University Of California Berkeley

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY The primary risk factor for prevalent diseases including cancer and neurodegeneration is aging. At the cellular level, aging manifests as an accumulation of conserved physiological defects that eventually cause functional decline, disease, and organismal death. Despite an extensive list of age-associated dysfunctions, we have a limited understanding of how aging becomes a major disease determinant. The traditional method in the field is to induce genetic modifications in a model organism before the aging process manifests itself, and to subsequently determine how these alterations affect lifespan. While these studies have been instrumental in identifying factors that impact longevity and healthspan, they lack the temporal resolution to distinguish the gene products that directly counteract age-associated damage from those that have indirect effects on lifespan, merely through delaying cell cycle progression, growth and/or development. The key challenge is the development of an effective system that allows identification of the underlying mechanisms of aging and manipulation of identified factors in a controlled manner. My lab has discovered that gametogenesis, the differentiation program that gives rise to reproductive cells, contains endogenous rejuvenation pathways. These physiological pathways have the ability to exclude and eliminate both cytoplasmic and nuclear pathologies that are associated with age. Therefore, mechanistic dissection of this program offers unique insights into the biology of aging as well as potential therapeutic avenues for age-associated diseases. This proposal seeks to provide a comprehensive understanding of the molecular and cellular events that are associated with meiotic rejuvenation. The experiments proposed in Aim 1 will determine how gametes are able to exclude and subsequently eliminate nuclear and cytoplasmic defects that accumulate with age. The experiments proposed in Aim 2 will take an orthogonal approach to identify and characterize the complete complement of meiotic genes that are capable of extending lifespan in vegetative yeast cells, akin to metazoan somatic cells. Further extension of these studies to C. elegans will identify conserved meiotic genes that can counteract organellar damage and will determine the effects of activating gametogenesis-specific rejuvenation pathways on tissue-specific as well as organismal healthspan. The combination of studies described in this proposal will reveal a mechanistic understanding of how meiotic rejuvenation occurs at the molecular level, determine which genes improve fitness and lifespan outside of meiosis, and reveal conserved pathways that can be leveraged to extend healthspan.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Abstract Mutation is the source of all evolutionary novelty and diversity shaping both the structure and sequence of genomes. Over evolutionary timescales changes to genome structure and content are associated with vast phenotypic changes between and within species. Throughout the lifetime of an organism individual cells accumulate somatic mutations that can also confer selective advantages. Our lab is interested in how mutations emerge and how these changes to genome sequence and structure are maintained and acted on by selection. We seek to understand at both the cellular and organismal level how cell-type, genotype, selective pressures, and evolutionary histories influence the structure and sequence of the genome. Ultimately, our research will further our understanding of the mechanisms underlying why specific cell types are more susceptible to disease as well as how genome structure influences phenotypic diversity within and between species. Patterns of somatic mutation have been extensively studied in the context of cancer tumor genomes in which clonal expansions amplify the signals of mutation to detectable levels. Far less is understood however about how “normal” cells accumulate mutations through time and how these dynamics are influenced by factors such as cell type and genotype. Furthermore, somatic mutations have proven challenging to identify due to the comparably high error rate of standard sequencing approaches. We propose to use novel genomic methods to investigate how different forms of somatic mutation accumulate and how somatic mutational processes are impacted by inherited genetic variation. In addition to discerning the contexts in which individual cells accumulate mutations, we propose to determine how genome structures have evolved in the context of different evolutionary histories, selective pressures, and life history strategies. While the size and structure of eukaryotic genomes varies tremendously spanning three orders of magnitude in vertebrates, the evolutionary and mechanistic bases of this variation remain unknown. We propose to study the evolution of genome architectures in the explosive adaptive radiation of rockfish to understand how extreme variation in lifespan can impact mutational processes and genetic diversity. We further propose to study how the structures of human and chimpanzee genomes have been shaped by local adaptations and the forces of selection. Identifying signatures of selection and adaption at structurally variable (SV) loci has been challenging in part due the tendency of SVs to emerge in complex repetitive regions of the genome. We propose to use long-read based genomics approaches and novel computational methods to assess these loci. Ultimately, our research will further our understanding of mutation, diversity, and genome structural diversity both within and between species as well as among the individual cells of organisms.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Recent studies have shown that population mixture (or `admixture') is pervasive throughout human evolution and has played a major role in shaping human genetic and phenotypic variation. Despite the ubiquity and importance of population mixture, we still lack adequate methods to characterize the impact of admixture on a genomic scale and leverage this information for effective gene mapping. Addressing these topics is the central focus of research in my lab. In this proposal, our goal is to develop new methods to reconstruct fine-scale genomic ancestry in admixed groups and leverage this information to identify novel disease and adaptive mutations and genes. The application of these methods to large genomic surveys will help to discover novel disease and adaptive variants. The first step in characterizing the genomic impact of admixture is to infer the ancestry of each chromosomal segment, referred to as local ancestry. Towards this goal, we are developing new methods for local ancestry inference using machine-learning approaches that are ideally suited for classification problems and computationally tractable for large datasets. Our preliminary results show that our method is highly accurate and applicable across a range of demographic models. With reliable local ancestry inference, we will be well placed to study the impact of admixture on disease architecture and evolution of complex traits. We propose to use Admixture Mapping, a method to identify disease associations by leveraging ancestry differences across the genome, between cases and controls or among cases alone. By applying Admixture Mapping to complex admixed groups like South Asians and Latinxs, we aim to discover new population- specific disease associations and advance our understanding of disease architecture. Further, we will develop a novel method to leverage the demographic history of admixed groups to identify adaptive variants. By applying the method to study selection at various timescales in human evolution, we will uncover candidate genes and pathways related to adaptive gene flow and characterize its role in shaping human genetic variation. Finally, we will build reference-free ancestral genomes by recovering chromosomal segments of our lost ancestors hidden in admixed genomes. We will use these genomes to reconstruct the demographic history of our ancestors, as well as understand the fitness effects of population mixtures and the phenotypic legacy of our extinct ancestors. The successful completion of the proposed project will provide new statistical tools to leverage patterns of admixture to perform effective disease mapping and evolutionary inference in diverse, admixed groups. Application of these methods to large-scale genomic datasets will provide insights into the genetic, evolutionary, and functional impact of admixture during human evolution. Algorithms proposed here will be implemented in freely available software for use by other researchers.

