Iowa State University

NIH Research Projects · FY 2024 · 2021-09

ABSTRACT This proposal addresses NIOSH sector of Healthcare and Social Assistance (HCSA) program (NAICS 62) and significant components of the Immune, Infectious and Dermal Disease Prevention (IID) cross sector. Over 21 million people are covered by the HCSA program in the United States and occupational IID diseases are of the most common illnesses affecting their safety and health. Personal protective equipment (PPE) as major intervention means is critical for healthcare workers’ (HCWs) health and safety. However, ineffective PPE can place HCWs and patients at risk of transmission of infectious agents, not only through direct contact with blood and body fluids, but also via microbial penetration through barrier fabrics, and aerosol and droplet transmission. Current PPE is not ideally suited to the needs of HCWs due to limitations in protection and comfort, such as self-contamination during doffing, poor fit and inward leakage risks offered by respirators, insufficient capture of airborne pathogens, difficulties in communication through materials, potential fluid penetration, and poorly executed fit and sizing. Limitations of current PPE have resulted in infections and mortalities of HCWs in fighting against recent outbreaks such as Ebola, SARS, and COVID-19. The overall goal of this project is to develop improved PPE for HCWs with an emphasis on self-decontaminating function. In this project, we will develop new textile materials with self- decontaminating property. Then, we will apply both the existing material and those materials developed in this project to isolation gown and respirator. A novel design approach will be applied to create a new, seamless, self-decontaminating PPE system with superior comfort, fit, and functionality compared with those of any currently available PPE. The evidence- based design process will incorporate three-dimensional (3D) body scanning and kinematic motion analysis to achieve greatly improved wearability and functionality. We will use the recently developed and validated Faceseal concept to develop a filtering facepiece respirator (FFR) made of biocidal material that will have a close-to-perfect fit. A Simulated Workplace Protection Factor (SWPF) will be determined for the new respirator. The overall PPE performance design will be evaluated using instrumented manikins, specifically designed human trials, and performance testing. The data obtained from these evaluations will be applied to further improve the newly-developed PPE components. The multidisciplinary team is well prepared to carry out the proposed work with established knowledge and successful track records in fiber/polymer science, textiles, PPE design and performance evaluation, respirator design, aerosol research, and exercise physiology and kinesiology. The state-of-the-art facilities and equipment across the collaborating institutes are ideally suited to fulfill the research aims. Clinicians and epidemiologists will serve as consultants on the project, offering insights and firsthand experiences of PPE implementation and utilization. This proposed project is significant since it embraces NIOSH r2p initiative by fundamentally directing novel PPE material and end products development and providing the technical basis in performance evaluation methods. The completion of the work will raise the horizon of PPE engineering in reducing occupational IID diseases and targeting the intermediate goal of addressing infectious disease transmission in the HCSA sector. The end outcomes of this proposed project will benefit millions of HCWs and save billions of healthcare costs.

NIH Research Projects · FY 2025 · 2021-09

There are significant advantages from translating genome sequences into proteins, where there is a large body of accumulated knowledge regarding their relationships among sequence, structure and function. Advances in genome sequencing are producing a deluge of data that can be used to train and test prediction methods to identify the characteristics of various mutants by building atop the large functional protein data. Clinicians need to know the functional behavior of mutants - whether they are neutral or deleterious - whether they affect protein structure – whether they affect protein dynamics - whether they affect protein binding specificity. Protein structures have local environments for each amino acid in the sequence, and usually amino acids at each position are compatible with their local environment. This leads to strongly correlated amino acids as manifested in the multiple sequence alignments. This project will combine protein sequence and structure data together with amino acid properties and their correlations to characterize each site in the protein structure to investigate the hypothesis that outliers in the distributions over the important amino acid properties for each position will negatively impact functionality, i.e. they will be deleterious mutants. The project will drill down deeply to learn what is the nature of the impaired mechanism. Two diverse approaches will be taken in the two aims: Aim 1 will investigate the amino acid property distributions to identify the properties that best characterize each position in the sequence and structure, and determine how the outliers negatively impact the functional structures, dynamics and binding characteristics. Preliminary results show that the deleterious mutants usually have a significantly broader range of single amino acid properties for the deleterious mutants. Data from these analyses will be fed into Aim 2 where two type of machine learning approaches – Extreme Learning Machines and Random Forests will be jointly applied. Preliminary results show that incorporating just one amino acid property yields significant gains over existing methods. One of the major strengths of this project is that results from the two Aims will be exchanged frequently to achieve improved predictions for both approaches. The project builds on the long experience of the PIs in datamining from protein structures and sequences, as well as previous machine learning applications. Important potential outcomes include a more reliable, more informed understanding of how mutants affect function. In addition, the project aims to predict connections of mutants to specific diseases. The results of the project will be important for drug development, because the specific part of the protein where function is impaired will be identified, to allow drug developers to narrow their focus onto more limited parts of a protein that is targeted for drug design. The predictors established by this project will also have the potential to screen for large numbers of previously unknown mutations that could be used to identify specific regions of a protein structure susceptible to further disease-related mutations.

