University Of California Berkeley

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract: The selective transformation of unactivated C–H bonds into C–C bonds has been a longstanding challenge for synthetic chemists, and the uranyl cation (UO22+) has been shown to catalyze these transformations on a variety of substrates14-20. The uranyl cation is the most common form of uranium in the environment, existing in sea water at a concentration of about 3.3 ppb10. Early studies on the photochemistry of the uranyl ion have shown that photoexcitation with visible light will populate a highly oxidizing (+2.6 eV vs SHE) triplet excited state of the uranyl unit, which has a lifetime of microseconds with free radical character on the oxo groups and the uranium center11-13. This oxyl radical abstracts H atoms to form an O-H bond that is sufficiently strong to enable abstrac- tion from unactivated C–H bonds. Recent publications on photocatalysis of the uranyl ion have reported the functionalization of a variety of C–H bonds to form C–F bonds, C–C bonds, and C–O bonds17-20. Due to the ubiquity of C–H bonds in organic molecules, site-selectivity transformations are often difficult to achieve. Fur- thermore, stereoselective reactions would require chiral ligands that have not yet been incorporated into uranyl complexes. Nature has developed a variety of metalloenzymes that functionalize unactivated C–H bonds with exquisite selectivities imparted by the enzymatic scaffold22-29. In many of these enzymes, the reactive metal-oxo intermediate abstracts a hydrogen atom from a C–H bond to generate a carbon-centered radical. Functionaliza- tion of the generated radical, however, is largely limited by the rapid rebound of the radical onto the metal hy- droxide, providing hydroxylated, halogenated, or pseudohalogenated products35-42. Abstraction of a C-H bond by the uranyl ion, however, is not followed by transfer of the hydroxyl radical, due to the strong U–O bonds, thereby enabling functionalization of the radical intermediate in different ways. Thus, one should be able to achieve site- selective and stereoselective C–H bond functionalization with the uranyl ion by incorporating a photocatalytically active uranyl ion in place of a natural ion in the active site of an enzyme. The Hartwig group has experience in creating and developing artificial metalloenzymes (ArMs) with non-native metallocofactors for abiotic chemistry and catalysis51-61, and the Arnold group has extensive experience studying the uranyl ion and the photochemistry of this unit14-16,21. Through a collaboration between these two groups, I propose to develop novel ArMs containing an unnatural uranyl cofactor for the site- and stereoselective functionalization of C–H bonds to form C–C bonds. This will be accomplished by following two complementary strategies: 1) design of a photocatalytically active uranyl complex and bioconjugation of this complex into an enzyme scaffold; 2) incorporation of the unnatural amino acid phosphotyrosine or phosphosyrine into an enzyme active site and binding the uranyl ion within the unnatural site containing a phosphate group. These metalloproteins will then be used for intra and intermolecular Giese-type reactions initiated by hydrogen-atom abstraction, followed by addition to electron-poor alkenes.

NSF Awards · FY 2024 · 2024-01

As cities grow everywhere, and urban roadways become overburdened, efficient strategies are required for improving urban mobility. With the emergence of autonomous cars, there is an opportunity to reclaim urban mobility provided that a proactive control and planning approach is taken. In this project, our goal is to develop fundamental theory and domain-driven techniques for leveraging the opportunities that autonomous cars provide to achieve mobility-efficient smart cities. We consider mixed-autonomy traffic networks which are road networks that are shared by human-driven and autonomous cars. We will develop a set of algorithms for socially-aware control of autonomous cars in these systems. More precisely, we develop control algorithms that take into account the social implications of the co-existence of human-driven and autonomous cars and guarantee overall societal good. Our project enables the transformation from the current reality to the brighter future, in which autonomous cars harmoniously interact with the road and network traffic control systems to increase mobility. To unlock the mobility potentials of autonomous cars, a key control challenge is to account for how humans adapt and respond to autonomous cars’ actions such as routing decisions. We focus on travelers’ routing decisions and develop design and control algorithms that induce efficient routing decisions. We use routing games for modeling travelers’ route choices over traffic networks and tackle research challenges associated with controlling the core components of a mixed-autonomy network. (i) We will develop algorithms for altruistic control of autonomous cars’ routing decisions, that is, autonomous cars that are routed in the favor of society. (ii) We will develop pricing algorithms that affect humans' routing decisions such that the network is steered towards desirable states. (iii) Finally, we will develop planning strategies for network topology design and capacity regulation that induce efficient routing decisions by travelers. Our proposed control strategies will also be validated through extensive real-world experiments and traffic simulations. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-01

Summary Inhibitory interneurons in cerebral cortex show rapid plasticity of intrinsic excitability in response to sensory experience and learning. The molecular mechanisms and functions of such plasticity, and its potential role in disease, are poorly understood. We study this in mouse somatosensory cortex (S1), where brief sensory deprivation drives a rapid reduction in parvalbumin (PV) interneuron intrinsic excitability, which acts to stabilize pyramidal cell firing rates. We recently found similar plasticity in somatostatin (SST) interneurons, suggesting that rapid intrinsic plasticity is a common property of MGE-derived interneurons. Here, we characterize the mechanisms for intrinsic plasticity in interneurons, and test the novel hypothesis that deficits in this process play a major role in inhibitory circuit dysfunction in autism. In Aim 1, we identify the molecular signaling pathways that mediate intrinsic plasticity in PV neurons induced by brief sensory deprivation. Prior work shows that deprivation rapidly increases Kv1 potassium currents, which elevates PV spike threshold. We will identify the molecular pathways for this plasticity, using a combination of immunohistochemistry and qHCR-FISH to detect alterations in protein and gene expression, and pharmacological and genetic tools to test causal involvement of candidate signaling pathways. We focus on candidate pathways that are known to regulate Kv1.1 channels. We also use single-nucleus RNAseq for unbiased discovery of regulated genes. Preliminary results implicate the Er81-KCNA1 pathway, which drives increased expression of Kv1.1. This work will yield molecular understanding and molecular markers of PV intrinsic plasticity, which we will use to characterize its prevalence and properties. Aim 2 tests the novel hypothesis that deficits in PV intrinsic plasticity are the underlying cause of PV circuit dysfunction in some genetic forms of autism, specifically for autism genes that regulate activity- dependent gene expression and/or Kv1.1 function in PV cells. We propose that due to loss of PV intrinsic plasticity, neural coding is destabilized in autism. We will test this hypothesis in Fmr1, Tsc2, and Cntnap2 transgenic mouse models of autism. As part of this work, we will test whether restoring gene expression selectively in PV cells rescues PV intrinsic plasticity and stabilizes pyramidal cell coding. If so, this would suggest a new therapeutic approach to autism in restoration of PV intrinsic excitability. Aim 3 tests for plasticity in SST circuits, which is little studied. In preliminary data, deprivation alters SST intrinsic excitability and other aspects of SST circuit function. This demonstrates that SST plasticity exists. We will characterize SST circuit plasticity and identify molecular mechanisms for SST intrinsic plasticity, to test for possible common mechanisms with PV intrinsic plasticity. Together, this grant will develop and test the novel hypothesis that plasticity of intrinsic excitability is a major form of plasticity in PV and SST interneurons that plays important roles in regulating cortical function and disease.

