics within a restricted temporal window, during which stiffness cues are processed by a signaling network that includes RhoA GTPase, actin/myosin, angiomotin, YAP, and β-catenin. Our discoveries raise two important questions of general interest to the stem cell field, which will serve as the foundation for our renewal application. First, what molecular mechanisms govern NSC mechanosensitive lineage commitment in three-dimensional (3D) ECMs, and how do these mechanisms differ from two-dimensional (2D) ECMs? We will build on our exciting recent discovery that the transcription factor Egr1 is a critical, 3D-specific regulator of NSC mechanosensitive lineage commitment. Second, how do stem cells dynamically integrate mechanical inputs on the time scale of minutes to hours to trigger functionally important signaling events? Here we will leverage our preliminary studies in which we have probed the timing of mechanosensitive signaling events with mismatched DNA-crosslinked viscoelastic hydrogels and optogenetic reagents that allow timed activation of RhoA and Cdc42 activation. We have two specific aims: In Aim 1, we will investigate mechanisms through which Egr1 controls mechanosensitive lineage commitment in 3D matrices using a combination of 2D and 3D engineered biomaterials, candidate-based molecular studies, and screens to identify Egr1 targets relevant to neurogenesis. In Aim 2, we will investigate how stiffness cues from 2D ECMs are triggered on the minutes-to- hours time scale are integrated over hours to days to control lineage commitment. Work in this aim builds on our observation that timed optogenetic stimulation of RhoA and incorporation of viscous (loss) properties into elastic ECMs both suppress NSC neurogenesis. We will identify critical regulatory time scales for both perturbations and determine if they influence lineage commitment through common mechanisms. To integrate aims, we will investigate the time-dependence of mechanosensitive lineage commitment in 3D ECMs and ask if RhoA stimulation and stress relaxation act through Egr1 to suppress neurogenesis. Our work will accelerate the field’s understanding of how stem cells sense and act upon mechanical signals to guide fate decisions, a problem of high fundamental and translational value. We will also marry several innovative approaches, including optogenetics, viscoelastic ECMs, genome editing, and sequencing/screening technologies. We expect that our studies will provide an intellectual roadmap that can be applied to other ECM and stem cell systems.

NIH Research Projects · FY 2025 · 2011-07

Abstract A large body of literature now indicates that antenatal and neonatal antibiotic exposure is associated with adverse childhood outcomes due to disruption of the developing microbiome. In premature infants, the standard of care for many decades has included the administration of broad-spectrum antibiotics in the first hours of life as treatment for a presumptive diagnosis of early onset sepsis. However, nearly all preterm infants receiving these antibiotics do not actually have sepsis. This grant renewal application proposes an ancillary microbiome study linked to the NANO (NICU Antibiotics and Outcomes) Trial, a recently launched clinical trial that will challenge this longstanding practice of immediately prescribing antibiotics to newborn preterm infants. NANO will test the hypothesis that antibiotics at birth worsens outcomes in preterm infants that are clinically stable. This multicenter trial, which is led by members of our research team, will randomize 802 premature infants to receive intravenous ampicillin and gentamicin or a saline placebo control. The study will measure the impact of variables including mode of delivery, gestational age, sex, and receipt of maternal milk, and administration of maternal antepartum antibiotics. Infant fecal samples in the first month of life as well as maternal fecal and vaginal swabs will be collected in NANO for basic microbiome profiling in the antibiotics and placebo groups using 16S rRNA gene sequencing. Here, we propose to augment microbiome analyses of NANO study subjects using novel strain-level metagenomic strategies and by analyzing samples beyond the first month of life. With this strategy, we propose to Aim 1. Test the hypothesis that empiric antibiotics (EA) disrupts mother-infant strain sharing in preterm infants. Aim 2. Test the hypothesis that EA increases the abundance of gut bacterial antimicrobial resistance genes in preterm infants. Aim 3. Test the hypothesis that EA delays the transition from a gut ecosystem dominated by facultative anaerobes to one dominated by obligate anaerobes. Because NANO is a first-of-its-kind clinical trial evaluating antibiotic therapy during the first days of life, this ancillary study will provide a rare opportunity to ask and answer a unique set of questions about the biology of early gut bacterial colonization.