NIH Research Projects · FY 2024 · 2021-09

No Abstract

NIH Research Projects · FY 2025 · 2021-08

Abstract The organophosphate nerve agent (OPNA) attacks in Tokyo, Syria, Malaysia, and England prove the real threat of OPNAs. Acute exposure to OPNA impacts human health globally, and we lack treatment for survivors. Until recently, preventing acute death due to OPNA exposure was a priority. However, addressing the long- term effects is also critical given that survivors, though hospitalized and treated with conventional therapy, developed seizures and cognitive, motor, and psychological impairments. OPNAs are cholinesterase inhibitors and potent seizurogenics. In animal models, acute OPNA exposure induces status epilepticus (SE) and other cholinergic symptoms. The current medical countermeasures (MCM) do not prevent long-term neurotoxicity and comorbidity, which are primarily due to persistent neuroinflammation and neurodegeneration. Our overarching hypothesis is that a neuroprotectant, in combination with MCM, will effectively counteract OPNA- induced long-term neurotoxicity and restore brain function. In the currently funded U01 project, we optimized the dose of two novel neuroprotectants, saracatinib (SAR/AZD0530, a Src kinase inhibitor) and 1400W (an inducible NO synthase inhibitor) and demonstrated their long-term disease-modifying effects in adult rat DFP and soman models. We have also shown the feasibility and pharmacokinetics of diet-incorporated SAR in long term studies in adult rat DFP and soman models. Based on the U01 project outcomes, we are preparing for a pre-IND meeting with the FDA to develop SAR for clinical trials. AstraZeneca (AZ), who discovered SAR, has supplied the test drug for all our preclinical studies through Open Innovation. AZ also supports the pre- IND process by providing critical preclinical and clinical data they have acquired from various clinical studies for many years. However, the significant gap in this process is the lack of pediatric data for SAR. In this Administrative Supplement project, we will test the long-term efficacy and tolerability of SAR in the diet in a pediatric rat DFP model (postnatal day 21) in both sexes as adjunct therapy. We will treat control littermates with a control diet (without SAR). We will maintain the animals on SAR in-diet throughout their adulthood, perform behavioral tests, and euthanize at 2 months post-DFP and some animals at one-week post-DFP. We will do multiplex cytokine/chemokine assays from serum and CSF as readouts for the effects of SAR and correlate with other outcomes. The brain tissues will be used for phosphoproteomics to identify differentially phosphorylated proteins and correlate these with behavioral outcomes. Based on the hits, we will validate the top 6-8 proteins of relevant pathways using the Westerns and confirm their cellular localization in brain sections for comprehensive molecular and cellular insights. Enrichment of specific subsets of the phosphoproteomes such as phosphotyrosine and phospho-motif enrichment of kinase substrates will reveal targets of SAR and DFP. This research is within the CCRP scope of current U01 (NS117284) and addresses NOT-AI-24-022, i.e., "to promote the development of pediatric chemical research models and MCM discovery."

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The long-term goal of this proposal is to improve outcomes of delivery of small molecules, peptides, and proteins to the human gut by engineered live biotherapeutic bacteria. To accomplish this, we will use two strategies to optimize biotherapeutic growth in the gut environment. First, we will match prebiotic compounds (complex carbohydrates not digested by the body) with heterologous expression of enzymes that allow cells to utilize these privileged carbon sources. Second, we will create genome-scale libraries in our candidate biotherapeutics, E. coli Nissle 1917 and Lactococcus lactis, to identify gene products that increase fitness in the gut and gut-like environments. The methods will be tested in in vitro culture, small intestinal organoid co-culture, and in vivo in the mouse gut to determine enrichment of the biotherapeutic population, enhanced production of the therapeutic small molecule, peptide, or protein of interest, and phenotypic outcome in systems of increasing verisimilitude to the human gut.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract The Shao group at Iowa State University aims to leverage their expertise in genetic tool development to assemble a comprehensive mitochondrial genetic toolkit. The causative role of mitochondrial DNA is implicated in a myriad of human diseases and disorders. However, human mitochondrial dysfunction studies suffer from the absence of an ideal eukaryotic model. The well-studied model yeast, S. cerevisiae, is unsuitable due to its mitochondrial physiology deviating too far from humans. We propose developing a new promising model to heighten knowledge of human mtDNA, yielding new insights on mitochondrial dysfunction and pathogenicity. We have identified that Yarrowia lipolytica strikes a perfect balance between practicality (i.e., low cost and quick timescale of genetic manipulations as a low eukaryote) and tractability (i.e., mirroring human's obligate aerobic needs). In addition, the overwhelming majority of current mitochondrial dysfunction studies focus solely on nuclear-encoded mitochondrial gene abnormalities. This is mainly attributed to the fact that the explosive progression of nuclear genome editing technology in the epoch of “post-CRISPR” has yet to translate to mtDNA editing. We propose leveraging a recently discovered stem-loop RNA motif to overcome nucleic acid import limitations – the largest technical barrier to the development of CRISPR-associated technologies. If this strategy proves effective, we envision that many mtDNA manipulation tools will be developed by research labs around the globe, following the same trajectory as the CRISPR nuclear genome manipulation revolution. Over the next five years, in addition to the foundational tool development, we will strive for elucidating mtDNA-phenotype relationships in the new model. The ability of Y. lipolytica to accumulate lipids makes it a particularly suitable model for human adipocytes. Mitochondrial dysfunction in adipose tissue is involved in a broad spectrum of epidemics plaguing human health. Mediated by our development of a mitochondrial genetic toolkit, we will reconstitute mtDNA-associated human pathologies in a precise manner, enabling tailored drug development and all the subsequent mechanistic studies. Moreover, we propose studying the impact of modulating the fluidity of the inner mitochondrial membrane on altering mitochondrial physiology, which will lead to the discovery of potential treatments of obesity-related diseases in the future. Lastly, along a side research branch, we will integrate the developed mitochondrial genetic toolkit with our previous efforts in metabolic engineering. We will leverage the multiplicity of mitochondria in a single cell as well as the high copy number of mtDNA in a single mitochondrion to boost the dosage of the gene encoding the rate-limiting step in a biochemical pathway. This strategy will revolutionize the metabolic engineering design of eukaryotic hosts to produce a wide variety of compounds derived from TCA cycle or whose biosynthetic mechanism requires a high ATP input. Altogether, this MIRA for ESI project will enable me to move into research areas distinct from my existing ones and grant me the flexibility to follow important new research directions as opportunities arise.

