University Of California Berkeley

NIH Research Projects · FY 2024 · 2024-06

PA-21-151 UCB PI: Seth M. Holmes PROJECT SUMMARY Medical and public health research has shown how economic and social structures negatively shape health outcomes in minority populations. This research has foregrounded culture and access to healthcare as the dominant determinants of health outcomes. However, an improved understanding of the ways larger social processes, including structural racism, impact minority health and health disparities would enhance health services to minority populations and reduce health disparities. Most health care practitioners are not trained in social science frameworks, and most social scientists who do related research do not know how to translate their research to make it accessible and actionable by clinicians. This project contributes to reducing health disparities by organizing an interdisciplinary conference to bridge social science and clinical knowledge of health in minority populations - especially immigrant populations. The conference will be attended by social scientists, clinicians, policy makers and the general public. The conference will include panels and keynotes delivered by expert clinicians and social scientists to thematically orient the conference and disseminate cutting-edge research on minority health and health disparities. Presenters come from diverse backgrounds and have specialized knowledge of the key health issues facing minority populations in the United States, including such topics as sexual gender minorities, rural residents, and immigrant communities. In addition to the keynotes and panels, clinicians will present real case studies of minority patients for rigorous co-analysis with social scientists in small groups. Cases will include maternal mortality/morbidity and infant mortality and infectious diseases. These workshop sessions will be designed to use diverse disciplinary knowledge, methods, and theories to understand cases previously considered intractable in new ways, sensitive to the unique structural and socioeconomic contexts of each case. Group discussions will maintain a translational focus so as to produce actionable interventions, health systems innovations, and original programs for equitable health policy and practice, with a focus on patient-clinician communication in primary care. Findings from this conference will have a wide-spread, lasting influence through its dissemination in the “Case Studies in Social Medicine for Health Equity,” which has already been accepted for publication in The Lancet. By fostering interdisciplinary collaborations to reduce health disparities, this project will cultivate cross-sector partnerships, produce original knowledge on health equity, and promote the dissemination and translation of this knowledge into novel interventions suited to ameliorate health injustices in the long-term.

NIH Research Projects · FY 2025 · 2024-06

SUMMARY Neonatal mortality remains high in sub-Saharan Africa, where 43% of global neonatal deaths occur. In rural, low-income health facilities, contaminated drinking water and environmental hygiene conditions put newborns and their mothers at risk for healthcare-associated infections, including antibiotic resistant infections. In Kenya, health facilities are typically limited to water treatment with manual, point-of-use products that require extensive effort to use and are insufficient for the volumes of safe water required to provide quality healthcare. Our team has developed a low-cost, in-line chlorination technology, the Venturi, that automatically doses chlorine without moving parts or electricity, and is able to use chlorine produced by an off-the-shelf electrochlorinator, which can produce chlorine disinfectant solution using only water, salt, and intermittent electricity. We propose to conduct a cluster-randomized controlled trial across 30 health facilities to generate rigorous evidence on the maternal and neonatal health benefits of chlorinated water supply paired with on-site generation of chlorine for disinfection. This study combines our team’s expertise in engineering, epidemiology, microbiology, and pediatric medicine to accomplish the following aims: 1) determine the impact of the intervention on pathogenic and antibiotic resistant bacteria contamination in water supplies, high-touch surfaces, and healthcare worker hands, 2) quantify intervention effects on gut colonization of mothers and neonates by a panel of pathogenic and antibiotic resistant bacteria species linked to neonatal infection, using molecular and culture- based methods, and 3) follow up with >20,000 mother-neonate dyads to measure intervention effects on symptoms of severe bacterial infection in the first week of life. Infection prevention through effective water, sanitation, and hygiene (WASH) has been cited by national action plans as a key tool in the fight against antimicrobial resistance and, while global data show dire WASH conditions in low- and middle-income (LMIC) health facilities, there exists very little guidance for implementing effective interventions. This proposal is a time sensitive opportunity to leverage substantial philanthropic funding to deliver a novel and scalable intervention to health facilities. The overarching goal is to generate actionable evidence to inform investments in chlorination at health facilities to improve neonatal health and reduce the threat of antibiotic resistant infections.

NIH Research Projects · FY 2025 · 2024-06

Project Summary/Abstract: The human transcriptome contains tens of thousands of long noncoding RNAs (lncRNAs). Although lncRNAs are involved in many basic biological processes and their dysregulation implicated in human disease, only a handful have been functionally and mechanistically studied to date. This is largely due to lack of efficient tools for lncRNA knockdown, imaging, and biochemical characterization, combined with evidence suggesting that many lncRNAs may be nonfunctional or function only in a context-specific manner. The overall goal of this proposal is to identify and characterize functional lncRNAs in human cells, deepening our understanding of fundamental RNA biology while laying the foundation for advances in disease diagnosis and treatment. To overcome prior technical limitations in studying lncRNAs, this work will take advantage of my newly developed RNA-targeting CRISPR-Csm methodology. This tool enables highly efficient knockdown of both nuclear and cytoplasmic RNAs with limited off-targets and cytotoxicity. When fused to GFP or other proteins, the tool also enables programmable live-cell RNA imaging or tagging without genetic manipulation. The specific aims of this project are thus to: 1) adapt CRISPR-Csm technology for high-throughput screening to enable investigation of thousands of lncRNAs in parallel; 2) perform an RNA imaging screen to identify candidate functional lncRNAs based on interesting morphology and subcellular localization; 3) perform an RNA knockdown screen to inform lncRNA function based on changes in single-cell transcriptome profile; 4) perform proximity labeling experiments to inform lncRNA mechanism based on interacting protein network. Significant findings relevant to the fields of CRISPR technology, noncoding RNA, and gene regulation are expected. Areas of additional scientific training that will enable successful completion of this work include experience conducting high-throughput CRISPR screens, in situ sequencing technique/image analysis, and single-cell RNA-sequencing analysis. The mentored phase of this award will be supervised by Dr. Jennifer Doudna, a world-renowned leader in RNA biology and CRISPR technology. Professional development activities centered around laboratory management, grant writing, and faculty search preparation will be completed to gain the remaining skills necessary before running my own lab. The excellent research environment of the Innovative Genomics Institute at UC Berkeley, combined with the expertise of my Scientific Advisory Committee, will ensure the successful and timely completion of this work and my transition to independence.