NIH Research Projects · FY 2026 · 2024-01

Project summary/abstract The long-term goal of my research is to understand how the kinetic and thermodynamic properties of enzymes maintain metabolic homeostasis. The function of metabolic homeostasis is to maintain appropriate levels of ATP and biosynthetic precursors. Understanding metabolic homeostasis is important as it is a fundamental property of all cells and its dysregulation leads to metabolic syndrome, which contributes to several common disorders, including diabetes, cardiovascular disease, cancer, and nonalcoholic fatty liver disease. Metabolic homeostasis is achieved by controlling enzyme activity through mass action and allosteric regulation. Studies of purified enzymes have yielded extensive knowledge of the structure, reaction mechanism, and allosteric regulation of enzymes in several metabolic pathways. However, metabolic homeostasis is the result of non-linear interactions between many enzymes and metabolites, which are difficult to fully understand by studying individual enzymes in isolation. As a result, the specific functions of most allosteric regulators is not well understood and it is largely unknown how allosteric regulation and mass action achieve metabolic homeostasis in cells. In this proposal, our main objectives are to 1) develop mathematical models to characterize metabolic homeostasis, and 2) develop experimental approaches to test model predictions by measuring and manipulating metabolic homeostasis in live cells and in vitro reconstituted pathways. We will use a combination of modeling and experiments to address key gaps in our understanding of the regulation of glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation. The big-picture questions that we plan to investigate are: How do glycolysis and respiration maintain cellular ATP homeostasis? How do cells resolve the conflicting demands of ATP production and biosynthesis? How do metabolic pathways that share products and substrates coordinate with each other? What is the role of compartmentalization and metabolite channeling in regulating metabolic homeostasis? Our lab is well-positioned to make advances in the understanding of metabolic homeostasis as we have extensive experience in developing and using LC-MS, fluorescence sensors, genetically-encoded tools for manipulation of metabolism, in vitro protein characterization, engineering cell lines using CRISPR-Cas9, and mathematical models to study metabolism.

NSF Awards · FY 2024 · 2024-01

Computing on personal data is a double-edged sword. On one hand, it enables revolutionary new applications such as personalized medicine and disease prediction. On the other hand, it runs the risk of revealing said personal data to unwanted parties. For example, using personal data on today’s processor chips can reveal that data through traces that the processor leaves behind. To make matters worse, different processors leave behind different traces, revealing different information, depending on how they were designed. This project will develop techniques to prevent data leakage through processors, for existing and future processor chips. The technical approach is to design a Distinguishability Set Architecture (DSA) for existing and future processors. DSAs are peers to existing Instruction Set Architectures (ISAs). Whereas the ISA specifies the functionality of each instruction, the DSA specifies under what conditions each instruction reveals secret information. With a DSA, programmers or compilers can tune sensitive programs to avoid leaking secrets. The first project thrust will develop DSA foundations, answering questions such as what should a DSA look like and how to capture leakage through various processor optimizations. The second thrust will develop compilers and hardware that use DSAs to improve program security. By precisely describing when and how processors reveal secrets, DSAs will unlock innovation on both software and hardware fronts. On the software side, programmers can focus on applications while DSA-aware compilers translate those applications to secure variants fit to run on different processors. On the hardware side, architects can use DSAs to reason about the privacy implications of hardware optimizations. The project will train a new class of students and researchers who can work across formal specifications, micro-architecture and compilers to build secure systems and, in the future, apply the lessons learned to other privacy-related problems. The DSA project will store all publications, code, and data-sets on public-facing websites, hosted at the University of Illinois for at least 3 years after the end of the project. This information will be made available via commercial websites. Links to these websites will be mirrored at http://cwfletcher.net/dsa. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-01