NIH Research Projects · FY 2026 · 2021-07

PROJECT SUMMARY/ABSTRACT Peptides are promising structures for drug development because they provide a modular scaffold with properties between small molecules and larger biologics. One of the most critical challenges in the field of bioactive peptides is developing proteolytically-stable versions that can enact their mode of action before breaking down. Proteolytic stability is therefore a fundamental component of peptide drug discovery and arguably of comparable importance to drug efficacy and toxicity. The research plans of this proposal will tackle this challenge from two orthogonal directions, utilizing different aspects of peptide-backbone chemistry: (1) Peptides that are cyclized head-to-tail display resistance to proteolysis due to conformational rigidity and a lack of any free N- and C-termini. Unfortunately, a key impediment to the synthesis of small peptide macrocycles is that the native conformation of the backbone amides is not conducive to cyclization. We will develop backbone modifications that promote conformations conducive to head-to-tail cyclization. These modifications can then be elaborated into a host of other backbone functional groups, including the native amide. These methods will permit the synthesis of diverse macrocyclic peptides and natural products that are otherwise impossible to access by conventional methods and will open new space for drug discovery. (2) We will install amidines at known sites of proteolysis. This single atom substitution along the backbone fundamentally alters the finely tuned interactions that are essential for enzymatic proteolysis. This modification does not alter the side-chain functional groups that may be responsible for engagement of the peptide with its target. Moreover, amidines have minimal impact on the native conformation of the peptide backbone, in contrast to other common strategies. The rationale for this approach is that promising peptide-drug candidates that failed due to metabolic instabilities can be resuscitated for further development. The proposed research is significant because it is expected to have broad importance in the development of bioactive molecules. The proposed research is innovative because it represents a substantive departure from the status quo by developing and employing new peptide-backbone chemistries to unlock new capabilities in peptide therapeutics.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Bacterial populations, including those that affect human health, are largely controlled by bacteriophages. Phages change the composition of bacterial ecosystems and strongly influence bacterial evolution. Defense mechanisms that protect bacteria from phages are important regulators of these host-pathogen interactions. Defining these mechanisms is crucial to understanding bacterial compositional dynamics and pathogenicity. CRISPR-Cas systems are sophisticated and diverse mechanisms that allow bacteria to memorize infection events and defend themselves upon reinfection. In addition to their important role in mediating bacteria- phage interactions, CRISPR-Cas systems have been harnessed for genome manipulation technologies that have greatly facilitated biomedical research and have enormous potential for human therapies. The goal of our research program is to fully define the mechanisms and specificities of a variety of nucleic acid-protein complexes that direct CRISPR-mediated immunity and have potential for CRISPR technology. Our program is divided between understanding the process of adaptation, during which a bacterial cell is immunized, and interference, during which the CRISPR-Cas system neutralizes an infection. We and others have recently discovered higher-order adaptation complexes containing poorly defined protein subunits that are essential for effective immunization. Our goal is to uncover the molecular steps that enable specificity and precision by higher-order adaptation complexes, ensuring productive immunization events. Following immunization, Cas effector complexes that neutralize infection during interference must quickly recognize pathogens, a task made even more challenging when phages evolve and evade detection. Our goal is to understand how Cas effectors can maintain effective immunity even in the face of pathogen evolution. Through this research program, we will contribute to the overall understanding of how CRISPR-Cas systems impact bacterial populations and help ensure that CRISPR-based research and therapeutic tools are used safely and effectively.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY / ABSTRACT Age-related defects of the immune response contribute to reduced efficacy of the influenza vaccine in older adults. Influenza A virus (IAV) infection results in greater risk of complications and higher hospitalization rates in older adults, with approximately 90% of deaths occurring in adults over age 65. Therefore, the development of a safe and effective vaccine that promotes protective immunity for the aged is an urgent public health need. The overall goal of this revised R01 application is to identify the effect of vaccine biomaterials and adjuvants on DC metabolism, and subsequent effects on antibody and T cell memory to develop a nanovaccine to overcome age-related immune impairments. Vaccines for older adults can be further optimized with biomaterials that enhance multiple arms of the immune system and provide a platform to expand antigen selection, broadening protection. Our studies will establish the contribution of specific biomaterials and adjuvants in improving B and T cell outcomes resulting in protection by enhancing vaccine efficacy. The goals are to: 1) develop an efficacious influenza nanovaccine for older populations; and 2) to understand the mechanisms by which rational selection of biomaterials and co-adjuvants in vaccines can enhance immune capabilities of aged individuals. Our two polymeric nanovaccine platforms, polyanhydride nanoparticles and pentablock copolymer micelles, have been shown to increase antibody titers, improve cell-mediated immunity, and prolong antigen delivery resulting in a protective immune response with reduced viral load upon delivery of recombinant hemagglutinin and nucleoprotein in an IAV challenge model. Compelling preliminary data demonstrates that these formulations differentially alter dendritic cell (DC) metabolic profile compared to traditional adjuvants. Aim 1 will identify how nanovaccine biomaterials and adjuvants that promote DC metabolic health augment the immune response in aged mice. Different vaccine formulations will compare adjuvants that produce high glycolytic responses with formulations that retain some oxidative phosphorylation and spare respiratory capacity to optimize DC function. In the second aim, we will optimize the nanovaccine formulation(s) that enhance B cell activation in aged mice and peripheral blood B cells from aged humans. Additionally, we will identify mechanisms by which our nanovaccine improves T follicular helper responses and the induction of protective immunity on an aging background. Traditional inactivated IAV vaccine will be used as a control so as to identify the formulation providing superior protection than the current vaccine. In Aim 3, we will determine how nanovaccine-induced metabolically-optimized DC-T cell priming contributes to T cell memory and heterologous protection against IAV in aged mice. Measures of viral load, serum antibody, and lung T cell responses will be evaluated in homologous and heterosubtypic IAV challenges in aged mice. The long-term goal of this research is to define the mechanisms responsible for induction of protective immune responses in aging populations, thus facilitating the rational design of improved vaccines for this underserved population.