NIH Research Projects · FY 2025 · 2024-06

Project Summary To accommodate a vast amount of sensory information, the cortex selectively distills perceptually and behaviorally salient features from stimuli. The visual system uses visual segmentation, the process of separating a visual scene into individual elements, to achieve this. Mechanistically, visual segmentation is enabled by “surround suppres- sion”, the attenuation of neural responses when a stimulus’ properties — such as orientation, contrast, or phase — are homogeneous. For orientation, neurons are maximally suppressed when a stimulus is iso-oriented with its surround and is relieved when they are orthogonal. Therefore, the orientation dependence of surround suppression is an important cortical computation that underlies accurate and efficient visual processing of stimuli. However, the exact mechanisms and organization of neural circuits responsible for generating oriented surround suppression is unknown. This proposal will describe and test, in vivo, a detailed mechanistic circuit model explaining how oriented surround suppression is generated in the cortex. The primary hypothesis is that orientation-dependent surround suppression results from feedback connections from higher visual areas onto inhibitory interneurons in the primary visual cortex. Specifically, I propose that neurons in the mouse higher visual area lateromedial (LM) synapse onto somatostatin-expressing (SST) interneurons in V1 — a key mediator of surround suppression — such that cells with similar orientation preferences are preferentially connected. In order to test this, I will exploit a novel two-photon holographic mesoscope that can record and manipulate neurons across multiple brain regions for the first time. With this microscope, I will holographically photostimulate excitatory neurons in LM, either sequentially or simultaneously, while recording from multiple cell classes in V1. First, I will use this technique to test the connectivity between single excitatory neurons in LM and their SST targets in V1 to determine how orientation preference governs connectivity. Then, I will activate ensembles of iso-oriented LM neurons to generate surround suppression when there should not be any. Together, these experiments will advance our fundamental understanding of how neural architecture governs cortical computations in surround suppression. Consequently, these results will also shed light on how neural circuits across brain areas coordinate to drive visual perception. The proposed work not only improves our understanding of neural architecture, but also how disruptions in these precise networks can lead to a number of psychiatric and neurological diseases.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract The genealogical structure for whole genomes can be described through Ancestral Recombination Graphs (ARGs). ARGs are summaries that contain all of the information in genomic sequencing data about processes such as demographic history, selection, and recombination. The primary objective of this research is to develop a suite of computational tools that use posterior sampling of ARGs in order to provide methods for testing hypotheses about the distribution and evolution of genomic variation, and in general, to provide improved quantification of mutation, recombination, selection, and demographic history. These methods will be full likelihood/Bayesian methods that can take advantage of the rich population genetic information in whole-genome sequencing data. We expect the methods to scale up to allow posterior sampling of ARGs from a coalescence prior for many hundreds, or perhaps thousands, of genomes. We will make an open-source, user-friendly, flexible, and integrated program available to other researchers that will allow them to test a wide range of demographic and evolutionary hypotheses on their own data. We will also develop associated methods for ancestral inference of past migration and the geographic location of ancestors of an individual. Additionally, we will develop improved methods for quantifying spatiotemporal patterns of natural selection affecting the genome. We will apply the methods to modern and ancient DNA to test hypotheses about the relative contribution of demographic processes and natural selection for shaping the landscape of phenotypic variation in Europe, including disease susceptibility. We will also use the methods to revisit an ongoing controversy of the relative importance of changing mutation patterns and changing generation times in shaping the pattern of human mutation variation. Finally, we will use the methods to develop more accurate human recombination maps and to test hypotheses about recombination rate variation. In addition to this, we will develop new Bayesian Markov Chain Monte Carlo methods for estimating Developmental Lineage Trees (DLTs) using mitochondrial heteroplasmies and single cell DNA sequencing. We will also develop methods that can jointly analyze single cell RNA sequencing and DNA sequencing data to make joint models of DLTs with associated transitions in expression state. Such explicit temporal models of cell differentiation will be central in the translational aspects of cell specific analyses, in particular for predicting the effects of various forms of medical intervention.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY The diarrheal disease shigellosis impacts 80-165 million individuals and causes an estimated 600,000 deaths worldwide each year, primarily among children, and no effective vaccines are available. The human gut microbiome is suspected to play a role in protection against Shigella infection, yet this is largely undefined. We propose to investigate the role of the human gut microbiome in protection against Shigella both in a novel mouse model and in culture. Human challenge studies with Shigella have identified individuals resistant to infection, even among people assumed to be naive to the pathogen. We will use stool samples collected from humans prior to Shigella challenge to characterize microbiomes that are sensitive and resistant to Shigella challenge. We will perform metagenomic sequencing to identify features of interest and transplant these communities into germ-free mice. We will use Shigella susceptible mice deficient in the NAIP-NLRC4 inflammasome to establish a novel humanized mouse model for shigellosis and identify factors driving microbiome-mediated protection. Our preliminary data suggests that specific human bacterial strains can decrease S. flexneri colonization, but are not as effective as complex human microbiomes, which can provide full protection. We will further characterize bacterial mechanisms inhibiting Shigella growth and virulence in culture using diverse gut bacterial culture collections and multiple experimental approaches. New therapeutic strategies are essential for future prevention and treatment of Shigella given the expansion of antibiotic resistant strains. This proposal will identify key bacterial species important for Shigella resistance that can pave the way for microbial therapeutics and prophylactic regimens in high-risk individuals, including children. It will also establish for the first time a Shigella susceptible mouse model with a human microbiome, enabling new avenues of research into gut microbiome-Shigella interactions.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY/ABSTRACT Humans form social attachments and develop friendships throughout our lifetimes, with fundamental consequences for wellbeing. Decades of research have established the importance of the neuropeptide oxytocin (OT) for social bond formation with reproductive partners. In contrast, very little is known about the mechanisms supporting selective affiliation between non-mate peers. Prairie voles exhibit a human-like social structure, forming specific and selective relationships with mates and same-sex peers, unlike other laboratory rodents. The goal of this project is to understand the roles oxytocin plays in shaping the selectivity of peer relationships in prairie voles, particularly its dual functions in promoting acceptance of familiar partners and avoidance of unfamiliar individuals. We will use complementary neuropharmacological and genetic manipulations to test the necessity and sufficiency of oxytocin signaling for partner approach and stranger avoidance and aggression. We make use of natural contrasts between reproductive (mate) and non- reproductive (same-sex peer) relationships in prairie voles, as well as species comparisons between peer relationships in prairie voles and prior studies in meadow voles to understand the specificity and generality of these mechanisms. By manipulating oxytocin across multiple circuits, we have a unique opportunity to determine how OT influences selectivity across brain regions and relationship types. This work has the potential to elucidate the neural basis of peer relationships, and to provide the foundation for understanding how prosocial and antisocial factors of relationships are related and mediated. Our long-term goal is to better understand how friendship-like relationships are established and maintained, and how the mechanisms underlying social relationships in prairie voles can be translated to human relationships.