NIH Research Projects · FY 2024 · 2021-07

SUMMARY/ABSTRACT G-protein–coupled receptors (GPCRs), the largest class of membrane signaling proteins, respond to a wide array of extracellular stimuli to initiate intracellular signaling via G proteins and arrestins. Recent studies have provided snapshots of GPCR structures in distinct conformations and revealed that they are extremely dynamic. The conformational dynamics appear to be central to ligand recognition, activation and signaling. Membrane receptors have evolved to respond to precise spatio-temporal concentration profiles of extracellular ligands. In the nervous system, neurotransmitter receptors encounter a wide range of neurotransmitter concentrations and spatio-temporal profiles. Key factors are the small extracellular volume of the synaptic cleft, pumps and/or enzymes that remove neurotransmitter, and diffusion. Additionally, neurotransmitter receptors can be localized within the synapse both pre- and postsynaptically, as well as extrasynaptically where they can encounter neurotransmitter released either locally, which briefly reaches low millimolar levels within the cleft, and spillover from nearby synapses, which reaches lower concentrations. Metabotropic glutamate receptors (mGluRs) are found pre- and postsynaptically at excitatory glutamatergic synapses, as well as on glia and at inhibitory GABAergic presynaptic nerve terminals, meaning that they are activated by both high local concentrations near the site of release and spillover. mGluRs of various kinds can be found together in presynaptic nerve terminals, even when they are all coupled to the same G protein. And they can dimerize, generating hybrid or in some cases totally unique properties and pharmacological profiles. To understand what each mGluR subtype does and develop effective drugs to treat the neurological disorders in which they are implicated, we need to understand how they function and how they are regulated. Our goal here is to define the molecular mechanisms that set and regulate the functional properties of homo- and heteromeric mGluRs at synapses and put into place assays that can be used to screen modulation in the nervous system.

NIH Research Projects · FY 2025 · 2021-07

SUMMARY Worldwide, over 3 billion people are at risk of infection and disease caused by dengue virus 1-4 (DENV1-4) and Zika virus (ZIKV), both potentially severe flaviviral diseases transmitted by Aedes mosquitoes. The devastating effects of endemic dengue across the tropics and subtropics are well documented. The recent Zika pandemic galvanized research as Zika swept across Latin America. Three years after the peak of the Zika pandemic, major dengue epidemics have started to re-occur; however, the future of flaviviral disease across areas with widespread ZIKV immunity is unknown. In this R01, we propose to develop new tools and address key knowledge gaps in flaviviral transmission and immunological interactions between DENV and ZIKV to understand how widespread ZIKV immunity impacts subsequent dengue disease and to inform evaluation of dengue and Zika clinical vaccine trials and post-licensure studies. Based on our serological, epidemiological, and clinical data to date, our overall hypothesis is that DENV1-4 and ZIKV are antigenically closely related and that immune interactions mutually affect transmission and disease severity. We will address this hypothesis with the ongoing Pediatric Dengue Cohort Study (PDCS, 2004-present), a community-based prospective cohort study in Managua, Nicaragua, following ~4,000 children, now in its 17th year. Samples from the PDCS, as well as companion studies in Managua, provide documented infection and disease data, as well as banked serum samples for over a decade before the arrival of ZIKV. The proposed study extends the cohort, ensuring that we are able to fully document the interactions of these viruses from the pre- to post-Zika eras. In Aim 1, we will develop innovative serologic tools based on glycan-fusion-loop-masked envelope proteins and new algorithms to distinguish DENV and ZIKV infection histories, critical for vaccination and epidemiological studies of dengue and Zika. We will then test our hypothesis that pre-existing ZIKV immunity can enhance disease severity caused by DENV3 but protect against DENV1. In Aim 2, we will measure changes in anti-DENV and anti-ZIKV antibody-mediated immunity over time, estimate annual changes in protective and enhancing population immunity to each virus, collect entomological data, and use modeling approaches to evaluate popula- tion susceptibility to DENV and ZIKV infection and the potential for future epidemics by incorporating immunolo- gical and entomological data. In Aim 3, we will identify determinants of protective and disease-enhancing anti- body-mediated immunity of prior DENV infection on Zika and prior ZIKV infection on dengue disease and severity. With support of expert collaborators, we will use state-of-the-art tools (e.g., new monoclonal antibodies, innovative flavivirus antigens, and antibody Fc profiling) to analyze specific infection histories and uncover potential immune correlates. Overall, this program will define new vaccine companion diagnostic assays, the dynamics of the antibody response to DENV and ZIKV, and correlates of protection and pathogenesis for dengue and Zika, which should be useful for the development and evaluation of dengue and Zika vaccines.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Regulatory T cells (Tregs) are critical for the maintenance of immunological tolerance but more recently their importance in regulating other aspects of tissue homeostasis has been an area of intense interest. Most relevant to this application are populations of tissue-resident Tregs that accumulate upon injury and facilitate tissue repair by producing factors such as the EGF family member amphiregulin (Areg). These repair functions are distinct from the suppressive function classically attributed to Tregs, but the signals that instruct Tregs to adopt these distinct functional modalities have not been well defined. The IL-1 family cytokines IL-18 and IL-33 are involved at some level, at least in certain tissues, but it remains unclear whether these cytokines act as the key initial determinants of Treg function. This proposal will test the hypothesis that Toll-like receptor 7 (TLR7) signaling in Tregs is a key determinant of Treg tissue repair function. We propose that TLR7 enables Tregs to sense pathogen-derived nucleic acids as well as self RNA released from damaged host tissues. Our hypothesis is based on our analysis of a panel of TLR reporter mice, which revealed that only TLR7 is expressed on Tregs, as well as strong preliminary data demonstrating that TLR7 can induce expression of the signature tissue repair gene, Areg, in both murine and human Tregs. Using newly generated mice with Treg-specific deletion of TLR7, we will examine the importance of TLR7 signaling in Tregs during lung damage (Aim 1). Single-cell RNA sequencing will identify which subsets of lung Tregs are controlled by TLR7 and will define TLR7-dependent genes in Tregs. We will also investigate the importance of IL18R and IL33R signaling in Tregs, using mice with Treg-specific deletion of these receptors, and will determine the extent to which TLR7, IL18R, and IL33R regulate distinct aspects of Treg expansion and/or differentiation in response to diverse lung damaging agents (Aim 2). Finally, we will build on our recent work that identified a mechanism by which the TLR chaperone Unc93b1 specifically dampens TLR7 signaling. Using Tregs from mice with mutant Unc93b1 that have enhanced TLR7 signaling, we will test whether adoptive therapy of Tregs with enhanced TLR7 responses to viral and self RNA can mediate more effective repair of lung damage (Aim 3). Altogether, these studies will define the signals that control Treg tissue repair functions and test the therapeutic potential of amplifying these signals in the context of lung damage, a key first step toward therapeutic manipulation of Treg function for clinical benefit.

