University Of California Riverside

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Development and reproduction of insects, including human disease vectors such as mosquitoes, are mainly controlled by two lipophilic hormones: ecdysone and juvenile hormone (JH). Although these hormones need to enter their target cells to exert their biological effects, almost nothing is known regarding molecular mechanisms that regulate hormone transport across cellular membranes. This is due to the prevailing dogma in endocrinology that lipophilic hormones enter and exit cells by simple diffusion across lipid bilayers. However, despite this dominant assumption, the simple diffusion model of lipophilic hormone transport is not supported by any conclusive evidence in any organism. Indeed, recent studies now suggest that some lipophilic hormones, including the insect steroid hormone ecdysone, require membrane transporter proteins to travel across plasma membranes. The overall objective of this project is to identify and characterize membrane transporters required for JH trafficking across cellular membranes, and to thereby challenge the conventional paradigm that lipophilic hormones freely enter and exit cells by simple diffusion. The approach will combine in vitro and in vivo approaches to characterize JH Transporter (JHT), which was discovered in preliminary studies using the fruit fly model system. In Aim 1, functions of the JHT ortholog in the yellow fever mosquito Aedes aegypti, the primary vector for Zika, yellow fever, chikungunya, and dengue viruses, will be thoroughly investigated in vitro using an arsenal of molecular genetic tools. In Aim 2, JHT functions will be further studied genetically in Aedes. As JH controls both growth and reproduction in Aedes and other mosquitoes, characterization of Aedes JHT is expected to aid our effort to combat these deadliest disease vectors for humans. Indeed, in Aim 3, in vitro chemical screening will be conducted to identify compounds that can inhibit functions of Aedes JHT, and their effects will be tested in vivo. The significance of this project is therefore not just to overturn the long-standing dogma in endocrinology, but also to provide a critical proof of concept as well as seed compounds for developing novel pharmacological tools to control mosquitoes and other deadly disease vector insects.

NIH Research Projects · FY 2026 · 2022-12

Project Summary Antipsychotic therapy is widely used in the treatment of psychiatric conditions including bipolar disorder, schizophrenia, and major depressive disorder. These conditions, which together affect more than 20% of the population, usually require lifelong medication. Atypical antipsychotics have superior therapeutic action and reduced adverse effects as compared with typical antipsychotics, but the use of atypical antipsychotics is also associated with dyslipidemia and an increased risk of cardiovascular disease (CVD) in patients. The underlying mechanisms responsible for these adverse effects remain largely unknown, which poses serious health challenges to patients undergoing long-term antipsychotic treatment. To this end, we recently identified several atypical antipsychotics including quetiapine that promote dyslipidemia, as potent agonists for the nuclear receptor pregnane X receptor (PXR). Our previous work revealed novel and unsuspected roles of PXR in lipid homeostasis and atherogenesis, and showed that PXR ligands increase dyslipidemia and atherosclerosis in atherogenic mouse models including PXR-humanized mice. Given intestine and lymphatic systems are essential for dietary lipid absorption and transport, our latest preliminary study using novel tissue-specific PXR knockout mouse models demonstrated that exposure to quetiapine fails to cause hyperlipidemia in intestine- specific PXR knockout mice. How PXR signaling in enterocytes regulates the intestinal lipid metabolism is an open and highly clinically relevant question. Furthermore, our pilot study revealed that ablation of PXR blunts VEGF receptor 3 signaling in lymphatic endothelial cells and reduces lymphatic button junction formation in lacteals of PXR-deficient mice. It is completely unknow how lymphatic PXR regulates lipid absorption and transport by gut lymphatic vessels. To unveil the aforementioned central mystery and to study the action mode of PXR in mediating antipsychotic-elicited adverse effects on lipid homeostasis and atherosclerosis, we propose the following specific aims to determine the molecular mechanisms of the atherogenic effects of atypical antipsychotics: 1) Define the enterocyte signaling through which PXR-activating antipsychotics regulate lipid homeostasis and atherosclerosis; 2) Determine the molecular mechanisms underlying PXR- regulated lymphatic lipid absorption and transport in atherosclerosis; and 3) Investigate the therapeutic potential of a naturally occurring PXR antagonist in preventing antipsychotic-induced dyslipidemia and atherosclerosis. Successful completion of the proposed work will fill in the void in uncovering novel molecular mechanisms underlying antipsychotic therapy-associated CVD risk. Our findings may also inaugurate new class of therapeutic strategies to treat dyslipidemia in patients undergoing long-term antipsychotic therapy.