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Filariases are a group of neglected tropical diseases produced by infection with microfilaria of Clade III parasitic nematodes that are transmitted by biting insects. One example is the lymphatic filariasis produced by Brugia malayi. Lymphatic filariasis is a debilitating and disfiguring disease which occurs in 120 million people worldwide. Other filarial diseases are River Blindness produced by Onchocerca volvulus and loiasis produced by Loa loa. Prevention and treatment of these nematode parasite diseases relies on the use of anthelmintic drugs because no effective vaccines are available. Prophylaxis using Mass Drug Administration [MDA] programs are limited by the efficacy of existing anthelmintics. Diethylcarbamazine is a mainstay for the treatment of lymphatic filariasis and loiasis in most parts of the world, except in areas where onchocerciasis is present because it is contra-indicated by risks of blindness. Diethylcarbamazine produces rapid clearance of microfilaria and causes ~40% mortality of adult parasites (macrofilaricide). A number of studies have suggested that diethylcarbamazine has an indirect host- mediated mode of action and that diethylcarbamazine acts by changing host arachidonic acid pathways. We have observed that diethylcarbamazine has direct effects on filarial nematodes. We present preliminary observations that show that diethylcarbamazine increases the opening of TRP-2 channels in Brugia malayi, and opening of calcium-activated SLO-1 K channels. The effect is a rapid, transient inhibition of motility followed by recovery: the response accommodates. Emodepside is an emerging and important cyclooctadepsipeptide class of anthelmintic that also has effects on microfilaria and adult filaria. Emodepside treatments could allow a major advance over existing mass drug administration (MDA) programs which require regular treatments to kill adult parasites. One of the sites of action of emodepside is on nematode SLO-1 K channels where opening of the channels inhibits motility, but it is not effective against all filaria. Here we propose to compare effects on filarial SLO- 1 K channels from Brugia, Onchocerca and Loa and to examine actions and interactions of these two drugs to explore their mode of action. We have 3 aims: Aim #1: Characterize, in vitro, the concentration motility-inhibition-response relationships of diethylcarbamazine and emodepside and their combination on: A) Brugia microfilaria; B) Brugia adult females; C) Brugia adult males. We will test the hypothesis that effects of diethylcarbamazine and emodepside are additive, synergistic or antagonistic and dependent of life-cycle stage and sex. Aim #2 Characterize the SLO-1 K channel current responses to diethylcarbamazine and emodepside in isolated Brugia malayi muscle flaps under patch-clamp We will test the hypotheses: a) that the effects of emodepside and diethylcarbamazine interact; b) that the interactions of diethylcarbamazine and emodepside are dependent on the presence of TRP-2 by knockdown of TRP-2 channels; and c) that TRP-2 and SLO-1 channel message & channel opening accommodates during prolonged exposure to diethylcarbamazine or emodepside. Aim #3: Characterize the comparative molecular pharmacology of: a) different SLO-1 K channels of Brugia malayi, Onchocerca volvulus and Loa loa and; b) TRP-2 channels of Brugia malayi and Loa loa expressed in oocytes. We will test the hypothesis that the pharmacology and potencies of emodepside and diethylcarbamazine on SLO-1 K channels and TRP-2 channels of, Brugia, Onchocerca and Loa are different and also different to a human channel homologue. We will examine channel desensitization. The proposal is innovative, using a combination of techniques to test the effects of diethylcarbamazine and emodepside on their putative target sites, SLO-1 K channels of filarial. The overall impact of using our mixture of techniques, will be discovery and comparison of effects of diethylcarbamazine and emodepside on different species of filarial TRP-2 channels and SLO-1 K channels. Knowledge of the molecular actions of these drugs is required for: a) molecular detection of sensitivity of different filarial species and resistance; b) designing new drugs and combination therapies; c) predicting and understanding sensitivities of different nematode parasite species; and d) predicting possible host toxicity.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Biomolecular condensates formed through phase-separation of proteins and nucleic acids are integral compo- nents of eukaryotic cells, playing key regulatory roles in various cellular processes. Recent studies have revealed a broad spectrum of dynamic behaviors in condensates, likening them to elastic gels, viscoelastic polymers, or soft, glassy materials. The dynamic and material properties of condensates impact the timing of numerous es- sential cellular processes, including the diffusion and retention of regulatory molecules, signaling, and catalytic reactions. Furthermore, the material properties of condensates change over time during aging, which can lead to irreversible transitions into solid-like pathological states such as amyloids. Although it has been shown that material properties in cells can be regulated and tuned through sequence and composition, the molecular rules governing the material properties of condensates remain poorly understood. Furthermore, there is a lack of mechanistic models that can elucidate dynamic regulatory relationships between condensate material properties and biochemical processes inside them. This proposal aims to decipher the molecular sequence grammar of condensate material properties and their impact on the diffusion and partitioning of regulatory molecules, irre- versible aging, and active biochemical reactions. By combining multi-scale computational techniques, we will create microscopically detailed models of viscoelastic properties of condensates as a function of sequence pat- terning, solvent and environmental conditions, and biomolecular composition. Subsequently, we will dissect the impact of foldable and rigid elements of biomolecules by investigating transcription factor-DNA and nucleosomal condensates with varying folding topologies and disordered linker sequences. Our detailed molecular simulations will ultimately be used to deploy physics-based machine-learning tools, enabling us to predict condensate mate- rial properties based on composition, sequence, and environmental conditions. In another proposed direction, we will utilize non-equilibrium models developed in our group to simulate condensate aging and explain the formation of pathological solid-like states with molecular-level detail. Lastly, we will use non-equilibrium particle and phase- field-based models to investigate dynamic regulatory feedback between material characteristics with active bio- chemical processes such as microtubule assembly and enzymatic reactions. Completing the proposed research program will establish sequence-material properties-function relationships, providing far-reaching insights into condensates' biological functions. Studying condensate material properties will also provide invaluable insights into human health because of the close connection of material properties to the propensity of condensate to age and form pathological states implicated in numerous neurodegenerative diseases. the acquired fundamental biophysical knowledge will enable the targeting of material properties of condensates, halting pathways toward pathological states of condensates.