University Of Illinois At Urbana-Champaign

NIH Research Projects · FY 2025 · 2021-09

Project Summary The overall aim of the research is Shukla group is to develop computational methods that facilitate investigation of rare conformational transitions in proteins and help guide the design of experiments to validate the in silico predictions. In par- ticular, we apply these computational methods to investigate functional regulation of membrane proteins such as membrane transporters and G-protein coupled receptors (GPCRs). Here, we propose development of transfer learning based methods to predict the effect of mutations on protein function and apply these methods to investigate monoamine transporters, sugar transporters and Class C GPCRs. Deep mutagenesis, whereby tens of thousands of mutational effects are determined by combining in vitro selections of sequence variants with Illumina sequencing, is an emerging technology for indirectly interrogating and observing protein conformations in living cells; the solving of an integrative structure of a neuronal class C G protein-coupled receptor in an active conformation by deep mutagenesis-guided modeling is one prominent example of this approach's success. Using deep mutagenesis and molecular dynamics simulations to inform each other, we plan to determine the mechanism of ion- coupled neurotransmitter import by monoamine transporters at atomic resolution. Fluorescent substrates have enabled us to use fluorescence-based sorting of libraries of transporter mutants to find mutations along the entire permeation pathway that increase or decrease substrate import. These comprehensive mutational landscapes will be used to interpret and support/reject hypotheses from simulations, including the role of ion-coupling in substrate transport regulation, proposed free energy barriers in the conformational-free energy landscape that limit import kinetics, and how sodium-neurotransmitter symport is coupled by a shared cytosolic exit pathway. Other notable features that arise from the deep mutational scans (e.g. putative regulatory sites) will be further explored, and a machine learning algorithm will be applied to transfer mutagenesis information to related transporters; the predicted mutational landscapes will then be validated by a small number of informative targeted mutants. We will further relate sequence to conformation and activity in metabotropic neurotransmitter receptors and sugar transporters. Finally, we plan to improve the proposed transfer algorithms by using deep learning techniques, which will facilitate integration of features derived from simulation datasets and multiple deep mutational scans to inform the effect of mutations on related proteins or tasks. The success of the proposed research program of results will be measured by development of algorithms that can accurately predict the variant effects on protein structure and function, elucidation of the mechanisms of ion-coupled regulation of neurotransmitter transport, selectivity mechanisms in sugar transporters and activation mechanisms of class C GPCRs.

NIH Research Projects · FY 2024 · 2021-08

Principal Investigator/Program Director (Last, first, middle): Bashir, Rashid Project Summary: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection (Sepsis- 3 definition), is the leading cause of death and most expensive condition in hospitals. Annually, > 30 million people affected worldwide, with at least 1.7 million adults developing sepsis (nearly 270K die) at a cost of $24 billion per year in the U.S. Patients diagnosed with sepsis and no ongoing sign of organ failure have about a 15-30% chance of death. However, the mortality rate can increase up to 40-60% for severe sepsis or septic shock patients. One in three patients who die in a hospital have sepsis. One major factor in these rising mortality rates is the inability to accurately and quickly diagnose potentially septic patients. Likewise, sepsis is a leading cause of hospital readmission (higher proportion than hospitalizations for heart attack, heart failure, COPD, and pneumonia in the U.S.). EDs and ICUs rely on monitoring extremely non-specific parameters (e.g. fever, low blood pressure, increased heart rate) to initiate a clinical diagnosis and begin treatment. These crude indicators cause doctors to mistake early stage sepsis with several other diseases. A positive diagnose of early onset sepsis is critical because mortality increases with delays in treatment. Survival rates have been reported to drop by 7.6% every hour that the proper antibiotics are not administered, and these delays compound unnecessary hospital costs. Over the last 30 years, clinics have used different criteria such as SIRS, LODS and SOFA or qSOFA as screening tools to assess the severity of organ dysfunction in a potentially septic patient. Common factors among these criteria are non-specificity and very high false positive rates. For patients with positive criteria, the final diagnostic test is a blood culture that may take up to 5 day for a negative result. Likewise, blood culture has a very high false negative rate (> 60%) and does not work for fastidious pathogens such as Chlamydia pneumoniae. More importantly, blood culture cannot be a gold standard method for sepsis diagnosis. This technique only detects the presence of bacteria in the bloodstream (bacteremia), which does not necessarily indicate illness. Many non-bacteremic infections can also cause life-threatening sepsis. In order to improve the accuracy and sensitivity of sepsis diagnosis, the Sepsis-3 definition underscores the requirements for both pathogen detection and information about the personalized state of the immune system of the patient. Therefore, we propose to focus our efforts on monitoring selective biomarkers of this immune response. However, no single, or even a combination of biomarkers has been validated for the diagnosis of sepsis. Because no single biomarker is specific enough to predict sepsis, we propose to develop a point-of-care microfluidic biochip for measuring cell-surface and plasma-proteins biomarkers that will be used for contributing in early sepsis diagnosis. The microfluidic biochip will provide a complete white blood cell count (WBC), as well as quantification of CD64 expression on neutrophil (nCD64), procalcitonin (PCT), C-Reactive Protein (CRP) and Interleukin 6 (IL-6). Multiple studies have demonstrated the high sensitivity of these biomarkers to sepsis. The proposed device will combine for the first time the analysis of cell-surface proteins and plasma proteins biomarkers from the same sample of blood. Such a device, combined with the routinely test performed in the hospitals, could significantly accelerate the diagnosis of sepsis and as consequence the clinical decision, to provide the correct treatment to the patients.

