Purdue University

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Ubiquitination, the post-translational attachment of ubiquitin or ubiquitin chains, controls the stability, interaction or activity of numerous key regulatory proteins in eukaryotic cells. Consequently, misregulation in protein ubiquitination can result in various human diseases, such as metabolic disorders, cancers, muscle and nerve degeneration. At the core of the ubiquitination process is the E3 ligase, which brings ubiquitin and the target protein together, and enables the transfer of the ubiquitin to its target. My lab investigates the largest family of E3 ligases, known as Cullin-RING ligases (CRLs). These enzymes are modular protein complexes, featuring a common cullin scaffold and an interchangeable substrate receptor that recruits specific target proteins for CRL- dependent ubiquitination and subsequent degradation. Seven cullins (Cul1-7) exist in human cells, each of which interacts with different sets of substrate receptors, yielding ~250 CRLs. We use a variety of approaches including biochemistry, biophysics, molecular genetics, quantitative proteomics, and mathematical modeling to study how CRLs work, how their activities are regulated, and what critical roles they play in cells and organisms. Given that a large number of substrate receptors compete for access to the same cullin, our current research focus is to uncover how the cellular repertoire of diverse CRLs is controlled to ensure ubiquitination of various CRL substrates at the right time. Using Cul1 based CRL1, we previously reported that CRL1s constantly undergo cycles of assembly and disassembly, which allows rapid recycling of Cul1 and timely formation of new CRLs when their target proteins emerge and demand ubiquitination. A crucial player in this highly dynamic process is Cand1, a protein exchange factor that promotes the exchange of substrate receptors associated with the same Cul1 core. Eliminating the Cand1 activity leads to impaired degradation of CRL1 substrates in human cells and severe developmental defects in multicellular organisms. In this application, we ask, how are the dynamics of other CRLs regulated? What role does Cand2, a homologue of Cand1 in human cells, play in regulating CRLs? What advantage does this evolutionarily conserved dynamic exchange mechanism provide for the CRL system? To answer these questions, we will use in vitro biophysical assays to quantify kinetic parameters for CRL and Cand1/2 interactions. We will apply our updated quantitative immunoprecipitation-mass spectrometry assay to characterize the impact of Cand1 and Cand2 on the cullin-associated proteome. We will employ genome-editing techniques such as CRISPR to examine the biological role of Cand1/2, using cultured human cells and the model plant Arabidopsis as our experimental systems. We will continue developing our mathematical model of CRL assembly and activity, to help understand the CRL network in different cell types or under changing cellular environment. Our efforts in understanding mechanisms regulating CRLs will help dissect the performance of these E3 ligases in normal, diseased, and drug treated cells, providing novel insights for the prevention, diagnosis, and treatment of human diseases.

NIH Research Projects · FY 2025 · 2020-08

Project Summary Emotion dysregulation is recognized as an integral but insufficiently characterized influence in child psychopathology. Attention-deficit/hyperactivity disorder (ADHD) is emblematic because wide variation in both cognition and emotion dysregulation exist and are related to impairment. Consensus is emerging that effective measurement of pathophysiological mechanisms underlying cognitive and emotional features of ADHD is essential for improvement of both pharmacological and non-pharmacological treatments, but these efforts are hampered by within-group heterogeneity. Thus, understanding ADHD mechanistically will require integrating cognitive and emotional accounts of the disorder. A central puzzle concerns the joint disruption of cognition and emotion— frequently observed but poorly explained mechanistically. Two fundamental questions are addressed here: what mechanisms drive emotion dysregulation in ADHD and how can variation in emotional features be incorporated into existing nosology? The current proposal builds on prior work that suggests three temperament-based emotional profiles in children with ADHD: 1) an emotionally-normative, “Mild” profile, 2) a positive dysregulation, “Surgent” profile, and 3) a negative dysregulation, “Irritable” profile. Here, that descriptive work is carried forward the next step into mechanistic and confirmatory study to advance the nosology and explanation of ADHD. Using a cross sectional, case-control design in 7-10 year-old children, Aim 1 validates and refines the proposed emotion-based phenotypes by examining their association with in vivo emotional experience using ecological momentary assessments. This is combined in Aim 2 with an experimental approach to identify reactive and regulatory mechanisms contributing to individual differences in emotion dysregulation in ADHD. Here, sequential sampling models are combined with time-locked measures of central and peripheral nervous system functioning adding novelty. Two types of low-cost, clinically-translatable measures are emphasized: eye-tracking/pupillometry and electroencephalogram-measured event-related potentials (ERPs). Finally, Aim 3 identifies biobehavioral correlates of emotion-related impairment that are directly relevant to development of novel treatments. The theoretically-driven, multi-level approach that is used directly addresses RDoC goals related to multi-method integration and defining mechanisms of complex behavior.