Project Summary The primary objective of this proposal is to map human retinal ganglion cell receptive fields in vivo, at cellular resolution, directly connecting light stimulation of individual retinal photoreceptors to both the activity of retinal ganglion cells and the conscious percept of retinal ganglion cell (RGC) activity. Performing this task in vivo with human subjects will have enormous implications for the study of the neural code leaving the eye. There are 120 million photoreceptors in the human retina that absorb and respond to light. The human optic nerve, however, the sole link between the retina and the brain, comprises a mere ~1 million axons. This bottleneck shapes the neural code of the retina, which has never been completely described1,2. Past the optic nerve, the transformation of information from the retina into a conscious percept involves neural interactions that can usually only be studied through relatively coarse methods (such as fMRI). To understand the organization of data leaving the eye, it will be essential to better connect the functional organization of human retinal ganglion cells (RGCs) to human visual percepts. It is difficult to study the neural code leaving the human eye because the optic nerve is inaccessible and dense. Psychophysics using adaptive-optics-stabilized microstimulation of the photoreceptor mosaic is one way to probe the information being sent from eye to brain3,4. This technique allows precise control of the input to the photosensitive layer of the retina. However, precise control of the inputs to the retina is not enough, since there are multiple parallel pathways from retina to brain5. A pulse of light falling on a single cone will cause many different RGC signals to be sent to the brain, most prominently ON and OFF of both midget and parasol, the most common RGC types6. Disentangling the percepts–what a human subject can report as "seen"—elicited by the different RGC types is a difficult problem. In order to probe the perceptual correlates of selectively stimulated groups of RGCs, this project will leverage perceptual desensitization with adaptive-optics-stabilized microstimuli. First a “desensitization” stimulus will habituate several RGC pathways with single cone stimulation, and then a “probe” stimulus will be used to target a single RGC type psychophysically, one cone at a time. In this way, a single RGC "perceptive field" can be mapped and investigated. Once mapped, RGC perceptive fields can be subsequently investigated. This work will be performed in Prof. Austin Roorda's adaptive optics psychophysics lab at the University of California, Berkeley. Single cone stimuli are unusual in the ex vivo literature. To confirm that desensitization and probe stimuli are driving the RGCs as expected, this project will also involve testing desensitization and probe stimuli using electrophysiological techniques in primate retina. Further, the design and evaluation of single cone pulsing light stimuli that maximally drive some RGC types over others will require ex vivo work. This pursuit will also be a potentially clinically relevant one, since stimuli that could maximally target RGCs that are implicated in the early stages of retinal disease, such as OFF RGCs in glaucoma7, could be used to design future diagnostic tools. The electrophysiology work will be performed in the lab of retinal physiologist Prof. Teresa Puthussery at UC Berkeley. If successful, this project could lead to novel clinical diagnostic tools for the treatment of retinal diseases and inform the understanding of how the neural code from the eye is interpreted by the brain.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY The mechanistic Target of Rapamycin Complex 1 (mTORC1) is master regulator of cell growth and metabolism. Dysregulation of mTORC1 is observed in sporadic and familial cancers. mTORC1 inhibition is an established treatment for renal cell carcinoma (RCC). Despite great efforts to target mTORC1 in cancer, adverse effects limit the use of mTORC1 inhibitors in the clinic. Recent work from our lab and collaborators has revealed the existence and structural basis of substrate-specific regulatory pathways that may be targeted with greater precision than heretofore. mTORC1 is activated on the surface of lysosomes in response to nutrient signals by conversion of the nucleotide state of the Rag GTPases from inactive (RagA/BGDP-RagC/DGTP) to active (RagA/BGTP- RagC/DGDP). The Rags are targeted to the lysosome by the Ragulator complex. Rag states are interconverted by the RagC/D GAP FLCN-FNIP and the RagA/B GAP GATOR1. Subunits of these complexes, and the Rags, are mutated in cancer. RagA/BGTP and GATOR1 inactivation is required for phosphorylation of all mTORC1 substrates. RagC/DGDP and FLCN-FNIP activity is only required for phosphorylation of non-canonical substrates, which include TFEB, the key transcriptional regulator of lysosome biogenesis and autophagy. Cryo-EM studies of the Rag, Ragulator, and FLCN-FNIP pathway from our laboratory provided a start-to-finish structural explanation for the repression and reactivation of FLCN GAP activity in starvation and refeeding. These studies contributed to the discovery that RagC/DGDP uniquely regulates TFEB and MiT-TFE transcription factors, which in turn explained the tumor suppressor activity of FLCN in Birt-Hogg-Dubé (BHD) syndrome. This pathway has now been linked to RHEB activity and Tuberous Sclerosis Complex (TSC). We then demonstrated the existence and determining the structure and function of the mTORC1-TFEB-Rag-Ragulator “megacomplex”, containing a full mTORC1 dimer, two copies of TFEB, and four copies of the heptameric complex of active Rags and Ragulator, showing how RagC/DGDP specifically recruits TFEB. In aims 1 and 2, we will explore the new avenues opened up the analysis of the megacomplex. We will determine how the megacomplex is turned over following TFEB phosphorylation, and whether the principles of substrate specific activation seen for TFEB and RagC also apply to canonical substrates. Findings will be followed up in BHD and TSC cell lines and a BHD mouse xenograft model. While we now have a start-to-finish structural mapping of FLCN/RagC/D pathway, the still mysterious regulatory mechanisms operating in the GATOR1/RagA/B pathway will be elucidated in aim 3, and the cancer implications explored in knock-out and Glioblastoma cell lines.

NIH Research Projects · FY 2025 · 2023-12

Project Summary/Abstract Allostery, the phenomenon describing how the state of one site in a protein is coupled to the state of a distal site, is a fundamental driver of functional evolution in protein families. It is especially impactful in multimeric and multidomain proteins – those that arise from the recombination of protein domains that are structurally and functionally distinct. The goal of this proposal is to develop methods that combine computational and experimental approaches to understand the role of allostery in establishing new functions by coupling enzymatic activity to biological processes at the membrane. Insights gained in this work will enable us to better understand how domain recombination has expanded the functional repertoires of protein families, and will enable more efficient engineering of synthetic proteins. In Aim 1 of this proposal, I will leverage recent advances in machine learning and computational geometry to develop more accurate generative models of protein families that implicitly account for evolutionary processes that act upon them. In Aim 2, I will conduct a systematic investigation into sequence-function landscape of a dimeric bacterial bicarbonate transporter that couples proton transport across the membrane to enzymatic production of bicarbonate. Using deep mutational scans in the context of a suppressor screen, I will identify sequence positions that decouple enzymatic activity from proton transport and will use this knowledge to test structure-function hypotheses related to allostery in this protein system. In Aim 3, I will use machine learning models fit to protein families to rationally design focused deep mutational scans to explore allostery in human atrial natriuretic peptide receptors. These receptors directly couple ligand binding to secondary messenger production in a single polypeptide chain containing multiple distinct domains. Using information from evolution will help me make more effective use of an experimentally limited mutational budget and will allow me to interrogate the higher order interactions that are a hallmark of allosteric networks. My background in structural biology and subsequent training in biological machine learning give me a unique perspective and skillset to tackle these challenging problems. The engaging scientific environment at UC Berkeley, and the strong support of my mentors Dr. Yun Song and Dr. David Savage will enable me to more seamlessly operate at the interface of computation and experimentation in biology as I launch my independent research career.

NIH Research Projects · FY 2026 · 2023-09

Project Abstract Dysfunction of the vascular blood-brain barrier (BBB) and cerebrovascular leakiness are present during aging and in Alzheimer's disease (AD) and are associated with the onset of preclinical mild-cognitive impairment. Based on recent discoveries we have defined a highly explanatory biological pathway that directly causes neural dysfunction and cognitive impairment following BBB dysfunction. While it is intuitive that loss of function of the fundamental vascular interface that protects the brain would be expected to cause neurological complications that may contribute to AD, previously there has not been a clearly defined mechanism linking BBB dysfunction to AD pathology. Existing data in humans suffer from limitations related to possible regional differences in BBB leakage and the temporal characteristics of BBB disruption particularly in relation to the deposition of the two proteins that have been implicated in AD pathogenesis, -amyloid (A) and pathological aggregates of tau. Very few studies have examined how these pathological proteins are related to BBB disruption, and there is no exploration of the four crucially different scenarios: (1) that there is no relationship between AD pathological proteins and BBB disruption (2) that BBB disruption leads to increased accumulation of these proteins or (3) that increased accumulation of these proteins leads to BBB disruption. (4) AD protein pathologies and BBB disruption form a positive feedback loop that originates with either and are related via the exacerbation of transmission/spread of protein pathologies by conditions created by BBB disruption. In this study we will combine descriptive longitudinal data in cognitively normal humans using PET scanning to obtain tau and A measurements and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to obtain BBB measurements, with studies in transgenic mouse models of AD where we will manipulate the BBB. Together these studies will probe mechanisms of AD pathogenesis in mouse models that enable dissecting the individual contributions of BBB disruption, amyloid and tau by manipulating each separately, and human studies that translate these basic findings to observations in the human situation of aging and preclinical AD.