NIH Research Projects · FY 2025 · 2021-08

Quantitative approaches, including data-driven artificial intelligence, can significantly help improve human health by providing innovative diagnostic tools and effective therapeutic interventions. The proposed training program will enhance doctoral-level professional development in the quantitative biomedical sciences at the University of Illinois Urbana-Champaign (UIUC). Our program focuses on bioinformatics, computational biology, genomic biology and biophysics. To achieve our goal, we will accomplish the following objectives: (a) provide PhD students with a rigorous scientific environment to strengthen their background in the relevant computational, mathematical and biological sciences; (b) ensure the trainees’ timely Ph.D. attainment within 5 years; (c) train students in scientific rigor, reproducibility, and the responsible conduct of research; (d) devise effective career development plans that will help the trainees succeed as independent scientists; (e) provide research training specifically at the interface of mathematical, computational and biomedical sciences. As outcomes of our training, we expect that our seamless infrastructure will significantly enhance professional development in the quantitative biomedical sciences and train doctoral students who will contribute to the mission of improving human health.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Bacteria are everywhere around us, playing critical roles in our health and global ecosystems. Understanding how bacteria thrive is extremely important in keeping our bodies and environments safe and healthy. Bacteria can dynamically adapt to a wide array of conditions by modifying their gene expression program, for example, by boosting the production of proteins necessary for survival and limiting the wasteful production of others. The ultimate goal of my research is to gain an enriched understanding of bacterial gene regulation, thus I study the very fundamental question of how transcription, translation, and mRNA degradation are performed and regulated in physiological settings. Due to the absence of a nucleus in bacterial cells, these three processes occur in the same cytoplasmic volume without clear separations. Therefore, the cellular mechanisms enabling their coordination inside a single cell offer an important foundation for understanding bacterial gene expression programs. Here I describe projects in my group aiming to define the generalizable principles underlying the spatiotemporal coordination of transcription, translation, and mRNA degradation in bacterial cells. We plan to answer the following key questions: (1) What is the mechanism of transcription-translation coupling? (2) How is the interaction between RNA polymerase (RNAP) and ribosome dynamically regulated? (3) How is the rate of mRNA degradation regulated by the age of mRNA and the subcellular localization of RNase E, the major ribonuclease for mRNA degradation in Escherichia coli? We will answer these questions by imaging the dynamics of RNAP, ribosome, and RNase E at the single-molecule level in live cells. Combining these techniques with bacterial genetics, we will identify factors that can modulate the dynamics of RNAP- ribosome interactions and analyze the subcellular heterogeneity in the localization and function of RNase E. Collectively, our work will uncover new mechanistic principles of bacterial gene regulation and generate new methods for measuring, controlling, and modeling gene expression dynamics at the single-molecule level in live cells. The findings from our work have potential applications for a broad range of human health issues, such as promoting healthy microbiomes, killing pathogens, and improving industrial processes to reduce pollution.

NIH Research Projects · FY 2024 · 2021-07

Abstract Molecular chirality is at the heart of many chemical processes that determine life and drives significant research in development and disease. All life has chiral asymmetry with naturally occurring molecules and long-range assemblies being of distinct handedness. Many exogenous molecules, for example those useful as drugs, also have a distinct enantiomeric dependence for their efficacy in benefiting human health. Thus, measurement of molecular chirality is of critical importance across the medical sciences. Vibrational Circular Dichroism (VCD) spectroscopy has emerged as a powerful platform for quantifying chirality and molecular structure. However, imaging has not been demonstrated due to technological challenges. VCD measurements are largely of homogeneous materials, neat or in solution and probed with sensitive Fourier transform infrared (FT-IR) spectrometers. Microscopy would require ~105 reduction of the typical sensing volume and increase in speed that would make imaging feasible. Instead of utilizing FT-IR spectroscopy, we built a custom quantum cascade laser (QCL) microscope to demonstrate feasibility of a point scanning VCD instrument capable of acquiring spectra rapidly across all fingerprint region wavelengths in both transflection and transmission configurations. Moreover, for the first time, we also demonstrate the VCD imaging performance of our instrument for site-specific chirality mapping of biological tissue samples. However, the feasibility data also point to several technological and conceptual challenges that this project seeks to address in developing a practical prototype. The prototype to be developed here, termed vibrational circular dichroism imaging microscope or VIM, aims to record chirality from microscopically heterogeneous biomedical samples. We propose a design for VIM using a laser scanning approach to minimize artifacts and maximize signal. Starting from a de novo design, we will use commercial and custom optics, custom electronics for control and data management, and in-house software to develop the prototype. Next, we model the VCD image formation process and develop the analytical methods for VIM. The theoretical model developed here builds on our models of IR microscopy and will guide prototype development while ultimately provide greater accuracy, precision and assurance to data recorded. Finally, we validate the performance and broad utility of VIM using well-characterized samples. Together, the work will develop new VCD imaging technology that opens capability to measure and research a wide variety of biological problems.