NIH Research Projects · FY 2025 · 2021-07

Graduate education has traditionally been successful in educating students in either engineering or the biomedical sciences, but the disparate nature of the scientific and engineering backgrounds necessary to successfully move the gene and cell therapy field forward requires novel educational approaches and methods that integrate these disciplines. With small molecule and protein therapies well-established in the pharmaceutical and biotechnology industries, gene and cell therapy represent the next generation of therapeutics to address serious unmet medical need. Because these therapeutics involve the delivery of DNA – in the form of genes or entire genomes – they have the potential to provide long-term therapeutic benefit following a single administration. However, the gene and cell therapy field face complex biological and technological challenges. Delivery of genetic constructs, either in vitro or in vivo, must be improved, and in addition therapeutic payloads including CRISPR/Cas9 and other genome editing machinery requires improved potency. In addition, cellular targets including human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and adult stem cells are difficult to precisely control, since the repertoire of signals and cues that naturally control stem cell self-renewal and differentiation are not well understood, yet precise control is essential to unlocking applications in tissue engineering and regenerative medicine. The University of California at Berkeley has developed the highly interdisciplinary Biology and Biotechnology of Cell and Gene Therapy (BBCGT) Training Program. With the involvement and support of our 24 faculty, the Berkeley Stem Cell Center, Bioengineering Department, Molecular and Cell Biology Department, and Helen Wills Neuroscience Institute, we have designed and have been successfully implementing a program to support the education and training of predoctoral fellows in gene and cell therapy. This newly emerging discipline represents the convergence of the biological and biomedical sciences, physical sciences, engineering, and ethics. The primary objectives of our program have therefore been to formally organize the structure and scope of new training opportunities in this rapidly expanding discipline, to dissolve traditional academic barriers to interdisciplinary graduate science education, and to provide strong research training in academia and industry. As part of these efforts, we will immerse trainees in a Biology and Biotechnology of Cell and Gene Therapy curriculum, training in the responsible conduct of research, seminar series, annual retreat, interdisciplinary research, career development resources, and industrial internship experience. The resulting program will be highly effective in training young scientists to work at the interface of the biomedical sciences and engineering in a rapidly-evolving, impactful, and timely area of biomedical research.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract: Alzheimer’s disease (AD) and its related neuropathology are linked to a set of neuropsychiatric symptoms (NPS) that come with marked clinical, personal and real-life burden. However, the underlying neural mechanisms of NPS are poorly defined, hindering novel treatment approaches. Core among these NPS are: 1) sleep disturbance, and 2) anxiety. Moreover, both sleep impairment and anxiety are independently associated with a faster rate of longitudinal cognitive decline, reinforcing a particular focus on their overlap. However, the possibility that AD pathology, impaired non-rapid eye movement (NREM) sleep and the NPS feature of anxiety are actually inter-related, representing a novel brain mechanism explaining why Aβ is associated with increased anxiety, has not been examined. Here, we seek to test a core mechanistic hypothesis: The impact of Aβ burden on anxiety is orchestrated through beta- amyloid (Aβ)-related impairment of NREM slow-wave sleep, which in turn, amplifies next-day anxiety through impaired mPFC-amygdala emotional brain regulation. If supportive, these studies would (i) advance our basic understanding of how AD-related pathology mechanistically impacts the NPS feature of anxiety through impairment of NREM sleep, and (ii) establish sleep improvement as a novel modifiable therapeutic intervention target that may de-escalate anxiety linked with the AD disease state, and potentially the rate of longitudinal disease/cognitive decline.