NIH Research Projects · FY 2024 · 2022-09

Given the chronic and pervasive impairments associated with Attention Deficit Hyperactivity Disorder (ADHD), high rates of comorbidity with other mental health disorders, and heightened problems in child social interactions and relationships, these children remain at risk for poor outcomes despite readily available medical treatments. Moreover, the COVID-19 pandemic, which led to disruption in receipt of some services – particularly educational supports – may lead to increased mental health problems among children with ADHD and increased stress and conflict in their families, as they cope with increases in behavioral problems and the loss of social and institutional supports. Technology-enabled interventions can potentially fill this gap, but the availability of such interventions is limited, and research evidence is scarce. Effective treatment requires communication and collaboration between patients, providers, and caregivers at multiple points of care including clinics, home, and school. Current approaches to information-sharing depend on subjective recall, on-the-fly conversations, phone calls, and a variety of messaging applications. This often results in a lack of reliable and valid information sharing, a less targeted and effective treatment approach, and delays in initiation or titration of treatment or other needed interventions. Moreover, monitoring symptoms and adhering to treatment recommendations requires considerable self-regulation in children and parents; self-regulation is impaired in children with ADHD, and the multiple stressors associated with ADHD may challenge parent self-regulation. To address these critical barriers to progress, our DHI uses Patient-Centered Digital Healthcare Technologies to promote self-regulation (child/parent), capture patient data, support efficient healthcare delivery by improving communication and access to reliable data, and facilitate shared decision-making. In the proposed innovative and developmental work (R21), we will work with stakeholders to identify, refine, and add features to our prototype to support multiple points of care. This participatory design work will inform further development of the current system with additional design features that will: 1) reinforce mental health intervention, 2) address adherence to treatment for children with ADHD and their caregivers, 3) use sensors, self-reports, and caregiver reports to capture and create visualizations of daily health behaviors and symptoms, and 4) provide reporting options to facilitate communication, shared decision-making, and tracking of progress over time. In the subsequent phase (R33), we will conduct a randomized clinical trial (RCT) to evaluate the impact of the DHI on patient, parent/caregiver, and provider experiences and outcomes. We expect that this system will integrate treatment across multiple points of care and will enable health care providers, caregivers, and children to share reliable and targeted information that will facilitate collaborative decision-making, which in turn will improve patient experiences and outcomes, particularly among children at high risk for poor outcomes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Subarachnoid hemorrhage (SAH) is a devasting acute neurological disease that remains a major cause of premature mortality. SAH is most caused by incidental rupture of an intracranial aneurysm (ICA). The mortality rate of aneurysm rupture can reach as high as 40% within the first week of incidence. Even if the aneurysm is treated in a timely manner, the chance of moderate to severe brain damage is 20-35%. Endovascular coil embolization is the current gold-standard, minimally invasive therapy of ICAs; however, emerging clinical challenges of coil embolization are unsatisfactory aneurysm recurrence rates: ~44% by 5-6 years after the initial coil therapy (of which more than 50% requiring re-treatment), and suboptimal complete occlusion, especially for treating wide-necked ICAs and/or aneurysms with a complex 3D geometry. Thus, there is a need for a durable device to treat unruptured ICAs that targets patient-specific aneurysms and intra-aneurysmal circulation and provides long-lasting complete occlusion. Our research objectives of this project are to: 1) design and fabricate personalized embolic devices for treating saccular, bifurcated IACs using additive manufacturing and a combined experimental/biomechanical approach, and 2) provide a holistic biomechanical and hemodynamic comparison between our device and other selected endovascular embolic techniques. This proposal builds upon the assembled preliminary data, and leverages Dr. Lee’s experience with tissue biomechanics and in-silico modeling, in collaboration with polymer science and additive manufacturing researchers at the University of Oklahoma, clinical and neurosurgical expertise of clinicians at Indiana University – Medicine, and micro-device and catheter expert at Purdue. Specifically, we propose to design, develop, and evaluate patient-specific SMP embolic devices using 3D printing-based polymer fabrication. Our embolic devices are designated to target personalized aneurysm filling and maximize the rate of long-lasting complete occlusion. Next, through in-vitro flow loop testbed and in-vivo small animal studies, the efficacy and aneurysm occlusion of our personalized embolic devices will be systematically evaluated in comparison to the clinical gold standard as well as three other contemporary embolic methods. The endpoint of this project will be a cutting-edge solution for ICA embolization, that uses fundamental information on aneurysms based on holistic biomechanical and hemodynamic analyses – allowing individual-optimized aneurysm filling to achieve immediate & long-term complete occlusion and reduce aneurysm recurrence. Collectively, our developments will serve as a logical first step toward attaining our long- term goal to advance the state of the art in translational medicine by facilitating personalized, preventive management of unruptured ICAs and reduce aneurysm rupture-induced hemorrhagic strokes.

NIH Research Projects · FY 2025 · 2022-08

The University of California Riverside (UCR) and Riverside City College (RCC) propose to create a Bridges to the Baccalaureate (B2B) research education program to facilitate transfer of community college students into university biomedical and behavioral science majors, with the ultimate goal of increasing participation of underrepresented in medicine and science (URiMS) groups in research-oriented careers in these areas. Many URiMS students have less access to high quality science instruction, resources and enriching opportunities at all stages of public education. African-Americans, Chicano/Latinos, Native Americans, Hawaiian and Alaskan Natives, Pacific Islanders or socio-economically disadvantaged students make up a small percentage of science teachers in K-12 as well as higher education, and are even more underrepresented in biomedical and behavioral science fields requiring post-graduate degrees. The proposed B2B program will identify community college students at RCC committed to pursuing a career in science and facilitate their transfer and successful completion of Baccalaureate degrees in science, technology, engineering, and math (STEM) majors. The program contains interventions and support services designed to help B2B scholars achieve graduation in a normative two-year window following matriculation at UCR and to address the causes of URiMS attrition in STEM programs. Specific program components include: compensated research experiences to stimulate greater awareness of career options in the biomedical and behavioral sciences and to reduce the necessity of working in non-academic jobs; peer and faculty mentoring that begins before RCC/B2B scholars transfer to UCR; and a summer boot camp to familiarize incoming students with UCR’s course structure and student support services. By creating a program that bridges the community college and university education and provides enhanced preparation before and mentoring after transfer, we believe we can overcome these disadvantages and improve the success and persistence of URiMS transfer students in biomedical and behavioral sciences.