NIH Research Projects · FY 2024 · 2023-09

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract Cytochromes c are crucial in methane-metabolizing archaea for the production and consumption of methane coupled to growth and energy conservation. Overall, methane-metabolizing archaea mediate the net flux of methane released into the atmosphere and thus, significantly impact the global methane cycle and climate change. Based on genomic studies, Cytochromes c have been hypothesized to play an important role in methane metabolism however the underlying molecular mechanisms remain elusive, primarily due to the absence of a well-developed genetic model system. Even attempts to study archaeal cytochrome c using an alternative approach such as heterologous expression in well-established bacterial systems have not proven successful so far. The main focus of the proposed research is to develop a genetically tractable methanogenic archaeon, Methanosarcina acetivorans, as a platform to functionally characterize archaeal cytochrome c and gain physiological insights into the role of cytochrome c in methane metabolism across different archaeal species. Cytochromes c are ubiquitous electron transfer proteins that require a covalent attachment to its heme co-factor, a process called cytochrome c biogenesis. Coordination between the cytochrome c biogenesis pathway and the cytochrome c of interest is critical for the successful production of a functional cytochrome c in a heterologous host. Using genetic and biochemical tools, I have recently characterized the cytochrome c biogenesis pathway in the model methanogenic archaeon, Methanosarcina acetivorans. Using this knowledge, the project aims to develop M. acetivorans as a genetic chassis to produce and functionally characterize archaeal cytochrome c from diverse archaeal species. Aim1 of the project will functionally characterize the crucial cytochromes c belonging to methane-producing archaea or methanogens, and Aim2 will study these proteins from methane-consuming archaea using both in vitro and in vivo analyses. This research will improve our understanding of methane metabolism and lead to the development of an archaeal host to study cytochrome c proteins from archaea. Ultimately, the knowledge garnered from these studies can be used to develop sustainable solutions for the global climate crisis and mitigate its harmful impacts on human health.

NIH Research Projects · FY 2025 · 2023-09

Overview. It is crucial to uncover the biological features underlying disease mechanisms to develop effective treatments and therapies. Typically, this is done via a two-step process: in stage 1, statistical analyses are used to recommend candidate variants/genes for follow-up investigation. In stage 2, researchers conduct costly experiments, clinical trials, or external studies via independent cohorts to validate or establish causality between candidate features and disease traits. To minimize costs, recommendations should lead to high-yield experiments and be replicable. These recommendations are often generated through GWAS methods, based on linear mixed models. Despite the successes of GWAS, there still exists a substantial heritability gap limiting the applicability of these associations in clinical practice. A number of key issues can contribute to missing heritability including: the need for more informative, multi-modal features; unidentified non-linear and epistatic effects; linkage disequilibrium among variants; and heterogeneous sources of variability. To confront these challenges, we propose a reality-checked stability-driven feature recommendation system based on decision trees that aims at efficient discoveries for high yields in experimentation. We build upon iterative random forests (iRF) and the veridical data science framework based on the principles of Predictability, Computability and Stability (PCS) developed by the PI to propose a number of novel advances for stage 1. We propose: (1) generalized MDI (gMDI) a stability-driven non-linear feature important measure for improving iRF recommendations; (2) dependence-aware feature and interaction discovery; (3) supervised local feature importance for heterogeneous mechanistic discoveries; and (4) validation through gene-silencing experiments. Importantly, we generate multi-modal features to extract information across the genome. Intellectual Merit. Our proposals: improve MDI-based methods by addressing drawbacks of MDI and tailoring to problem settings; incorporate gMDI and dependence structure in iRF; and detect heterogeneous sources of noise. Each aim will be vetted and follow the veridical data science framework. In the case study, we will recommend genes and interactions for gene-silencing experiments. These will supply valuable insights into genetic mechanisms underlying traits related to cardiac hypertrophy. Results of this work will impact mechanistic discovery for complex diseases and advance statistical methodology.

NIH Research Projects · FY 2025 · 2023-09

Abstract Magnetic Particle Imaging (MPI) is a new tracer imaging technology that could soon allow MDs to tell whether a CAR-T cell immunotherapy is actually targeting a tumor— with zero radiation, and in just 3 days. This is much safer and much faster than today’s practice of a followup PET/CT, which is too slow to allow for personalized immunotherapy care. MPI uses no radiation and yet it matches even the sensitivity of advanced nuclear medicine scans. MPI could soon revolutionize the early diagnosis of life-threatening Strokes, Traumatic Brain Injuries, Cardiovascular disease, Pulmonary Embolisms (PE), Gastrointestinal (GI) Bleeds and Cancer. In essence, MPI offers the the sensitivity of Nuclear Medicine with the safety of MRI. MPI is quantitative and robust everywhere in the body and it uses no radiation. MPI also is ideal for emergency diagnoses because MPI tracers can be bound to a targeting agent in the factory and then injected directly from the refrigerator. Hence, emergency MPI scans can be done in just 5 minutes with MPI, much faster than the 2 hours typical for nuclear medicine scans. We aim to remove the only technical weakness holding back MPI from clinical adoption, its weak spatial resolution. Our key innovation is to improve the spatial resolution of MPI by 10-fold in each di- mension. Our innovations span hardware, pulse sequences, reconstruction algorithms and the nanoscale physics and chemistry of innovative high-resolution MPI tracers. Our preliminary experiments show 10- fold (routinely) and 30-fold (occasionally) boost in resolution and SNR compared to the MPI standard, VivoTrax. Once our new tracers are made biocompatible they will pave the way for unprecedented 10- cell sensitivity at 2mm resolution in humans with a safe and affordable human MPI scanner, at roughly $100,000 hardware costs—a truly enabling advance. Our Specific Aims are to (1) Develop high-resolution MPI tracers shared between Industry and Academia; (2) Develop Engineering Tools for High Resolution MPI shared between academia and industry; and (3) In vivo scanning to prove the efficacy of high-resolution MPI. This Bioengineering Partnership with Industry is the ideal mechanism to foster this breakthrough in MPI resolution by a collaboration between three labs at UC Berkeley and a startup out of the PI’s lab (Magnetic Insight).