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract: Inhibitors of the PD-1/PD-L1 axis has been successful across multiple diseases. However, only a small subset of patients respond to these regimen and identifying patients likely to benefit from these therapies remains challenging. Current clinical standard relies on histopathology that fail to accurately predict PD-L1 due to spatial and temporal heterogeneities among patients. Further, screening patients for PD-L1 alone is not predictive of treatment response due to significant variabilities in PD-L1 assays across labs necessitating simultaneous detection of multiple immunomarkers. This establishes our scientific premise that an urgent need exists for accurate noninvasive diagnostic tools that enables detection of both PD-L1 and other markers involved in immune modulation directly in vivo. Whereas ImmunoPET (positron emission tomography) imaging has transformed our ability to detect single immunomarkers in vivo, multiplexing cannot be achieved with PET as signal between radionuclides cannot be distinguished. Without the ability to multiplex, patients would undergo multiple radiotracer dosing and repeated radiation exposure. Further, dynamic changes in immunomarkers during treatment would be missed as sequential dosing of different radiotracers would require >1 week wait time between doses to allow for decay of the radiotracers. Our objective is to address the limitations of current approaches and enable multiplexed detection of both PD-L1 and CD8+ T cells in vivo with an innovative nanoprobe, immunoactive gold nanoparticles (IGNs). IGNs labeled with antibodies, Raman reporters, and 89Zr radiotracers synergistically integrates the merits of immunoPET with surface-enhanced Raman spectroscopy (SERS). SERS, an optical technique, uses near-infrared light to enhance the vibrational signal of Raman reporters enabling narrow spectral features amenable for multiplexing. Our approach is unique because clinically-translatable IGNs seamlessly combine the depth-resolved whole body imaging of PET with the high resolution and multiplexing ability of SERS enabling simultaneous detection of both immunomarkers in vivo with high specificity. Detection of both immunomarkers in vivo is important because dynamic changes occur in both PD-L1 and CD8 during and after treatment that are not captured by static measure of receptors or by single biomarker imaging. Whereas immunomarker detection with IGNs is relevant to many diseases, we will use mouse models of breast cancer (BC) since PD-L1 and CD8 immunomarkers play a critical role in BC treatment response. IGNs will detect both PD-L1 and CD8 in orthotopic BC mouse models (Aim 1), monitor response to immunotherapies (Aim 2), and validate in clinically-relevant humanized mice (Aim 3). IGNs is a generalizable platform and ultimately our strategy can be mapped onto other diseases including infection and autoimmunity where PD-L1 and CD8 biomarkers also play a key role. Further, IGNs can also be targeted to a number of other biomarkers via antibodies facilitating treatment response in multiple disorders with unprecedented accuracy not achievable with current clinicopathological approaches.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY / ABSTRACT Overview of research in the laboratory – The Reuel Group is focused on sensor and probe development to provide data necessary for machine learning (ML) enabled optimization of biologic products and processes. Active learning techniques, such as reinforcement learning, can discover new materials and methods but are reliant on large, standardized data sets that can be efficiently and iteratively collected in the lab. For the optimi- zation of protein products (MIRA project focus) fluorescent nanoprobes built off single walled carbon nanotubes (SWCNT) are used to rapidly characterize proteins prototyped in cell free expression. For the optimization and control of enzyme production in fermentation (BioMADE project) SWCNT probes are used on sampled fermen- tation broth. For the optimization of cell therapy manufacturing (NSF CAREER and EPSCoR projects) radio frequency based resonant sensors and imaging are used to track cell expansion and differentiation. To make progress in these fields, the group has built up capabilities to test and establish whole workflow solutions, includ- ing the capacity to make and design new sensors, conduct high throughput cell and cell free biologic experiments with robotic fluid handlers, and apply the latest active learning algorithms to these data to generate new protein sequences and control cell production process parameters. Goals for the next five years – The Reuel Group aspires to produce materials and methods for ML assisted biologic product discovery and manufacturing that are simple, robust, and straightforward to use, such that any lab wishing to make a new custom protein or cell production process would be able to use these powerful tech- niques. The methods and workflows will be described in publications, but most of the resources will also be embedded into accessible web interfaces where one would specify a target, be directed to the necessary exper- iment to run, upload data for analysis, and then receive generated next experiments until convergence on a new optimized product is achieved. To support the experiments charted through this web interface, the reagents from this work will be made commercially available through licensing or new company formation. Overall vision of the research program – Biology is the solution to many of the world’s pressing health and sustainability needs. We firmly believe that new cell and protein-based products can be used to cure diseases, improve nutrition, track health, and provide sustainable fuels, food, and materials. Natural and directed evolution has provided a wealth of cell and protein products that are used currently with great impact. Now, with the advent of high-performance computing and AI techniques, new design spaces, yet explored by Nature, can be effectively searched. The Reuel Group envisions the need for large, iterative data sets to support this exploration and is working to that end through this MIRA support to design the foundational materials and protocols necessary. These will then be leveraged into follow-on, collaborative research projects by our group and made available to the world to support new protein design.