NIH Research Projects · FY 2025 · 2011-03

Project Summary The long-term goal of this project is to identify the genetic circuitry regulating tooth formation and replacement. As 30 percent of people worldwide over the age of 65 have no natural teeth, understanding how teeth regenerate is a major goal in biology. Furthermore, teeth, like most organs, form through repeated reciprocal signaling between epithelia and mesenchyme. Thus, understanding the genetic basis of tooth formation and replacement is important both for understanding organogenesis in general, as well as for understanding how teeth can be regenerated in vitro and ultimately in vivo. Teeth are homologous to other vertebrate skin appendages including mammalian hair, and shared genes regulate both tooth and hair formation. Although genetic studies in humans, mice and other vertebrates have identified signaling pathways involved in tooth formation, less is known about how genes regulate tooth replacement. In contrast, how genes regulate mammalian hair regeneration is much more understood. One parsimonious hypothesis is that teeth and hair regenerate using similar genetic circuits. Fish retain the ancestral jawed vertebrate condition of constant tooth replacement throughout adult life. Fish also fertilize their offspring externally in large numbers, providing powerful systems for developmental biology and genetic analyses. Threespine stickleback fish (Gasterosteus aculeatus) offer a new and powerful system to learn the genetic basis of tooth formation and replacement. Relative to low-toothed marine ancestors, derived freshwater populations evolve major heritable increases in tooth number and tooth replacement rates. The different forms can be crossed in the lab, enabling detailed and unbiased forward genetic analyses to map factors controlling the changes in tooth number. Genetic and genomic experiments have mapped one genomic region controlling tooth number to a cis-regulatory intronic tooth enhancer of the Bone Morphogenetic Protein 6 (Bmp6) gene in one high-toothed population. Relative to the marine enhancer, the freshwater enhancer displays expanded tooth epithelial expression, and reduced tooth mesenchymal expression, suggesting these spatial shifts in enhancer activity underlie evolved increases in tooth number. Furthermore, in mice, BMP signaling negatively regulates hair regeneration, and in fish BMP signaling negatively regulates tooth replacement. Together these data support the hypothesis of shared genetic circuitry regulating tooth and hair regeneration. To test this hypothesis, three specific aims are proposed. First, transgenic and genome editing experiments will determine which mutations in the freshwater Bmp6 enhancer affect expression differences and tooth number. Second, genome editing experiments will determine Wnt ligand function during tooth formation and replacement. Third, vital dye pulse-chase experiments will test whether tooth replacement is coordinated within a tooth field. Together these aims will reveal fundamental knowledge of the developmental genetic circuitry regulating tooth formation and replacement, and provide further tests of the hypothesis that teeth regenerate similar to mammalian hair.

NIH Research Projects · FY 2026 · 2010-05

ABSTRACT Kaposi sarcoma-associated herpesvirus (KSHV) is the predominant etiologic agent of AIDS- associated cancers. It is endemic in many areas of Africa where, due to the extraordinarily high HIV burden, Kaposi sarcoma has emerged as one of the most common cancers. During AIDS-induced immunosuppression, KSHV replication is no longer effectively controlled, and, together with a large latently infected population of cells, contributes to disease progression and transmission. During lytic replication, KSHV dramatically remodels the host gene expression environment. A key player in this remodeling is the virally encoded, messenger RNA (mRNA) endonuclease termed SOX, which degrades cytoplasmic mRNA. SOX activity plays diverse roles in the gammaherpesvirus lifecycle and immune evasion. However, little is known about how the SOX protein is functionally regulated, either by the virus or by the host cell. Our preliminary data indicate that it is regulated both spatially and kinetically to balance the need for host shutoff against viral gene expression. In Aim I, we will define how these regulatory features impact SOX targeting of cellular and viral RNA transcripts throughout the course of KSHV lytic infection. SOX-induced cytoplasmic mRNA decay activates a feed forward loop that suppresses cellular but not viral transcription by RNA polymerase II (Pol II), effectively magnifying the depletion of the cellular mRNA pool. We previously showed that transcriptional repression requires the nuclear influx of a pool of cytoplasmic RNA binding proteins, which is triggered by mRNA. Thus, SOX profoundly alters both cellular RNA stability and synthesis and serves as a model for understanding how pathogenic stress impacts transcription. In Aim 2, we will define the sub-nuclear changes that underlie transcriptional repression during lytic KSHV infection, including how specific RNA binding proteins that are relocalized upon mRNA decay contribute mechanistically to this phenotype. Findings derived from this proposal should have a sustained impact on the field of gammaherpesvirus biology and reveal how stress or virus-induced alterations to mRNA stability influence seemingly distal components of the gene regulation circuitry.