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract An important goal in biology is to link genotype with phenotype for traits that affect fitness. The unique adaptations found in animals that sequester neurotoxins are a useful model for understanding the genetic underpinnings of simple and complex traits that are relevant to human medicine. Specifically, neurotoxins target ion channel proteins that are critical for nervous system function. In humans, mutations in single ion channel genes can cause diseases such as epilepsy, myotonia, cystic fibrosis, migraines, and diabetes. However, animals often resist neurotoxins through mutations in these same ion channels, usually without suffering from disease phenotypes. Understanding how diverse organisms fine tune the function of ion channels without causing disease provides important information regarding the genetics of ion channel function and disease. Animals that not only resist but also sequester toxins likely modulate multi-gene pathways underlying toxin metabolism and transport, ultimately leading to selective toxin accumulation into specific tissues at high concentrations. The few known genes involved in toxin sequestration also play critical roles in drug resistance (e.g., multi-drug transporters) and metabolism (cytochrome p450s) in humans. Thus, resistance and sequestration mechanisms parallel pharmaceutical goals to efficiently deliver drugs to specific targets and/or tissues while avoiding drug breakdown or insensitivity. The proposed research aims to further our understanding of the genetic basis of toxin resistance (simple) and sequestration (complex) mechanisms by leveraging state-of-the-art approaches in model and non- model systems. In amphibians, tetrodotoxin resistance has been traced to mutations in ion channels, and tetrodotoxin is thought to be sequestered from symbiotic bacteria. The proposed research will determine whether Harlequin toads obtain toxins from bacteria through bacterial culturing and inoculation experiments. Researchers will then use transcriptome sequencing to determine whether Harlequin toads and Pacific newts modulate production and storage of TTX through specific protein activity in skin tissue. In another project, researchers will identify genes and pathways involved in epibatidine sequestration using toxin-feeding experiments, RNA sequencing, and whole-genome sequencing in poison frogs that can and cannot sequester epibatidine. Finally, researchers will experimentally evolve nicotine sequestration in fruit flies to identify genes and pathways underlying toxin sequestration with unprecedented detail. Understanding the mechanisms used by animals to modulate toxin accumulation and clearance will provide insight into the suite of genes that interact with toxins as they are ingested, transported, stored, or excreted. Given that neurotoxins target critical nervous system proteins and interact with several biological pathways targeted by human medicine, the proposed research has translational implications for pharmacology and the biology of disease.

NIH Research Projects · FY 2025 · 2023-09

Project Summary My research takes a unique approach in which the development of next-generation microscopy methods progresses in parallel with fundamental discoveries in cell biology. On the method-development front, besides earlier success in achieving sub-10 nm resolution for super-resolution microscopy, my recent work has pioneered the concept of multidimensional and multifunctional super-resolution microscopy, in which intracellular functional parameters, including local chemical polarity, pH, diffusivity, and protein activity, are mapped out at nanometer resolution and single-molecule sensitivity. Empowered by such capabilities, my lab has been highly successful in unveiling hidden subcellular structures and processes, as well as their underlying biophysical rules, for diverse systems ranging from the mammalian cytoskeleton, intracellular transport, organelle morphology and biogenesis, to membrane biology. Our future research continues to push forward the synergy between method development and biological discoveries. Major directions include charge-modulated protein interactions and effects on diffusion inside the organelle lumen, superdiffusion and subdiffusion in the living cell, organelle pH dynamics and role in protein trafficking, and the structure and physical properties of the ER exit site in relationship with the biogenesis of transport carriers. Moreover, by integrating super-resolution microscopy with FIB-SEM, we will obtain holistic pictures of the unusually thin tubular organelles we recently discovered and further substantiate their functions and biogenesis. Separately, we are developing a new tool, single-molecule electrophoresis microscopy, to quantify protein charge states at the super-resolution level. Together, through the continued development of empowering microscopy tools and their tactical application to fundamental biological questions, we will continue shifting the paradigms of how we understand the complex, dynamic behavior of the cell.

NIH Research Projects · FY 2025 · 2023-09

The field of computational psychiatry seeks to understand the symptoms and causes of neuropsychiatric diseases as dysfunctional learning processes. The learning algorithms used by the brain fall along a continuum between two extremes. At one end of the continuum is model-free learning, an automatic process that relies on trial-and-error, storing the values of past actions, and inflexibly repeating those actions that led to higher values. On the other end is model-based learning, which generates predictions via a computationally expensive, deliberative process that models the environment, which endows flexibility to respond to environmental changes. Dysfunction of these algorithms can produce maladaptive behaviors. For example, compulsive behavior is argued to arise from disruption of model-based learning, which biases patients towards more inflexible model-free learning mechanisms. Although a great deal of progress has been made in understanding the neural mechanisms underlying model-free learning, we have limited understanding of how the brain uses models to generate reward predictions. The grant aims to test the hypothesis that interactions between hippocampus (HPC) and orbitofrontal cortex (OFC) implement model-based learning. Specifically, we predict that HPC is responsible for constructing a cognitive map that instantiates a neural representation of behavioral tasks, and OFC is responsible for using the cognitive map to generate reward predictions that can be used to generate flexible decision-making. The current grant will test key predictions of this hypothesis. Our first aim uses a novel task that temporally separates the presentation of information about states and values. We will use high-channel count recordings from HPC and OFC and closed-loop microstimulation to examine how the putative HPC state representation affects the coding of value in OFC. In addition, we will examine whether this interaction occurs through the synchronization of the theta rhythm between the two areas. In the second aim, we will examine how a more complex map involving multiple distinct states might be used to enable rapid readjustments to reward changes. Dysfunction of pathways between HPC and frontal cortex are implicated in several neuropsychiatric disorders, including schizophrenia, major depression, and post-traumatic stress disorder. Medication-based treatments have failed to show significant reduction in the prevalence or severity of these disorders. An alternative approach is to use electrical stimulation, but to date this has also yielded mixed results. Our goal is to develop more sophisticated devices that will interact with neural circuits in a more principled way to treat neuropsychiatric disorders, such as using neural activity to detect symptoms and microstimulation to intervene. An impediment to this approach is that the neural coding in many of these circuits remains poorly understood. The aim of the current grant is to understand the neuronal properties of HPC and OFC to help lay the groundwork for future potential therapeutic approaches based on closed-loop microstimulation.