NIH Research Projects · FY 2025 · 2021-07

The World Health Organization reports that nearly a half billion people suffer from a disabling hearing loss worldwide. Hearing loss is typically measured in the standard audiometric frequency range, 250 to 8,000 Hz. However, the young, healthy human frequency range of hearing extends to about 20,000 Hz. Extended high- frequency hearing loss (>8,000 Hz) is rarely assessed in the clinic but is a widespread, natural, age-related condition that begins as early as young adulthood. It is unknown to what extent extended high-frequency hearing loss affects daily functioning. This proposal assesses the ecological value of extended high-frequency hearing for speech perception and spatial awareness in realistic settings, which will include spatially separated talkers having different head orientations in reverberant conditions. There are three specific aims. Aim 1 will generate a high-fidelity multi-directional anechoic speech database with a representative cohort of adult native speakers of American English. This database is designed to be used for speech perception experiments and will be made publicly available. Speech spectral energy across the full range of human hearing will be examined, as well as the nature of speech radiation toward different directions around the talkers. Aim 2 will test the effect of simulated loss of extended high frequencies on speech perception and spatial awareness for listeners with normal hearing out to 16 kHz. These experiments are based on our recent observation that extended high frequencies in speech provide a benefit for speech-in-speech recognition. We will determine the magnitude of this benefit in realistic settings and the factors responsible for it. Aim 3 will test the effect of natural loss of extended high frequencies on speech perception and spatial awareness. Forms of natural loss that will be studied include hearing loss at extended high frequencies and the wearing of face coverings (masks) by talkers. The proposed studies will establish the ecological utility of extended high frequencies for speech communication. Valuable insight will be gained as to how extended high-frequency hearing loss impacts daily living for otherwise normal-hearing listeners. It is expected that results of the proposed studies will aid in ongoing efforts to (1) improve clinical measures of hearing loss and (2) improve hearing-aid processing techniques.

NIH Research Projects · FY 2025 · 2021-07

Abstract Colorectal cancer (CRC) is one of the leading causes of death in the US. Active screening and early intervention in risky cancers can lead to good outcomes; however, a bottleneck in rapidly delivering appropriate patient care is the long time period for histologic assessment and lack of precision in predicting disease severity. Morphological assessments prevalent in histology are useful but resource intensive and not predictive enough. Molecular techniques to complement traditional pathology are emerging but often require much more effort and time, without being especially compatible with histologic assessments. Here, we seek to develop a technology that measures the chemical content of tissues, does not require reagents, is entirely compatible with clinical workflows and leverages modern artificial intelligence (AI) techniques to provide real-time histologic assessment. The foundation of our approach is a new design for an infrared spectroscopic imaging system that is faster than any reported, offers a higher spatial and spectral quality and uses a solid immersion lens with a fixed focus at the sealed surface of the lens to enable use by a minimally trained person. In conjunction with the instrument, we develop AI algorithms that measure the chemical content of tissue and use it to provide (a) conventional pathology images without the use of dyes (“stainless staining”), and (b) histologic assessment based on molecular data, which can provide complementary composition, disease and risk of lethal cancer images akin to conventional pathology. The instrument will be usable by laboratory technicians, without the need to prepare thin sections from excised tissue and will provide information in minutes. Using preliminary data from human patients on over 850 tissue microarray (TMA) samples from 8 TMAs and 30 surgical resections, we validate the use of technology in providing complete histologic and disease grade assessment. Statistical methods will be used to assess the results rigorously and quantitative milestones guide the entire approach. We then translate the results to fresh tissue chunks, providing histology minutes after tissue is extracted from the body. Finally, we use the detailed tumor and microenvironment information available from the tissue to segment patients into a “high risk” and “low risk” group. The availability of rapid histologic assessment can help prevent delays in providing care, provide intraoperative assessment, and add more information to morphologic assessments following screening, enabling a wide use in CRC and other cancer pathologies.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY / ABSTRACT A key goal of biophysics is to predict the behavior of living systems. This goal is hampered by the fact that, when examined at the single-cell level, this behavior appears largely unpredictable: Genetically identical cells, within a uniform environment, exhibit heterogeneous phenotypes in terms of gene expression, signaling, and consequent fate choice. This cellular individuality is observed throughout biology, from the emergence of antibiotic resistance among bacteria, to cell differentiation in the early mammalian embryo, and numerous other examples. Studies over the last two decades have pinpointed the stochastic origins of cellular individuality, by demonstrating that the inherent randomness (“noise”) of single-molecule events can be amplified into protein number fluctuations at the cellular level. The picture that emerged from those studies is of living cells as “noisy machines”, incapable of high precision, whose fate choices are subject to significant randomness. But the widespread success of the “noise” concept in describing cellular heterogeneity also points to its weakness: It is easy to describe single-cell properties as “stochastic” and map them into statistical distributions, but doing so does not mean that we understand the underlying cellular process. On the contrary, by creating a façade of understanding, a stochastic description may impede our efforts to uncover the deterministic factors that drive single-cell behavior. Recent years have seen a growing awareness of this caveat and an increase in efforts to identify the deterministic drivers (so-called “hidden variables”) of cellular individuality, but it is fair to say that we still lack a satisfactory picture for what drives single-cell behavior even in the simplest systems, to say nothing of more complex ones. Research goal. The choice between rapid cell death (lysis) and viral dormancy (lysogeny), following infection of E. coli by bacteriophage lambda, serves as a paradigm for the way genetic networks drive cell fate decisions, and for the purported role of molecular randomness in this process. Building on our work over the last decade, we will use lambda infection to identify hidden drivers of cellular individuality in gene regulation and fate choice. By revealing how deterministic the decision process is, we aim to establish lambda as a paradigm for precise— rather than “noisy”—cell fate choice. In parallel to the work on lambda, we will continue to develop tools for the manipulation, imaging, analysis, and modeling of individual cells, and apply them in collaborative projects addressing cellular individuality across diverse biological contexts.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Defects in craniofacial bones of the skull occur congenitally, after high-energy impacts, and during the course of treatment for stroke and cancer. Autologous bone or alloplastic implants are the current gold-standards for surgical reconstruction. However, limited quantities and time-intensive intraoperative fitting of autologous bone, the non-regenerative nature of alloplastic implants, and surgical challenges that stem from irregular defect margins and the quality of the surrounding bone all contribute to poor healing and high complication rates. A biomaterial that could be shaped precisely and quickly like an alloplastic implant but that works in a regenerative fashion like autologous bone would be transformative for craniofacial reconstruction. The objective of this proposal is to potentiate regeneration of the structure, composition, and mechanical properties of craniofacial bone using an innovative scaffold-mesh composite biomaterial. We have generated extensive proof-of-principle data for a surgically-practical composite biomaterial for craniofacial bone regeneration. Our core technology is a porous mineralized collagen scaffold to expand MSCs in vivo. We have identified microstructural features of this material to activate mechanotransduction and BMP receptor signaling to accelerate MSC osteogenicity and secretion of osteoprotegerin (OPG), a soluble glycoprotein and endogenous inhibitor of osteoclast activity. As a result, this material increases osteogenicity and transiently inhibits osteoclast activity to accelerate regenerative healing of craniofacial bone defects osteogenic supplements or exogenously-seeded stem cells. We have independently developed a millimeter-scale polymeric mesh that can be integrated into the scaffold, à la rebar in concrete, to form a modular composite that can be shaped intraoperatively to conformally fit irregular defects. Excitingly, prototype scaffold-mesh composites generated using a mesh printed from an advanced Hyperelastic Bone® material increases MSC OPG secretion. These findings suggest the exciting possibility to co-optimize scaffold microstructural properties as well as the composition and architecture of the integrated polymer mesh to both passively aid surgical-practicality and actively accelerate regenerative healing. Our central hypothesis is that a multi-scale scaffold-mesh composite will accelerate MSC recruitment and retention, increase osteogenesis while inhibiting osteoclast activity, and facilitate vascular remodeling to improve regeneration. To do this we will first define the contribution of scaffold anisotropy on the recruitment and activity of osteoprogenitors and endothelial cells (Aim 1). We will establish topology parameters of a scalable mesh to aid surgical practicality and regenerative potential (Aim 2). Lastly, we will demonstrate in vivo efficacy of a scaffold-mesh composite in a confined calvarial defect model (Aim 3). Our unified effort to develop craniofacial regenerative technologies will generate significant preclinical data to support an FDA IDE application essential for accelerating this technology towards clinical use as a material-only regenerative therapy for craniofacial bone injuries.