NIH Research Projects · FY 2026 · 2024-04

The survival of neuronal cells critically depends on the correct function of their lysosomes, catabolic organelles that play key roles in disposing damaged and harmful cellular components. Niemann-Pick type C (NPC) is a neurodegenerative and metabolic disease triggered by mutations of the NPC1 gene, which encodes a lysosomal membrane protein involved in the trafficking and partitioning of cholesterol. In cells lacking NPC1, cholesterol accumulates aberrantly inside the lysosome, triggering a cascade of downstream events that impact mitochondria, ultimately leading to cellular dysfunction and death. Understanding how the molecular details of the primary lysosomal dysfunction, and the downstream processes that impact mitochondrial homeostasis and overall cell metabolism is key to the design of more effective and targeted strategies to restore neuronal cell homeostasis in NPC. Recently, the PI (RZ) and co-Investigator (PO) laboratories have made foundational discoveries that deepened the current understanding of NPC pathogenesis. In particular, through organelle immunoisolation and profiling we uncovered a profound impairment in lysosomal proteolysis, hydrolase content and membrane stability, coupled with defective delivery of damaged mitochondria to the lysosome during autophagy. Additionally, through initial studies in iPSC-derived patient-specific and NPC1-deleted neurons, we uncovered a direct connection between alterations of lysosomal function, mitochondrial metabolism and neuronal failure. Finally, we established that mTORC1, a nutrient-sensing pathway based at the lysosome, becomes dysregulated in NPC, and that its pharmacological manipulation corrects multiple organelle defects. Combined, these findings provide a rich and detailed understanding of molecular aspects of NPC pathology. Specifically, they lead us to hypothesize that the lysosomal cholesterol-mTORC1 pathway we discovered drives loss of neuronal cell homeostasis and may be a therapeutic target in NPC. In order to effectively target the cholesterol- mTORC1 axis for neuronal resilience and NPC therapy, we propose to i) mechanistically and structurally elucidate the molecular mechanisms connecting cholesterol levels to mTORC1 regulation, ii) determine how selectively disabling the cholesterol-mTORC1 sensing pathway impacts mitochondrial metabolism and neuronal survival in NPC disease models iii) discover new pathways that control organelle function and cellular metabolism, and evaluate their role as candidate NPC disease modifiers. To accomplish these goals, we will combine biochemical, cellular and unbiased approaches, including functional genomics and metabolomics in both neuronal and non-neuronal models of NPC disease. We will rigorously prioritize and validate the most promising targets, and elucidate their role within the framework of organelle function and neuronal cell homeostasis. This proposal will deepen our understanding of NPC drivers and disease modifiers, with relevance to neurodegenerative conditions linked to cholesterol imbalance, such as Alzheimer Disease.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY During the last two decades, nearly half a million people died each year from heat-related causes; climate change is expected to exacerbate the burden of adverse health outcomes. Heat stress has been associated with an increase in all-cause mortality, cardiovascular disease and mortality, chronic respiratory disease, lower respiratory infection, chronic kidney disease, diabetes, adverse pregnancy outcomes, and poor mental health. In this RO1, we propose to determine personal heat stress of low-income individuals who do not have access to air conditioning, evaluate the effectiveness, acceptability, feasibility, and scalability of building-level cooling strategies to reduce indoor heat stress among vulnerable individuals, and evaluate the impact of these interventions on heart rate. A disproportionate burden of heat-related death and disease is borne by low-income communities because they do not have access to cooling and suffer from comorbidities that exacerbate the adverse impacts of heat stress. South Asia faces the greatest current and predicted loss in disability-adjusted life years due to heat stress, and heat stress is particularly strong in informal settlements. As such, we plan to conduct this study in informal settlements in Dhaka, Bangladesh. Our overall hypothesis is that individuals who live in homes with corrugated iron roofs and walls are at elevated risk of heat stress and that it is possible to modify homes to prevent increases in heart rate associated with heat stress, ultimately reducing cardiovascular morbidity and mortality. Aim 1 will characterize personal heat stress in individuals across age ranges, occupations, sex, and housing types, and examine heterogeneity in the effect of existing building variation on heat stress. Aim 2 will model building-level interventions, test their ability to cool indoor spaces, and evaluate their impact on heart rate in a randomized-controlled trial. In Phase 1, energy modeling will be used to evaluate the cooling potential of 12 passive or active building-level infrastructure modifications, material additions, and technologies to identify six strategies with maximum effectiveness for households in informal, low-income settlements in Dhaka, Bangladesh. In Phase 2, we will implement each of the six strategies in 17 homes to experimentally assess their impact on indoor thermal conditions and determine their feasibility and acceptability. The two most cost-effective interventions will be tested in a randomized controlled trial in 459 houses in Phase 3. Phase 3 will evaluate the impact of the interventions on residents’ heart rate (primary outcome), blood pressure, self-reported thermal comfort, wellness, productivity, fatigue, and indoor thermal conditions (secondary outcomes).