NIH Research Projects · FY 2025 · 2022-08

My group is working to develop NMR-assisted crystallography – the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry – as an atomic-resolution probe of enzyme active sites, capable of defining the position of all atoms, including hydrogens. By locating hydrogen atoms, this technique provides the often critical missing chemical information necessary to link structure and mechanism, as well as providing crucial information for the rational design of therapeutics. The approach is three-fold: X-ray crystallography is used to provide a coarse structural framework upon which chemically-detailed models of the active site are built using computational chemistry, and various active site chemistries explored; these models can be quantitatively distinguished by comparing their predicted NMR chemical shifts with the results from solid-state NMR experiments. Provided a sufficient number of chemical shift restraints are measured within the active site, NMR-assisted crystallography can uniquely identify the structure. The targeted systems include pyridoxal-5’-phosphate (PLP)-dependent enzymes, which have been implicated in numerous health conditions and as targets for treating diseases, and the β-Lactamases, which mediate antibiotic resistance to β-lactam antibiotics. The family of PLP-dependent enzymes are involved in the metabolism of amino acids and other amine- containing biomolecules. This single cofactor can participate in a diverse array of chemical transformations, including racemization, transamination, α/β-decarboxylation, and α/β/γ- elimination and substitution. Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of this proposal. To accomplish this understanding, NMR-assisted crystallography is employed to characterize these enzymatic transformations with atomic resolution. In tryptophan synthase, this allows us to peer along the reaction coordinates into and out of multiple intermediates. Here the protonation states complete the chemical picture for why, for example, specific inhibitors such as benzimidazole are unable to react to form a covalent bond as it is held in the wrong orientation by hydrogen bonds to βGlu109 and the charged ε-amino group of βLys87. A second goal is to extend the successes in characterizing enzymatic transformations in PLP-dependent enzymes to the β-lactamases, starting with the Toho-1 β-lactamase. Here we build on our initial chemical shift assignments and characterization of dynamics in solution to study the chemical mechanism used to inhibit antibiotics. In this application, NMR-assisted crystallography will be developed at the interface with neutron crystallography, which to date has been unable to solve the structure in the presence of an inhibitor, but where understanding the mechanism at the chemical level requires that we assign the protonation states of the key active site acid/base catalytic residues.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract: Background. Sepsis, a severe and life-threatening condition, is one of the most common causes of death in hospitalized patients. Sepsis is generally caused by bacterial infection, including both Gram-negative and positive bacteria. In the United States, the hospital mortality rate of patients with sepsis could be as high as 41.1%, which accounts for more than 250,000 deaths and $20 billion loss annually. Due to the inadequate sensitivity and specificity of the current technologies, there is no global standard for sepsis diagnosis. In this project, the PI has the ambition to address the critical bottlenecks specifically of concern in sepsis testing using: 1) hybrid bio-inorganic nanobots, 2) CRISPR-based devices, and 3) CRISPR-equipped engineered phages. Goals for the next five years. Our first goal is to engineer phage M13 with nanobodies on the capsid protein pVIII and his-tags on the tail fiber protein pIII. After binding cobalt-coated magnetic nanoparticles, the resulting hybrid bio-inorganic nanobots will be used to concentrate and purify pathogens from blood samples. Capture efficiency will be investigated using spiked samples and then proceed to clinical ones. Taking advantage of CRISPR and microfluidic technologies, the second goal is to fabricate portable devices to detect sepsis-related pathogens, which can be used in resource-limited settings. The last goal is to engineer phages with different CRISPR systems, that can be used to detect and combat sepsis-related bacterial pathogens. Towards the end of the fifth year, we will have integrated these technologies as a robust tool for sepsis diagnosis. Overall vision of the research program. The technologies we are developing will have a broad impact on the biomedical research communities to detect and treat sepsis, even for other diseases. Our developed technologies can also advance pathogen detection in other fields, such as food safety and environmental monitoring.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Defects in Hedgehog (Hh) signaling is widely implicated in various birth defects and cancers. The transduction of Hh signaling relies on the primary cilium, a miniature cell surface organelle known as the antenna of the cell. To activate Hh signaling, almost all protein transducers need to transit through the cilium. However, the molecular mechanisms of these protein transportation are not completely understood; and the signaling cascade within the cilium also remains unclear in the field. Our goal is to fill this gap with mechanistic studies of novel cilium proteins in the regulation of Hh signaling in vitro and in vivo. In preliminary studies, we have leveraged a new proximity-labeling tool and built an experimental platform to discovery new signaling proteins in the cilium with quantitative proteomics. We discovered surprising cilium localization of a molecule involved in receptor internalization during neural development. In this proposal, we aim to 1) decipher how the new cilium molecule control protein transport in the cilium during Hh signal activation; 2) determine its role in the Hh-controlled neural progenitor proliferation in the developing brain; and 3) apply the proximity biotinylation approach to identify new proteins involved in Smo trafficking and signaling during the time course of Hh activation. Our approach is innovative because it will reveal a new function of an old protein in the cilium during Hh transduction, and it employs a newly developed biotinylation tool to gain the systematic view of signaling proteins during Hh signaling activation. Upon completion, our study will shed light on long standing questions in the Hh pathway and may highlight new methods for the intervention of Hh related developmental disorders.

NIH Research Projects · FY 2026 · 2022-01

SUMMARY Cellular morphology is one of the most distinctive features of somatic cells in multicellular organisms and is intimately linked with cellular function. How neurons and other polarized cells commit to their morphologies is poorly understood, but spontaneous morphogenesis of dissociated cells in culture suggest that the basic instructions for morphology are often intrinsically encoded. We previously identified a sequence-specific RNA- binding protein, Unkempt, as a factor that is essential for the establishment of the early neuronal morphology and as a protein that is capable of endowing a similar shape to cells of nonneuronal origin. Unkempt recognizes a unique binding motif predominantly within coding regions mRNAs the translation of which it suppresses. It is unclear how Unkempt regulates translation, and whether translation or another functional modality of Unkempt is critical to its induction of cell polarization. Here, we seek to solve this problem by deciphering the mechanistic basis of Unkempt-driven remodeling of cellular shape. Our preliminary studies indicate that in the broad protein-protein interaction network of Unkempt, the interaction between Unkempt’s low-complexity domain (LCD) and the CCR4-NOT complex is exquisitely required for the induction of cell morphogenesis. We propose three specific aims to investigate the molecular and cellular roles of the CCR4- NOT complex as a critical effector of Unkempt-controlled cell morphogenesis. First, we will investigate the nature of Unkempt – CCR4-NOT interactions and their impact on the fate of the targeted messages, focusing in particular on their poly(A) tail length, stability, and translation. Second, we will interrogate the recruitment and function of CCR4-NOT in Unkempt-induced cell morphogenesis. Third, we will determine the effect of post-translational modifications of Unkempt on its interactions with the CCR4-NOT complex and RNA, as well as its control of local protein translation. This study will shed light on the molecular underpinnings of the early neuronal morphogenesis and contribute to our general understanding of the cues that control cellular morphology in development and disease.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Mutations in key factors of nonsense-mediated mRNA decay (NMD), including Upf2, Upf3a, Upf3b, and Smg6, are enriched in various neurodevelopmental diseases. In additional to ensuring transcript quality by degrading aberrant transcripts with a premature stop codon, NMD modulates stability of selective mRNAs to fine-tune transcript abundance. Whether and how NMD influences brain development remains elusive. Our long-term objective is to understand the functional role of NMD regulation for the complicated and dynamic process of neurogenesis and how its mis-regulation leads to neurodevelopmental disorders. We determine the requirement of NMD for neural development through selective genetic ablation of Upf2 and in vivo manipulation of other NMD factors. Our preliminary data show that deletion of UPF2 in neural stem and progenitors results in microcephaly. UPF2 loss specifically affects the cell cycle and lineage progression of radial glia cells (RGCs), the major neural progenitor cells in the developing neocortex. We will combine cutting edge molecular cellular ribogenomics approaches, mouse genetics, and developmental neurobiology to dissect the mechanisms of NMD regulating neurogenesis. We propose three independent and interrelated aims to investigate possible variables underlying the microcephaly phenotype. In Aim 1, we will determine the cell cycle behaviors of RGCs in NMD knockout mice qualitatively and quantitatively and unveil the underlying regulatory mechanisms. In Aim 2, we will determine the lineage progression of RGCs and the resulting neuronal outputs per time unit in NMD knockout mice. By characterizing these molecular cellular defects, we also aim to provide mechanistic insights to transcriptomic regulation of RGC’s lineage transitions. NMD may regulate cell fates either independent of cell cycle controls or as the consequence of affecting the cell cycle. In Aim 3, we will test these two hypotheses and leverage our results to reexamine the relationship between cell cycle and cell fate. Successful completion of these studies will provide fundamental insights into how selective mRNA stability underlies the highly regulated cortical neurogenesis process in the mammalian brain. The proposed studies will also shed light on some fundamental questions about the control of cell cycle, cell fate, and their relationship during neural development.