NIH Research Projects · FY 2025 · 2021-04

Tissue damage caused by trauma or chronic illness reduces quality of life and shortens life expectancy. The ability to regulate the endogenous response to damage, and to induce regenerative growth, would have profound implications for the field of regenerative medicine. The Smith-Bolton lab has developed innovative techniques to: 1) induce tissue damage in hundreds of animals simultaneously, enabling the use of powerful Drosophila genetics to identify mechanisms that regulate tissue regeneration, and 2) isolate the regenerating tissue, ena- bling high-throughput genomic approaches to characterize the molecular mechanisms that underly regeneration control. The long-term goal of the Smith-Bolton lab is to understand how damaged tissue regenerates a func- tional structure. During the past five years, funded by NIH R01GM107140 “Regulation of Cell Fate and Patterning during Regenerative Growth”, the lab has used genetic and genomic techniques to 1) demonstrate that regen- eration signaling and unconstrained expression of regeneration growth factors have deleterious side effects, 2) identify several protective factors that prevent these unwanted outcomes, and 3) identify multiple mechanisms through which the magnitude and duration of regeneration signaling are tightly controlled. The purpose of this R35 MIRA application is to obtain stable and flexible funding to continue our successful and innovative work identifying the intricate pathways that control tissue regeneration. Important questions remain unanswered, such as 1) How do tissue-damage signals induce the changes in gene expression that carry out each step in regen- eration? 2) How does the regenerating tissue switch back to its normal patterning and gene expression profile? 3) Does regeneration recapitulate development, or are there regeneration-specific patterning controls? The re- search programs in the Smith-Bolton lab over the next five years will seek to achieve specific goals, including using genetic, genomic, and molecular techniques to: 1) identify the transcription factors and the genetic targets of those factors that constitute the gene regulatory networks that control individual steps in regeneration, 2) provide a detailed understanding of how key genomic loci are regulated after tissue damage, 3) elucidate the mechanism through which regenerating tissue returns to normal, and 4) identify additional regeneration-specific regulators of cell fate and patterning. When this work is complete, we will have a mechanistic understanding of how regeneration can derail cell fate, and the variety of mechanisms used to prevent catastrophic changes in gene expression after tissue damage. This work will have a critical positive impact because strategies developed to induce medically relevant regrowth of tissue after acute injury or chronic illness must account for deleterious side effects of pro-regeneration signals and incorporate protective factors to prevent aberrations. Furthermore, this work will identify candidate genes that can be targeted to manipulate specific aspects of the tissue damage response, while avoiding unwanted effects such as overstimulation of the wound response or unregulated pro- liferation, both in model systems and in humans.