NIH Research Projects · FY 2024 · 2023-09

The field of computational psychiatry seeks to understand the symptoms and causes of neuropsychiatric diseases as dysfunctional learning processes. The learning algorithms used by the brain fall along a continuum between two extremes. At one end of the continuum is model-free learning, an automatic process that relies on trial-and-error, storing the values of past actions, and inflexibly repeating those actions that led to higher values. On the other end is model-based learning, which generates predictions via a computationally expensive, deliberative process that models the environment, which endows flexibility to respond to environmental changes. Dysfunction of these algorithms can produce maladaptive behaviors. For example, compulsive behavior is argued to arise from disruption of model-based learning, which biases patients towards more inflexible model-free learning mechanisms. Although a great deal of progress has been made in understanding the neural mechanisms underlying model-free learning, we have limited understanding of how the brain uses models to generate reward predictions. The grant aims to test the hypothesis that interactions between hippocampus (HPC) and orbitofrontal cortex (OFC) implement model-based learning. Specifically, we predict that HPC is responsible for constructing a cognitive map that instantiates a neural representation of behavioral tasks, and OFC is responsible for using the cognitive map to generate reward predictions that can be used to generate flexible decision-making. The current grant will test key predictions of this hypothesis. Our first aim uses a novel task that temporally separates the presentation of information about states and values. We will use high-channel count recordings from HPC and OFC and closed-loop microstimulation to examine how the putative HPC state representation affects the coding of value in OFC. In addition, we will examine whether this interaction occurs through the synchronization of the theta rhythm between the two areas. In the second aim, we will examine how a more complex map involving multiple distinct states might be used to enable rapid readjustments to reward changes. Dysfunction of pathways between HPC and frontal cortex are implicated in several neuropsychiatric disorders, including schizophrenia, major depression, and post-traumatic stress disorder. Medication-based treatments have failed to show significant reduction in the prevalence or severity of these disorders. An alternative approach is to use electrical stimulation, but to date this has also yielded mixed results. Our goal is to develop more sophisticated devices that will interact with neural circuits in a more principled way to treat neuropsychiatric disorders, such as using neural activity to detect symptoms and microstimulation to intervene. An impediment to this approach is that the neural coding in many of these circuits remains poorly understood. The aim of the current grant is to understand the neuronal properties of HPC and OFC to help lay the groundwork for future potential therapeutic approaches based on closed-loop microstimulation.