NIH Research Projects · FY 2025 · 2021-09

Abstract Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of liver-specific gene expression, is regulated by two promoters (P1 and P2) which drive expression of two groups of HNF4α isoforms referred to as HNF4α1 and HNF4α7. HNF4α is a known regulator of gluconeogenesis and mutated in maturity onset diabetes of the young one (MODY1). Conventionally, it was thought that HNF4α1, but not HNF4α7, is expressed in the normal adult liver, while HNF4α1 is downregulated and HNF4α7 is upregulated in liver cancer. Now, research in our lab reveals a previously undescribed role for HNF4α7 in the normal adult mouse liver – one involved in the diurnal variations of lipid and carbohydrate metabolism. More specifically, HNF4α1 appears to be a major driver of gluconeogenesis while HNF4α7 is a driver of ketogenesis: we propose that alterations in the levels of the HNF4α isoforms during the day flip the molecular switch between the two. Our preliminary data also show that HNF4α7 is required for increased levels of circulating ketone bodies in female mice. AMP-Activated Protein Kinase (AMPK), an energy-sensing enzyme, has been shown to phosphorylate HNF4α1 in vitro, but effects in vivo and on HNF4α7 are not known. SIRT1 is a deacetylase that works with AMPK to regulate glucose and lipid metabolism. HNF4α1 is known to be acetylated and our preliminary data suggest that HNF4α7 but not HNF4α1 interacts with SIRT1. Here, we propose to use HNF4α1-expressing (α1HMZ) and HNF4α7-expressing exon swap mice (α7HMZ) to determine the physiological function of the HNF4α isoforms in the switch between gluconeogenesis and ketogenesis, and to characterize the impact of sex on those functions. In Aim 1, we will determine whether intermittent fasting and a ketogenic diet increase the levels of HNF4α7 in the liver, and whether the increase occurs in all hepatocytes, or just a subset. We will determine the consequences of HNF4α7 on gene expression. Kidney and intestines will also be explored. In Aim 2, we will determine whether the AMPK pathway acts in a differential fashion on the HNF4α isoforms to help flip the metabolic switch. Phosphorylation by AMPK and deacetylation by SIRT1 will be explored. Finally, in Aim 3, we will determine whether the estrogen pathway impacts the HNF4α isoforms in female mice and determine the consequences for the metabolic switch. Our compelling preliminary data that the HNF4α isoforms are involved in the switch between gluconeogenesis and ketogenesis shed new light on this basic metabolic process that occurs on a daily basis and under conditions of feeding and fasting. The results from this proposal will illuminate not only the molecular mechanism underlying the switch but also how that mechanism is impacted by sex. The proposed studies have the potential to impact our understanding of numerous metabolic diseases, including diabetes, obesity, fatty liver disease and cancer. Finally, given the fact that ketone bodies serve as a source of fuel for the brain, our results could have a broader impact, including on neurological diseases, such as dementia.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Single-cell RNA sequencing (scRNA-seq) is currently at the forefront of biotechnological innovation. scRNA-seq experiments enable gene expression measurement at a single-cell resolution, and provide an opportunity to characterize the molecular signatures of diverse cell types, states, and structures in tissue development and disease progression. However, it remains a substantive challenge to construct a comprehensive view of single- cell transcriptomes in health and disease, due to the knowledge gap in properly modeling the high-dimensional, sparse, and noisy scRNA-seq data. While the development of new data science methods, including our recent work, has facilitated the design and analysis in scRNA-seq studies to identify and annotate distinct cell populations, there is a critical need for computational methods that can accurately evaluate biological hypotheses for these diverse cell populations. To address this knowledge gap and critical need and thereby enable a systematic understanding of transcriptional and post-transcriptional mechanisms across biological scales (from cells to genes to RNA molecules), the objective of our MIRA research program is to develop novel statistical methods and bioinformatics software for multiscale analysis of single-cell transcriptomes. We will pursue three parallel but complementary research directions: (1) to develop novel statistical methods for quantifying and comparing gene regulatory associations from single-cell gene expression data; (2) to develop the first statistically principled methods for identifying, quantifying, and comparing alternative polyadenylation usage from 3’-end scRNA-seq data; and (3) to develop a novel statistical model for jointly analyzing and comparing scRNA-seq data from heterogeneous biological samples, such as multiple patients, developmental stages, or related species. The proposed research will be built on the foundations of our recent studies in developing interpretable statistical methods and user-friendly software for quantifying, denoising, integrating, and comparing genomic data at various biological scales. Throughout the program, we will work closely with experimental biologists at Rutgers Cancer Institute of New Jersey and Wistar Institute, and use our proposed methods to identify and study transcriptional mechanisms in intestinal biology, neurobiology, and cancer biology. Together, this concerted effort will provide efficient and broadly applicable statistical and bioinformatics tools for generating substantial insights into identifying key cells, pathways, gene interactions, and RNA transcripts associated with various biological contexts, including human disease. The proposed program also aligns with my team’s long-term goal to develop a statistically principled understanding of transcriptional and post- transcriptional regulation in single cells, thus improving our ability to define, interpret, and predict cellular commitment and functionality in health and disease.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Metalloproteins perform chemical transformations with rates and selectivites that have yet to be achieved in synthetic or designed systems. These differences in reactivity are directly linked to the environment produced by the protein matrix. To test our understanding of how metalloproteins function, I aim to design de novo metalloenzymes from scratch. Proteins that bind porphyrin-like cofactors are of particular interest, as heme proteins are known to perform a variety of reactions. Recently, I designed a protein to bind the abiological porphyrin, Mn-diphenylporphyrin (MnDPP), that provided the first crystallographic structure of a de novo designed porphyrin-binding protein (MPP1). MPP1 was also capable of stabilizing a Mn(V)-oxo species, a powerful oxidant that can perform sulfoxidation of thioether substrates. The proposed research seeks to elucidate design features necessary to control the reactivity/stability of this high-valent species through rational mutagenesis of my designed protein. This will allow direct correlation of changes in reactivity to changes in structure. To gain greater control of substrate orientation and, therefore, product distribution, I will design a 5-helix bundle that has a large pocket for substrate binding. Using the design strategy for MPP1 and in-house developed computational methods, the 5-helix bundle will be parameterized from scratch and designed to bind MnDPP. A library of sequences will be expressed and screened using high-throughput methods for binding and sulfoxidation activity. Promising scaffolds will then be redesigned to include substrate-specific interactions to bind the anti-inflammatory drug, diclofenac. Using COMBS, a recently developed bioinformatics method for designing backbone specific polar interactions, I will design two proteins to control the orientation of diclofenac to direct the hydroxylation to yield 5-hydroxydiclofenac or 4’- hydroxydiclofenac. This work would be a breakthrough in protein design and will directly impact the fundamental understanding of the effects of protein environments on the function of metal centers in metalloproteins.