NIH Research Projects · FY 2025 · 2021-02

Project Abstract Cryptosporidium is a leading cause of diarrheal disease (cryptosporidiosis) and death among young children living in resource-poor settings. In the US, Cryptosporidium is the major cause of waterborne outbreaks linked to recreational water use. Currently, there is no fully effective drug and no vaccine to treat or prevent cryptosporidiosis. The only available US FDA approved drug, nitazoxanide has no proven efficacy in young children with weak immune status and immunocompromised individuals. Therefore, there is an urgent need to develop new drugs and vaccine to reduce the burden of cryptosporidiosis. Progress in anti-cryptosporidial drug and vaccine development has been hampered due to our limited understanding of parasite biology. The underlying reasons for this slow progress have been the unavailability of a robust method to continuously propagate Cryptosporidium, and the absence of molecular genetics to manipulate the parasite genome. We have overcome these hurdles by developing a powerful technology to manipulate the Cryptosporidium genome and propagate these genetically modified parasites in an immunocompromised mouse model system. The key advantage of this genetic system is that the entire life cycle of Cryptosporidium (both asexual and sexual stages) is completed in the mouse intestine, allowing us to unravel parasite biology (Vinayak et al 2015, Nature 523:477). We lack an understanding of the molecular signaling mechanisms that control development of parasite stages for successful completion of the complex life cycle. Signaling pathway components such as the plant-like calcium-dependent protein kinases (CDPKs) have emerged as attractive drug targets in Cryptosporidium and related parasites, due to the absence of their homologues in human host. Taking advantage of our genetic system, we have demonstrated the efficacy of selective bumped kinase inhibitors against calcium-dependent protein kinase-1 (CDPK1), thus indicating a critical role of this signaling kinase in C. parvum. Utilizing the conditional protein degradation system recently developed in our laboratory, we have demonstrated the essential role of CDPK1 in asexual proliferation and parasite survival. Moreover, we have compelling preliminary evidence that sheds light on the role of two signaling kinases in sexual developmental stages. The goal of this project is to elucidate the mechanistic role of these signaling proteins in regulating development of asexual and sexual stages in C. parvum required for parasite proliferation and transmission. Elucidation of these mechanisms will provide novel insights into the fundamental biology of Cryptosporidium, and open new avenues for development of effective therapies.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY / ABSTRACT Binaural hearing provides substantial benefits in complex listening environments, improving the ability to understand speech and providing the ability to localize sounds. However, in order to take advantage of binaural cues, sounds from the two ears need to be integrated (binaural integration). Binaural integration does not fully occur for some populations of listeners, such as cochlear implant (CI) users. Whether, and the degree to which, binaural integration occurs depends on two aspects of the acoustic signal. One aspect is the statistical similarity between the waveforms in the left and right ear (interaural correlation). The second is the symmetry in terms of the place of stimulation in the two ears (physical interaural symmetry). Our overarching hypothesis is that interaural correlation and interaural symmetry both play a role in binaural integration, with interaural correlation also driving adaptation, altering the functional interaural asymmetry to counter the effects of the physical interaural symmetry. The proposed study will manipulate the interaural correlation and interaural symmetry of the signal as well as the cochlear region to which the signals are delivered. These experiments will provide insight into both the functioning of the auditory system and the critical factors to consider when developing device programming techniques for bilateral CI users (Specific Aim 1). While adaptation, reducing the effects of physical interaural asymmetry, has been well documented for pitch-matching tasks, we hypothesize that interaurally correlated signals drive adaptation across the entire binaural auditory system, but the magnitude and/or time-course of the effects differ across different binaural cues. These experiments will provide critical insight into the relative importance of interaurally correlated and physically interaurally symmetric signals for driving adaptation. They will also provide critical guidance as to when it is crucial to address the common issue that bilateral CI users chronically receive interaurally correlated signals at interaurally asymmetric locations (Specific Aim 2). The proposed studies will provide fundamental insight in to how the binaural auditory system combines signals from the two ears. This research will also provide insight into the factors that will influence bilateral CI users’ binaural abilities, both directly after activation, and over time. This will lay the groundwork for a paradigm shift in terms of how and when clinicians program bilateral CI users’ devices to maximize binaural benefits.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY Despite the global rise in fungal infections, including those by endemic sp ., there is no licensed fungal vaccine available. This is mainly due to poor understanding of mechanisms of vaccine immunity, and lack of a functional phenotypic marker associated with vaccine efficacy. Opportunistic fungal infections, including those caused by dimorphic fungi, Histoplasma, Coccidiodes and Blastomyces, are rising at an alarming rate in such at-risk individuals. A major limitation in the development of tailored fungal vaccines for the at-risk population is poor understanding of requisite elements of CD8+ T cell responses to mediate vaccine-immunity. Recent advances in the understanding of immune correlates against fungal infections has helped in advancing vaccinology in parallel. T-cell derived IL-17A, IFNγ, GM-CSF, IL-22, and TNFα are primarily involved in protection against fungi. Identification of potential targets on host cells can provide novel efficacious vaccine platforms, including for immunocompromised. We have established a mouse model of CD4+ T cell lymphopenia, where CD8+ T cells can be stimulated to produce protective cytokines IL-17A (Tc17) and IFNγ (Tc1) to execute a sterilizing immunity against lethal pulmonary fungal infection. We have shown that vaccine-elicited antifungal CD8+ T cells persisted as long-term functional memory. In this proposal, we present seminal findings: 1. GM-CSF+ Tc17 cells bolster vaccine-immunity without pathology; 2. Anti-fungal CD8+ T cells preferentially express O-glycosylated Sialophorin; and 3. Sialophorin is required for differentiation and expansion of CD8+ T cells. Therefore, our central hypotheses are that (1) Sialophorin acts as a co-stimulator for CD8+ T cell responses, (2) retention of Sialophorin is essential for memory CD8+ T-cell homeostasis and recall responses, (3) Sialophorin signaling potentiates cross-presentation to augment CD8+ T cell responses. Our specific aims are to: 1. Determine and dissect the role of Sialophorin for CD8+ T-cell fungal vaccine responses. We will decipher and delineate the cell- intrinsic role of Sialophorin for vaccine-induced Tc17 and Tc1 cell responses using adoptive transfer and bone- marrow chimera experiments, and using TCRα KO, congenic and crosses of Sialophorin KO mice. 2. Elucidate the role of Sialophorin for memory T cell homeostasis and recall responses during fungal pneumonia. We will define the role of Sialophorin for vaccine-induced memory CD8+ T-cell homeostasis, recall responses, and vaccine-immunity. We will use bone marrow chimera, adoptive transfers, CRISPR-Cas9 gene-editing, in vivo stimulation, and E-selectin blocking to delineate the role of Sialophorin on memory CD8+ T-cell responses. 3. Dissect the role of Sialophorin on dendritic cells for CD8+ T-cell fungal vaccine responses. We will identify the role of CD43 on antigen-presenting cells for activation of CD8+ T cells following vaccination . Our findings will uncover the functional role of Sialophorin for fungal CD8+ T-cell responses and immunity to guide in the design of novel vaccine platforms and test the efficacy of vaccines.