NIH Research Projects · FY 2026 · 2021-06

SUMMARY Interactions between central nervous system (CNS)-resident cells are highly heterogeneous; astrocytes and microglia nourish and protect neurons, while inflammatory subsets drive demyelination and neurodegeneration in neurologic diseases. However, the molecular mechanisms that control CNS-resident immune cell interactions remain mostly unknown because methods for defining the specific cell types, pathways and molecules involved are limited. In this project we apply two novel approaches that we developed to study astrocyte-microglia interactions during inflammation: 1) an in vivo barcoded rabies tracing strategy that analyzes cell connections and transcriptomes with single cell resolution, and 2) a droplet-based platform for genome-wide, unbiased CRISPR/Cas9 screening of interacting cell pairs. These methods provide a unique opportunity to study pathways used by CNS-resident immune cells to communicate with each other and control inflammation and neurodegeneration. We propose to: SPECIFIC AIM 1: Define the transcriptomes of single cells in pro-and anti-inflammatory networks. We will simultaneously sequence connections and single cell transcriptomes of CNS cells in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS). These studies will identify pro- and anti-inflammatory cellular networks and the molecular mechanisms that regulate disease-relevant cell-cell interactions within these networks. SPECIFIC AIM 2: Identify novel astrocyte-microglia interactions using a droplet-based platform for CRISPR/Cas9 forward genetic screens. We will perform unbiased genome-wide screens for genes that participate in microglia-astrocyte crosstalk. We will co-incubate and microfluidically sort millions of picoliter water-in-oil droplets containing single microglia harboring a CRISPR/Cas9 library mutation and single astrocytes carrying a fluorescent reporter. Independent droplets do not mix, providing a powerful platform for the identification of immune interactions mediated by cell surface and soluble molecules.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract Candidate: The goal of this K01 Award is to support the training and research of Dr. Cassondra Marshall, who's long-term goal is to advance reproductive and maternal health equity through the translation of evidence into practice using tailored interventions that address the needs of racial/ethnic minority women with chronic medical conditions. Her research has examined young women's contraceptive decision-making and has explored the ways in which health care providers and systems can deliver patient-centered contraceptive care. Research Plan: Women with diabetes prior to pregnancy have elevated rates of maternal and neonatal morbidity and mortality. Type 2 diabetes is increasingly prevalent among women during their childbearing years, disproportionately impacting Black and Latina women. To prevent maternal and infant health complications, there is an urgent need for enhanced efforts to improve reproductive health outcomes among reproductive-aged women with type 2 diabetes, particularly racial/ethnic minorities. However, there are currently no evidence-based strategies to implement clinical guidelines for improving reproductive outcomes among adult women with type 2 diabetes. Patient decision support tools in primary care represent a promising implementation strategy. The research aims are: Aim 1: Elucidate reproductive decision-making processes and reproductive health care preferences among Black and Latina women of reproductive age with type 2 diabetes, using person-centered and structural competency frameworks. Aim 2: Adapt an existing patient decision support intervention to support reproductive decision-making and reproductive health counseling delivery in primary care to meet the needs of Black and Latina reproductive-aged women with type 2 diabetes. Aim 3: Assess acceptability and feasibility of the adapted intervention among patients and providers and examine the implementation context for the intervention. Career Development and Training Plan: The candidate's training plan capitalizes on an interdisciplinary mentoring team with expertise in person-centered family planning care, diabetes care, implementation science, and organizational research in primary care. Through coursework, workshops, apprenticeships, and mentored research experiences, Dr. Marshall will receive training in Person- Centered and Structurally Competent Reproductive Health Interventions, Advanced Mixed Methods, Implementation Science and Stakeholder Engagement, and Designing and Implementing Trials in the Primary Care Setting. UC Berkeley and close partners UCSF and Kaiser Permanente Northern California provide an excellent research infrastructure and training environment for Dr. Marshall. Summary: The training and research proposed in this application position Dr. Marshall to be competitive for R01 funding to support a multisite effectiveness-implementation trial of the adapted intervention and facilitate her transition to independence as a researcher.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Social relationships develop between individuals with a social network. Difficulties in mediating social relationships with other individuals is strongly associated with severe mental disorders ranging from depression, chronic stress, autism and other. Thus, understanding the neural underpinning of social relationships is paramount. To gain insight that would inform of real-life behavior, I propose to study the nervous system under real-life conditions in which social interactions in humans and animals typically occur. In particular, I focus on the fact that social interactions typically involve multiple participants, employ the usage of a flexible repertoire of communication signals, and occur between individuals of varying social bonds and personality traits. Furthermore, social relationships evolve over prolonged periods of time in a dynamic fashion. In this proposal we focus on the anterior cingulate cortex (ACC). We do so because activity in this area has previously been strongly associated with social behaviors across a wide range of mammalian species, including humans. However, much less is known about the neural computations in the ACC with respect to social relationships, especially during real-life and multi-dimensional social conditions. To do so, we use the Egyptian fruit bat, a highly social, long-lived mammal that is accustom to group living and where individuals engage in relationships that extend over many months/years. We further develop advanced behavioral measurements that allow us to monitor the social interactions of individuals within our colonies continuously and characterize their social relationships between group members. To study the neural circuits that underlie social relationships we develop wireless neurophysiological tools that enable monitoring neural activity from entire colonies of bats simultaneously at cellular and millisecond resolutions (electrophysiology) and over prolonged periods of time (calcium imaging). This novel approach allows us to consider the true complexity of real-life social interactions and consider the social bonds between the individuals, the dynamic structure of the social relationships as well as the individual variability in personality traits. Specifically, we aim to achieve the following aims: (1) We start by describing the basic neural dynamics in the ACC during semi-natural, dyadic, social interactions and communication. (2) We next describe the ACC neural dynamics during interaction occurring within real-life, stable, social networks while considering the relationships between individuals (3) We describe the evolution of ACC neural dynamic in parallel to the dynamical changes that occur in real-life social networks. (4) We use optogenetics tools to disrupt neural activity in the ACC during group social interactions in order to assess its causal role in real-life social relationships with other individuals. Combined, these experiments will provide a detailed description of ACC neural computations underlying the mediation of social relationships within a social network. In doing so, we aim for these results to provide important insight that could be used in clinical future application in patients.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Natural navigation is an important skill that engages many sensory, motor and cognitive systems. Because aging and degenerative brain disease both diminish the capacity to navigate in the real world, a better understanding of the brain mechanisms mediating navigation will improve diagnosis and monitoring of neurological and neurodegenerative diseases. Neurophysiological studies in animals have led to fundamental insights about the neural mechanisms mediating navigation. However, due to methodological limitations neuroimaging studies of navigation in humans have generally been less compelling than the animal work. We propose to overcome these limitations by using the NexGen 7T MRI scanner recently installed at UC Berkeley to measure brain activity during a naturalistic driving task. Driving is an excellent target for fMRI studies because is a common human navigation task that unfolds across a large and varied landscape, and on a timescale commensurate with fMRI; it engages many navigational brain systems; and it is impacted by aging and neurological diseases. Data will be analyzed by means of an innovative and powerful voxelwise modeling framework developed in PI Gallant's lab over the past 10 years, and validated in many publications. Computational models reflecting 33 different types of navigational features will be fit to the fMRI data separately for each voxel and in each individual subject. Model prediction accuracy and generalization will be cross-validated using separate data sets and subjects reserved for this purpose. The results will be used to test dozens of specific hypotheses about navigation drawn from the theoretical and experimental literature on both rodents and humans. These results will also be used to obtain a detailed functional parcellation of navigational representations in each individual and across the group, and to identify functional networks that represent specific navigation-related features. By combining naturalistic experiments, large-scale computational modeling, multiple hypothesis testing, data-driven functional parcellation and functional network analysis, this research will provide fundamental new information about the human brain mechanisms mediating navigation and their relationship to prior findings from the animal literature.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Hyaluronic acid (HA) is the most abundant component of the human brain, where it serves essential structural, mechanical, and cell-instructive functions. Adhesion between HA and its receptor CD44 critically regulates development and homeostasis, and dysregulation of HA and/or CD44 causally drives many brain pathologies, including invasion of the deadly brain tumor glioblastoma (GBM). Despite the clear biological significance of HA-CD44 adhesion, comparatively little is known about either the biophysical mechanisms through which HA- CD44 interactions drive cell adhesion, migration, or matrix remodeling or how HA composition (e.g. molecular weight) influences adhesion and migration. Over the past decade, our team has made seminal contributions to addressing these questions, including introducing and refining synthetic 3D HA matrices as a culture model for studying GBM invasion. We also discovered that CD44 transduces HA-based mechanical signals to regulate cell shape, cytoskeletal assembly, and motility. Most recently, we discovered that GBM cells engage HA using “microtentacles” (McTNs), CD44-dependenent processes that extend tens of microns from the cell body, are associated with HA digestion, and mechanically couple to the cytoskeleton through a complex that includes IQGAP1 and CLIP170. McTNs bear important similarities to structures that have been observed in invasive GBMs in vivo, and overexpression of McTN components is predictive of with aggressive progression and poor survival in GBM. In this R01 application, we will leverage these discoveries and biomaterial platforms to advance the field’s understanding of how HA and CD44 contribute to cell adhesion, migration, and invasion. In our first aim, we will investigate how McTNs facilitate adhesion, invasion, and matrix remodeling. In our second aim, we will determine how biophysical features of the HA network in brain tissue contributes to 3D migration, using GBM as a model system. Our approach is distinguished by tight integration of engineered biomaterial culture models, mouse models featuring human GBM stem/initiating cells, and analysis of biopsies obtained from specific anatomic regions of human GBMs. Our multi-institutional team also uniquely combines expertise in biomaterials, mechanobiology, neurosurgery, and cancer biology. Successful completion of these studies will yield unprecedented insight into the biophysical basis through which HA and CD44 contribute to adhesion and invasion, a problem of high fundamental interest that may lead to novel therapeutic targets in GBM.