NIH Research Projects · FY 2025 · 2023-09

Abstract Action potential propagation through nodes of Ranvier is central to nervous system function. Understanding this process is essential for developing improved treatments for nodal pathologies of electrical signaling including multiple sclerosis, Guillain-Barré syndrome, stroke, spinal injury, and glaucoma. Saltatory conduction—the jumping of the action potential from one node to the next—has been described since its discovery as a purely electrical phenomenon. This proposal aims to investigate whether it is also fundamentally mechanical in nature. The mechano-activated two-pore domain potassium channel TRAAK is exclusively expressed at nodes of Ranvier. TRAAK is insensitive to voltage, but acutely tuned to membrane tension, with cell swelling increasing TRAAK-mediated potassium currents up to one hundred-fold. Still, whether mechanical activation of TRAAK is relevant to spike propagation is unknown. Using a combination of organic chemistry, molecular biophysics, and neurophysiology, this proposal will examine how mechanically activated TRAAK currents contribute to action potential propagation, speed, and reliability. To selectively control TRAAK channels, photoswitchable tethered ligands (PTLs) will be designed, synthesized, and optimized for maximal spatiotemporally precise block of TRAAK current. Screening of PTL tethering sites in leak and mechano-activated open TRAAK channels will enable the identification of state-specific PTL·Cys-TRAAK pairs and the precise modulation of basal and/or mechano-activated TRAAK currents. Using these tools, TRAAK's contributions to action potential propagation will be characterized in myelinated optic nerve under typical conditions and in response to mechanical perturbation. These experiments will both elucidate the role of TRAAK in spike propagation and, potentially, demonstrate that mechanical force is central to node repolarization, with broad implications for the treatment of nodal pathologies and the field of neuronal communication as a whole.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Mitochondria are essential organelles that supply cells with ATP and metabolic building blocks, but also play key roles as signaling hubs that orchestrate the immune response or cell survival. Mutations in mitochondrial proteins impede development and cause diseases, such as neurodegeneration, and a decline in mitochondrial activity is considered a hallmark of aging. To prevent such consequences, cells employ conserved signaling pathways that detect and alleviate mitochondrial dysregulation. It is a major goal of this proposal to dissect the regulation of a mitochondrial signaling pathway, the reductive stress response, which safeguards activation of the electron transport chain (ETC) through sensing an invariant ETC byproduct, reactive oxygen species (ROS). The reductive stress response is built on a Cys redox switch: in healthy cells, Cys residues in FNIP1 are oxidized, which stabilizes this protein and allows it to downregulate ETC activity. When cells run out of ROS, however, the FNIP1 Cys residues become reduced and FNIP1 is recognized by the E3 ligase CUL2FEM1B. The subsequent ubiquitylation and proteasomal degradation of FNIP1 removes this mitochondrial gatekeeper to re- activate the ETC and re-supply cells with ROS. FNIP1 and CUL2FEM1B are therefore the sensory module of the reductive stress response. Importantly, mutations in FEM1B that hyperactivate this E3 ligase cause syndromic developmental delay, showing that tissue formation and homeostasis require tight regulation of the reductive stress response. This proposal will dissect three crucial modes of regulation that ensure accurate reductive stress signaling. We will first investigate spatial control of reductive stress signaling. As with all ubiquitylation reactions, FNIP1 modification by CUL2FEM1B takes places in the cytoplasm, yet how cells can modulate the oxidation state of the critical FNIP1 Cys residues in this already reducing environment is unclear. We found that substrate and enzyme of the reductive stress response are anchored on the outer mitochondrial membrane via the TOM complex, a channel that connects the oxidative mitochondrial intermembrane space with the reducing cytoplasm. In our first aim, we will dissect how this localization impacts reductive stress signaling. We expect that this work will reveal a novel function of a membrane channel as an E3 ligase co-adaptor. Moreover, it will likely allow us to pinpoint the source of ROS that mediate reductive stress signaling, thereby revealing a sought-after physiological role for ROS in signaling. We will next focus on the regulation of reductive stress signaling by the cell cycle. ROS have long been suggested to control cell division, and we had indeed found that hyperactivation of CUL2FEM1B inhibits proliferation. This result implied that ROS control the cell cycle via the reductive stress response. In line with this notion, we identified the cell cycle regulator RNF187, which promotes cell cycle progression downstream of growth factor signaling, as an inhibitor of CUL2FEM1B. Our preliminary data suggest that RNF187 and CUL2FEM1B collaborate to restrict another E3 ligase, AMBRA1, which drives cyclin D degradation and thereby prevents initiation of DNA replication. In our second aim, we will dissect the mechanistic underpinnings of this E3 ligase crosstalk to reveal how ROS signaling is integrated into the cell cycle program. We expect this work to explain how redox stress can its exert negative consequences onto development or onto tissue homeostasis during tumorigenesis. While our first aims address physiological modes of regulation, we will finally develop methods to exert pharmacological control over reductive stress signaling. As the reductive stress response tunes the ETC, activating the reductive stress E3 ligase CUL2FEM1B provides us with a unique opportunity to increase ETC output in pathologies driven by mitochondrial decline or inhibition. Moreover, because CUL2FEM1B acts on mitochondrial surfaces, compounds that target this E3 ligase could be converted into localized proteolysis-targeting chimera for more efficient and more specific focused degradation of pathological proteins. In our last aim, we will build on our discovery of compounds that displace protein inhibitors from CUL2FEM1B,, thereby activating both FNIP1 degradation and ETC function. This work will lay the foundation for mitochondria-associated protein degradation as a new modality to provide therapeutic benefit during aging or in neurodegenerative disease. Our proposal takes an integrated genetic, biochemical, and pharmacological approach to dissect the regulation of the reductive stress response as a conserved mitochondrial stress response. This work will reveal fundamental principles of redox signaling and may lead to the development of a new therapeutics that could benefit patients of neurodegenerative diseases that currently have few, if any, treatment options.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Mitochondria are endosymbiotically-derived double membrane-bound organelles which provide cells with energy via oxidative respiration. Mitochondria also serve as a major hub for cellular metabolism and are involved in numerous vital pathways, including cell signaling, innate immune response, and apoptosis. Dysfunction of mitochondria is implicated in aging and many diseases and is a potential causative factor in neurodegenerative diseases. Most of >1,000 mitochondrial proteins are encoded by the nuclear genome and thus are imported from the cytosol shortly after being synthesized as precursors on cytosolic ribosomes. Thus, mitochondrial protein import is an essential process required for biogenesis and functional maintenance of mitochondria. The import process is mainly mediated by two universally conserved membrane complexes, the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. The TOM complex mediates the initial translocation of precursor proteins across the outer mitochondrial membrane, and the TIM complex further translocates the precursor proteins across the inner mitochondrial membrane. The TIM complex is also responsible for integration of many integral membrane proteins to the inner membrane. Currently, it is poorly understood how the TOM and TIM complexes mediate these translocation processes. In the current proposal, we aim to address central outstanding questions about protein import mechanisms by the TOM and TIM complexes, using structural, biochemical, and biophysical approaches. These questions include how the translocase complexes specifically recognize their client proteins, how they form a path for protein translocation in the membranes, what are the molecular interactions and forces driving protein translocation, and how the translocase complexes are regulated. In particular, we will perform several cryo-electron microscopy (cryo-EM) studies to visualize the translocase complexes in different functional states, including substrate-engaged states, and gain insights into their mechanisms for substrate engagement and conformational changes. The outcomes of these studies will fundamentally advance our understanding of mitochondrial biology and provide new insights to develop novel approaches to treat mitochondrial-associated diseases, such as neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2023-09

Our understanding of primate evolution was transformed by the sequencing and assembly of the human reference genome which identified the content and context of genes shared amongst the many different taxa in the primate lineage. Subsequent efforts to assemble the genomes of chimpanzees, macaques, and other nonhuman primates further accelerated our under- standing of these and related species. Problematically, these genomes were of substantially lesser quality than the human genome and often relied on the human genome for long-range scaffolding. Recently however, the advent of long-read sequencing technologies have enabled de novo con- struction of complete, accurate vertebrate genome assemblies. These technological advances have supported renewed assemblies of a handful of apes and monkeys which are vastly improved providing unique insights into the evolution and biology of these species. Nevertheless, complete, accurate, reference quality genomes are lacking for the vast majority of the ~500 extant species of primates. Here we propose to construct complete reference genomes from 50 diverse primate species spanning 75 million years of evolution. Using state-of-the-art long-read sequencing and assem- bly methods we will sequence, assemble, annotate, align, and disseminate these genomes as a resource for the community. Source DNA will be derived from the Integrated Primate Bioma- terials and Information Resource (IPBIR) at the Coriell Institute for Medical Research (Coriell), from a high quality, low-passage, cytogenetically verified collection of cell lines. This resource will greatly enhance the potential for research that will advance our understanding of primate evolutionary history, population genomics, infectious disease, human origins, and primate biodiversity and conservation.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY During postnatal developmental stages known as critical periods (CPs), sensory experience acts upon a genetically-hardwired connectivity map to sculpt the neocortical circuitry that enables mammalian functioning. Neurodevelopmental disorders such as autism disrupt this experience-dependent plasticity and compromise the development of social, cognitive, and physical function. Since autism spectrum disorder (ASD) patients suffer from tactile hyper- or hypo-sensitivity that may reflect abnormal development of sensory circuits, ASD is commonly studied in primary somatosensory cortex (S1). In addition, the mouse whisker S1 is a somatotopic map of the mouse whisker pad, so manipulation of specific whiskers induces observable functional changes in the corresponding barrels of S1. Morphological and physiological studies of experience-dependent plasticity in S1 have revealed several CPs and elucidated the influence of ASD on their emergence in mouse models of autism. However, the gene expression programs underlying experience-dependent plasticity and the influence of ASD on it remain unknown at the resolution of S1’s 100+ transcriptomically distinct cell types. Since these cell types form the circuits that carry out sensory function, it is important to study the influence of experience and ASD on their maturation. This project combines single-nucleus mRNA sequencing (snRNA-seq) and computational biology approaches rooted in machine learning with temporally resolved whisker manipulations and a mouse model of ASD to test two hypotheses. To test the hypothesis that whisker experience is required for cell type development in S1, snRNA-seq will be performed at several time points spanning two established CPs in whisker-deprived and control mice. Unsupervised and supervised machine learning approaches such as dimensionality reduction, clustering, graph embedding, and classification will be used to identify transcriptomic cell types at each time point and assess the influence of whisker experience on their maturation. Hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) will enable the validation of cell type-specific development patterns. To test the hypothesis that ASD disrupts experience-dependent cell type maturation, snRNA-seq will be performed on Fmr1 KO mice under whisker deprivation and control conditions. Fmr1 KO models Fragile X syndrome, the most frequent monogenic cause of intellectual disability and ASD in humans. While Fmr1 deletion has been shown to delay the maturation of circuits in S1 during a CP, its influence on experience-dependent maturation of S1 cell types remains unknown. Comparing gene expression profiles and cell types between KO and wild-type mice with and without whisker-deprivation will reveal transcriptomic signatures of ASD and pinpoint the cell types in which its effects are localized. Knowledge generated from this study about the manifestation of ASD in transcriptomic cell types will improve understanding of ASD pathology and reveal candidate cell types for targeted treatment.