NIH Research Projects · FY 2026 · 2021-08

Summary Project The majority of traumatic brain injury (TBI) is mild in nature but is known to elicit long-term consequences, including emergence of dementia and accelerated age-related declines. The highest-at-risk group are children whose brains are still undergoing development. This proposal will investigate the short- and long-term, cellular and molecular changes in the brain following juvenile mTBI (jmTBI) with the goal to intelligently develop new therapeutic options. Caveolin-1 (Cav-1) is an abundant structural protein involved in caveolae formation and cell signaling which is expressed in cerebral endothelial cells and in astrocytes, key components of the neurovascular unit (NVU). Recent development of a compound to target the Caveolin Scaffolding Domain (CSD), a complex that compartmentalizes structural proteins (e.g. claudin-5) and signaling molecules (e.g. eNOS), has provided tools to explore the role of Cav-1 in acquired neurological disease. After stroke, we found increased Cav-1 expression and Cav-AP treatment was beneficial for post-injury recovery. However, consensus is lacking whether Cav-1 exhibits beneficial or deleterious actions in other acquired brain disorders, such as jmTBI. Our model of jmTBI exhibits accelerated loss of cognition associated with decreased vascular function over their lifespan. We therefore will test the hypothesis that dysfunction in neurovascular coupling after jmTBI can be prevented by modulation of Cav-1 signaling, blunting accelerated hippocampal and cortical aging. Aim 1 will demonstrate that Cav-1 is critical for maintaining NVU functionality. We examine the role of vascular Cav-1 in male & female jmTBI mice in normal (WT), vascular Cav-1 deficient mice (Cav-1-/-) and in Cav-AP treated mice. We believe that jmTBI mice treated with Cav-AP will exhibit vascular recovery, whereas the loss of Cav-1 will worsen NVU outcomes. In Aim 2 we will examine how Cav- 1 in reactive astrocyte processes influences progression of jmTBI. We will modulate Cav-1 expression directly in astrocytes by injecting AAV-GFAP-Cav-1-shRNA and AAV-GFAP-synCav-1 in control and injured mice and quantify vascular recovery and behavioral outcomes. Increased astrocytic Cav-1 will be associated with improved NVU properties and cognitive outcomes. In Aim 3 we will examine male & female mice over their lifespan and examining if increased Cav-1 blunts accelerated brain aging that we have observed after jmTBI. We will assess behavioral, neuroimaging and histological outcomes. jmTBI mice treated with Cav-AP will exhibit improved outcomes related to enhanced NVU function and integrity. In sum, the proposed research is a critical first step in examining the role of Cav-1 in jmTBI and if therapeutic intervention can lead to enhanced NVU stability and function and thereby moderate accelerated aging.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Background: Sepsis is a life-threatening emergency, normally caused by the body’s response to a bacterial infection. Without early treatment, sepsis can lead to septic shock with approximately 50% mortality rate. Rapid and accurate diagnosis of sepsis is the key to decrease the mortality rate. However, there is no global standard for sepsis testing due to the inadequate sensitivity and specificity of the current technologies. The PI has the ambition to address the critical and far-reaching bottlenecks specifically of concern in sepsis testing: 1) a rapid and simple method to isolate and concentrate bacteria from whole blood sample, 2) a sensitive and one-step CRISPR microfluidic chip to detect the nucleic acid biomarkers of the pathogens, and 3) a multiplexing and miniaturized optofluidic waveguide platform to enhance the fluorescence based detection. Overview of the laboratory: The PI established his own lab at RIT in 2018. With the overwhelm start-up support by the home department, the PI is leading an active and interdisciplinary research group with 1 postdoc researcher, 4 Ph.D. students, 2 Master students, and a couple of undergraduate students. Within 2 years, the lab has published ~10 journal articles in the fields of bacteria/virus isolation, CRISPR biochemistry assay, and optofluidic sensing. Exploiting interdisciplinary approach, the lab is working on technologies to quickly identify antimicrobial resistant bacteria in whole blood sample. As more strains become resistant to available therapies, the risk for people developing life-threatening sepsis is increasing. The research topic we are working on will be the key for clinicians to provide quick clinical decisions and increase the chances of survivals. In addition, the technologies developed in our lab will also lay the foundation for the diagnosis and treatment of many different kinds of diseases such as cancer, viral infection, and neurological diseases. Goals for the next 5 years: Our first goal is to develop a fully automated nanodevice that can collect and concentrate bacteria from whole blood with a retrieval efficiency of 99% and a concentration factor of 10,000. We will begin with spiked sample and then proceed to clinical sample. Leveraging the unique properties of nanomaterials and nanostructures, the second goal is to develop a one-step and isothermal CRISPR chip for low concentration (1 CFU/mL) bacteria detection without front end target amplification. Towards the end of the fifth year, we will integrate the sample preparation chip and the CRISPR detection chip as a single and compact unit for the testing of clinical samples. The third goal is to develop a liquid-core, superhydrophobic nanostructure cladding waveguide platform for multiplexing bacteria detection. The high fluorescence collection efficiency will enable sensitive sepsis detection with microliter level sample consumption. Overall vision of the research program: The technologies we are developing will have a broad impact to the biomedical research communities to understand and engineering small molecules, cells, and tissues. The proposed work will also advance disease diagnosis, prevention, and treatment.

NIH Research Projects · FY 2025 · 2021-05

Project Summary The overarching goal of the present proposal is to understand how individual differences in the structure and function of Locus Coeruleus (LC) moderate perception and memory in an older adult population. There is substantial evidence that the LC circuit plays a central role in cognitive processes and neuronal loss in LC is known to occur in neurodegenerative disorders such as ADRD and PD. Integrity of LC neurons is hypothesized to mediate the preservation of cognitive abilities during normal aging as well. To date, however, there exists a dearth of research that either characterizes differential effects of LC integrity or details relationships between LC integrity and cognitive function in older adult humans. More generally, the link between LC activity and cognitive processes has not been well characterized in humans. Historical reasons for this is that the LC has been difficult to image due to its small size and thus most human research makes inferences about LC function by using pupil dilation as a surrogate measure. To overcome existing limitations in the field, we propose a series of detailed psychophysical and MRI-based studies in older adults aimed to characterize how LC structure and function moderates behavior and in turn how this is mediated by activity in intermediate brain regions known to be involved in perceptual and memory processes. We further propose computational approaches to characterize individual differences in how LC circuit integrity relates to different patterns of cognitive performance across tasks, and advanced neuroimaging methods to localize and image the LC, which have been pioneered by our group. Using MRI-based methods, we will examine LC integrity using high-resolution neuromelanin-sensitive structural imaging, tractography and functional connectivity. This approach will allow us to identify candidate biomarkers of LC circuit integrity. We will use a series of within-subject designs where we manipulate LC activity and examine whether relationships between LC and behavior and brain regions thought to mediate those behaviors are consistent or not between different perceptual modalities and memory tasks. Overall this study will provide an important and much needed understanding of how LC integrity underlies cognitive declines in older adults. By combining advanced neuroimaging, well-controlled behavioral assessment, and computational analysis, we expect to uncover previously inaccessible in vivo mechanisms of LC modulation and generate a unique dataset to address fundamental mechanistic questions of how the LC integrity moderates cognition, how this varies across older adults and the extent to which relationships between LC and cognition are generalized or individualized to particular domains. The resulting understanding of LC circuit can help explain how dysfunctional modulatory circuits may generate cognitive declines or be implicated in normal aging and age- related disorders such as Alzheimer's and Alzheimer's related disorders. This, in turn, has potential to support non-invasive methods for diagnosing pathologies associated with LC decline and developing new treatments.