NIH Research Projects · FY 2025 · 2021-03

Project Abstract – no change from original proposal Computational modeling can help us formalize how choice behaviors can be optimally adapted to different situations and investigate the ways in which individuals deviate from optimal behavior. Both anxious and depressed individuals report difficulties with decision-making; these difficulties have consequences for social interactions and occupational function. Understanding whether anxiety and depression are associated with common or unique deficits in decision-making has been hampered by studies focusing on either anxiety or depression alone and overlooking issues of comorbidity. This is important to address to better identify which aspects of decision-making should be targets for intervention in different patient groups. The separate investigation of anxiety- and depression-related deficits in decision-making has also led to a lack of equivalence of tasks and limited use of both reward-related and aversive outcomes within the same study. In the proposed research, we will conduct bifactor analysis of item-level responses to anxiety and depression questionnaires and use participant scores on the dimensions obtained to interrogate whether deficits in decision making under second-order uncertainty are common to both anxiety and depression or unique to one or the other. We focus upon second-order uncertainty as this characterizes many of the situations we encounter in every-day life but there has been limited investigation of whether anxiety or depression are linked to deficits in adjusting decision-making to second-order uncertainty. Second-order uncertainty arises both when the probability of our actions resulting in certain outcomes changes across time (volatility) and when information needed to estimate how likely a given action is to lead to a given outcome is not fully available (ambiguity). In the proposed studies, we will use volatility and ambiguity manipulations to examine whether deficits in decision-making under second-order uncertainty are common to both anxiety and depression or unique to one or other and whether such deficits are domain general or domain specific (vary by outcome type: aversive, reward gain or reward loss). On-line studies will be used to conduct replication work and to examine if impaired decision-making under second-order uncertainty is primarily linked to internalizing symptomatology or common to a broader range of psychopathology. These online studies will also enable us to test exploratory hypotheses pertaining to other dimensions of psychopathology. Understanding the extent to which alterations in decision making under second order uncertainty are unique to anxiety or depression, common to both anxiety and depression (i.e. a transdiagnostic marker of Internalizing psychopathology), or associated with psychopathology more broadly is important to clarify so that we can better tailor cognitive and psychoeducational interventions to different patient groups. It may also help clarify whether existing interventions developed in relation to anxiety (e.g. CBT focusing on ambiguity aversion) might valuably be applied to other forms of psychopathology.

NIH Research Projects · FY 2026 · 2021-03

PROJECT SUMMARY/ABSTRACT Microbial communities are ubiquitous on earth, inhabiting nearly every niche in the environment and on the human body. The composition and metabolic activities of the human microbiome are known to impact health. However, much remains unknown about how these communities assemble and function, in part because it is not well understood how individual microbial species, each unique capabilities, interact with each other to form complex microbial communities. To address this gap, we have established the corrinoid model to investigate microbial interactions across scales of complexity, from individual genes, enzymes, and microbes to binary interactions to complete communities. Corrinoids, the vitamin B12 family of cofactors, are ideal for this approach because they are shared nutrients, synthesized by a fraction of the microbes that use them. Furthermore, different microbes have distinct preferences for the nearly 20 corrinoids found in nature. Our previous work focused on identifying the molecular factors underlying corrinoid specificity. We found that corrinoid structure significantly influences microbial composition in communities of different origins and complexity – soil, human gut-derived laboratory enrichment cultures, and a synthetic consortium of human gut bacteria – despite the presence of endogenously produced corrinoids in these environments. This project focuses on identifying the mechanisms underlying these changes in an effort to understand how individual microbes contribute to community function. Employing top-down approaches, we will determine the effects of changing corrinoid composition and inhibiting corrinoid-dependent metabolism on community composition. These results will be combined with genomic prediction and experimental characterization of corrinoid metabolism in individual microbes to inform the design of bottom-up microbial consortia. This bidirectional approach focusing on a single nutrient class across scales will reveal how microbial communities assemble from their constituent parts, providing a path toward the rational development of interventions to improve microbiome function.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Today’s youth suffer from unprecedented levels of physical inactivity and poor physical fitness, which contribute to myriad health complications, including increased risk for cardiovascular disease. School physical education (PE) is one of the most valuable tools for increasing physical activity and improving youth fitness and shows potential for reducing related health disparities. However, despite laws in 43 states mandating that schools offer PE, compliance is extremely low in elementary schools, and disparities based on school-level student race/ethnicity and income exist. Evidence points to a lack of accountability as a major factor in low PE law compliance, but measures of accountability are inconsistent (or non-existent) across states, and best practices for increasing compliance with PE mandates remain unknown. This study proposes to examine two novel approaches for increasing PE law compliance by first, determining the impact of the New York City Department of Education’s multi-level school physical education (PE) intervention on PE law compliance and student cardiorespiratory fitness, as well as its cost-effectiveness for increasing student physical activity in order to inform replicability in other school districts. Secondly, one component of New York’s intervention – a PE audit and feedback tool – will be piloted in Oakland, CA schools to determine the effectiveness, adaptability, and scalability of this potential cost-effective approach for increasing PE law compliance and student physical activity. I am pursuing the NHLBI K01 Mentored Research Scientist Development Award to fill critical training gaps in (1) causal inference from observational data; (2) cost-effectiveness analysis; and (3) implementation science. This award will build upon my significant experience in school-based physical activity research, epidemiologic methods, and participatory action research. My long-term career goal is to develop and rigorously evaluate policy and programmatic approaches to improve youth health through physical activity. The skills I seek to obtain will be critical in achieving this goal. My detailed training plan includes formal coursework at UC Berkeley and UCSF, NIH courses, mentored experience, meetings, seminars, and directed readings. The research component of this project will provide opportunities to integrate new knowledge into practical research experience. Together, the training and protected time provided by the K01 award, combined with the rich collaborative environment and strong institutional support at the UC Berkeley School of Public Health and the Nutrition Policy Institute, will facilitate my transition into an independent investigator who can successfully compete for R01 funding. It will also provide the means to help me become a leader in the development and testing of scalable school physical activity environment and policy interventions that will reduce population risk of inactivity-related chronic diseases.

