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NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY In multicellular organisms, development and homeostasis require exquisitely precise, dynamic, and coordinated deployment of complex regulatory programs. As many genes are under the control of vast regulatory regions – with some enhancers located over a million base pairs away from target genes – we and others have explored to what extent the 3D organization of the genome facilitates the establishment of specific regulatory interactions. Additionally, regulatory regions are pervasively transcribed to generate a striking diversity of long noncoding (lnc)RNAs, which are emerging as important chromatin and transcriptional regulators. However, the molecular determinants and regulatory roles of genome organization remain unclear, and only a minute fraction of known lncRNAs have been ascribed any function. My recent work used innovative single-cell live imaging approaches to show that genome organization and cis-regulatory lncRNAs both play a key role in regulating the temporal dynamics of gene expression. My findings also suggest that lncRNAs may exploit – or even regulate – genome organization to exert long-range transcriptional control. The goal of my research is to understand how genome organization and lncRNAs regulate gene expression, and to investigate their mutual interplay. My lab will use our cutting-edge approaches to measure transcriptional dynamics in Drosophila embryos to investigate how long-range interactions are established; how lncRNAs operate in the context of long-range regulation and utilize genome organization; and how in turn lncRNAs can control genome organization. In my prior work, I identified novel “tethering elements” that foster specific long-range interactions to enable fast gene activation during development. In Project I, I will investigate the role of a well-known epigenetic repressor, Polycomb Repressive Complex 1, in facilitating transcriptional activation by mediating long-range interactions between tethering elements. Through a combination of genomics and live imaging, I will identify the molecular mechanisms establishing long-range interactions and determine their impact on gene expression. My recent work also showed that both long-range enhancers and tethering elements are pervasively transcribed genome- wide. In Project II, using methods I developed to visualize the transcription of endogenous lncRNAs in living embryos in combination with complex genetic perturbations, I will begin to determine the regulatory functions of select lncRNAs associated with important developmental genes and dissect their interplay with genome organization. In particular, I will investigate how lncRNAs regulate the recruitment of architectural factors and the establishment of long-range interactions. Finally, in Project III, I will use novel single-cell live imaging approaches that I developed to investigate how lncRNAs dynamically control local regulatory microenvironments to regulate genome organization and gene expression in vivo. Collectively, these three synergistic projects will enable my future research program by identifying guiding principles that will inform our understanding of the dynamics of gene regulation and be examined in future studies in other model systems and cellular and temporal contexts.

Economic Development Initiatives 16-17

Dynamic Systems Working Capital

U.S. National Science Foundation

Dynamics, Control and Systems Diagnostics

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Immune-mediated protection against malaria, termed clinical immunity, develops over many years of repeated exposure to Plasmodium, requires continuous exposure to Plasmodium, and correlates with circulating titers of Plasmodium-specific antibodies. Why Plasmodium infections fail to induce long-lasting protection following one or few infections, thereby contributing to the chronic pathogenesis of malaria, remains unknown. The objective of this proposal is to determine the effect of Plasmodium infections on germinal center (GC) B cells and bone marrow (BM) homeostasis towards inefficient generation of long-lived plasma cells (LLPCs). Many infections and vaccines elicit long-lasting antibody titers after one or just a few exposures. The half-lives of circulating antibody titers for viral antigens following infection or vaccination have been shown to range from 10 years to >300 years. In contrast, half-lives of circulating Plasmodium-specific antibody titers are reported to be in the range of several days. Long-term maintenance of circulating antibody titers comes from GC-derived plasma cells (PCs) that migrate to the BM where they receive survival signals and become LLPCs. To investigate why Plasmodium infection induces short antibody half-lives, we compared spleen GC responses and development of BM LLPCs in mice following infection with Plasmodium yoelii 17XNL (Py) to mice immunized systemically with NP-CGG plus the adjuvant AddaVax. Our preliminary data demonstrate that despite the induction of robust spleen GC responses during Py infection, Py-induced GC B cells are inefficient at generating LLPCs in the spleen and BM and that Py-specific IgG-secreting cells in the spleen and BM exhibit decreased IgG production and affinity. These data suggest Py infections exhibit dysregulated humoral immunity by impacting the functional programming of spleen GC B cells or PCs. Py-induced splenic PCs were able to migrate towards the BM homing cytokine CXCL12, suggesting inefficient generation of LLPCs following Py infection could be attributed to functional deficiencies in the BM microenvironment that support engraftment and survival of LLPCs. Consistent with this possibility, there were decreases in the number of BM cells that provide recruitment signals for PCs and survival signals for LLPCs during Py infection that was associated with an increase in multiple cytokines in the BM, including IFN-γ. Blocking IFN-γR signaling during Py infection partially prevented the loss of BM cells and increased BM PC numbers. These observations collectively lead to the hypothesis that failure to engender LLPCs following Plasmodium infection is attributed to intrinsic differences in spleen GC B cell programming that affect PC functionality and changes in the BM microenvironment that fail to support LLPCs. The hypothesis will be tested through the following aims. Aim 1. Identify the functional deficits in spleen GC B cells and PCs following Py infection responsible for inefficient generation and function of LLPCs. Aim 2. Define the cellular and molecular mechanisms by which Py alters the BM microenvironment to impede the development of LLPCs.