NIH Research Projects · FY 2025 · 2021-04

Project Summary: Neurological disorders such as epilepsy and memory loss develop several years after traumatic brain injury (TBI) and are a major source of physical disability and economic burden. The delay between the initial trauma and eventual disability results from progressive neuropathology that could be limited by early interventions. However, mechanisms by which TBI impacts memory and seizure susceptibility are not fully understood. The hippocampal dentate gyrus, a circuit critical for memory processing, a key regulator of information transfer from entorhinal cortex to hippocampus, and a niche region for adult neurogenesis, is a focus of neuronal damage and increased excitability after TBI. Although adult born granule cells (abGCs) are implicated in memory processing, the contribution of abGCs to dentate spikes which represent entorhinal cortex to dentate information flow and support memory consolidation is not known and how injury-induced changes in neurogenesis affect memory processing is not fully understood. Unexpectedly, we find that suppressing injury-induced increase in neurogenesis reduces dentate excitability one week after TBI, during the same period when posttraumatic increase the innate immune receptor, toll-like receptor 4 (TLR4) augments dentate excitability. TLR4 is known to suppress neurogenesis in naïve animals and paradoxically increase neurogenesis in stroke. While the molecular mechanisms by which TLR4 regulates excitability and neurogenesis are unknown, recent findings that TLR4 enhances the endopeptidase, matrix metalloproteinase- 9 (MMP-9), a critical player in synaptic plasticity and neurogenesis provides a promising molecular link between trauma, TLR4 and aberrant network plasticity. In an integrative approach spanning molecular to cellular to network function, we propose that early increase in neurogenesis and excitability after TBI disrupt dentate regulation of cortico-hippocampal throughput and contribute deficits in memory processing by TLR4- dependent persistent elevation of MMP-9 activity. Using the fluid percussion injury model in mice and current in vivo and ex vivo electro- and optophysiological techniques, Aim 1 will determine the role of TLR4 signaling in altered development, maturation and circuit integration of abGCs born after injury. Aim 2 will test if altered DG excitability and neurogenesis after TBI compromise oscillatory coupling between dentate and hippocampus which can be prevented by blocking TLR4 early after injury. Finally, Aim 3 will use a combination of histological, biochemical, physiological, and behavioral assays to test if aberrant TLR4 signaling after TBI results in persistent increase in MMP-9 which can be targeted to limit aberrant neurogenesis, deficits in oscillatory coupling and memory deficits after TBI. Such preventive strategies will greatly improve the quality of life of patients after TBI and address the NINDS mission of decreasing the long-term health care burden posed by post-traumatic neurological diseases.

NIH Research Projects · FY 2025 · 2021-04

Abstract CRISPR-Cas9 is the core of a transformative genome editing technology that is innovating life science with cutting-edge impact in basic and applied sciences. By enabling the correction of DNA mutations, this technology promises to treat a myriad of human genetic diseases, as shown for the first cancer patients treated with CRISPR-Cas9–modified T-cells. This technology is based on the endonuclease Cas9, which associates with guide RNAs to recognize and cleave complementary DNA sequences. Ceaseless development and engineering of CRISPR-Cas9 tools has opened novel intriguing hypotheses that grant in-depth investigations of the system. Here, the PI will implement unconventional multiscale approaches, combining a variety of state-of-the-art theoretical methods, to clarify the metal-dependent catalysis, the allostery in the selectivity mechanisms, as well as the inhibition of the system. We will pursue three specific aims, characterizing: (Aim 1) the DNA cleavage dependency on alternative divalent metal ions other than Mg2+ and the conformational effects associated with their binding; (Aim 2) the allosteric modulation witnessed in newly engineered Cas9 variants with enhanced specificity; (Aim 3) the inhibition mechanism by naturally occurring anti-CRISPR proteins to implement control over gene regulation. Toward these aims, we will leverage classical and enhanced sampling molecular dynamics (MD) simulations, high-level ab-initio MD (using the Car-Parrinello and Born- Oppenheimer approaches) and mixed quantum mechanics/molecular mechanics (QM/MM) approaches. Moreover, combination of ab-initio MD with graph theory will implement a synergistic approach capturing instantaneous sub-nanosecond signaling transfers. This will reveal how long-range allosteric effects impact the dynamics through evolving catalytic steps, elucidating the role of allostery in aiding catalysis. These multiscale approaches will offer a computational framework for the biophysical analysis of not only CRISPR-Cas9, but can also be extended to emerging CRISPR systems that are promising for genome editing and viral detection. Theoretical studies will be performed in close collaboration with experimental scientists, providing kinetic measurements and biophysical characterization, assisting in the interpretation of the experimental data and enabling testable predictions. Overall, this proposed research will expand the repertoire of mechanistic knowledge regarding the CRISPR-Cas9 function and lay the framework for novel engineering rationales toward improved genome editing.