NIH Research Projects · FY 2025 · 2021-02

Abstract Alzheimer’s disease (AD) affects over 5 million Americans and is expected to affect 2-3-fold more in the next few decades. AD is associated with aggregation of amyloid-beta (Ab) and phosphorylated tau proteins. Curiously, burden of Aβ, classically considered the most important AD pathological hallmark is not enough to indicate clinical decline or progression. For example, cognitive-normal elders may also carry high levels of Aβ and recent clinical trials aiming to reduce Aβ have generally failed to improve patients’ conditions. Therefore, developing biomarkers that can better predict clinical outcome and progression are needed. Confluent evidence shows that regional brain magnetic susceptibility measured by MRI differs between AD patients and healthy controls, and importantly such changes may predict cognitive decline. However, it is unclear what causes these susceptibility changes in AD. While iron deposition has been widely suspected as the underlying cause, our recent study has discovered that aggregation of Ab and tau by itself produces strong diamagnetic susceptibility, opposite of the paramagnetic susceptibility generated by iron deposition. The opposing magnetic susceptibility of iron and aggregated pathological proteins poses a significant challenge as current MRI-based magnetic susceptibility mapping algorithms cannot differentiate iron from other colocalizing diamagnetic susceptibility sources within the same voxel. Our goal is to develop a novel technique that can differentially quantify molecular sources of magnetic susceptibility and test whether the resulting susceptibility components can serve as markers of progressive AD pathology. We will test our techniques and hypothesis utilizing a unique capability that combines in cranio MRI at autopsy with histological examinations. We have developed innovative histological processing methods that allow voxel-to-voxel matching between MRI and histology in 3D, thus permitting the examination of the relationship between magnetic susceptibility components and the neuropathology underlying AD. If successful, our techniques and findings might ultimately allow the detection of AD-related neuropathology at much earlier stages, permit intervention before neurons become irretrievably damaged and non-invasively assess disease progression. These techniques, once standardized, will be highly cost-effective, widely accessible and readily implementable in non-specialized clinical imaging centers, thus better serving the growing population of AD patients.