NIH Research Projects · FY 2026 · 2009-07

The development of neural circuits involves a rich interplay between molecular cues and neural activity. This is perhaps most well studied in the visual system, where several molecular interactions have been identified as being critical for early establishment of coarse visual maps while both early spontaneous activity called retinal waves prior to eye opening and visual deprivation after eye opening leads to map refinement. We study this question in a direction-selective ganglion cells of the retina. Direction selective ganglion cells respond strongly to an image moving in the preferred direction and weakly to an image moving in the opposite, or null direction. Direction-selective ganglion cells are critical for driving ocular-motor reflexes that stabilize images on the retina as we move through a visual scene as well as for sensing the movement of objects within the visual scene. The preferred directions of direction selective ganglion cells cluster along four directions that align along two optic flow axes, an organization we refer to as the direction selectivity map. The mechanisms that instruct the development of this direction selectivity map are unknown. Here we propose to use a combination of state-of-the-art two-photon calcium imaging, electrophysiology, and transgenic mouse strategies to determine the mechanisms that underlie the development of the direction selectivity maps. In particular, we will determine if neural signaling, either through gap junctions or retinal waves, play a critical role in the formation of these direction selectivity maps. Finally, we will test candidate synaptogenic molecules identified in an RNA-seq screen that may instruct the emergence of the functional inhibitory synapses that underlie direction selective responses.

NIH Research Projects · FY 2025 · 2008-07

This is a MERIT extension of R37 AI075039. In the previous funding period we addressed the fundamental mechanisms by which pathogen-encoded virulence activities are detected by the innate immune system. We focused on an innate immune sensor called the NLRP1 inflammasome. This sensor was known to be activated by a protease toxin, called lethal factor (LF), that is secreted by the bacterial pathogen Bacillus anthracis. We demonstrated that LF activates NLRP1 by direct proteolytic cleavage of NLRP1. We found that this cleavage results in the N-end rule-dependent degradation of NLPR1 by the proteasome. Unexpectedly, however, we found that LF-induced degradation does not inactivate NLRP1. Instead, we found that NLRP1 underwent “functional degradation”, a unique process by which the signaling competent C-terminal CARD domain of NLRP1 was released and thereby able to undergo oligomerization and inflammasome formation. Our work identifying the molecular basis by which NLRP1 is able to sense pathogen-encoded enzymatic activity led us to ask whether NLRP1 might sense additional pathogen- encoded activities and, moreover, whether additional pathogen-encoded effectors might be detected by other innate immune sensors. Indeed, we discovered that a pathogen-encoded ubiquitin E3 ligase called IpaH7.8, secreted into host cells by the bacterial pathogen Shigella flexneri, is also sensed by NLRP1. In the extension period, we propose to continue to investigate the molecular mechanisms by which the enzymatic activities of pathogen-encoded virulence factors are detected by the innate immune system. We are conducting screens to identify novel virulence factors that stimulate host immune responses. We will then conduct mechanistic studies to identify the sensors of these virulence factors and the molecular basis of sensor activation. Together our studies will illuminate fundamental molecular mechanisms by which pathogens are sensed by the innate immune system. RELEVANCE (See instructions): Human health depends on the accurate and sensitive detection of pathogenic microbes by the innate immune system. We propose to determine fundamental mechanisms by which the virulence activities of bacterial pathogens are sensed by the innate immune system.