NIH Research Projects · FY 2025 · 2020-09

Project Summary/Abstract 1 Detection and surveillance of antimicrobial resistance among enteric bacteria from raw 2 retail meat and seafood in Iowa: A NARMS Retail Food Surveillance Project 3 Antimicrobial resistance (AMR) is considered a silent pandemic threatening the well-being of 4 humans, animals, and the environment. Robust and comprehensive surveillance systems 5 embracing the “One Health” approach are necessary for effectively combatting AMR. In the 6 United States, the retail arm of the National Antimicrobial Resistance Monitoring System 7 (NARMS) under the Food and Drug Administration (FDA) performs surveillance of AMR in 8 select enteric bacteria (Salmonella, Campylobacter, Enterococcus and Escherichia coli) from 9 retail raw meat (chicken, turkey, beef, and pork) across the nation. In addition, surveillance of 10 AMR in retail seafood (tilapia, shrimp, salmon) for select bacteria (e.g., Vibrio, Aeromonas, 11 Enterococcus) is also included. The NARMS program has contributed significantly to 12 monitoring the sources and trends of AMR in the food chain. To enhance the effort on combating 13 AMR, FDA launched a cooperative agreement program to expand the NARMS surveillance 14 network throughout the U.S in 2016. Under the initiative, Iowa State University (ISU) became a 15 NARMS network laboratory in late 2016. Since then, the ISU site has contributed substantially 16 to strengthening the capacity and effectiveness of the national AMR surveillance program in 17 retail food. In this application, we propose to continue the collaborative partnership with 18 NARMS to address the updated goals of the Retail Food Surveillance Program as described in 19 FON PAR-25-014 “NARMS Cooperative Agreement Program to Strengthen Antibiotic 20 Resistance Surveillance in Retail Food Specimens”. Specifically, we will continue to collect and 21 process raw meat and seafood samples from grocery stores in Iowa and culture them for isolation 22 of bacteria of interest as defined by NARMS, and ship the isolates to FDA on a monthly basis. In 23 addition, we will continue to perform whole-genome sequencing (WGS) of Salmonella, 24 Campylobacter and E. coli from meat samples (and start WGS of select isolates from seafood) 25 and submit sequences to the NCBI per GenomeTrakr guidelines. This application is built on 26 existing experience and is a natural extension of our current effort with NARMS. Trained 27 personnel and infrastructure required for successful performance of the planned work are already 28 in place. We strongly believe that ISU’s continued participation in this collaborative network 29 will contribute significantly to mitigation of AMR in the food chain and ultimately to the 30 enhancement of food safety and public health.