NIH Research Projects · FY 2025 · 2020-08

PROJECT SUMMARY For effective management of the COVID-19 pandemic and its second wave, the design and implementation of multiple intervention approaches are crucial. They include the development of effective antivirals, high-affinity SARS-CoV-2-neuralizing human or humanized monoclonal antibodies, rapid diagnostic assays, immunogenic and protective vaccines, strategies to mitigate virus transmissibility, and enhancing capacity related to trained medical personnel, facilities, and supplies. Due to the possibility of antibody-dependent enhancement (ADE) of COVID-19, vaccine efforts should consider the use of a novel vaccine platform and design of a relevant antigen strategy. It is essential to note that the elderly are the most vulnerable segment of the population that is at a higher risk of COVID-19 severity; the vaccine development efforts should, therefore, consider the decline in the immune competence in the elderly. We have developed a novel replication-defective (E1 & E3 deleted) bovine adenovirus (Ad) type 3 (BAd3)- based vaccine platform, which is better than the currently available Ad vector systems for providing heterologous influenza protection with dose sparing and is not impacted by the pre-existing human Ad vector immunity. Recently, we have revealed that the BAd vaccine platform provides the expression of significantly higher levels of the immunogen and innate and adaptive immunity-related factors compared to that of human Ad vectors in mice. This work suggests that the BAd vector system could serve as an excellent delivery vehicle for the development of recombinant vaccines against emerging pathogens for the elderly and other segments of the population. We have also identified a 22 amino acid residues Autophagy-Inducing Peptide (AIP) C5 (AIP-C5) from the CFP10 protein of M. tuberculosis that enhances robust T cell immune responses in mice to NP of H7N9 influenza virus when delivered through an Ad vector. It conferred complete protection against H1N1, H3N2, H5N2, H7N9, and H9N2 influenza viruses. The proposal is based on the hypothesis that immunization with the autophagy-inducing replication-deficient BAd vector expressing relevant antigen/s of SARS-CoV-2 will strengthen an effective mucosal (lung) and systemic anti-COVID-19 immunity. Under Aim 1, we will evaluate the immunogenicity and protective efficacy of a novel vaccine platform and antigen design in animal models for developing an effective COVID-19 vaccine. Whereas under Aim 2, we will investigate the vaccine-induced antibody-dependent enhancement (ADE) of SARS-CoV-2 infection, the quality of memory innate, B and T cell responses, and the durability of protective immunity in the best animal model. We believe that the use of a unique nonhuman Ad vaccine platform and novel antigen design containing AIP-C5 will yield an effective COVID-19 vaccine for all segments of the population. This effort will be of significant value to effectively flatten the COVID- 19 pandemic's trajectory and its second wave.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT The lack of diversity (91.2% White) in the veterinarian-scientist workforce is a very serious concern and impacts the profession’s ability to serve a society undergoing dramatic demographic shifts. Veterinary colleges and professional organizations have put forth significant effort to recruit underrepresented students; however, most efforts target high school students, a time when racial and ethnic disparities are already evident. Meanwhile, young children are ruling out veterinary careers because of a lack of relatable role models. The overall vision of this team is to establish a diverse veterinary workforce by developing a far-reaching, inclusive veterinary STEM ecosystem consisting of US veterinary colleges (28/30 (93%) participating), veterinary work settings (practice, research, industry, government), and community entities serving disadvantaged youth. By working towards this critical vision, See Us-Be Us will be able to meet the goals of the 2014 NIH Physician- Scientist Workforce Working Group and the 2018 Committee on STEM Education of the National Science and Technology Council. As a step toward this vision, See Us-Be Us will provide a supportive role model network and veterinary STEM educational resources to inspire and prepare underrepresented youth to pursue careers in veterinary medicine. This goal will be attained through three specific aims. The focus of Aim 1 is to establish an army of diverse, equity-minded veterinarians who will provide disadvantaged K-4 students direct, supportive veterinary role models. Veterinary medical students will be certified to deliver STEM lessons to children in communities surrounding veterinary colleges, while in school, and then after graduation, will be equipped and supported to continue this effort in their home communities. Aim 2 is focused on developing SuperPower Packs, self-guided veterinary STEM educational experiences that feature underrepresented veterinarians, for disadvantaged children lacking access to direct supportive veterinary role models. Aim 3 develops a Web-based role model networking hub and podcasts, for middle and high school students to learn about relatable veterinary professionals, find specific career information, and take advantage of experiential and mentoring opportunities. Combined, these aims will enhance opportunities for students from underserved communities, beginning in elementary school, to have authentic veterinary STEM experiences while being supported by diverse veterinary role models, as they advance through the educational pipeline. Elementary school teachers will serve as consultants to ensure that educational materials are consistent with Next Generation Science Standards, and will assist in training role models to better communicate the societal impact of their work. The ecosystem will continue to use the successful model of engaging students in STEM activities by communicating the impact of veterinary medical research on public health and animal health challenges. Ultimately, this project will educate and facilitate career exploration and experiential learning opportunities for K-12 students, particularly those from underserved communities, to careers as veterinary scientists.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by employing guide RNAs and endonuclease effectors to specifically recognize and cleave invasive nucleic acids. The specific DNA targeting and cleavage activities of CRISPR-Cas systems have been adopted and developed for genome editing and various other applications, which are revolutionizing biomedical research and beyond. However, safety concerns are raised because of off-target genome editing and the dependence of these systems on endogenous host DNA repair pathways, hindering clinical application. Exploration of alternative CRISPR-Cas systems in nature not only offers an opportunity to overcome those challenges but may also inspire new applications. Structural and biochemical characterizations of CRISPR-Cas systems are critical for understanding their mechanisms and repurposing them for precise genome editing. Our long-term goals are to unravel the mechanisms underlying target nucleic acid recognition and cleavage mediated by type V and transposon-associated CRISPR-Cas systems, which provide essential knowledge for safer and more reliable application in treating human disease. In this proposal, we will work on the molecular mechanisms for four newly discovered CRISRP-Cas systems, covering DNA targeting (Cas12i), RNA targeting (Cas12g), and CRISPR RNA-guided DNA transposition (type I-F Cascade and Cas12k). As revealed in our preliminary data, Cas12i accommodates a longer crRNA-DNA heteroduplex than currently used Cas effectors, thus potentially improving specificity for genome editing. The RNA-guided RNase Cas12g is compact and thermostable, highlighting its potential for RNA editing and RNA targeting. Furthermore, type I-F Cascade and Cas12k direct transposition machinery for RNA-guided DNA transposition, opening a new paradigm for genome editing independent of DNA repair pathways. 1