NIH Research Projects · FY 2025 · 2021-03

The family of flavivirus consists of over 90 vector-borne, single-stranded RNA-containing viruses, including Dengue virus (DENV) and Zika virus (ZIKV), which cause major epidemics among humans and pose a serious threat to global public health. No vaccines or antivirals exist to prevent or treat infections caused by DENV, ZIKV, and some other flaviviruses. To establish infection, flaviviruses need to overcome the antiviral state induced by type 1 interferon (IFN-1), the first line of host defense. In this regard, flaviviruses have encoded several antagonists to suppress IFN responses. For example, the nonstructural NS5 proteins of DENV, ZIKV, and some other flaviviruses have been shown to be potent suppressor of IFN signaling, targeting different steps of the IFN signaling pathway. Like DENV, ZIKV NS5 protein bind human signal transducer and activator of transcription 2 (hSTAT2) protein and trigger its proteasomal degradation, albeit using different downstream mechanisms. To elucidate the mechanistic basis of flavivirus NS5-mediated hSTAT2 suppression, we propose to provide structural insight into the ZIKV NS5-hSTAT2 and DENV NS5-hSTAT2 complexes, which, in turn, will guide interrogation of the consequence(s) of the flavivirus NS5-hSTAT2 interactions in proteasome-mediated degradation of hSTAT2 and suppression of IFN signaling. Toward this goal, we will use structural, biochemical, molecular, cellular and virology approaches to investigate the structural basis of the ZIKV NS5-hSTAT2 and DENV NS5-hSTAT2 interactions and their functional consequence. In Aim 1, we will establish the structural basis of the ZIKV-hSTAT2 interaction by using X-ray crystallography and cryo-electron microscopy and validate our observations with mutational and in vitro pull-down analyses. In Aim 2, we will examine the ZIKV NS5-hSTAT2 interaction at a cellular level and investigate the functional consequence of the ZIKV NS5-hSTAT2 interaction through evaluation of the mutational effects of ZIKV NS5 on hSTAT2 degradation, IFN response and viral infection. The results of these studies will provide critical structural and functional insights into the virus- and species-specific ZIKV NS5-hSTAT2 interaction, thereby establishing a mechanistic link between flavivirus NS5 proteins, hSTAT2 degradation, suppression of the IFN response and viral infection. Results from the proposed studies will ultimately benefit development of novel antivirals and live vaccines against flaviviruses infection.

NIH Research Projects · FY 2025 · 2021-02

Understanding how animals make behavioral decisions is one of the biggest problems in neuroscience. The proposed study aims to understand molecular and neural mechanisms underlying innate defensive behaviors elicited by chemical cues from predator species. Defensive responses to predatory threats are made through a pre-programmed defensive brain circuit, which has an ability to instantly make an appropriate behavioral decision upon sensing predator-derived sensory stimuli. It is widely appreciated that olfaction is one of the major sensory modalities through which predator-derived chemical cues trigger behavioral responses in prey species. When prey animals detect immediate danger in predator cues, they exhibit acute defense behaviors such as freezing or flight. On the other hand, when prey animals detect only potential danger in predator cues, they exhibit vigilance and risk assessment behaviors such as repetitive stretched sniffing. An important question in behavioral neuroscience is whether these defensive decisions for predatory threats are made through distinct neural circuits or by a shared neural population. Our preliminary data establish a framework of the proposed study to dissect defensive behavioral circuitries activated by predator cues through the vomeronasal chemosensory organ (VNO), which trigger either freezing or risk assessment behaviors in mice. In this proposal, we aim to identify the freezing- and risk assessment-inducing predator cues and their sensory receptors in the VNO, and to assess whether the sensory signals induce behavioral outputs through independent, parallel circuitries, or they are integrated in the brain to induce an appropriate behavior. Our central hypothesis is that different predator cues are detected by distinct sensory receptor circuitries and elicit distinct defensive behavioral outputs in parallel. To test this hypothesis, we will investigate mechanisms of the predator cue sensation at molecular levels; more specifically, we will first identify the sensory stimuli (Aim 1) and the sensory receptors (Aim 2). Moreover, using freezing- and risk assessment-inducing sensory cues as tools, we will further examine whether the defensive decision towards predator cues is made by independent neural circuitries or not (Aim 3). The results from these experiments will provide new insights into the molecular mechanisms underlying the sensory processing in predator cue sensation, and will reveal an operational principle of decision making circuitries for emotional behaviors. This will critically contribute to our understanding of pre-programmed brain machinery that underlies multiple levels of fear and stress processing in response to threat.

NIH Research Projects · FY 2025 · 2021-01

Project Summary Reactivation of toxoplasmosis is a significant health threat to people chronically infected with this parasite and is life-threatening to infected individuals that are or become immunocompromised. Millions of people face this threat as it is estimated one third of human populations are infected with this pathogen. Recrudescence of the Toxoplasma bradyzoite tissue cyst is the cause of toxoplasmosis reactivation, which can not be prevented as there is no current treatment that eliminates the dormant tissue cyst in chronically infected individuals. Approaches to find therapeutic solutions to treat and prevent chronic toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. Our goal in this proposal is to breakthrough these impasses. We have developed a new innovative ex vivo model of bradyzoite recrudescence that we will utilize to define the host cell specificity (Aim 1a), whole-cell gene expression (Aim 1b) and metabolic changes (Aim 2) that unfold when a bradyzoite converts back to the tachyzoite and also in a newly discovered alternate pathway where bradyzoites directly replicate to reform the tissue cyst. This information is critically needed in order to understand how we might prevent toxoplasmosis reactivation. The loss of developmental competency in vitro that is exacerbated in current protocols producing transgenic strains is also a major impediment to understanding the molecular basis of tissue cyst reactivation. In this proposal, we will implement and optimize an innovative approach to generate developmentally competent transgenic strains (Aim 3a), and use this new protocol to define cyclin and other protein mechanisms (Aim 3b) that have critical roles in regulating bradyzoite recrudescence and tissue cyst re-formation

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Challenges exist in bioremediation of halogenated contaminants, including low donor utilization efficiency and slow dehalogenation, low dehalogenation activity and degree for the emerging per- and polyfluorinated substances, as well as the difficulty in simultaneously treating co-contaminants. To address those challenges, this project integrates advances in materials sciences and microbial reductive dehalogenation and proposes a synergistic materials-microbe interface that can achieve faster, deeper, and air-tolerant reductive dehalogenation. Charge transfer mechanisms in the proposed electricity-driven materials-microbe hybrid will be investigated, which will guide the design and optimization of novel nano- and micro-scale materials to enhance the mass-transport efficiency and accelerate dehalogenation. The local electron donor levels can be stably maintained at low levels, favoring dehalorespiring microorganisms over methanogens and homoacetogens, leading to enhanced electron donor utilization. A systems-level understanding of microorganisms enriched in the bioelectrochemical system and genes/enzymes responsible for deeper defluorination will be obtained with omics techniques. Novel reductive defluorination products/pathways and synergistic interactions between microbial and electrochemical defluorination will be elucidated using advanced analytical tools such as high-resolution mass spectrometry. Furthermore, an air-tolerant materials-microbe framework for reductive dehalogenation will be developed using a recently designed microwire array electrodes and implemented to achieve concurrent oxidation of the co-contaminant 1,4-dioxane in an open system. This project will significantly advance the mechanistic understanding of the accelerated and deeper reductive dehalogenation at the synergistic materials- microbe interface. This hybrid framework is powered by electricity that can be generated from sustainable solar energy and may lower the cost by reducing the requirement of fermentable organics and by combining the anaerobic and aerobic remediation processes. The successful demonstration of this new paradigm of bioremediation will potentially lead to future applications for cleaning up the halogenated contaminants and co- contaminants in subsurface environments. The developed materials-microbe framework is also highly transformable to the bioremediation processes of other environmental contaminants.