Easton

E-Citation PD RMS System

National Institutes of Health

Early Immune System Development and Ontogeny (R01 Clinical Trial Optional)

The East Ballard Community Association

On May 20, 2017 from 10am to 2pm the East Ballard Community Association , along with Urban Systems Design, and neighborhood residents are coming together to restore the rain garden that is located at NW 58th and 14th Ave NW. This project will provide a great opportunity to improve the urban landscape, and learn about rain gardens to the East Ballard neighborhood residents.

Waterbury

East Mountain Golf Course Irrigation System Upgrades and Repairs

United Community Centers, Inc.

Development of sustainable systems and community education programs for two urban farms and a farmers' market.

U.S. National Science Foundation

ECosystem for Leading Innovation in Plasma Science and Engineering

National Institutes of Health

Effectiveness Trials to Test Mental Health System Interventions (R61/R33 Clinical Trial Required)

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY More than 38 million people are living with Human Immunodeficiency Virus (HIV) infection worldwide which continues to be a global public health problem. Despite the widespread use of efficacious combination of antiretroviral therapy (cART), up to 70% of HIV-positive individuals suffer from cognitive and behavioral deficits collectively known as HIV associated neurocognitive disorders (HAND). Although acute viral replication has been well controlled with cART in the early stage of HIV infection, long-term HIV-1 viral protein exposure within the CNS causes dopaminergic deficits and neurocognitive impairments despite the advent of cART. Substance abuse such as methamphetamine (METH) have been shown to increase the incidence of HAND and exacerbate its severity. Among the viral proteins, HIV-1 transactivator of transcription (Tat) protein plays a significant role in viral replication in the early stage of HIV infection and the pathophysiological effects on development of HAND. Dopamine (DA) transporter (DAT) transports the extracellular dopamine (DA) into cytosolic space of the synaptic terminals, whereas the vesicular monoamine transporter2 (VMAT2) transports the cytosolic DA into synaptic vesicles, whereby both DA transporter and VMAT2 are critical for normal DA homeostasis. Our published studies have demonstrated that Tat protein increases extracellular DA concentration by directly inhibiting DAT, however, the cellular mechanisms underlying Tat-induced inhibition of VMAT2-mediated DA release/uptake remains unexplored. We hypothesize that HIV-1 Tat, acting via the unique binding sites, perturbs the VMAT2 regulatory network that normally sustains concentrative DA transport and potentiates METH’s effect on VMAT2 function, resulting in DA-linked neuropsychiatric dysfunction prominently featured in HAND. Studying single Tat protein allow us to identify targets for Tat binding and develop therapeutic approaches to prevent Tat-mediated neurological damages. The purpose of the proposed investigation is to understand the molecular mechanisms of dysregulation of VMAT2-mediated DA transmission induced by HIV-1 Tat and METH and explore the potential of novel compounds for the prevention of HAND. The specific aims to be pursued in the proposed investigation are: (1) through computational modeling and experimental validation, identify the recognition binding pockets on human VMAT2 for Tat, METH, or novel VMAT2 inhibitors and explore the potential interactions of the inhibitors with Tat and METH, (2) accelerate the pathophysiological roles of VMAT2 in Tat- and METH-dysregulated DA system, and (3) perform proof-of-concept studies using pharmacological and genetic approaches as biological probes to establish their potential for therapeutic application in HAND in concurrent METH users.

El Centro de la Raza

The El Centro de la Raza(ECDLR) located at 2524 16th Ave S will replace the outdated boiler control system, the thermostats and the fan motor system. These improvements will provide the community with updated facilities for community gatherings and event activities.