NIH Research Projects · FY 2025 · 2023-08

Abstract: The choice of developing T cells to adopt the CD4 helper or CD8 killer cell fate is a valuable model for understanding mammalian cell fate decisions and is a key step in shaping the adaptive immune response. While it is known that TCR recognition of self pMHC ligands in the thymus controls cell fate, the molecular links between TCR signaling and the activation of the lineage-defining transcriptional networks remains unknown. Guided by a high-resolution single cell map of T cell development, we propose to define the molecular links between different branches of the TCR signaling pathway and the transcriptional network that specifies the CD4 fate (Aim 1). We will also probe the factors that determine why thymocytes that recognize MHC-2 fully activate this network, whereas thymocytes that recognize MHC-1 do not (Aim 2). The proposed studies will reveal fundamental principles underlying mammalian cell fate decisions and help to understand the molecular mechanisms that link the killer versus helper T cell effector functional programs to the recognition of MHC 1 versus 2.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Sensory representations are influenced by an animal’s external context, internal state, past experiences, expectations, and future goals. Prior information – including the history of recent stimuli, actions and rewards – plays an important role in guiding ongoing behavior, and can modulate the neural code even at the level of primary sensory cortex. The involvement of sensory cortex in mediating history- dependent shifts in behavior, and the contributions of specific cell types to these effects are not well understood. Using a novel whisker-based behavioral paradigm, I have demonstrated that mice can flexibly and selectively enhance sensory processing of recently rewarded whisker stimuli based on history cues. Here, I propose experiments to uncover the cell type-specific mechanisms in for history-based modulation in primary somatosensory cortex (S1), and test for their causal role in behavior. In Aim 1 (K99), I will use behavioral modeling approaches to systematically quantify history-based perceptual biases during goal-directed behavior in mice. I will examine modulation of pyramidal (PYR) cell activity in S1 of behaving mice while tracking trial-by-trial behavioral shifts in sensory detection performance guided by recent history. I will then empirically test the necessity of S1 in mediating history effects on behavior using reversible inactivation techniques. Two cortical interneuron classes, namely VIP cells and NDNF cells, are widely theorized to play a role in selective enhancement of sensory processing in cortex, since they receive a wide range of glutamatergic and neuromodulatory inputs and boost sensory responses in PYR cells through local disinhibition. Both these cell types are activated in different active behavioral states and learning contexts. In Aim 2 (K99/R00), I will test the role of VIP and NDNF interneurons in gating history-related signals using 2p imaging to monitor their neural activity, and through targeted activation or inactivation of these cell types using optogenetic techniques. VIP and NDNF interneurons are both recruited by acetylcholine, a neuromodulator that is necessary for stimulus-specific enhancement of sensory processing in primates, and behavioral state-based modulation of sensory cortex in rodents. In Aim 3 (R00), I will test the role of basal forebrain cholinergic projections in conveying history-related signals to S1. I will perform two-photon imaging of cholinergic terminals in S1 and use selective optogenetic activation to test whether the locus of prioritized processing on the whiskers can be artificially shifted in behaving mice. Together, the proposed studies will provide new insights into the local cortical circuits that facilitate prioritized processing of behaviorally relevant stimuli in sensory maps.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY The "NexGen" 7 Tesla MRI scanner at UC Berkeley is a unique resource that we wish to make available for neuroscience collaborations across the globe. It was specifically designed for extremely high resolution structural and functional neuroimaging at the scale of cortical laminae and columnar neurocircuit organization ("meso-scale"). To achieve this, the NexGen scanner builds upon existing standard 7T scanners and integrates a number of technological advances, creating synergistic improvements and gains in speed, resolution and signal. Such advances include: currently the highest performance one-of-a-kind head-only magnetic gradient coil designed to safely create more signal at faster time scales (higher gradient amplitudes and slew rates without peripheral nerve stimulation; PNS), high-density receiver array coils (e.g. up to 128 channel receive array integrated into a 7T) that enable unprecedented spatial resolution and image-forming accelerations. As part of this collaborative endeavor, we will be creating novel advances in pulse sequences for novel neuroscientific applications imaging to take advantage of the scanner hardware’s higher performance. The scanner has been validated in reproducible mesoscale fMRI studies using GE-EPI and VASO fMRI at isotropic resolutions between 0.39 mm and 0.6 mm, that is a 4.6 to 23-fold higher volumetric resolution over typical 1 mm isotropic resolution in fMRI on conventional 7T scanners. The stronger gradients offer considerable improvements in diffusion imaging reaching higher b-values with shortened TEs. These hardware advances also enable larger areas of brain coverage, higher temporal resolutions and reduced distortions at mesoscale resolutions. Thus, with the NexGen 7T, meso-scale neuroscience experiments can be greatly expanded from traditional zoomed, small volume imaging approaches. Such a one-of-a-kind instrument has great potential to continue to advance the field and will be optimally used by a diverse group of neuroscientists and clinician scientists. Funding of the current U24 proposal would facilitate engineering and scientific personnel to support and maintain the use of the scanner; enables efficient data transfer and analysis, as well as the subject recruitment, user training and guidance of scientific collaborations nationally and internationally. While the main goal of this project is to provide an innovative resource for higher granularity in functional and structural human brain research, the research resource also holds potential for better understanding neurological diseases, such as Epilepsy, Alzheimer’s and mental disorders.