NIH Research Projects · FY 2025 · 2024-04

-Project Summary- As neurons grow, their dendrites develop a unique structure and the ability to perform computations that are essential for nervous system function. Research studying dendritic maturation has largely focused on mechanisms that shape the morphology of the cell but understanding how this structural development relates to the functional development of the dendrite is critical. To that end, I propose investigating dendritic development in a model that allows me to measure functional and structural maturation independently: starburst amacrine cells (SACs) in the mouse retina. SACs are axonless interneurons that have radially symmetric dendrites extending out from the soma. Each branch has a primary dendrite proximal to the soma that receives glutamatergic input from bipolar cells, then branches out in the distal regions where neurotransmitter is released at output synapses marked by varicosities. Functionally, each branch acts as a direction of motion detector; varicosities preferentially release neurotransmitters in response to visual stimuli moving away from the soma. Thus, each branch is an independent computational unit that relays directional information to postsynaptic partners. When and how SAC dendrites develop this functional property is unknown, but there is some evidence to suggest it may happen independently of the morphological development of the cell. Thus, I propose to test the hypothesis that distinct mechanisms underlie structural and functional maturation of SAC dendrites. In Aim 1, I will map the time course of both structural and functional development of SACs and determine the extent to which structural maturity predicts functional maturity. In Aim 2, I will use targeted manipulations to identify mechanisms that dictate this developmental timeline. In Aim 2.1 I will test the hypothesis that SAC functional maturation relies on spontaneous activity during development (retinal waves) and an intracellular protein called FRMD7. Mice without retinal waves and mice with mutations in the FRMD7 gene both show deficits in direction selective circuit function that relies on proper SAC function. Additionally, both models lack normal optokinetic reflexes, a phenotype that was shown to arise from faulty retinal direct selective circuits and is shared with human patients who have FRMD7- related nystagmus. Thus, both models are clinically relevant and promising candidates to reveal mechanisms of functional SAC development. FRMD7 is thought to interact with proteins involved in activity-dependent synaptic protein trafficking, and the location of glutamate synapses on SAC dendrites contributes to their direction-selective output. In Aim 2.2, I will use glutamate uncaging to test the hypothesis that disrupting retinal waves and FRMD7 expression is decreasing direction selectivity by interfering with the trafficking of excitatory receptors to their correct location on the SAC dendrite. Together, these experiments provide insight into how dendrites acquire the ability to compute information and contribute to circuit function, as well as elucidating how changes in such developmental mechanisms can lead to circuit dysfunction.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The past decade has witnessed a breakthrough in cancer immunotherapy, from checkpoint inhibitors to adoptive T cell therapies, a new pillar in our armament of anti-cancer drugs now exists. However, our current therapies are based on the premise that adaptive immunity alone, mediated by activated T cells, can eliminate tumors. While the generation of anti-tumor T cells is paramount, many additional factors must be considered in an immune response, including access of therapies to solid tumors, the role of innate immunity, and the suppressive tumor microenvironment (TME). Microbial-based cancer therapies have the potential of addressing all of these challenges that can impede the success of immunotherapy. This proposal is based on extensive preclinical and clinical experience using an attenuated (DactA) strain of Listeria monocytogenes as a therapeutic cancer vaccine. In this proposal, we focus on the direct impact of L. monocytogenes (Lm) on shaping the immune phenotype of the TME. We find that injection of Lm intratumorally (IT) results in profound changes in the TME, including the reduction of Tregs which we trace to activation of TLR2. Surprisingly, bacteria injected IV also localize to tumors, but while bacteria in the liver and spleen are eliminated, bacteria injected both IT and IV, persist indefinitely in the tumors, although neither treatment results in reduction of tumor volume. However, mice previously immunized with Lm followed by IT injection, dramatically reduce the tumor burden, which we show requires CD8+ T cells. Based on our preliminary data, we hypothesize that Lm injected either IT or IV localizes and persists in tumor and increases the inflammatory milieu. Upon prior immunization, influx of Lm-specific CD8+ T cells mediate clearance of both the remaining bacteria and substantial shrinkage of the tumor, although the mechanism remains to be determined. Collectively these data reveal the importance of both innate and adaptive immunity in mediating a productive response to the tumors.

NIH Research Projects · FY 2026 · 2024-04

Molecular regulation of fluid pressure homeostasis in the inner ear Project Summary A significant portion of hearing and balance disorders are caused by the unregulated accumulation of endolymph fluid and pressure within the inner ear. A key challenge in treating these diseases of elevated endolymph pressure is identifying new strategies to regain pressure homeostasis. We previously discovered a tissue-scale pressure relief valve in the epithelial tissue of the endolymphatic sac whose behavior is consistent with long observed physiologies that lacked explanations. Despite its importance in maintaining an internal environment within the ear, the molecular and cellular mechanisms by which the endolymphatic sac forms and functions remain unclear. The long-term goal of this research is to define how molecular signals during development and during homeostasis control the pressure relief valve's setpoint. Using a combination of advanced live imaging, genetics, and cell and molecular biological technologies like genome editing, we study these systemic processes in zebrafish embryos and larvae whose inner ears are optically accessible in the living animal instead of buried within the temporal bone as in mice and humans. Our prior generation of a single-cell gene expression atlas of the zebrafish inner ear identified molecular leads of signaling pathways in the endolymphatic sac. Synthesis with past work motivates our central hypothesis which posits that cells within the endolymphatic duct maintain a strong adhesive interaction, while adhesion strength at distinct subsets of cell-cell interfaces in the endolymphatic sac is reduced by adhesion protein turnover. This regulatory mechanism allows these interfaces to temporarily separate, facilitating the release of excessive pressure. We will pursue how the integration of molecular signals regulates these local cell behaviors to determine the pressure setpoint within the entire inner ear. First, we will determine how regulation of spatiotemporal differences in Wnt signaling regulate turnover rates of cell-cell adhesion complexes in subregions of the endolymphatic duct and sac. Second, we will determine the molecular responses in the endolymphatic sac that are regulated by vasopressin and how these responses integrate into physiological circuits. Third, we will determine how mechanical and calcium signals contribute to tissue contractions in the endolymphatic sac to regulate resistance to stretch. These studies will uncover regulatory mechanisms that determine the inner ear's pressure setpoint that are essential for our ability to sense sound for hearing and body acceleration for balance.