Association of Beloved Communties

Association of Beloved Communities will capture the history of Seattle's El Centro in a fifteen minute film. It will share the inspiring story of the formation and legacy of this iconic institution, where a small nonviolent movement won a victory for justice. The video will be shot in summer 2019 and final product be distributed through media partners and select events focused on inclusive ecosystem-building strategies.

U.S. National Science Foundation

Electrochemical Systems

TYLER TECHNOLOGIES INC

Information Technology Broadcasting and Telecommunications

Union Settlement Association, Inc.

Implementation of a modern State of the Art EMR system for their Mental Health program. This system will be shared by all clinic staff, through internet or a proprietyary network, allowing centralized, accessible, secure record- keeping and collaboration

Track Group Inc.

Engineering and Research and Technology Based Services

KNOWINK LLC

Distribution and Conditioning Systems and Equipment and Components

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary / Abstract This is a R01 application from Dr. Daniel Lingwood (PI) an Associate Professor of Medicine at the Ragon Institute of MGH, MIT and Harvard and Dr. Batista (co-PI), a Professor of Medicine at the same institution. These investigators define immunological decision making by B cells to inform vaccine design and have built humanized mouse systems that recapitulate human antibody responses. Dr. Andrew Ward (co-I) is Professor in the Department of Structural and Computational Biology at Scripps and specializes in high resolution cryoEM of antibody:antigen complexes. The application goal is universal influenza vaccine development, centered on their discovery of a human broadly neutralizing antibody (bnAb) pathway that the investigators can vaccine-elicit to protect against all influenza A viruses (IAV), the major source influenza disease and all pandemic events. Differences in N-glycosylation generally prevents antibodies from engaging the otherwise conserved hemagglutinin (HA) stem of group 1 versus group 2 IAV. To solve this issue, the investigators engineered nanoparticle immunogens that elicit cross-group IAV immunity by selectively triggering and maturing germline B cell receptors (BCRs) encoding VH1-18 QxxV class bnAbs, a rare but genetically reproducible or ‘public’ category of human pan-IAV bnAbs that accommodates N-glycan diversity on the HA stem. The investigators successfully elicit this cross-group bnAb response using a single shot within a humanized vaccine model containing the VH1-18 QxxV bnAb precursors at physiologically relevant human frequency within the naïve B cell pool. The immunogens select for key affinity enhancing mutations, including N55T in the CDRH2, a hallmark of VH1-18 QxxV bnAbs. The investigators show that N55T alone provides cross-group IAV protection by a novel antibody tilting mechanism that accommodates N-glycan diversity on the HA stem. The investigators will now test the central hypothesis that this ‘molecular switch’ endows humans with an exceptionally simple vaccine- expandable pathway for eliciting broad spectrum IAV immunity. In Aim 1, the investigators will apply their modular human vaccine model to define the number of naive VH1-18 QxxV B cells needed for pan-IAV vaccine protection; if these bnAb precursors are absent, the germline stimulating nanoparticles no longer elicit pan-IAV bnAbs, revealing a human B cell repertoire effect encoding for unprecedently broad IAV immunity. In Aim 2, the investigators will define whether their nanoparticle immunogens can co-expand multiple classes of cross-group IAV bnAbs within their human vaccine model. Critically, their engineered nanoparticle immunogens also bear germline stimulating affinity for the naïve BCRs encoding the other known classes of genetically reproducible pan-IAV bnAbs produced by humans, potentiating multiclass bnAb elicitation via pan-germline stimulation. In the Aim 3, the investigators will define how prior exposure to IAV modulates (and enhances) germline stimulation and vaccine elicitation of pan-IAV bnAbs via imprinting effects. Collectively, this proposal will exploit novel genetically hardcoded templates for eliciting cross-group IAV immunity in humans.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract. The Bacillus Calmette-Guerin (BCG) vaccine has been in use for over a century and is currently administered in more than 200 countries to protect against tuberculosis (TB). Despite this, TB remains one of the leading causes of death due to an infectious disease. Efforts to improve or enhance BCG has been met with limited success in part due to the lack of an immune correlate that is associated with vaccine-induced protection in the human population. Although it is unclear why BCG remains ineffective against adult pulmonary TB, traditional animal models such as the C57BL/6 (B6) mice display a relatively homogenous Th1 response following BCG vaccination and fail to replicate the heterogeneous clinical response observed in human populations. Using the Collaborative Cross (CC) mice, we have demonstrated that host genetics is a critical determinant of both primary susceptibility and BCG-induced protection against Mtb, with the hope of using