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Torsades de pointes (TdP) is a ventricular tachycardia associated with prolongation of the corrected QT (QTc) interval, and which may be caused by > 150 widely used drugs. TdP results in catastrophic outcomes, including sudden cardiac death. Older age is a risk factor for drug-induced TdP, possibly due to declining serum progesterone and testosterone concentrations in postmenopausal women and men, respectively. The ECG biomarkers J-Tpeak and Tpeak-Tend, represent early and late repolarization, respectively, as well as dispersion of repolarization (Tpeak-Tend). Preclinical evidence and preliminary data from our group indicate that progesterone and testosterone exert protective effects against drug-induced prolongation of ventricular repolarization. Effective means of reducing the risk of drug-induced QTc interval prolongation and TdP in high risk populations requiring therapy with QTc-prolonging drugs have not been identified, and the effects of sex hormones on early vs late ventricular repolarization and dispersion of repolarization are unknown. The objectives of this research are to evaluate novel therapeutic approaches to attenuate drug-induced QTc lengthening. Our central hypothesis is that drug-induced QTc lengthening is attenuated by administration of oral progesterone and transdermal testosterone. Specific Aim 1: Determine the efficacy of oral progesterone as a preventive method to attenuate drug-induced QTc interval lengthening in postmenopausal women. Specific Aim 2: Determine the influence of oral progesterone on drug-induced lengthening of early and late ventricular repolarization in postmenopausal women. Specific Aim 3: Determine the efficacy of transdermal testosterone as a preventive method to attenuate drug-induced QTc interval lengthening in men ≥ 65 years of age. Specific Aim 4: Determine the influence of transdermal testosterone on drug-induced lengthening of early and late ventricular repolarization in men ≥ 65 years of age. Specific Aims 1&2 will be achieved via a prospective, randomized, double-blind, placebo-controlled two-way crossover study in postmenopausal women age ≥ 50 years (n=48). Each subject will take oral progesterone 400 mg or matching placebo daily for 7 days (≥ 14-day washout period between phases). On day 7, each subject will receive a single dose of the QTc-lengthening drug ibutilide 0.003 mg/kg. Specific Aims 3&4 will be achieved via a prospective, randomized, double-blind, placebo-controlled two-way crossover study in men ≥ 65 years of age (n=35). Each subject will apply transdermal testosterone 1% 100 mg or transdermal placebo once daily for 3 days (≥ 7-day washout period between phases). On day 7, each subject will ibutilide 0.003 mg/kg. In both studies, post-ibutilide QT, J-Tpeak and Tpeak-Tend intervals and serum ibutilide concentrations will be determined serially. Primary outcome measures: 1) Maximum post-ibutilide QTc intervals, 2) Maximum post-ibutilide % change in QTc intervals, 3) Area under the QTc interval-time curves, and 4) J-Tpeak and Tpeak-Tend intervals. This research will identify effective approaches for reducing the risk of drug-induced QTc interval prolongation in high-risk patients.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY It was long taken for granted that obstructive coronary artery disease (CAD) is the primary driver of angina and major adverse cardiac events. However, recent landmark studies have shown that up to 50% of the patients referred for diagnostic testing have ischemia with no obstructive CAD (INOCA). A large proportion of INOCA patients have coronary microvascular dysfunction (CMD), which even in the absence of flow-limiting stenoses can lead to myocardial ischemia and carries a high risk of adverse events. The reference standard for assessment of CMD is the functional coronary reactivity (CR) test, which is invasive. Despite key studies showing value of stratifying therapy based on CR testing, the practical utility of CR testing in the INOCA population is limited by its invasive nature, which carries serious risks even at experienced centers. Hence, a noninvasive approach that can detect and stage the severity of CMD would be invaluable for managing INOCA patients. Driven by this unmet need, prior studies have employed imaging approaches to index myocardial perfusion reserve (MPR) against CR; however, the association shown to date between MPR and CR impairment has been weak, likely due to the suboptimal sensitivity of MPR to subendocardial myocardial blood flow (MBF) deficits which is a hallmark of CMD. Studies using invasive microsphere-based methods have established a stress subendocardial-to-subepicardial (endo-epi) MBF gradient of larger than 1.0 in healthy animals, and shown that it decreases well below 1 under abnormally elevated microvascular resistance. However, noninvasive detection of endo-epi MBF gradients using existing imaging strategies is challenging because of the need to resolve MBF transmurally. We have developed new MRI strategies aimed at overcoming key barriers for accurate evaluation of endo-epi MBF gradients and applied them in preliminary animal and patient studies. Based on our preliminary data, we hypothesize that in the setting of CMD, impaired microvascular CR manifests as a stress-induced endo- epi MBF gradient, and the magnitude of this gradient significantly correlates with CMD severity. To test this hypothesis, we propose 3 specific aims. In Aim 1, we will develop a free-breathing artifact-free MRI technique optimized for high-resolution imaging of endo-epi MBF gradients, combined with a machine learning approach for fully-automated objective quantification of MBF gradients. In Aim 2, we will test the hypothesis that CMD severity can be staged on the basis of MRI-derived stress MBF gradient in a pig model of CMD. In Aim 3, we will test the hypothesis that CMD severity in INOCA patients is highly correlated with MRI-derived stress MBF gradient. This project brings together multiple interdisciplinary investigators with a strong collective track record in developing cardiac imaging strategies to advance a noninvasive approach for determining CMD severity based on the MRI-derived stress MBF gradient. Hence the proposal is a major step towards improving the management of INOCA patients and towards imaging-guided evaluation of novel therapies aimed at CMD.

NIH Research Projects · FY 2024 · 2020-07

Reactive oxygen species (ROS) can act as signaling molecules mediating physiological functions in immunity, cell proliferation, differentiation, and migration. Whether ROS have a major signaling function as second messengers in axonal growth and guidance is currently unclear. The neuronal growth cone is a highly motile structure at the tip of neuronal processes, guiding them to appropriate target cells during development and regeneration of the nervous system. The growth cone integrates molecular information from the environment and transduces it via multiple signaling cascades to affect underlying cytoskeletal dynamics. Whereas most major second messenger systems have been implicated in regulating directional growth cone movement, such a role has not been established for ROS. The present study has two major objectives focusing on ROS produced by nicotinamide adenine dinucleotide phosphate-(NADPH) oxidase (Nox): (1) to determine the cellular and molecular mechanism by which ROS control neurite growth; and (2) to determine whether ROS act as second messengers downstream of specific guidance cues to control axonal growth and guidance. The four central hypotheses state that (1) a physiological level of ROS is optimal and required for adhesion-mediated neurite growth; (2) Src tyrosine kinase is a key target of ROS signaling in neuronal growth cones; (3) neuronal Nox2-derived ROS regulate axonal pathfinding; and (4) specific axon guidance cues such as slit2 control axonal pathfinding via Nox2-derived ROS both in vitro and in vivo. This project will take advantage of two excellent model systems to test these hypotheses: large Aplysia growth cones for quantitative live cell imaging of growth cone motility and intracellular ROS in vitro and developing zebrafish embryos for imaging and manipulating axonal development in vivo. In vitro growth cone guidance assays, novel fluorescent dyes and biosensors specific for hydrogen peroxide and Src activity, respectively, advanced imaging techniques, chimeric analysis of Nox2-deficient zebrafish lines as well as retinal ganglion cell-specific Nox2-mutant fish lines will be used to address the following two Specific Aims: (1) The first aim is to determine the cellular and molecular mechanism by which ROS in control neurite growth. (2) The second aim is to determine the role of neuronal Nox2 in axonal pathfinding of retinal ganglion cells. The proposed work is highly innovative because it investigates ROS as a novel group of signaling molecules in axonal growth and guidance and develops several new zebrafish lines suitable for studying Nox function in the nervous system. In summary, these studies have the potential of leading to breakthrough findings in the field of neuronal development and regeneration. Furthermore, since basic mechanisms of axonal growth and guidance are highly conserved across species, these studies will impact the development of antioxidant treatments for neurodegenerative diseases and central nervous system injuries.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY/ABSTRACT Natural products from the soil-dwelling bacteria Streptomyces have been a rich source of medicines. Additionally, the enzymes that produce natural products perform unique and challenging chemistry, providing inspiration for novel biocatalysts. One important class of natural products is cyclic nonribosomal peptides. Unfortunately, the discovery of novel cyclic peptides using traditional techniques is often unsuccessful. Also, chemical synthesis of cyclic peptides, especially small strained tetrapeptides, remains a challenge, resulting them being underexplored as potential therapeutics. Genomics data suggests a plethora of novel Streptomyces cyclic peptides and their biosynthetic enzymes remain to be discovered. However, the biosynthetic machinery responsible for producing these novel natural products is often cryptic (i.e. transcriptionally inactive). A significant gap remains in the strategies available to discover new bioactive cyclic peptide natural products and to synthesize these peptides, especially cyclic tetrapeptides, for further analysis. Our long-term goals are to 1) Discover biosynthetic enzymes that perform unique and challenging reactions and develop these enzymes as biocatalysts, and 2) Develop bioinformatics and synthetic techniques that allow us to directly access natural products from cryptic biosynthetic gene clusters. Our current research objectives are to 1) Determine activities and substrate scopes for previously identified, as well as bioinformatically predicted, tetrapeptide cyclases and develop the most promising ones as biocatalysts and 2) Utilize bioinformatics methods to identify nonribosomal peptides of interest followed by direct chemical synthesis and biological testing to identify bioactive leads. Our recent discovery of the first standalone tetrapeptide cyclase and our development of the Synthetic Natural Product Inspired Cyclic Peptide (SNaPP) methodology, make us well positioned to complete these objectives. The central hypothesis of the first project is natural product biosynthetic enzymes will be efficient biocatalysts for the generation of cyclic tetrapeptides that otherwise are very challenging to access. The objectives of the first project are to 1) better understand the mechanism of Ulm16, a known tetrapeptide cyclase 2) bioinformatically identify new tetrapeptide cyclases and 3) apply these tetrapeptide cyclases to the synthesis of bioactive cyclic tetrapeptides and the development of cyclic tetrapeptide libraries for screening. In the second project, we are directly chemically synthesizing natural products that are bioinformatically predicted from nonribosomal peptide synthetase biosynthetic gene clusters. While our previous work in this area has resulted in promising bioactive leads, challenges remain including accurate predictions of off-loading methods, unnatural amino acids, and tailoring enzymes. The objectives for the second project are to address these challenges by incorporating predictions of these three areas into our predicted peptides and then synthesizing them. This work will provide access to many novel natural product scaffolds that are currently inaccessible and that are likely to have interesting bioactivities.