NIH Research Projects · FY 2025 · 2021-01

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Abstract is unchanged. Parental care is critical to the health of both parents and offspring, yet little is known about how molecular substrates act within brain areas and neuronal circuits to alter parental behavior, and this gap is especially wide for paternal care, i.e. the care-giving behavior of fathers. The proposed research capitalizes on natural variation in paternal care within a single species (threespined stickleback fish) to uncover the biological basis of paternal care in vertebrates. The current models for paternal care have biparental care, which makes it difficult to disentangle the behavior of mothers and fathers. Three-spined stickleback fish are a promising new model for paternal care because fathers are solely responsible for providing care. Moreover, the molecular mechanisms related to care are deeply conserved in vertebrates, the stickleback genome is small and compact and there are a growing number of functional tools available to study them. The goals of this proposal are to define the molecular elements responsible for differences in paternal care within a single species and to characterize the neurobiological pathways involved in such variation in behavior. There are two objectives: 1) Identify genes that contribute to differences in paternal care and test their function; 2) Define the ways in which neuropeptide signaling regulates paternal care. The project combines brain gene expression profiling at the bulk and single cell level, viral mediated transgenesis experiments to establish mechanism and pharmacological manipulations of oxytocin and arginine vasopressin signaling. The proposed work offers an innovative solution to the challenge of dissecting the biological basis of paternal care by using a powerful new model for fathering, and by capitalizing on natural variation in paternal care within a single species.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT Healthy subjects rapidly shift their attention in response to a dynamic sensory environment and changing cognitive demands. Failure to quickly shift perceptual resources between auditory and other modalities has been hypothesized to be a core deficit in both dyslexia and autism. Precisely how the brain shuttles information between sensory systems is not known, but recent work suggests that the thalamus contains circuits that have the capacity to rapidly transition between different sensory modalities. Specifically, the thalamic reticular nucleus (TRN), a thin shell of GABAergic neurons surrounding the thalamus, may serve as a link to allow communication between different areas of the sensory thalamus, a phenomenon we refer to as thalamic “cross- talk.” Based on recent data and our preliminary findings, we hypothesize that thalamo-TRN- thalamic circuits provide critical connections between thalamic nuclei and therefore permit rapid switching between auditory and other thalamocortical pathways. We propose to test this hypothesis using a novel combination of anatomical, chemogenetic, optical stimulation and optical imaging approaches in the mouse, using both slice and in vivo approaches. Specifically, we will determine which of multiple potential circuit pathways is/are used to permit auditory, visual and auditory thalamic nuclei to communicate with each other. Next, we will determine whether and how such thalamic cross-talk influences synaptic responses at the level of the auditory cortex. Finally, the impact of the TRN on cross-modal processing will be directly examined by optically modulating the TRN while imaging cortical responses to combined sensory stimulation in awake mice. Successful completion of this project will provide the first circuit-level characterization of the role of the TRN in communication between the auditory thalamus and other thalamic regions. In addition, this work will lay the groundwork for a greater understanding of how thalamoreticular systems break down in disorders of communication.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT Glioblastoma (GBM) is the most common and lethal form of brain cancer. Standard of care is surgical resection followed by treatment with the alkylating agent temozolomide (TMZ). However, two major challenges make GBM currently untreatable: 1) its diffuse invasion beyond the surgical margin; and 2) TMZ resistance that is tightly linked to expression of the DNA damage repair protein MGMT. While perivascular niches (PVNs) extending from the tumor into the surrounding parenchyma are believed to regulate invasion, recurrence, and poor survival, the majority of animal glioma models are sensitive to TMZ and most do not express MGMT, making it difficult to assess novel therapeutics in animal models that don’t display TMZ resistance. This Cancer Tissue Engineering Collaborative project will develop and thoroughly characterize a multidimensional engineered PVN biomaterial, study pathophysiological processes driving GBM invasion and TMZ resistance, and accelerate the evaluation of novel TMZ derivatives created to target diffuse GBM cells regardless of MGMT status. We will use advanced microfluidics to create libraries of miniaturized gelatin hydrogels containing margin-mimetic hyaluronic acid (HA) and an embedded perivascular network. We also use a novel synthetic pipeline to create TMZ derivatives that generate alternate DNA modifications that cannot be removed by MGMT that we hypothesize work in an MGMT- independent fashion. Merging these technologies, we will benchmark an engineered PVN platform formed using primary brain neurovascular cells for rapid evaluation of GBM invasion, MGMT expression, and TMZ resistance amenable to analysis of cell lines and patient-derived GBM specimens with disparate MGMT profiles. To do this, we will first construct and thoroughly characterize an engineered perivascular niche (Aim 1). We will use this novel biomaterial to benchmark patterns of invasion and MGMT expression in GBM cell lines (Aim 2). Finally, we will establish predictive efficacy of TMZ variants in an engineered perivascular niche (Aim 3). Together, we will develop, characterize, and benchmark a tissue engineered PVN to examine the role of microenvironmental selection pressures in the tumor margin on behaviors related to invasion, MGMT-mediated TMZ resistance, recurrence, and poor survival. Consistent with score-driving criteria of the CTEC program, we will develop and thoroughly characterize an engineered PVN biomaterial, show it fits within the continuum of existing cancer models, use it to examine phenomena underlying the failure to achieve durable survival, and gain actionable insight regarding novel TMZ derivatives with potential to effectively target GBM cells in the margins independent of MGMT status.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT Adaptation of living organisms to constantly changing environments depends on the plasticity of the nervous system. Neuronal plasticity often requires activity-dependent translation to rapidly supply selected proteins, for example, through activation of Group 1 metabotropic glutamate receptors (Gp1 mGluRs). Gp1 mGluRs, including mGluR1 and mGluR5, mediate translation-dependent synaptic plasticity, including long-term synaptic depression (LTD). Dysregulated Gp1 mGluR signaling is observed with various neurological and mental disorders, including Fragile X Syndrome (FXS) and autism spectrum disorders (ASDs). Although pharmacological correction of Gp1 mGluR activity reverses many of the phenotypes in animal models of those diseases, the molecular and cellular mechanisms underlying Gp1 mGluR-mediated synaptic plasticity have been elusive. Our published and preliminary data introduce the ubiquitin E3 ligase Murine double minute-2 (Mdm2) as a novel translational repressor and a “switch” that permits Gp1 mGluR-induced protein translation (Liu et al., Hum Mol Genet., 2017). In our proposed research, we aim to characterize the role of Mdm2 in Gp1 mGluR- dependent synaptic plasticity (Aim 1) and determine the mechanism by which Mdm2 mediates activity-dependent protein translation (Aim 2). Our new data also show that Mdm2 is molecularly altered and unresponsive to Gp1 mGluR activation in the Fmr1 knockout (KO) mouse, the commonly used animal model for studying FXS (Tsai et al., Hum Mol Genet., 2017). In Aim 3 we will characterize the mechanism by which Fmr1 interconnects Gp1 mGluR signaling to permit translational activation through de-repressing Mdm2. Successful completion of this proposal will greatly facilitate the understanding of Gp1 mGluR-mediated synaptic plasticity through a novel mechanism of translational control. Building on the deep knowledge of Mdm2 in cancer biology, our research will also open a new avenue for the study of neurological disorders associated with abnormal Gp1 mGluR signaling.