NIH Research Projects · FY 2025 · 2021-01

Cilia are microtubule-based cellular protrusions with diverse biological functions, including fluid movement, cellular locomotion, environmental sensing, and signal transduction. Traditionally, most cilia are classified based on differences in ciliary ultrastructure, biological function, and ciliary motility, with primary cilia and motile cilia as the major categories. The primary cilia functions as solitary sensory hubs to transduce extracellular stimuli into intracellular signaling pathways, and the motile cilia exhibited coordinated beating to generate directional fluid movement. Choroid plexus epithelial cells contain multi-sensory cilia that regulate the production of cerebrospinal fluid (CSF) to support neuronal development and physiology. Using serial transmission electron microscopy (TEM) and focus ion beam scanning electron microcopy (FIB-SEM), our preliminary results suggest that the multi-sensory cilia of choroid plexus represent a distinct type of cilia, exhibiting unique ultrastructural features, while resembling aspects of both primary cilia and motile cilia. Defective ciliogenesis in choroid plexus causes hydrocephalus, at least in part, due to CSF overproduction. Choroid plexus cilia are likely to play an important role in Shh signaling, as FoxJ1 deficient choroid plexus cilia no longer respond to Shh treatment in explant culture. We discovered a functional connection between Shh signaling and Aqp1 expression. Hence, we hypothesize that choroid plexus cilia are a unique type of multi-sensory that mediate Shh signaling to regulate CSF production, at least in part, by regulating the expression of water channels and ion transporters. Here, using a combined approach of advanced imaging techniques, mouse genetics, imaging studies, cell biology and molecular biology, we propose to study the ciliary ultrastructures, ciliogenesis mechanisms and biological functions of the multi-sensory cilia of choroid plexus. First, using electron microscopy, FIB-SEM and super-resolution imaging, we will characterized the ultrastructure of choroid plexus cilia, and define their developmental dynamics at different developmental stages. Second, we will employ mouse genetics and genomics studies to identify and characterize the ciliogenesis machineries of choroid plexus cilia. Finally, we will elucidate the molecular mechanisms through which dynamic choroid plexus cilia mediate the Shh signaling to regulate CSF production. Taken together, the proposed studies will structurally and functionally define a new type of multi-sensory cilia in choroid plexus, and will generate important insights on the molecular basis for the regulation of CSF production.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Goals: Peri-Centromeric Heterochromatin (PCH) is required for genome stability/DNA repair, chromosome pairing, nuclear architecture, and transposon and gene silencing. Previous studies suggested that histone H3 lysine 9 methylation (H3K9me2/3), Heterochromatin Protein 1 (HP1) binding, HP1-interacting protein recruitment and chromatin compaction are sufficient to explain PCH formation and function. In 2017, my lab and the Narlikar lab published complementary studies suggesting that 3D PCH domains form via liquid-liquid phase separation (LLPS), generating membrane-less condensates with an immobile HP1a core surrounded by a liquid. We proposed that novel properties associated with highly networked, phase separated systems (e.g. liquidity) are critical to understand how PCH, and other chromatin domains, form and regulate essential nuclear functions. However, we lack a mechanistic understanding of the organization, dynamics and biophysical/material properties of PCH components and condensates in a cellular and organismal context. In addition, we need to determine if and how biophysical properties regulate genome functions such as repair, replication and transcription, a current major challenge for the whole field of condensate biology. Approach: This MIRA will interrogate how LLPS and biophysical properties impact the in vivo organization and function of heterochromatin and other associated nuclear bodies. We will capitalize on our preliminary results and knowledge of PCH biology, combined with advanced imaging, biochemical, and experimental and theoretical biophysical approaches, to elucidate 1) the molecular interactions responsible for PCH domain formation; 2) the architectural, biophysical and chemical properties of the domain; and 3) whether or not phase separation and liquidity regulate PCH functions and interplay with other nuclear bodies. Innovation: Although LLPS and biological condensates have become a popular topic for study and discussion in recent years, we know little about in vivo mechanisms and relevance to function in the complex but important cellular and organismal contexts. This is an emerging field, with unique challenges, and an interdisciplinary approach is required to address these key questions. Thus in this MIRA proposal we will combine our decades of experience in PCH biology with the expertise of collaborators in experimental and theoretical biophysics, and advanced bioimaging. Testing our hypothesis will elucidate important information about the organization and function of heterochromatin in cells and animals, potentially providing a paradigm- shifting foundation for understanding how chromatin domains in general form and function. Health Relatedness: Defective PCH causes genome instability and altered gene expression, contributing to cancer, birth defects, and aging. Understanding how biophysical properties that underlie PCH formation and function are altered in human diseases will likely result in novel approaches to diagnosis and treatment.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Goal-directed navigation often occurs in complex, large environments where the same goal can be reached from different starting point and through different routes which are often self-selected. The hippocampus is believed to play a central role in navigation yet it remains poorly understood how it supports the planning and execution of naturally emerging navigation patterns during goal-directed behaviors. The Egyptian fruit bat is a powerful model for bridging this gap due to specialization for spatial navigation and in particular, its natural desire to converge onto self-selected, stereotyped and highly structured navigation patterns. Here, we leverage the bat natural behavior and significantly extend the arsenal of tools and approaches to study how the hippocampus contributes to the planning, emergence and sustainment the goal-directed, structured navigation behavior. To do so, we develop novel fully automated environments aimed at engaging the bats in self-paced and self-selected, natural navigation under closely monitored laboratory conditions. In doing so, we find that the bats readily engage in goal directed foraging behavior which resembles that observed in the natural setting. To study the neural dynamics, computations and involvement of the hippocampus in this behavior we integrate a wide range of wireless neuro-technologies, many of which are entirely novel for the bat model system. These include, wireless electrophysiology, wireless cellular resolution calcium imaging and wireless optogenetics in freely flying bats. Our preliminary results provide a detailed account of the bat's navigational strategies during goal-directed behavior and are beginning to reveal the neural computations in the hippocampal formation that could facilitate this function. These includes functionally discrete set of neurons that are participating during discrete stages of the foraging behavior including, planning, execution and evaluation of goal-directed navigation as well as neuronal sequences that are specific to distinct navigational routes. Combined, we marry the development of the controlled, yet ethologically-relevant, behavioral setup with a wide range of cutting- edge neurophysiological methods to thoroughly examine the role and computations in the bat hippocampus that can subserve that bat's natural ability for structured goal-directed navigation behavior. In doing so, this research aims to provide a new model the hippocampal neural computations that can support self-selected and complex goal-directed navigation.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Human genome engineering has widely anticipated promise as a healthcare strategy, but current technologies are unlikely to provide the safe, efficient, and broadly useful implementation of transgene introduction essential to complete the next big leap forward for gene therapy. CRISPR-based approaches for transgene integration have major impediments, including the need for donor DNA delivery, the propensity of that DNA to undergo non-specific integration, and the low efficiency of repair by homologous recombination relative to sloppy rejoining of the broken DNA ends. Also severely limiting is the fact that slowly proliferating cells are rarely in a cell cycle phase favorable for homologous recombination, and just the presence of a DNA break can be toxic. The alternative approach of adeno-associated virus introduction of a transgene also has limitations, among others including the small transgene size permitted by the virus capsid and the challenges of engineering virus uptake into different cell types. It remains an unmet need to have a non-mutagenic, non-toxic approach for gene introduction to the human genome. Therapy for many loss-of-function pathologies hinges on this missing technology. Also, only transgene introduction offers the opportunity for non-native control of protein expression, isoform selectivity, and myriad other clinically useful outcomes. Starkly missing from current efforts to develop transgene introduction techniques is an approach exploiting the gene insertion strategy widespread endogenously across eukaryotes: cDNA synthesis. The ancestral, evolutionarily persistent type of eukaryotic LINE/non-LTR retroelement integrates by nick-primed reverse transcription that is rigorous both it its sequence specificity of target site selection and in its specificity for use of an RNA transcript with the retroelement 3’ UTR as template. The biochemical activities required for target site selection, introduction of precisely positioned nick, and cDNA synthesis are carried out by a single protein. Any RNA sequence flanked by 5’ and 3’ regions of the retroelement genome should assemble with a favorably modified retroelement protein, and this RNP would then seek its native insertion site. Because several LINE/non-LTR retroelement families target highly conserved, repetitive sequences invariant across multicellular eukaryotes, there is no need to re-engineer DNA site-specificity of these retroelement proteins, although that may become of interest to undertake. The simple architecture of the non-LTR retroelements begs to be exploited for developing an approach to human genome supplementation with genes of therapeutic impact. The novelty of this approach demands continuous innovation and obliges high risk of failure to reach the goal of delivering an engineered RNP capable of transgene introduction into human cells. Success of this strategy would usher in a new modality of therapeutic treatment for loss-of-function diseases.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT This is an application for a K01 award for Dr. Mark Fleming, a medical anthropologist at the University of California, Berkeley (UCB). Dr. Fleming is establishing himself as an investigator focused on health system interventions to address social determinants of health for vulnerable populations. For this K01 award, Dr. Fleming proposes a novel mixed-methods study of patient navigation (PN) for emergency department (ED) high utilizers. ED high utilizers typically have multiple chronic illnesses and significant unmet social and behavioral health needs. PN is a promising intervention for health systems to address this combination of medical, social and behavioral health needs for high utilizers, but its effectiveness is poorly understood. Dr. Fleming will conduct a mixed-methods evaluation of an ED PN program implemented at the University of California, San Francisco (UCSF). He will determine the effect of ED PN on subsequent medical, social services and behavioral health utilization using an existing composite database (Aim 1). This will be the first study to measure whether ED PN for high utilizers actually changes utilization of social and behavioral health services situated outside of the medical system (including housing services, income support, and mental health and substance use treatment). This will enable Dr. Fleming to test the hypothesis that linkage to and use of these services mediates the effect of ED PN on subsequent reductions of acute care utilization. Dr. Fleming then will conduct a qualitative study to identify the tasks and processes that constitute “navigation” and explain how ED-based PN works to achieve its effects (Aim 2). Finally, Dr. Fleming will use stakeholder-engaged, design engineering methods to develop an evidence-based, optimized PN program for high utilizers (Aim 3) and will subsequently propose an R01 to implement and evaluate the optimized intervention. This K01 award will provide Dr. Fleming with the support and research experience necessary to build on his training in medical anthropology and develop a mixed-methods research program in health services. To achieve this goal, this award will provide targeted training the following areas: 1) quantitative methods for health services research, 2) implementation science, and 3) professional development to become an independent health services investigator. Dr. Fleming’s training plan includes formal coursework, individual tutorials from his multi-disciplinary mentorship team, and participation in health services research and evaluation groups. Dr. Fleming has assembled a mentoring team comprised of primary mentor, Dr. Steven Shortell, national leader in health systems research and Director of the joint UCB-UCSF health services training program (AHRQ-T32), Dr. Urmimala Sarkar, expert in implementation science and innovative design methods for health services interventions, Dr. Nancy Burke, expert in mixed methods research in health systems, Dr. Maria Raven, Medical Director of UCSF ED PN program and expert in emergency medicine health services research, and Dr. Laura Gottlieb, national leader in health system social needs interventions.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Shigella species are highly infectious and important pathogens of humans. In 2016, there were an estimated 269 million cases and 212,000 deaths due to Shigella. Humans are typically infected with Shigella after oral ingestion of a minimal inoculum, consisting of as few as 10-100 bacteria. A major roadblock in Shigella research is the lack of an in vivo oral infection mouse model that recapitulates key aspects of human disease. Mice resist oral doses of Shigella as high as 100 million bacteria, but the reason for this resistance remains poorly understood. In our preliminary data, we describe our discovery that the reason mice are resistant to Shigella is because of a robust and mouse-specific innate immune inflammasome response in intestinal epithelial cells. Mice lacking inflammasomes are thus susceptible to oral Shigella infection and provide the first opportunity to use the full repertoire of mouse genetic and immunological tools and methodologies to dissect Shigella pathogenesis in a physiological infection model. Importantly, our data suggest that inflammasome-deficient mice are a highly relevant model because, in humans, we find Shigella inhibits or evades the NAIP/NLRC4 inflammasome. We propose three Specific Aims. In Aim 1, we will characterize innate immune and bacterial factors responsible for shigellosis in vivo. In Aim 2, we will characterize the adaptive immune responses of mice to wild-type and mutant Shigella. In, Aim 3, we will test the hypothesis that Shigella encodes effectors to inactivate the human NAIP/NLRC4 inflammasome. By exploiting the experimental tractability of our new model, we hope to identify the key factors mediating immunity and disease during Shigella infection, thereby providing a foundation of knowledge to inform the development of safer and more effective vaccines.