NIH Research Projects · FY 2024 · 2020-06

Project Summary While treatments for eradicating some infectious diseases have been successful, there is still a large gap in the treatment of infections caused by bacteria, viruses, fungi and parasites, in particular as drug resistance continues to grow as a problem. To address this evolving issue, scientists are needed who are trained to understand how pathogens interact with the host, in order to better uncover opportunities and strategies for drug discovery. This training program’s goal is to address this need by providing qualified graduate students with broad training in infectious diseases and drug discovery, complemented by professional development opportunities, that prepares them for a long productive career in infectious disease related research. The training program includes 21 faculty members from 6 departments across 4 different colleges at Purdue University. These faculty members have diverse and complementary research interests in infectious disease, drug discovery and drug delivery, structural biology, and molecular biology. All of these faculty members are part of the Purdue Institute of Inflammation, Immunology and Infectious Disease and the Purdue Institute for Drug Discovery. These institutes bring faculty and trainees together through shared research space, a seminar series, journal clubs, and social events, resulting in a unique and supportive training environment for the “Drug Discovery in Infectious Disease Training” program. In this program, students will develop expertise both in infectious disease and in drug discovery. Specifically, trainees will take a course focused on: 1) infectious diseases and drug discovery and 2) biological membranes (a common theme of pathogen entry, exit and replication). To promote responsible, rigorous and reproducible research practices, students will complete courses in statistical analysis and the responsible conduct in research. To develop written communication skills, trainees also will take a grant-writing course, where they participate in a collaborative, active learning environment to gain feedback on the development of an NIH F31 style proposal. Additional training activities include participating in a seminar series, attending regional/national scientific meetings, and completing a capstone project in the area of drug discovery in infectious disease. In completing this semester-long capstone project, trainees will build teamwork, critical thinking and data analysis skills, all while working in the context of drug discovery and infectious disease. The capstone project will be complemented with networking and learning opportunities with industrial partners who are developing new drugs for infectious diseases. Further, fellows have the opportunity to engage in entrepreneurial training, an important aspect of academic drug discovery. Using a jointly developed Individual Development Plan, the trainees and mentors will create a training plan that is tailored towards the students’ training needs and career goals. In the end, the training program will have helped students work towards their desired career goals, while also developing a workforce with a unique skillset for addressing the challenges of discovering therapies for infectious disease. !

NIH Research Projects · FY 2025 · 2019-07

The mission of the Purdue University Molecular Biophysics Training program (MBTP) is to bring together outstanding graduate students from multiple departments and colleges across campus into a cohesive training program that (i) provides enhanced training in the rigorous and reproducible application of molecular biophysics to modern problems in human health and disease, (ii) fosters effective mentorship and teamwork, and (iii) offers career development opportunities tailored to individual trainees. Trainees are typically appointed for two years at the beginning of their second year of study after they join one of 32 preceptor labs. They choose from a rigorous palette of biophysics courses appropriate for their individual thesis projects and participate in hands-on workshops provided by local and/or national experts. They help develop and host an interdepartmental biophysics seminar series called the Frontiers in Biophysical Sciences Seminar, which showcases trainee-selected external speakers as well as the research of trainees across campus. They also independently plan and implement Purdue’s annual biophysics symposium called the Hitchhiker’s Guide to the Biomolecular Galaxy. Trainees also benefit from exercises in teamwork built into the program coursework and symposium planning, active development and implementation of detailed individual development plans, personalized teaching opportunities in preceptor classes, a grant-writing class tailored to biophysical topics designed to launch F31 proposals, and training in the responsible conduct of research including lectures from MBTP preceptors. By leveraging Purdue’s investment in Biology Education and self-assessment, the training program and its individual activities are evaluated and refined annually to ensure that the program is meeting trainee needs and program objectives, with the long-term goal of producing excellent biophysicists who can meet the needs of an increasingly complex world.

NIH Research Projects · FY 2026 · 2018-09

Project Summary/Abstract Our research program involves the use of DNA-encoded chemical libraries (DELs) and DNA-linked enzyme activity probes, which are new approaches for biomedical research that capitalize on the power of DNA analysis techniques. Specifically, this work involves development of in vitro selection assays for both DELs and enzyme activity probes. It is the in vitro selection that encodes transduces information (drug molecule activity or biochemical activity of a sample) into DNA sequences to facilitate analysis. This work advances these techniques into new areas, particularly for medicinal chemistry applications, to provide tools for biological discovery and development of new therapeutics. We are using DELs in a directed, targeted way (on-DNA medicinal chemistry) to produce inhibitors to the chromodomains in the CBX family and to several bromodomains. The homology of the chromodomains in the eight chromobox (CBX) proteins and of the family of bromodomains (61 in humans) makes selective inhibition challenging. Inhibitor probes generated will be used to the decipher roles of these proteins in transcriptional regulation and in disease states. DELs are now routinely used for de novo discovery of compounds that bind to a drug target to initiate a drug development campaign. The selection assay used for this discovery is a simple affinity purification with a purified protein on a solid support. The requirement of a pure and active protein for this assay severely limits the target scope of DELs, particularly for membrane bound protein targets, which constitute a large portion of drug target space. We are developing selection assays to enable use of DELs to protein targets both on and within live cells. These assays rely on bioluminescence resonance energy transfer (BRET), which is a common modality for detection of interacting molecules in cells. We will apply these unique assays with highly diverse (>109), commercially available DELs to challenging protein targets including Nrf2 (potential cancer target) and adenylyl cyclase 1 (a potential target for pain). In addition, we are developing selection assays to identify molecules that not only bind to a protein receptor but activate downstream signaling pathways. We are applying this selection to the opioid family of GPCRs to identify novel agonists. We will advance to use DNA-linked enzyme activity probes for the proteomic profiling of tyrosine kinase activities and for drug binding assays amenable to high throughput screening of traditional (off-DNA) compound collections. We are implementing our DNA-based kinase activity profiling to further understand the mechanism of drug resistance to tyrosine kinase inhibitors in cancer therapy. Also, the high sensitivity of the approach (enabled by DNA amplification) will be used to assay kinase activities in single cells, which will provide a greater understanding of cellular complexity within tumors.