NIH Research Projects · FY 2026 · 2024-04

SUMMARY Natural products are important small molecules for studying, treating, and even causing human diseases, and they typically have unique functional groups that are critical for their biological activities. By exploiting the biosynthetic machinery by which these functionalities are synthesized, it is possible to enhance, vary or diminish the biological activities of parent compounds and apply the biosynthetic machinery to new systems for functional group installation. Toward this goal, the chemical logic and enzymatic machinery underlying natural product biosynthesis need to be fully characterized and understood. Our lab has been focused on the biosynthesis of unusual pharmacophores of natural products, including but not limited to terminal alkene, alkyne, isonitrile, and N-hydroxytriazene. These moieties often serve as the warhead of bioactive NPs and have distinct physical properties that enable molecular tracking such as Raman- or IR-imaging based cellular uptake and dynamic studies. More importantly, they are often called “clickable” and utilized in bio-orthogonal chemical transformations for various chemical biology applications. Specifically, our program has and will continue to make contributions in the following four areas: 1) Novel enzyme discovery. “Bio-orthogonal” suggests that these functionalities are rare in nature, but structures of a few rare NPs have already indicated the prospect of novel enzyme discovery for these functionalities. 2) Enzyme mechanism interrogation. New and fundamental insights into the catalytic mechanisms of these new enzymes will be obtained. 3) Biocatalysis and biosynthetic pathway engineering. We plan to examine the substrate scope of new enzymes in detail and explore the utilization of these biosynthetic machinery to install “clickable” functionalities on various biomolecules on demand. 4) Leveraging the unique properties of these functionalities to promote natural product research, such as visualization, identification, enrichment, quantification, diversification, and biological target identification. Our program employs multidisciplinary approaches including bioinformatics, genetics, heterologous reconstitution, organic synthesis, biochemical and structural analysis, spectroscopic analysis, bio-orthogonal chemistry, protein engineering, and pathway engineering, with support from world-renowned collaborators providing complementary expertise in structural biology, bioinorganic chemistry, computational chemistry, enzymology, chemical biology, biophysics, and microbiology.

NIH Research Projects · FY 2026 · 2024-04

The importance of membrane potentials is most widely recognized in electrically excitable cells in organs like the brain, the heart, and muscles. However, all cells maintain a membrane potential. Small differences in these electric fields have been linked to enhanced cell signaling, differentiation, development, control of circadian rhythms, modulation of ultradian rhythms, regulation of cell volume, proliferation, cell cycle progression, metastatic potential of cancerous tumors, and more. Across all cell types, up to 50% of the cellular ATP budget is spent on setting the membrane potential via the action of the ATP-powered Na+/K+ exchanger. Despite the massively expensive energy expenditure put out by cells to maintain this electrical potential, a clear picture of the details of how bioelectrical signals and potentials shape physiology outside of the brain remains elusive. The lack of readily implemented methods to reliably measure membrane potential values has limited progress in the field of understanding how bioelectrical signals shape cellular physiology outside of the context of the brain. A vast ecosystem of electrical signals outside of the brain exists between and within cells. This interconnected network has been previously inaccessible. Electrodes fall short when measuring multiple cells or accessing intracellular membranes. Existing dyes are prone to serious artifacts that confound a straightforward interpretation. We are developing optical methods to address these existing shortcomings and visualize and quantify bioelectrical signals and potentials in cells and in organelles outside of the context of the brain. We combine approaches and methodologies from synthetic chemistry, physiology, and cell biology to make contributions to understand the chemistry of fluorophores for live cell imaging, uncover new vistas of cellular physiology across cells and within cells, and take translational approaches with implications for understanding drug-ion channel interactions.

NIH Research Projects · FY 2026 · 2024-03

Project Summary The oxidation of carbon-hydrogen (C-H) bonds is a central biochemical process required by all aerobic organisms. The microbial conversion of these bonds endows potent bioactivity on natural products and enable pathogens to thrive on otherwise inert host-derived biomolecules. These reactions are currently known to be accomplished by a short list of cofactors that include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation. We recently discovered that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme revealed a redox- active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single electron transfer or stoichiometric protein oxidation. Since the AibH1H2 cofactor is supported by a protein fold distinct from typical, well-studied bimetallic oxygenases, our proposed research centers on the characterization of ill-defined members of this protein family. Our preliminary results indicate that many of these proteins harbor two active-site metal ions, behave as monooxygenases and, in some cases, display enhanced enzymatic activity when manganese ions are preferentially incorporated within the active site. The core scientific hypothesis for this study is that the unique sequence and structural features of this emergent class of monooxygenases endows embedded manganese ions with otherwise unknown biological reactivity. To investigate this hypothesis, the specific aims of this study are to (1) understand the signature amino acid motifs that differentiate these monooxygenases from their ancestral, structurally-related amidohydrolases, (2) characterize the geometric and electronic structures of mixed iron/manganese-containing enzymatic intermediates, and (3) evaluate the thermodynamic and kinetic competence of cambialistic Mn/Fe monooxygenases. The resulting enzymatic information, methods, and structures will be of specific interest to those in the fields of metallo-enzymology, structural biology, and synthetic bioinorganic chemistry, and of broad interest to microbiologists in general.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The ability to install precise genetic changes is a longstanding goal in biology. While there are over 6,000 known disorders with monogenic origins, estimates are that only 10% of these are currently treatable. Most genetic variants associated with disease are single point mutations which are potentially correctable via systems that exchange a single DNA base (base editors). Base editors treat point mutations by appending a deaminase enzyme that catalyzes single nucleotide changes to a programmable DNA binding protein (Cas9) that localizes the editor to its target. This technology has seen overwhelming success with adenine base editors (ABEs) entering the clinic within just five years of their initial report. Notably, ABEs do not generate double-stranded breaks, making them an ideal candidate for genome editing—especially in stem cells which suffer low genome editing efficiency and large rearrangements or deletions in DNA in response to DNA cleavage. The correction of point mutations in the genomes of stem cells has the potential to provide essential cell-based therapies for immunodeficiency and neurodegenerative diseases. However, the capacity of this approach is limited by editing promiscuity at neighboring bases. This constrains target selection to a narrow range of mutations where nearby off-target edits would not negate the effect of the edit or induce additional pathogenic mutations. For instance, mutations causing severe combined immunodeficiency (SCID) and Hurler syndrome are potentially reversible by adenine targeting base editors, yet inaccessible by current methods due to the proximity of another editable adenine bases to the target site. Therefore, there is an unmet clinical need for the advent of precision editors capable of precisely targeting mutations in hematopoietic stem and progenitor cells (HSPCs) to produce edited cells for autologous transplantation. My proposal describes a two-pronged approach to developing precision base editors that both extends existing technology and creates an entirely new kind of editing enzyme with intrinsic properties that prevent off-target edits. Our approach provides key insights into genome editing mechanisms that can be harnessed for treatment of a wide range of diseases.