NIH Research Projects · FY 2024 · 2020-09

The overarching goal of our research program is to elucidate how nature produces polyether natural products. Polyethers are a subgroup of polyketide natural products and, as a class, they possess a wide range of useful activities, including antibacterial, antifungal, and anticancer properties. However, polyether drug development is hampered by our inability to quickly and efficiently synthesize natural polyethers and their derivatives for medicinal chemistry and drug optimization studies. This is due to the unusually complex structure of natural polyethers. An attractive solution to this problem is to biosynthesize complex polyethers using engineered laboratory-friendly organisms such as bacteria or yeast. This approach is expected to make countless new polyethers accessible for drug research. In order to create a robust and reliable polyether bioproduction platform, we must first achieve a detailed and comprehensive understanding of how polyethers are produced in living organisms. More than 100 different polyether natural products have been discovered so far, and examination of known polyether biosynthetic gene clusters show that all polyethers are generated via a common three-stage biosynthetic scheme. Stage 1: construction of the polyketide backbone by modular polyketide synthases. Stage 2: stereoselective epoxidation of the polyene intermediate by a monooxygenase. Stage 3: formation of the hallmark cyclic ether groups by one or more epoxide hydrolases. The universal nature of this scheme ensures that investigation of any one particular polyether biosynthesis pathway and its associated enzymes will lead to a general understanding of how nature generates polyethers. In this project, we will study the biosynthetic enzymes from the lasalocid A biosynthesis pathway from Streptomyces lasaliensis. Lasalocid A biosynthesis pathway is an excellent model system for studying how nature produces polyethers because it consists of just nine enzymes, yet it possesses all the hallmark chemical transformations of polyether biosynthesis.

NIH Research Projects · FY 2024 · 2020-09

The study of gene expression and possible role of condensates in regulating gene expression have largely ignored known nuclear structures. This proposal is significant because we propose a novel model for the role of nuclear organization in regulating gene expression: 1) Nuclear speckles and still unknown nuclear compartments/bodies help organize other phase-separated condensates to modulate gene expression; 2) Nuclear speckles together with surrounding nuclear compartments/bodies and associated phase-separated condensates together represent active nuclear niches which may have different functional properties; 3) Small distances matter: gene movements of only a few hundred nm between repressive and these different active nuclear niches may differentially regulate gene expression; 4) Action-at-a distance: component flux into and out of these nuclear compartments will have global effects on gene expression; 5) These same nuclear compartments/bodies may similarly modulate RNA processing and organize nuclear export. Here we propose to: 1) Identify multiple components of known and still unknown nuclear “active niches”; 2) Map genome-wide the positions and predicted movements of genes relative to these active niches during physiological transitions; 3) Visualize nuclear body/compartment dynamics and fluxes of proteins between nuclear bodies in steady-state and through physiological transitions; 4) Visualize movements of reporter transgenes, endogenous genes, and rewired chromosome loci relative to these nuclear bodies/compartments and temporally correlate changes in gene expression with their dynamic movements and compartment associations; 5) Visualize movements of pre-mRNAs and nuclear mRNAs during RNA processing and export; 6) Measure fluxes of nuclear body components to and from adjacent transcribing chromatin. Additionally, we propose developing relatively low-cost, novel microscope platforms and software specifically designed to facilitate these live-cell imaging goals in our laboratories as well as others. Our Aims will be to: 1. Map proteins, genes, RNAs relative to active nuclear compartment(s) using iterative rounds of TSA-MS-Ratio, validation by light microscopy, and TSA-Seq; 2. Measure dynamics of bodies, components of nuclear bodies using live-cell imaging; 3. Measure temporal correlation between changes in gene expression and gene movement relative to nuclear bodies and visualize the export path of expressed transcripts; 4. Design and deliver two novel microscopes designed to facilitate Aims 1-3 at a modest cost. Successful completion of these Aims should significantly change our current understanding of the role of nuclear organization in regulating gene expression with impact across a wide range of research fields.