NIH Research Projects · FY 2025 · 2018-07

There is a national need to advance the understanding of hearing in both healthy patients and those with various causes of hearing loss, which contribute to reduced quality of life and are correlated with much poorer health outcomes. The objective of this continuing training program is to train the next generation of faculty who will populate colleges of science, engineering, and health sciences, as well as to send graduates into industry prepared to work toward creative solutions for treating hearing loss. Specifically, in order to advance auditory neuroscience training, this graduate training program leverages faculty expertise in both basic hearing science and technology development, from three Purdue University colleges (Science, Engineering, and Health & Human Sciences) and 8 doctoral admissions programs. Two types of investigators are included: 10 hearing scientists with focused research programs related to auditory-system neuroscience, and 12 technology innovators trained in other disciplines (e.g., biomedical and electrical engineering, computer science). Collectively and collaboratively, the program expands knowledge about mechanisms at the molecular, cellular, and systems levels that underlie auditory information processing. This fundamental knowledge can then be applied to better understand the changes that lead to pathologies of the auditory system due to damage, disease, aging, and congenital disorders, as well as understanding how hearing evolved and influences behavior and natural selection. Technological approaches to these questions include, but are not limited to, super-resolution microscopy, biological implants for neuromodulation, high-resolution four-dimensional calcium imaging deep in the mammalian brain, optogenetics and robotics (automated patch-clamping) for brain circuit analysis, and multimodal brain imaging methods, coupled with advanced approaches in statistical and data analysis and model development. TPAN is unique in that it is specifically designed to serve students with undergraduate degrees in the disparate disciplines of life science, physical science or engineering, and merge them into a unified cohort focused on auditory neuroscience. Three fellows are selected for a 2-year term on the training grant, ideally beginning at the start of their second year. The training curriculum includes 4 core courses (one each in neuroscience, the auditory periphery, central/behavioral auditory science, and signal processing), a required grant-writing and statistics course, a weekly Hearing Science seminar series where students and faculty present, and yearly attendance at extramural training courses and/or auditory-neuroscience conferences. Vertical and horizontal mentoring provide training and support at all levels, to enhance scientific networks, and to foster a sense of community among all hearing-science students (current and recent TPAN fellows, plus TPAN affiliates) that promotes success in the PhD and afterwards. Support is provided, in collaboration with Purdue’s Institute for Integrative Neuroscience, for an individual data-management consultant for each Fellow, individually budgeted training-related expenses, as well as for administrative support.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY Like the challenges and skepticism that faced the antibody therapeutics field over a decade ago, RNA therapeutics is facing the same. And, like the antibody therapeutics field, we are beginning to realize the clinical impact of RNA therapeutics amiss these challenges. This is most clearly highlighted with the recent approval of two mRNA vaccines to prevent against SARS-CoV-2 and the first three FDA approved RNAi drugs targeted to the liver. Unfortunately, RNA-based drugs targeted to cancer cells is lagging behind, even with countless years of work that has revealed the power of using RNAi for treating oncological diseases. Lack of success in this space is attributed to inability to deliver RNAi safely and effectively. We previously developed a method that can safely deliver therapeutic microRNAs (miRNAs) to tumors that overexpress the folate receptor. However, the anti-tumor response was short-lived due to instability of the miRNA and poor pharmacokinetics, necessitating frequent dosing. To overcome these insufficiencies requires a stabilized miRNA that retains targeting activity. Recently we screened a panel of fully modified versions of miR-34a (FM-miR-34a) and identified one with >400- fold increased stability and outstanding in vivo efficacy when conjugated to folate. Treatment of mice implanted with breast cancer xenografts with folate-FM-miR-34a resulted in complete cures in two out of six mice and significant tumor regression in the remaining four. Based on this exciting data, here we propose to advance FM- miR-34 forward in two ways. In Aim 1 we will evaluate the activity, efficacy, and safety profile of FM-miR-34a in in vivo models of lung and prostate cancer. FM-miR-34a will be conjugated to: i) folate for delivery to lung cancer, and ii) PSMA-617, a ligand that targets prostate specific membrane antigen (PSMA) for delivery to prostate cancer. In Aim 2 we propose to capitalize on the stability afforded by FM-miR-34 to increase the circulation ½ time of folate-FM-miR-34 and PSMA-617-FM-miR-34a though incorporating an albumin binding moiety (ABM) into the ligands. Using these ligands we will evaluate serum albumin binding and stability of the new ligands. We will also verify that conjugation to ABM does not alter the activity of miR-34a nor cell binding and internalization kinetics. Finally, we will assess in vivo distribution of ligand-ABM-miR-34a conjugates. At the completion of this work we expect to have an all-encompassing miRNA delivery vehicle that can target a stabilized tumor suppressive RNAs specifically to NSCLC and prostate cancer. We will also have new ligands with increased circulation ½ life. The data obtained will ultimately have a significant impact in cancer treatment by providing new opportunities to advance the next phase of miRNA-based therapeutics. While proposed for NSCLC and prostate cancer, based on the utility of miR-34a for treating other cancers and overexpression of the folate receptor alone on many epithelial cancers, including ovary, kidney, and colon cancers, successful completion of this study could have far-reaching positive consequences.