the CC model to identify immune signatures associated with protection that is not observed in the traditional B6 mice. Within several CC strains, we have identified a hybrid-Th1/17 population that is correlated with BCG-induced protection against Mtb, suggesting that this is a signal that is associated with vaccine- induced protection across multiple host genotypes. Using CC037, which is the strain that is the most protected against Mtb following BCG vaccination and has the strongest hybrid-Th1/17 signal, we will ask how BCG vaccination modifies host immunity in CC037 to generate this population in response to Mtb infection. As dendritic cells (DC) are considered the primary cell type that bridges the innate and adaptive immune systems, our overarching hypothesis is that modification of the DC compartment by BCG is leading to the generation of hybrid-Th1/17 response in CC037 mice. We will ask whether the ability to generate a hybrid-Th1/17 response is inherent to the host T cells or is due to certain signals produced by DC in response to BCG vaccination. We will also use a combination of scRNA-Seq and ATAC-Seq to examine how BCG modifies host T cell and DC responses to Mtb in CC037, from both a transcriptional and epigenetic approach. CC037 represents an important tool for understanding how hybrid-Th1/17 responses are generated and how they contribute to protective immunity against Mtb. This study aligns with the NIH NOT-AI-24-054, where we expect our study to identify novel mechanisms that will lead to the induction of this response and enhance TB vaccine development in the context of diverse genetic backgrounds.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY / ABSTRACT This proposal, “Elucidating biomolecular dynamics using magnetic resonance augmented by machine learning and quantum technologies” addresses the need for experimental methods to probe the dynamics of large biomolecules in near-physiological conditions in a straightforward and rapid manner. Indeed, many post- translational modifications employed by cells to regulate fundamental biological processes remain poorly understood. Magnetic resonance techniques are well-suited to elucidate these mechanisms, but their application to large biomolecules is limited by sensitivity and resolution constraints. Dr. Seetharam proposes a two-pronged approach to overcome these limitations: (i) develop a novel analysis workflow for magnetic resonance experiments that can extract information from data uninterpretable by humans, and (ii) develop protocols for a novel nanoscale magnetic resonance platform that can operate at high molecular weights and low concentrations. This approach will be conducted through three specific aims that leverage machine learning, quantum computing, and quantum sensing methods. Successful completion of these aims will greatly expand the application of magnetic resonance techniques by enabling an interpretability to information density trade-off in protocol design, bypassing the molecular weight bottleneck, and circumventing the sensitivity limitation, thereby opening a high-resolution window into biomolecular dynamics in near-physiological conditions. Dr. Seetharam is uniquely positioned to perform the proposed work given his interdisciplinary background bridging research and policy, physics and engineering, theory and experiment. He is currently a postdoctoral researcher under the mentorship of Prof. Mikhail Lukin, a pioneer in quantum computing and nanoscale quantum sensing, at Harvard University and Prof. Haribabu Arthanari, a leading expert in biomolecular magnetic resonance, at the Dana-Farber Cancer Institute (DFCI). The proposed research will be conducted at Harvard University and DFCI with support from an exceptional scientific advisory committee experienced in machine learning for magnetic resonance, nanoscale magnetic resonance, single-molecule biophysics, biochemistry, cell biology, and systems biology. This K25 award will provide Dr. Seetharam with the requisite training in machine learning and biological techniques needed to successfully transition to an independent career in magnetic resonance while simultaneously addressing an unmet need in the study of biological processes.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Hepatitis B virus (HBV) chronically infects more than 250 million people and puts them at risk of developing liver cirrhosis and hepatocellular carcinoma. Every year over 800,000 people die due to HBV-related complications. The natural history of chronic HBV infection goes through several phases characterized by the presence of HBV e antigen (eAg) in serum. Most chronically infected individuals are initially eAg positive and later in life become eAg negative, which is associated with enrichment of viral mutations in the basal core promoter (BCP) and precore (PC) regions. Some individuals spontaneously control HBV and lose surface antigen, termed a functional cure. There are two approved types of medications for chronic HBV infection, nucleoside analogs (NA) and interferon-alpha (IFNα). While NA are well-tolerated, work across genotypes and irrespective of eAg status, they rarely lead to a functional cure. By contrast, IFNα therapy can functionally cure a subset (<10%) of patients but comes with significant adverse effects. It