NIH Research Projects · FY 2024 · 2020-09

Summary/Abstract Crimean-Congo hemorrhagic fever virus (CCHFV) is a ssRNA (-) nairovirus that produces fever, prostration, and severe hemorrhages in humans. Fatality rates associated with CCHFV range from 5- 80% based on phylogenetic variation of the virus, transmission route, and different treatment facilities. Originally identified in Russia and the Congo, CCHFV has rapidly spread across large sections of Europe, Asia, and Africa. Recently, CCHFV has illustrated its continued ability to spread into previously naive regions. At the same time, U.S. citizen traffic has increased substantially to the regions endemic with CCHFV, specifically South-Central Asia. As a result, there is a substantial risk for transmission of CCHFV and/or its tick vector to the United States. Intriguingly, CCHFV is not the only nairovirus that threatens the public. Nairobi Sheep Disease virus (NSDV) as well as nairoviruses Issyk-kul, Dugbe and Erve can cause human disease of varying severity and economic distress. There is no vaccine or prophylactic currently available for treatment of CCHF or any other nairovirus related disease. Reports have identified a viral homologue of the ovarian tumor protease (vOTU) located within the nairovirus genome. Recently, vOTUs’ ability to reverse post-translational modification by proteins ubiquitin (Ub) and Ub-like interferon-simulated gene 15 (ISG15) on a narrow subset of host pathways has been illustrated to be critical to pathogenesis. Also, vOTUs from CCHFV and other nairoviruses have been found to be sensitive to species-species variations in ISG15 and their specificity includes at least the species that disease is most prominently identified. This proposal will determine the identity of specific host proteins within those pathways targeted by vOTUs. This will enable therapeutic approaches that protect, or elevate, specific host inhibitory factors for these viruses. The proposal will also seek to evaluate the correlation between the in vitro activity/substrate species-specificity of these nairovirus vOTUs and overall virulence and zoonotic range of the nairoviruses in question. Additionally, the efficacy of using CCHFV vaccine candidates with altered CCHF vOTU functions will be assessed. Together, the resulting information will provide critical insight into the role of vOTUs play in pathogenesis and host restriction as well as advance the development of prophylactics targeting vOTUs.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Fungal pathogens infect humans, animals, and plants and cause severe consequences on global human health and crop production. Communication between hosts and pathogens is essential for host defense and pathogen virulence, but the underlying mechanisms are not well understood. Previous studies in my lab discovered that some non-coding regulatory small RNAs (sRNAs) from fungal pathogens, such as Botrytis cinerea, which causes grey mold disease on more than 1000 plant species, can be transported into host plant cells and suppress host immunity genes, a mechanism called “Cross-Kingdom RNAi”. Recently, we discovered that such sRNA communication is bi-directional. Plant hosts have also developed the ability to deliver sRNAs, mainly using extracellular vesicles, to fungal cells and induce cross-kingdom RNAi of fungal virulence-related genes. Such sRNA communication was also observed between mammals and parasites. Although more and more studies across diverse systems demonstrate that mobile sRNAs are key regulatory molecules in host and pathogen interactions, the field of cross-kingdom/cross-species RNA communication is still in its infancy. This proposal is designed to use plant Arabidopsis and fungal pathogen Botrytis as a model system to address the outstanding questions in this field, including, how host cells control sRNA transport upon infection, how specific classes of small RNAs are sorted into extracellular vesicles, how fungal cells deliver sRNAs into host cells, what are the mechanisms of RNA and vesicle uptake in the host cells and fungal cells, and whether other classes of RNAs, such as mRNAs and long non-coding RNAs, move between host and fungal cells, how they function in the counter party, etc.. A combination of genetics, genomics, biochemical and molecular biology approaches will be used. This project is expected to provide unprecedented insight into the underlying mechanisms of cross-kingdom/cross-species RNA communications, which will ultimately help develop innovative and eco-friendly disease control strategies and RNA-based fungicides or antifungal drugs.

NIH Research Projects · FY 2024 · 2020-08

Regulated protein synthesis, or translation, is essential for life, and allows the cell to flexibly respond to external stimuli and stress. Conversely, dysregulated translation is a hallmark of diseases including cancer, viral infection, and developmental disorders. Translation is regulated principally through its initiation phase, where a crucial regulatory function of the initiation machinery is to ensure selection of the correct translation start site on messenger RNA. Failure to do so compromises the proteome by permitting synthesis of elongated, truncated, or nonsense proteins. In eukaryotes, start-site selection requires a directional search beginning at the 5’ end of the message. This search must move the megadalton ribosomal pre-initiation complex (PIC) efficiently through mRNA leader sequences that can span tens, hundreds, or even over a thousand nucleotides, and then halt this motion at exactly the correct start site. A linear “scanning” mechanism was first proposed over 40 years ago for this remarkable biophysical feat. However, fundamental properties of scanning have never been directly validated experimentally, and alternative mechanisms have been proposed. The importance of motion through the mRNA leader in translational control has also been brought into renewed sharp focus in recent years with the discovery that many mRNAs contain upstream open reading frames in their leaders that control translation of the main open reading frame; the scanning mechanism lies at the heart of how these are utilized. A critical barrier to progress is the remarkable molecular complexity and dynamism of the scanning machinery, whose numerous transient intermediates have made it challenging to characterize experimentally. Directly visualizing scanning in real time would allow many key unsolved questions to be addressed. Single-molecule methods are uniquely positioned to do this with the molecular resolution required to dissect mechanism. We have developed a single-molecule fluorescence assay for scanning of a reconstituted yeast PIC on full-length mRNAs. Here we will apply this assay to address the scanning mechanism. In Aim 1 we will directly determine the physical mechanism of motion in scanning, establishing the contributions of mRNA sequence and structure to the scanning rate. In Aim 2, we will elucidate how scanning directionality is established and maintained, focusing on the central translational helicase, eIF4A. We will distinguish between proposed mechanisms for how eIF4A transduces the chemical potential of ATP to bias scanning direction. In Aim 3, we will define the roles of pre- initiation complex components in scanning, with experiments that isolate their contribution to scanning specifically, rather than their aggregate functions throughout initiation. These studies will establish a physical- mechanistic model for scanning that will deepen understanding of translational control in health, and inform ongoing efforts to understand and reverse dysregulation in disease.