NIH Research Projects · FY 2025 · 2018-01

The pathogenic bacterium responsible for Legionnaires’ disease, Legionella pneumophila, uses SidE family of effectors (such as SdeA) to target several host proteins through a noncanonical ubiquitination mechanism radically different from the ATP-driven, E1-E2-E3 ubiquitination of eukaryotes. This mechanism involves an all- in-one ubiquitination machinery in SdeA which employs, first, mono-ADP-ribosylation (mART) of ubiquitin (Ub) at Arg42, catalyzed by its mART domain, to produce ADP-ribosylated ubiquitin (ADPR-Ub), which is then subjected an additional catalytic step, executed by the phosphodiesterase (PDE) activity embedded in a separate domain, resulting in phosphoribosyl (PR) ubiquitination of serine residues of host targets. Essential to the pathogen’s intracellular life cycle, SdeA and its orthologs target numerous host proteins involved in a range of processes, from vesicular trafficking to nutrient acquisition and autophagy. While resistant to host deubiquitinases, the PR ubiquitination is regulated at multiple levels at the hands of other effectors: the SidJ effector (and its paralog SdjA) can shut off mART activity by modifying a key catalytic residue though a pseudokinase-based polyglutamylation activity; whereas the DupA and DupB effectors can reverse PR- ubiquitination by restoring host targets (such as Rab33) to their native form. This sort of deubiquitination activity, while releasing the native host target, still leaves Ub as a modified derivative, with a phosphoribosyl appendage at Arg42 (PR-Ub). Accumulation of such a Ub derivative, that cannot be used in host ubiquitination pathways, has the effect of poisoning the cellular Ub pool which could be detrimental to Legionella’s replication. In this proposal we explore regeneration of free, functional Ub from PR-Ub through a two-step process involving an unusual AMPylation reaction catalyzed by a novel S-HxxxE motif-containing, actin-activated AMPylator, called LnaB, producing ADPR-Ub, which is then further processed by a macrodomain (ADP- ribosyl)hydrolase, MavL, returning Ub to its native form. Using single particle cryo-EM we will provide structural basis of actin activation, PR-Ub recognition and the ATP binding site of LnaB. The EM studies will be complemented with x-ray crystallography of apo LnaB and its ATP-bound form. Together with biochemical studies aimed at capturing enzyme intermediates, our work will provide key insights into the novel AMPylation reaction. The MavL effector, while using macrodomain for deADP-ribosylation, features a unique motif which we found was shared by a group of previously uncharacterized proteins in the DUF4804 family of the Pfam database. Such a motif appears to confer residue-level selectivity for arginine de-ADP ribosylation, a novel aspect of macrodomain function. We seek to provide structural basis of ADPR-Ub recognition, while elucidating the basis of arginine selectivity across the newly found MavL-like enzymes. Collectively, our study will reveal a novel mechanism for AMPylation, that appears to be employed by a large family of toxins of unknown function from diverse pathogens, while expanding the scope of macrodomain hydrolases.

NIH Research Projects · FY 2026 · 2017-09

PROJECT SUMMARY Like the challenges and skepticism that faced the antibody therapeutics field over a decade ago, RNA therapeutics is facing the same. And, like the antibody therapeutics field, we are beginning to realize the clinical impact of RNA therapeutics amiss these challenges. This is most clearly highlighted with the recent approval of two mRNA vaccines to prevent against SARS-CoV-2 and the first three FDA approved RNAi drugs targeted to the liver. Unfortunately, RNA-based drugs targeted to cancer cells is lagging behind, even with countless years of work that has revealed the power of using RNAi for treating oncological diseases. Lack of success in this space is attributed to inability to deliver RNAi safely and effectively. A successful delivery agent requires multiple features. First, the agent must deliver the RNA specifically to the intended cells. Second, the agent must have a large therapeutic window, meaning that toxicity, if observed, should occur at doses that are orders of magnitude higher than the therapeutic dose. Third, if delivery of the RNA is by way of a specific ligand and receptor pair, as is the case herein, the RNA must successfully escape the endosome. Simply swelling the endosome is not enough if noncovalent interactions between the ligand and the receptor cannot be disrupted. Fourth, the RNA should include appropriate stabilizing modifications to increase intracellular half-life that will reduce dosing and cost. Through hard work and dedication in this space, we have come up with an inclusive, easily synthesized, intramolecular molecule that will achieve all of these essential features. Moreover, the ligand used to achieve successful delivery is also being evaluated for imaging tumors localized in the central nervous system. The premise for this work is based on conjugating the tumor suppressive microRNA, miR-34a to 5- methyltetrahydrofolate (5-MTHF), a ligand that is superior for the intended needs, in this case, release from the receptor when an endosomal escape agent is present. Our preliminary data and strong scientific premise supports our objective to i) advance 5-MTHF as a specific and non-toxic therapeutic ligand for delivery of therapeutic miRNAs to triple negative breast cancer (TNBC) and ii) to characterize and prepare 5-MTHF-nigericin conjugated to a fully modified version of miR-34a for clinical trial. To support these objectives, the following Aims will be conducted: 1) To test the hypothesis that both in vivo and intracellular biodistribution of 5-MTHF conjugates are superior to folate conjugates, and 2) To evaluate activity, efficacy, toxicity, pharmacokinetics, dynamics, and combinatorial effects of 5-MTHF-nigericin conjugated to fully modified miR-34a in vivo. At the completion of this work we will have the first an all-encompassing RNAi delivery vehicle that can deliver a stabilized RNA to the intended cells, into the correct subcellular location, with limited toxicity for the treatment of TNBC.