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Habits make up an essential part of humans' everyday behavior. Habits enable us to perform routine activities, such as driving to work, in an automated, effortless way, freeing up our cognitive resources for more effortful concomitant tasks, such as conversing with our passenger. Successfully establishing good habits, such as regular hand washing, sleep times, or exercise, is essential for a healthy lifestyle. However, because they are automated and inflexible, habits can also lead us astray, causing us to inadvertently drive to work when we meant to go to the doctor or eat snacks when we're not hungry, and in extreme cases, promoting drug seeking behavior and addiction. Overcoming habits when they are not appropriate for our current goal requires goal-directed control. While the balance between habits and goal-directed control has been well-studied in non-human animals, cognitive neuroscientists have struggled to translate the standard approaches in this domain into human ex- perimental research. Specifically, humans are more efficient than non-human animals at exerting control that overrides habits, making it difficult to reveal habits in lab experiments, even after thousands of training trials. Furthermore, it has proven difficult to attribute habit-like behavioral patterns (such as “slips of action” like go- ing to work instead of going to the doctor) to the strength of the habit, as opposed to the weakness of control processes. This project will develop a new human protocol that will allow researchers to 1) induce habits in hundreds of trials, making it a practical, single-session task for future research, 2) disengage control enough to reveal habit behavior, and 3) reveal engagement of control in a way that is separable from the strength of habit expression. To do so, our approach steps away from successful rodent task designs, and instead relies on the insight that human decision-making is hierarchical. Specifically, we will leverage the hypothesis that humans make more abstract choices (e.g. driving, vs. walking to work) in a controlled, goal-engaged way, then naturally disengage control and execute their high-level decision with habitual routines. Aim 1 will develop and test the validity of the protocol with regards to the three targets above; aim 2 will directly test the role of hierarchical decision-making in expressing habits. If successful, this project will open the doors to a wealth of downstream research on habits and goal-directed decision-making, including probing their neural substrates in functional imaging, and their role in individual dif- ferences and clinical conditions. If someone is unusually prone to habit-like mistakes (e.g. finding themselves at work when planning to go to the doctor), it is currently difficult to attribute this to strong habit expression as opposed to weak control. Our protocol will enable us to pull this apart, with important implications for the study of psychiatric conditions such as obsessive-compulsive disorder, eating disorders, and addiction.

NIH Research Projects · FY 2026 · 2024-03

Project Summary Perturbation of mitochondrial proteostasis, a form of mitochondrial stress, activates the mitochondrial unfolded protein response (UPRmt), a retrograde signaling pathway leading to transcriptional up-regulation of mitochondrial chaperones and stress relief. Recent advances in mitochondrial biology link UPRmt to lifespan extension independently of oxidative stress and damage in model organisms. These observations beg the questions of whether mitochondrial protein folding stress and the UPRmt regulate healthspan and lifespan in mammals. We have recently identified a novel regulatory branch of the UPRmt. We will elucidate the physiological significance of this pathway in regulating stem cell aging, tissue degeneration, and degenerative diseases, such as Alzheimer’s disease. Using a gain-of-function approach, we will test the feasibility of activating these molecules to extend healthspan during natural aging and ameliorate Alzheimer’s disease. Collectively, these studies highlight a novel defense program that improves mitochondrial integrity and tissue homeostasis. Successful completion of the proposed studies will provide a critical mechanistic understanding of how mitochondria and metabolism regulate tissue homeostasis. In addition to generating important basic knowledge, these studies will provide new targets for developing potential interventions for aging and aging- associated diseases, including Alzheimer’s disease.

NIH Research Projects · FY 2026 · 2024-03

Usability remains a significant barrier to broader adoption of cutting-edge bioinformatics tools, due to a lack of user-friendly web interfaces. The Generic Model Organism Database (GMOD) provides standards-based software components from which web-facing genome databases can be quickly assembled. JBrowse, the genome browser of GMOD, aims to "democratize" genome informatics by making genome annotations and sequence analysis tools more accessible to the broader community of biologists, using JavaScript and the dynamic web. JBrowse is now in active use by thousands of websites and over a hundred thousand users, with over a million hits per month. Its annotation editing plugin, Apollo, is used by many NIH-funded projects to coordinate contributions from professional biocurators and motivated experts in the community. In this phase of the JBrowse project, we plan to build machine learning capabilities into JBrowse: to make automated recommendations to users that help them more rapidly review evidence and form hypotheses, to help developers of AI tools assemble and curate genomic datasets, and to present the results of machine learning analyses of genomic data dynamically to the user on demand. We also propose to enhance JBrowse's capabilities for using genomic synteny as a navigation tool to move between related genomes, view evolutionarily conserved gene structures and their associated alignments, and visualize genome annotations in their phylogenetic context. We will also significantly improve JBrowse's speed and design via series of systematic performance and usability benchmarks, and by user testing at design sprints. Finally, we will continue GMOD's outreach/helpdesk efforts with focused workshops, training materials, and documentation to maximize the utility of GMOD/JBrowse to the broader community.