NIH Research Projects · FY 2026 · 2020-09

Project Summary Large biomolecular assemblies constitute much of the molecular machinery necessary for cellular function and are often targeted by therapeutics. Other large molecular assemblies, including virus-like particles, lipid nanoparticles, and some large synthetic particles are used as drug delivery agents. These molecular assembles are large (masses in the range of MDa to GDa) and heterogeneous, making them challenging to accurately analyze using conventional analytical methods. Electrospray ionization can transfer these large complexes intact into the gas phase, but both the high mass and heterogeneity prevents analysis using conventional mass spectrometers due to overlapping and unresolved charge states. Charge detection mass spectrometry (CDMS) weighs individual ions, circumventing interference between charge states and has been used to analyze heterogeneous complexes with masses up to a GDa. However, individual ion measurements can be slow, typically requiring minutes to hours to analyze a sample with a statistically relevant number of ions. A new CDMS instrument with an electrostatic ion trap is proposed that in combination with a new data analysis method, has the potential to increase the speed of analysis of CDMS by up to 25x. This will be done using a unique differential detection method that reduces noise and increases signal while not adversely affecting ion trapping times. Additional gains in performance will be achieved by developing a new method of data analysis to determine the phase of individual ions to make possible absorption mode (as opposed to magnitude mode that is typically used) for the first time in CDMS. By reducing the data acquisition time required to obtain mass spectra to several seconds, new applications will become possible, including combining CDMS with in-line separation methods, rapid measurements of protein aggregation kinetics, variable temperature electrospray ionization, etc. Aerodynamic acceleration currently limits the mass range of trapped ion CDMS to a few GDa. Two new innovations, a reduced pressure electrospray ionization source and a two-stage funnel will extend these measurements to the 100s of GDa mass range (500+ nm particles), making accurate mass measurements possible for a wide range of particles that are challenging to rapidly characterize by other methods. The performance of the instrument will be rigorously tested using well characterized particle standards and developments of this technology will be guided by information learned from a variety of sample types of importance to human health. These experiments include characterizing distributions of mRNA packaging in lipid nanoparticles, antibody binding to adeno-associated viruses for prediction of adverse immune response in gene therapy, and analysis of extracellular vesicles that could lead to new methods of disease detection at early stages.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT: Dengue, Chikungunya, Zika and other mosquito-borne diseases continue to pose a major global health burden through much of the world, despite the widespread distribution of insecticide-based tools and antimalarial drugs. Consequently, there is interest in novel strategies to control these diseases, including the release of genetically sterile male mosquitoes, mosquitoes transfected with Wolbachia, and mosquitoes engineered with gene drive systems. The safety and effectiveness of these strategies and considerations regarding trial design and implementation are critically dependent upon a detailed understanding of mosquito movement at both fine and broad spatial scales, yet there are major gaps in our understanding of these movement patterns. The declining cost of genome sequencing and novel methods for analyzing geocoded genomic data provide opportunities to address these knowledge gaps. In this project, we propose to devise a robust approach for inferring fine-scale mosquito dispersal patterns and their impact on innovative vector control strategies. We propose to use in silico simulations of mosquito ecology and preliminary geocoded mosquito genomic data collected from Fresno, California to determine sampling routines capable of quantifying dispersal patterns, population sizes and mating patterns using genetic kinship analyses (Aim 1). Results from these analyses will iteratively inform sampling schemes for two rounds of subsequent collections of Aedes aegypti, the mosquito vector of dengue, Chikungunya and Zika viruses, in Yishun, Singapore (Aim 2). Genome sequencing and kinship analyses will be used to quantify Ae. aegypti movement patterns, population sizes and mating behaviors at this location, and to parameterize spatially-structured 3D models of Ae. aegypti population dynamics. The resulting models will be used to explore biosafety, trial design and implementation considerations for novel vector control strategies including: i) population suppression systems such as Wolbachia-infected males and genetically sterile males, and ii) population replacement systems such as population transfection with Wolbachia, localized systems such as chromosomal translocations, and non- localized systems such as homing-based gene drive (Aim 3). We expect the proposed research to lead to the development of greatly enhanced surveillance strategies to infer fine-scale mosquito movement patterns and other demographic parameters, and to help inform the safe application of several novel and highly promising strategies for the control of dengue, Chikungunya and Zika viruses and other devastating mosquito-borne diseases.