NIH Research Projects · FY 2026 · 2017-09

Autism spectrum disorders (ASDs) are characterized by altered sensory processing and intellectual disability. Atypical sensory processing has been recognized as an important diagnostic criterion for autism and is predictive of social communication deficits later in life. ASDs are also associated with impaired structural and functional connectivity within and between neocortical areas. However, how impaired neural connectivity, which is present in ASDs, leads to impaired sensory processing and learning is not understood. Recently, we have discovered new visual familiarity-evoked theta oscillations in the primary visual cortex (V1). In Fmr1 KO mice, a mouse model of Fragile X syndrome, these oscillations are weaker, shorter, and frequency shifted. Furthermore, we have shown a similar emergence of visually cued theta oscillations in the anterior cingulate cortex (ACC), an area connected to V1 and involved in social interaction, decision making and error detection. We have also started mapping the neural circuit underlying these oscillations. Our prior studies suggest that these familiarity-evoked theta oscillations and the underlying changes in the neural circuit connectivity may be the cause of impairments in visual learning and perception. Our preliminary data also suggest that Fmr1 KO mice demonstrate impaired theta oscillations in a visual discrimination task. We have developed a computational model to reproduce theta oscillations in the cortex. This proposal builds on foundational advances by dissecting the mechanisms of theta oscillations in WT and Fmr1 KO mice. Using an integrated approach combining mapping of neuronal connectivity in brain slices, in vivo extracellular recordings, and behavior, we will: 1) map the circuitry necessary to form a theta oscillator in V1 and identify altered connectivity patterns within V1 and between V1 and ACC in Fmr1 KO mice, 2) examine how the strength of theta oscillations in V1 and ACC correlates with behavior in WT and Fmr1 KO mice following learning, and 3) rescue theta oscillations by restoring FMRP expression selectively only in the specific neuronal groups in Fmr1 KO mice guided by the computational model. Based on our prior studies, preliminary data, and computational model, we expect to see familiarity-evoked theta oscillations correlate with successful behavior in a visual discrimination task. We also expect to identify the critical parts of the neural circuit required for the generation of theta oscillations and its impairment in Fmr1 KO mice. Results from this proposal will help inform the development of targeted neural circuit-based behavioral and pharmacological therapeutics to enable personalized medicine for individuals with ASDs.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY Embryogenesis from a fertilized egg into an individual is a precisely controlled process. One of the critical aspects of embryogenesis is developmental patterning, which determines organ size and shape by orchestrating many developmental and cellular events. Any abnormality from this patterning process will lead to congenital diseases in humans. However, understanding the mechanisms that pattern embryos remains a central challenge in developmental biology. The status quo of embryonic developmental patterning centers on the conceptual framework that development is governed predominantly by morphogenetic proteins that activate transcription factor networks in responding cells. The roles of bioelectricity in regulating embryonic developmental patterning have just started to be recognized as a new mechanism of cellular signaling. Given many ion channels and solute carriers are frequently involved in human congenital diseases, there is a critical need to understand ion channel-mediated bioelectricity in developmental patterning. The lack of information about this bioelectric patterning mechanism is a significant obstacle to understanding fundamental biological sciences and developing therapeutic strategies for many human congenital diseases. To address the demanding need, we will investigate the embryonic patterning mechanism of bioelectricity in the zebrafish model using newly developed technologies for neuroscience, such as chemogenetic tools and genetically encoded voltage indicators. Aim1. To elucidate the roles of bioelectricity in regulating zebrafish fin patterning. Aim 2. To reveal the roles of bioelectricity in pigment cell patterning. With this long-studied pigment system, we will further demonstrate bioelectricity as a general patterning mechanism in vertebrate embryogenesis. The expected outcomes will elucidate a less recognized developmental patterning mechanism by bioelectricity in both zebrafish fin and skin pigment. This knowledge will establish a new concept for patterning in developmental biology, provide the foundation for understanding vertebrate morphological diversity in evolution, and principles for developing prevention or therapeutic strategies for congenital diseases.

NIH Research Projects · FY 2025 · 2017-08

Project Summary/Abstract Carbenes are versatile reactive intermediates capable of engaging in cycloaddition, bond insertion, rearrangement, and coupling reactions. Previous efforts to develop catalytic variants of carbene transfer reactions have largely focused on the use of diazoalkanes. The primary limitation of this approach is that diazoalkanes generally must be stabilized with electron-withdrawing or aryl groups in order to avoid the spontaneous, exothermic elimination of dinitrogen gas. The overarching goal of this program is to study transition metal catalyzed reductive carbene transfer reactions that use readily available gem-dihalo reagents as precursors to non-stabilized carbenes. Catalytic turnover can be achieved using chemical reductants, such as metal powders. Alternatively, the use of photoredox or electrocatalytic reduction makes these reactions compatible with emerging flow synthesis platforms. A broad scope of catalytic cycloaddition reactions will be developed. A particular focus will be on generating odd-membered rings, which are challenging to access by conventional thermal pericyclic processes. For example, transition metal catalysis will allow current limitations of the Simmons–Smith reaction to be addressed, such as the enantioselective synthesis of dimethyl-, spiro-, and methylenecyclopropanes. Asymmetric [4 + 1]-cycloadditions of vinylidenes and 1,3-dienes will generate complex cyclopentene derivatives. Finally, three-component [n + m + 1]-cycloadditions will be developed for the synthesis of five- and seven-membered carbocycles and heterocycles. Some of these reactions will use dinuclear metal catalysts, which provide a unique active site environment to mediate carbene and vinylidene transfer reactions. Transition metal-catalyzed additions of vinylidenes to alkenes can also be diverted to non-cycloaddition pathways by intercepting metalacyclic intermediates prior to ring closure. Reaction design is based on promoting β-X elimination or transmetalation reactions of these metalacycles. Based on this concept, novel carbon–carbon coupling reactions will be developed for the synthesis of chiral alcohol and amine products. The catalysis concepts developed in this project will impact human health by providing access to complex C(sp3)-rich frameworks that can be incorporated into biological probes and therapeutics.

NIH Research Projects · FY 2025 · 2017-08

The ubiquitin network regulates virtually every host processes, particularly membrane trafficking and immunity. The bacterial pathogen Legionella pneumophila extensively modulates host processes using hundreds of effectors translocated into the host cytosol by its Dot/Icm transporter. The use of activity-based probes, bioinformatics analysis coupled with careful biochemical and structural analyses has identified at 28 Legionella effectors involved in co-opting ubiquitin signaling. These proteins function either as canonical E3 ubiquitin ligases, deubiquitinases or as ubiquitin ligases that defy the catalytic mechanism of canonical ubiquitination. To explore additional effectors involved in co-opting the host ubiquitin network, we have created mutants that lack a specific set or each of the known effectors involved in ubiquitin signaling. Using biotin ligase-mediated proximity labeling, we have identified additional Dot/Icm substrates capable of catalyzing ubiquitination. We have also obtained evidence for the existence of Legionella proteins that directly modify ubiquitin. The goal of this project is to determine the function of these proteins, their catalytic mechanism and their cooperation with host ubiquitination components by biochemical and structural analyses. We will also study the regulation of their activity by factors from the bacterium and determine how such activity contributes to the biogenesis of the phagosome supportive of intracellular bacterial replication. Finally, we will design experiments to address the issue of the potential functional redundancy among Dot/Icm effectors (with or without E3 ubiquitin ligase activity) in the recruitment of Sec22b to the bacterial phagosome. Results from these experiments will reveal not only novel mechanisms of host function exploitation by intracellular pathogens, but also insights into the regulation of host ubiquitination pathways, both of which have the potential to be capitalized to develop novel methods for diagnosis and treatment of diseases.

NIH Research Projects · FY 2025 · 2017-08

Project Summary/Abstract As a Vet-LIRN Laboratory the Indiana ADDL will continue to participate in Vet-LIRN activities including monthly calls and laboratory testing and exercises. This includes analysis of samples submitted through this cooperative agreement in both the bacteriology and toxicology laboratories in cases of surveillance and outbreak testing. The ADDL will contribute to the surge capacity in outbreaks as requested and in accordance with State requirements. The ADDL will provide analytical data for potential regulatory use through use of standardize methods, equipment platforms, and reporting methods. The ADDL will participate in proficiency testing and method training provided by the VPO as well as implementation of standardized quality management systems. The ADDL will participate in small scale method development, method validation and matrix extension work as determined by the VPO, and will participate in the AMR project as a source laboratory.