NIH Research Projects · FY 2026 · 2024-02

SUMMARY Natural products, often known as specialized secondary metabolites, are used by microbes to control complex processes such as fitness, biofilm formation, nutrient acquisition, stress response, and virulence. Recent genomics and transcriptomics analysis of human oral microbiota has indicated that oral microbes have great potential to synthesize diverse natural products which are correlated with oral health or disease. However, among thousands of predicted oral natural products, less than 1% have been known. This knowledge gap prevents thorough study of natural product-mediated microbe-microbe and microbe-host interactions and hampers our understanding of the interplay between oral microbiota and host at a molecular level. There is currently an urgent need for experimental characterization of these abundant, yet poorly understood, molecules and the downstream socio-chemical relationships they mediate, which impact human oral health and disease. Our long-term goal is to harness the medical benefits that are offered by understanding chemistry and biology of oral natural products. This project focuses on the identification and characterization of bioactive natural products from the two important oral microbial species: Streptococcus mutans, a key etiological agent of human dental caries, and Streptococcus salivarius, a probiotic widely available over the counter for oral health. Following our extensive preliminary data, particularly the recent discovery and mechanistic interrogation of mutanofactin from S. mutans, we here organize our efforts into three independent specific aims to scrutinize three unique families of bioactive natural products. These oral metabolites have been predicted to play important roles in biofilm formation, acid production, or chemical defense from omics analysis and phenotypic correlations, but their chemical structures, direct biological activities and molecular mechanisms remain elusive. Using a combination of bioinformatics, microbiology, analytical chemistry, biochemistry, metabolic engineering, and molecular biology, three major questions related to these bioactive natural products will be addressed: “what is the chemical structure”, “how it is biosynthesized and regulated”, and “why it is produced”. The research strategy is both innovative and significant, because innovative multidisciplinary approaches are adopted to reveal new knowledge on oral microbial metabolism, gene functions, and molecular mechanisms of bioactive metabolites. This project is expected to reveal new targets to inform the design of prophylactics and treatments for oral disease and infection. Ultimately, new therapies may be developed to complement or synergize with traditional treatments for oral diseases such as dental caries.

NIH Research Projects · FY 2025 · 2024-02

Project Summary Targeted covalent inhibitors have empowered the potent and selective targeting of driver oncogenes in cancer. Between 2011 and 2021, 16 new covalent drugs (12 with cancer indications) were approved by the US FDA, with the recent notable example of the first KRAS inhibitor sotorasib. However, most of current covalent drug discovery efforts are directed toward cysteines, which is the least abundant amino acid in the proteome (2%). Meanwhile, about 6% of mutants in cancer lead to serines. While some of these mutations directly contribute to oncogenic signaling (e.g. KRAS G12S), others confer clinical resistance to existing drugs (e.g. EGFR C797S, BTK C481S). To date, direct engagement of these mutant serines have not be achieved. Methods to selectively engage amino acids beyond cysteine are highly desirable, as they will bridge the gap between the prevalence of serine-acquiring mutations in human cancer and the lack of chemical tools to target them. This project aims to develop new chemistry that enables the covalent engagement of serine residues, especially those acquired in cancer such as EGFR(C797S) (Aim 1) and BTK C481S (Aim 2). Successful targeting of these proteins will provide new avenues for targeted cancer therapy. The chemistry developed in this project will also serve as a platform for the discovery of new covalent ligands for currently “undruggable” cancer targets (Aim 3).