NIH Research Projects · FY 2025 · 2020-02

Project Summary/Abstract: The promise of stem cell therapies has not been fully realized, due in part to a paucity of appropriate pre-clinical models. The pig is an excellent model of human biology, due to similarities of size, physiology, and genetics, and in some cases may be superior to rodent models, which often fail to provide data which effectively translates to human clinical trials. Thus, pig models that more accurately model humans are critically needed to improve research outcomes of regenerative medicine therapeutics and maximize safe translation to the clinic. As SCID pig models show promise in xenograft studies, this establishes further needs for humanized pigs as second-generation models and improved methods to routinely rear immunodeficient pigs for measurement of survival, safety, and potential efficacy of implanted therapeutic cells. We are focused on generating such new knowledge and demonstrating SCID pig use in preclinical regenerative medicine research. We have developed biocontainment facilities and protocols to raise several genetic lines of SCID pigs. We have demonstrated inability to reject xenografts through successful engraftment with human induced pluripotent stem cells (iPSC), cancer cell lines, and human skin. We have generated both SCNT-based and zygotic mutagenesis-based (non-cloned) RAG2-IL2RG models and have successfully demonstrated partial human immune system development in these pigs. We have also demonstrated engraftment and function of human iPSC-derived cardiomyocytes into the SCID pig heart. Thus we have taken pioneering steps to establish a pig SCID model, but improvements in xenograft safety/efficacy testing and humanization remain prerequisites to harness these research models for translational medicine. The specific aims of this application are to: 1.Determine the extent of human hematopoiesis in existing and newly developed SCID pig models and delivery systems; 2.Validate existing and novel SCID pig models for their value in preclinical safety and efficacy testing of cell and/or tissue xenografts; and 3.Refine second-generation SCID pig model management protocols and delivery systems of the SCID pig model at client locations. The rationale for the proposed research is that our outbred SCID pig may more accurately reflect how proposed stem cell derived therapies will function in humans compared to mice. This project is innovative because we will use an integrated approach to combine research on the model’s xenograft potential with research focused on protocols for improving the use of immunodeficient pigs, including humanization methods. We expect that the successful completion of this project will create genetic resources, data on xenograft and humanization rates, and improved husbandry. All of these will be highly desirable for SCID based modeling on the safety and efficacy of stem cell therapeutics. These unique resources are expected to have a significant impact in accelerating the translation of regenerative medicine research into the clinic.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY/ABSTRACT Investigating the functional aspects of RNA structure is significant as it provides fundamental knowledge of RNA biology and advances emergent technologies that seek to use, and target, RNA therapeutically. Major obstacles to such efforts arise from our limited knowledge of the extent of functional RNA structure encoded within human pathogenic viral genomes. This renewal seeks to advance upon our previous work that developed a methodological pipeline for structured RNA discovery. This approach divides the discovery process into two parts: a scanning step, where long sequences are decomposed into overlapping analysis windows from which structure-related metrics are predicted and a folding step, where consensus structures across overlapping windows are generated revealing the most sequence-ordered, thermodynamically stable structures (an indication of an evolved property). Our approach has been very successful at identifying highly ordered and functional structures across an array of human pathogens, e.g., SARS-CoV-2, where discovered motifs were used to develop therapeutic leads. Our overarching hypothesis is that many regulatory RNA structures remain to be found in the highly restricted space of viral genomes and that they can be uncovered using a holistic research approach that combines computational and experimental methods. In this renewal, our goal is to make improvements to our discovery pipeline to overcome its current limitations (most significantly to address higher order RNA structure) and apply it to medically important viral genomes. The outcomes of this proposal will be: (i) the generation of a robust methodological pipeline for functional RNA 2D and 3D structure discovery; (ii) insight into novel regulatory features of pathogenic human viruses; and (iii) leads for future work to therapeutically target RNA structure.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY/ABSTRACT Enzymes are remarkable nanomachines that play a myriad of essential functions in cellular metabolism. Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of regulation of enzyme function, yet our understanding of the fundamental mechanisms by which concerted protein motion facilitate enzymatic activity is still largely incomplete. Indeed, while several studies have appeared in the past two decades describing how conformational dynamics mediate the biological function of small proteins, our understanding of how the coupling among multiple conformational equilibria determines the activity of large multidomain systems continues to lag. The overall goal of this proposal is using and developing integrated approaches combining NMR with complementary biophysical and biochemical tools to reveal how modulation of local disorder upon cofactor/substrate binding affects concerted motions and regulates the activity of high molecular weight enzymes that are essential for human and bacterial metabolism. This combination of tools sensitive to protein motion brings a newly detailed picture of high molecular weight enzyme function. The enzymes characterized in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the AlkB family of nucleic acid demethylases – together these distinct classes of enzymes show how the relationship between local disorder and concerted domain motions can be probed by this combination of tools and demonstrate how essential these mechanisms are across diverse enzyme classes. In particular, the EI enzymatic activity depends upon the synergistic action of four conformational equilibria that results in a series of large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding. Therefore, our efforts to uncover EI function at atomic level will reveal how modulation of local disorder mediates long-range interdomain communication and, ultimately, regulates the activity of this essential bacterial enzyme. The AlkB dioxygenases are flexible enzymes that are known to undergo modulation of their internal dynamics upon substrate binding. Our studies will visualize conformational disorder in apo and holo AlkB enzymes with unprecedented atomic-resolution details, and will reveal how residual disorder at the active site determines substrate selectivity. In addition, we will investigate a number of complexes formed by AlkB proteins with their inhibitors. We expect these results to indicate new strategies, based on selective perturbation of conformational disorder, to develop AlkB inhibitors with subfamily selectivity. In summary, my research program will elucidate the coupling between large scale conformational changes and function in two distinct classes of high molecular weight multidomain enzymes, providing new insights for future therapies for obesity and cancer as well as novel antibiotic targets. Moreover, these efforts pushing the frontier of the application of biophysical tools to study with atomic resolution the relationship between disorder and functional concerted motions in complex enzymes provide a template transferrable to mechanistic investigations of uncharted multi-domain systems.