NIH Research Projects · FY 2025 · 2017-08

SUMMARY Fluorination of an organic compound affects physicochemical properties, which in medicinal settings perturbs pharmacodynamic, pharmacokinetic, distribution, and/or metabolic profiles both in vitro and in vivo. Thus, the ability to selectively install fluorinated groups under mild conditions is essential for accessing new therapeutics and biological probes. However, the unique physical properties of fluorinated substrates and/or reagents typically perturb fundamental organic reactivities, which can complicate synthetic sequences to access fluorinated compounds. Thus, many routine organic reactions simply do not work in the presence of fluorinated reagents or with fluorinated substrates. Additionally, the unique properties of fluorinated substrates enable new reactivities that cannot be achieved by the respective non-fluorinated counterparts, which provides opportunities to develop innovative reactions and strategies for accessing medicinally relevant substructures With this R35 program, the Altman group has a long-term goal of developing innovative catalyst systems, reagents, and/or synthetic strategies for accessing medicinally relevant fluorinated substructures. In this area, we develop fluorination and fluoroalkylation methodologies using innovative strategies (e.g. electrochemistry, C– H functionalization, deoxyfluoroalkylation, transition metal catalyzed reactions) that enable synthetic chemists to convert simple and readily available functional groups (e.g. alcohols, carbonyls, fluorinated alkenes) into a broad spectrum of highly valuable fluorinated analogs. Additionally, we explore synthetic transformations in which fluorinated substructures react through distinct mechanisms and/or deliver products with distinct selectivities relative to analogous reactions of nonfluorinated substrates. Development of the proposed strategies will enable medicinal chemists to access new and unique biological probes and therapeutics. A second long-term goal is to explore physicochemical perturbations imparted by fluorinated substructures that might influence drug stability, distribution, metabolism, and/or ligand-protein interactions, and to apply such principles in the design of next- generation fluorinated therapeutic candidates with improved drug-like properties. In the next phase of our work, we will apply modern innovative synthetic reactions to deliver next-generation fluorinated analogs of natural products that will retain the therapeutically valuable pharmacodynamic action and also improve stability and distribution relative to the parent compounds.

NIH Research Projects · FY 2026 · 2017-07

Project Summary. Childhood adversity is a potent risk factor for depression, increasing lifetime risk of this common and burdensome disorder by at least two-fold. While the association between adversity and depression is well documented, the mechanisms explaining this relationship are poorly understood. In a BRAINS R01 award, we made several new discoveries about how childhood adversity could become biologically embedded to shape depression risk through DNA methylation (DNAm), a major type of epigenetic modification. We showed that DNAm associations with adversity may not merely be molecular records of adversity exposure, but rather, possibly function as a biological mediator linking childhood adversity to depression risk. We also identified potential sensitive periods after birth and in the first five years of postnatal life when adversity exposure imparted more enduring effects on the epigenome and in shaping depression risk. However, these analyses were limited to mostly European-ancestry samples of children with low/moderate adversity exposure and only 2 time points of blood DNAm. In this renewal, we build on our prior work by exploring these relationships in a population-based longitudinal sample of children in South Africa, who are part of the Drakenstein Child Health Study (DCHS). Relative to our prior work and the field of epigenetics at large, the DCHS birth cohort provides an unprecedented opportunity to study these associations within an established group of more racially/ethnically diverse children, many of whom have experienced considerable early adversity directly or indirectly through their families own exposure. We will capitalize on existing, repeated adversity markers collected by the DCHS during early childhood and derive epigenetic data from stored blood samples collected at ages 1, 3, and 5. With these rich longitudinal data, we will identify the genetic and social drivers and outcomes of chrono-epigenetics, a newly coined term to describe the temporal dynamics of epigenetic processes, across the early life course. In Aim 1, we will characterize the effects of genotype on DNAm levels at specific ages and DNAm trajectories across time. In Aim 2, we will investigate the role of repeated adversity exposure measures before age 5 on DNAm patterns using a two-stage structured life-course modeling approach that our interdisciplinary team developed for high-dimensional epigenetic analyses. In Aim 3, we will use statistical mediation and causal inference approaches (e.g., Mendelian Randomization) to evaluate the extent to which these DNAm patterns explain the relationship between adversity timing and children’s internalizing symptoms at age 8, one of the earliest signs of depression risk. In sum, this renewal project will identify specific genetic and social factors shaping DNAm patterns, determine the ages when adversity is most likely to affect this biomarker, and generate biological insights that may lead to new intervention strategies to prevent depression, ensuring these findings apply to diverse samples of youth.

NIH Research Projects · FY 2025 · 2016-09

This proposal is a competing renewal application of R01 GM117675 that uses a multidisciplinary approach to enhance our understanding of protein α-N-terminal methylation. The α-N-terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. The increasing occurrences of α-N-terminal methylation on the canonical X-P-K/R motif and noncanonical motifs highlight the importance of this underexplored modification. However, there are major gaps remain in our understanding of these fundamental biological processes. Filling these gaps is essential to elucidate the α-N-terminal methylation-mediated pathways and to enhance the opportunity to devise novel therapeutic approaches. The objective of this research is to develop novel chemical tools and apply them to understand the pathway and functions mediated protein α-N-terminal methylation. Meanwhile, we will elucidate the molecular basis for substrate specificity of human methyltransferase like 13 that methylates the the eukaryotic elongation factor 1 alpha containing the new GKEK motif at the α-N-terminus. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein α-N-terminal methylation. Accomplishment of the proposed work will provide new chemical tools for both basic biology research and has the opportunity to enhance the development of novel therapeutic approaches to target α-N-terminal methylation-involved pathways.

NIH Research Projects · FY 2025 · 2016-09

Children with developmental language disorder (DLD; also referred to was children with specific language impairment) experience a significant deficit in language ability that is longstanding and harmful to the children’s academic, social, and eventual economic well-being. Word learning is one of the principal weaknesses in these children. This project focuses on the word learning abilities of four- and five-year-old children with DLD in an effort to understand the nature of these difficulties. The goal of the project is to build on our work accomplished in the first five years to determine whether, as we have found thus far, special benefits accrue when these children must frequently recall newly introduced words during the course of learning. The planned studies seek to increase the children’s absolute levels of learning while maintaining the advantage that repeated retrieval holds over comparison methods of learning. We also adapt our learning procedures to an illustrated storybook format to promote children’s engagement with the materials to be learned. Finally, we will determine if our repeated retrieval procedures continue to prove superior when the words to be learned move from the single-word level to appearing in sentences from the outset. Of special interest will be whether repeated retrieval activities narrow the differences between children with DLD and their typically developing peers relative to other word learning procedures. If the planned studies reveal larger word learning gains than current methods, repeated retrieval activities can serve as the basis for the development of new methods of treatment for children with word learning difficulties.