NIH Research Projects · FY 2025 · 2024-02

Project Summary The development of the human brain is a highly coordinated process involving the controlled expression of thousands of genes, where deviations from this genetic code can result in neurological conditions or deficits in cognition and behavior. Thus, it is crucial to understand the complexities of gene expression variation of the cortical transcriptome in the human brain across all life-stages. Recently, with advancements in sequencing technology, researchers can now examine the transcriptomic heterogeneity in humans and correlate these variations to neuroanatomical and functional properties of the brain. Prior research in adults has shown that a small subset of genes is indeed related to regional variability in function and neuroanatomy, but it is still presently unknown how this translates across development. Thus, this project aims to identify which genes are highly variable across all developmental stages, what neuroanatomical properties these genes are associated with, their localized and cell type expression, and which are specific to human development. Findings from this project will complement those from early prenatal stages, which has been heavily researched, and will provide novel insights for later stages of development, primarily across childhood and adolescence. To carry out the proposed research, the applicant will implement a novel pipeline established in the lab that identifies genes most differentially expressed across development in both humans and macaques, carry out gene enrichment and network analyses, spatially localize identified gene expression patterns, and determine related neuroanatomical properties through cross-modal neuroimaging analyses. Both aims will take advantage of publicly available transcriptomic atlases. Aim 1 will examine the transcriptomic variability across human development using bulk RNA sequencing analyses and in situ hybridization. Aim 2 will then apply the same methods from Aim 1 on transcriptomic data from macaques in order to make cross- species comparisons and to identify which genes from Aim 1 are human-specific. Findings from these approaches will identify novel sets of human-related genes that are crucial to various stages of development and ultimately, will be beneficial to applications in mental health. The two sponsors of this project, Profs. Kevin Weiner and Silvia Bunge, along with thesis committee member Dr. Mercedes Paredes, will provide relevant expertise and guidance in the field of neuroanatomy, development, and transcriptomics. Additionally, support from consultants and resources from UC Berkeley and the Helen Wills Neuroscience institute will ensure that the applicant successfully completes the dissertation and is prepared for a competitive post-doctoral fellowship and research career investigating the transcriptomic basis of human brain development.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Formula provides nutrition to infants as a partial or complete substitute for human milk for working mothers or in situations like lactation failure or poor maternal health. The heavy reliance on infant formula in the U.S. is reflected by a National Immunization Survey which found that only ~25% of infants (< 6 months) were exclusively breastfed between 2018-19, a number that was even smaller in rural areas and low-income families. The fat composition of formulas serves a vital role in replacing human milk fat (HMF), which provides >50% of energy to the infant during early lactation. However, derived from vegetable oils, the fat in commercial formulas often lacks the unique fatty acid composition and lipid structure of HMF, resulting in reduced dietary benefits and poorer health outcomes for infants. The distinct features of HMF include but are not limited to 1) a substantial portion of medium-chain (C8:0-C14:0) fatty acids and 2) the prevalence at the sn-2 position of palmitic acid (a C16:0 fatty acid) on triacylglycerols (TAGs). Medium-chain fatty acids are desirable as a quick source of energy for infants because of their higher absorption rates compared to longer fatty acids. C16:0 esterification at the sn- 2 position prevents its release during digestion by the sn-1,3 lipase. This is important to avoid 16:0 precipitation, which chelates essential calcium ions and causes hard stools. Present methods for correcting fatty acid composition and TAG regioisomeric structure in formula are expensive and because of their reliance on palm oil, undesirable from an environmental perspective. This project addresses these challenges through the development of an HMF substitute in metabolically engineered algae: the goal is to generate a healthy, environmentally friendly, and low-cost product for US consumption. Auxenochlorella protothecoides (A.pro) presents an ideal platform for this goal because of 1) its exceptional biomass and oil accumulating capabilities compared to traditional oil crops, 2) convenient genetic and genome modification tools to facilitate metabolic engineering, and 3) the potential for producing the product at commercial scale. In this context, a fatty acid substrate pool resembling the fatty acid composition of HMF will be built by heterologous expression of chain length-determining fatty acyl-ACP thioesterases with desired substrate specificities (e.g., C8:0-C14:0) in A.pro, followed by GC-MS product analysis for assessment (Aim 1). To achieve TAG products with the correct regio- selectivity (C16:0 at sn-2), the C16:0-specific lysophosphatidic acid acyltransferase will be introduced, followed by stereochemical analysis of TAGs by sn-1,3 lipases treatment and GC-MS analysis of the isolated monoacylglycerols (Aim 2). Finally, the endogenous polycistronic gene expression system, discovered recently by the sponsor, will be exploited for simultaneous expression of rate-limiting enzymes in fatty acid biosynthesis, which will streamline the engineering process and enable more efficient production of HMF in A.pro (Aim 3). In summary, this research will explore the feasibility of producing HMF in oleaginous green algae for infant formula and also pave the way for developing a promising synthetic biology approach for other bioproducts in A.pro.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract The scalable and sustainable biosynthesis of terpenoid drugs like cyclopamine and diverse libraries of terpenoid drug candidates will transform the treatment of disease. There are >30,000 unique plant terpenoids and a valuable subset of these natural products have been approved by the FDA approved or are currently in clinical trials for cancer treatment, malaria treatment, symptom relief, and immune system activation. Terpenoids can be biosynthesized in plants through expensive cultivation (i.e., time-, labor-, and land-intensive), but these approaches can exhibit low and hypervariable yields. Plant terpenoids can also be produced by chemical total synthesis and semi-synthesis. However, terpenoids can contain many chiral centers (e.g., 10 in cyclopamine), resulting in complex syntheses with low overall yields. In contrast, the production of terpenoids in engineered microorganisms is scalable, renewable, and inexpensive, often producing complex terpenoids from simple sugars and salts. For example, an artemisinic acid-producing yeast has resulted in 51 million treatments of artemisinin, contributing to the low current cost of artemisinin anti-malarial combination therapies. This proposal aims to establish methods to accelerate the future microbial biosynthesis of medicinal terpenoids. The full 30-step biosynthetic pathway for cyclopamine will be constructed step-by-step in the brewer’s yeast (Saccharomyces cerevisiae) to produce cyclopamine from simple sugars and salts and solve the challenge of sourcing this drug for the treatment of cancer. In addition, S. cerevisiae will be engineered to produce sets of new-to-nature triterpenoids through the combinatorial screening of enzymes with well-characterized activity. The studies will accelerate the microbial biosynthesis of medicinal terpenoids and diverse libraries of terpenoid drug candidates.

NIH Research Projects · FY 2025 · 2024-01

Project Summary Pharmaceutical technologies are inherently limited by the synthetic methods available to medicinal chemists. Novel transformations in organic chemistry have the potential to enhance human well-being through the prevention of human suffering through pharmaceutical therapies. Modern innovations in synthesis have shown the ability to furnish new molecular scaffolds, especially through the employment of catalytic methods. Past efforts in supramolecular catalysis have offered strategies to more traditional methods by enabling orthogonal selectivity through non-covalent interactions which are imparted via encapsulation. In this way, supramolecular hosts behave similarly to enzymes, inspiring further study due to the allure of the rate enhancements offered by nature’s catalysts. Utilizing supramolecular hosts pioneered by the Raymond, Bergman, and Toste groups, we plan to enable several transformations relevant to the production of pharmaceuticals. Previous work with these privileged clusters has shown powerful reactivity through the coencapsulation of two reagents within the host cavity. Trends in these reactions have shown the ability to enhance the electrophilicity of electrophiles through protonation and increased reactivity from nucleophiles through p-p stacking interactions. These motifs will be leveraged for various pharmaceutically relevant transformations, including N-heterocycle reduction and functionalization, regio- and diasteroselective olefin epoxidation. Through the combination of supramolecular catalysis and Pt catalysis we also hope to provide methods for an enantioselective C(sp3)-H oxidation of alkanes. These methods all involve the use of widely available or readily synthesized reagents that may be rapidly constructed into valuable pharmacores for use in drug discovery investigations with the ultimate goal of improving human health. The specific aims in this proposal provide a detailed plan for the production of these potent technologies. Under the tutelage of Professors Toste, Bergman, and Raymond I will develop these methods with expert guidance in asymmetric reactivity, physical organic chemistry, and organometallic cluster synthesis. Conducting this research at the University of California Berkeley provides an optimal environment for both the success of this project and for my personal and professional development as a researcher. Providing the tutelage of brilliant faculty, ambitious colleagues, and stunning state-of-the-art facilities, Berkeley offers no limit to the resources for the practicing scientist.