has long been appreciated that IFNα responses differ by eAg status and by HBV genotype. eAg positive patients are cured at a higher rate than eAg negative patients, and HBV genotype A (HBV-A) is more susceptible to IFNα than HBV-D. In a recent North American trial studying the effects of NA withdrawal after four years of therapy a small number of eAg positive HBV subgenotype A2 (HBV- A2) patients responded very well, with 67% functional cure rate in the monotherapy group and 100% cure in the NA + IFNα group. This contrasted with HBV-A2 eAg negative and HBV-A1 or HBV-D patients, none of whom where cured. These results suggest that eAg positive HBV-A2 is highly susceptible to currently approved therapies and offers an opportunity to understand the mechanisms by which IFNα leads to a cure. IFNα has broad antiviral mechanisms and can induce hundreds of human interferon-stimulated genes, depending on the pathogen and cell type. The antiviral mechanisms of IFNα against HBV are poorly understood. They involve the induction of interferon-stimulated genes in hepatocytes, the liver cell type infected by HBV, as well as effects on immune cells. We here aim to use eAg positive HBV-A2 to understand the antiviral mechanisms of IFNα in human hepatocytes. Our recent advances in launching HBV infection from recombinant DNA and our primary human hepatocyte cell cultures and animal models can now be exploited and combined with multiomics approaches and novel CRISPR validation tools. These systems will be used to investigate the antiviral mechanism of an interferon-stimulated host gene against HBV-A2 and other genotypes, map regions of patient- derived HBV-A2 genomes conferring susceptibilty to IFNα, and compare IFNα responses between isogenic wild type and PC and BCP region HBV-A2 mutants. In all, these studies combine expertise from multiple fields with the goal of advancing insights into the antiviral mechanisms of IFNα, which may aid in the development of more effective therapies against chronic HBV.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary The somatosensory system has evolved to detect, process, and relay information from the body’s surfaces into the central nervous system. At the sensory periphery, each of a diverse repertoire of somatosensory neuron types housed in the dorsal root ganglia (DRG) expresses one or more of a small collection of sensory receptors that transduce mechanical, thermal, and/or chemical stimuli. While these cell types, the sensory receptors they express, and even the physiological responsivity of many cell types are relatively well-characterized in mice, it remains unclear whether other mammals exhibit the same cell-type-specific sensory receptor gene expression patterns and cell-type-specific physiology. My preliminary data indicate that even closely related species such as the mouse and rat frequently exhibit notably different cell-type-specific patterns of sensory receptor gene expression. Furthermore, some DRG neuron types have remained genetically inaccessible, even in mice, and thus have remained anatomically and physiologically uncharacterized. The proposed work will leverage single-nucleus RNA- and ATAC-sequencing datasets, viral engineering, and modern neuroanatomical and neurophysiological approaches to investigate the diversity, evolution, and function of DRG somatosensory neuron repertoires across different mammals. In Aim 1, I will describe interspecies differences in cell-type- specific sensory receptor gene expression patterns across mammals ranging from the widely used laboratory mouse to niche non-genetic model organisms such as the naked mole rat. In Aim 2, I will mine my single-cell ATAC-seq datasets to find enhancers that allow genetic access to specific DRG neuron types to develop new viral-genetic tools to genetically access and describe the anatomy, physiology, and function of DRG neuron types previously genetically inaccessible in mice; I will also extend these viral-genetic tools to describe the anatomy, physiology, and function of conserved DRG cell types that elicit pain and itch in rats and test whether the divergent patterns of sensory receptor gene expression indeed affect the function of these cellular orthotypes. These proposed experiments will be paired with a scientific training plan in bioinformatics and genomic analysis, enhancer- virus engineering, somatosensory neuron physiology, and animal behavior, as well as a career development plan preparing me to become an independent researcher. Technical and conceptual guidance on designing and performing bioinformatic analyses and physiological and behavioral experiments will be provided by the postdoctoral advisory committee I have assembled, including Drs. Gordon Fishell, Stephen Liberles, and Guoping Feng. My mentors, Drs. David Ginty and Michael Greenberg, will supervise the mentored phase of the award through regular meetings and ensure that my training fully prepares me to transition to an independent position. Together, the proposed research and training plan will enable me to make important discoveries concerning the diversity, evolution, and function of mammalian somatosensory neuron repertoires and prepare me to lead an independent research group addressing outstanding questions in somatosensory neuron evolution and function.