NIH Research Projects · FY 2026 · 2017-01

Project Summary The long-term goal of this project is to determine how growth, development and stress responses are coordinated in Arabidopsis, a model plant with extensive genetic, genomic and proteomic resources. This will be accomplished through detailed mechanistic studies that will provide insights into the fundamental conserved biological processes of steroid hormone signaling and autophagy. Brassinosteroids (BRs) are plant steroid hormones that promote growth and development and regulate stress responses. Autophagy occurs in all eukaryotes and degrades organelles and proteins, especially under stress conditions. We have identified multiple mechanisms by which BR signaling and autophagy are coordinated, and generated a regulatory network by combining multiple types of ‘omics data. Key points of interaction between the pathways include degradation of BES1, a transcription factor mediating BR responses, by selective autophagy to reduce growth; and phosphorylation of the TORC subunit RAPTOR1B by the BR-regulated GSK3-like kinase BIN2, leading to an increase in autophagy in response to stress via a switch in TORC substrate specificity. Our new data indicate that the MAP kinase MPK6 functions downstream of BIN2 to negatively regulate autophagy, BIN2 phosphorylates ATG13 to directly regulate autophagy, and BES1 transcriptionally regulates autophagy gene expression through histone 3 lysine 27 (H3K27) methylation, by recruiting H3K27 demethylase REF6 and methyltransferase CLF. We hypothesize that BR signaling regulates autophagy to coordinate plant growth and stress responses, through multiple mechanisms including BIN2 regulation of MAPK6, TORC and ATG13 and BES1 regulation of ATG gene expression. We propose the following Specific Aims to test the hypotheses: 1) to establish the mechanisms of BIN2 and MPK6 regulation of TORC in the control of autophagy and growth; 2) to investigate how BIN2 and TOR phosphorylation of ATG13 regulates its function by mapping phosphorylation sites and mutational analyses; 3) to establish the mechanisms and network through which BES1, together with histone H3K27 demethylase REF6 and methyltransferase CLF, regulates the expression of ATG12 and other genes to control autophagy. These studies will leverage the genetic and genomic resources in Arabidopsis and use multi-omics technologies to generate and integrate information ranging from transcriptome changes to phosphorylation sites and protein-protein interactions. These innovative approaches have the potential to provide transformative knowledge on the mechanisms of integration of growth and stress responses across eukaryotes. For example, autophagy is involved in many human diseases including neurodegenerative diseases, such as Alzheimer, Parkinson and Huntington, and links with steroid hormone action are now being identified, although mechanisms remain obscure. Autophagy also plays critical roles in cancer development, and drugs that modulate autophagy and/or TORC signaling are in clinical use or under development. The proposed studies can therefore provide important insight into processes related to human health.

NIH Research Projects · FY 2025 · 1999-01

Project Summary T cell development, activation, and differentiation are all dependent on TCR signaling, along with important contributions from costimulatory and cytokine receptor pathways. Many lines of evidence indicate that variations in TCR signal strength have a major impact on the outcome of these processes. For mature CD8 T cells, TCR signal strength determines proliferative capacity and effector versus memory potential following infection. We assert that there is a growing need to develop a more comprehensive understanding of the pathways regulating distinct T cell functions and differentiation processes. Moreover, manipulation of T cell responses underlies many therapeutic strategies currently in practice and under development. As this field has progressed, it has become clear there is a need for more nuanced manipulation of T cell signaling pathways. To achieve clinical efficacy, we need to understand how to tune T cell responses. The field presently lacks an understanding of how differences in TCR stimulation strength produce distinct gene expression patterns that steer T cell responses. We now have a body of data supporting a key role for the TEC kinase ITK in tuning T-cell signaling. ITK is not required for all TCR signaling; instead, in its absence, TCR signaling is significantly reduced. From these studies, the clear function of ITK has been difficult to discern, as some aspects of T cell activation appeared normal in the absence of ITK, whereas other T cell functions were greatly impaired. Our data now provide a framework to understand these apparent discrepancies, and suggest that variations in antigen density and in TCR affinity dictate the magnitude of ITK activity modulating subsequent gene expression responses. This proposal will study the contribution of specific ITK activation mechanisms (Aim 1) and costimulatory interactions (Aim 2) to understand how ITK mediates tunable responses to TCR stimulation. Biochemical and structural biology insights are directly transferred into primary T cell experiments to elucidate the molecular mechanisms controlling ITK regulation. We will also study the role of ITK regulation in human disease (Aim 3). Genetic mutations that activate ITK have been recently identified in patients with autoimmunity and there is evidence that the ITK-SYK fusion tyrosine kinase interacts with normal ITK in promoting oncogenesis in peripheral T cell lymphomas. The results of these studies will provide us with critical information about the nature of ITK regulation and how ITK activity influences T cell activation in response to TCR signal strength.