NIH Research Projects · FY 2025 · 2020-09

ABSTRACT Cell homeostasis relies on dynamic and effective protein pathways. Typically, cooperative interactions between partner factors drives selectivity, yet the inherent stability of such complexes can be a detrimental to cell function. Adding to the complexity is the nature of the cell interior, which is densely packed with biological molecules with each having multiple, potential binding partners. As these features challenge the effectiveness of biological pathways by increasing the occurrence of off- pathway or non-productive interactions, cell must have mechanisms to counter these challenges. We believe molecular chaperones, in part, help resolve these complexities. Specifically, we suggest that the Hsp90 molecular chaperone system has evolved to recognize select intrinsically disorder regions (IDRs) on native proteins to both recognize client polypeptides and regulate their actions. Furthermore, we believe a breakdown in the Hsp90 network results in a deterioration of numerous cellular pathways and a triggering of premature aging.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Textbooks teach us that actin filaments give cells their shape, and that a “parts list” of proteins drives actin remodeling when cells change shape. But what is missing from this simple telling is a holistic understanding of how upstream gene expression and signaling control actin remodeling, how different proteins work together to remodel actin, and how downstream cell shape change is converted into timely and reliable organismal out- comes. Because actin-based failures can stem from events before, during and after remodeling, we need an integrated understanding to make sense of actin’s critical role in health and disease. To obtain this kind of “whole picture” view of actin, my lab studies cellularization, the first tissue-building event in Drosophila embryos. We developed this simple experimental system so that we can study the actin remodeling that drives cellularization, while also relating that remodeling to upstream events at the level of gene expression and signaling, and downstream outcomes including morphogenetic fidelity and embryonic viability. Our methods combine Drosophila genetics and embryology with quantitative live-cell imaging of mRNAs, actin, and actin regulatory proteins, down to single-molecule resolution. Our long-term objective is to understand how the actin cytoskeleton interacts with subcellular processes (e.g. transcription) and systems (e.g. nucleus) to orchestrate cell shape change with “the right” kinetics, robust- ness and mechanical properties to achieve successful organismal outcomes. In the next five years, we will focus on three goals arising from our ongoing studies: Goal 1. Determine how gene expression regulates actin remod- eling – Gene expression instructs morphogenesis. Yet, we do not know how transcriptional dynamics inform actin remodeling. For cellularization, five genes that encode actin regulators must be transcribed. We will test a hypothesis that quantitative features of transcription of these genes underpin the global synchrony and uniformity of cellularization in embryos. Goal 2. Determine mechanisms of actomyosin contraction – Actomyosin contraction is essential to cell shape change, but its mechanism is controversial. During cellularization, actomyosin rings contract in back-to-back phases that are mechanistically distinct (Myosin-2 dependent versus independent). We will determine how actin binding proteins drive each mechanism. Goal 3. Determine how the actin cytoskeleton responds to environmental stress – Actin is increasingly recognized as a mediator of stress response. We re- cently identified a heat inducible Actin Stress Response (ASR) in embryos. We will test the hypothesis that ASR puts embryo viability at risk by altering homeostasis between free actin pools in the cytoplasm and nucleus. These goals build on each other so that we will understand how mechanisms before, during and after actin remodeling work together to determine outcomes for the embryo. Our efforts are facilitated by my lab’s proven ability to quantify phenotypes and relate events across scales and subcellular systems. The proteins and processes we study are conserved across organisms so our findings will be broadly relevant.

NIH Research Projects · FY 2024 · 2020-08

Paragons of Conformational Control in Metalloenzyme Reactivity Lisa Olshansky Project Summary. Recent decades have witnessed a revolution in what was once a static picture of biology. For example, the tenets of biochemistry once taught that sequence determines structure, but we now know that sequence and cellular environment determine conformational sampling. Evolutionary selection acts on dynamic rather than static features. The implications for this dynamic biochemical world permeate all aspects of human health. Therefore, it is essential that contemporary research explore the roles, mechanisms, and structure- function paradigms at play therein. However, the complex interplay between structural changes and changes in reactivity make exploring these paradigms in natural systems incredibly challenging. At the same time, simplified synthetic models typically fail to capture the key elements of control leveraged in Nature to regulate activity. My approach is to combine these tactics. By incorporating synthetically prepared metal complexes into proteins that have evolved to undergo allosterically driven conformational changes, we are preparing switchable artificial metalloproteins (swArMs) that represent paragons for conformational control in metalloenzyme reactivity. By creating artificial systems in which changes in structure are directly linked to changes in function, we aim to quantify the effects of conformational control in terms of thermodynamic and kinetic parameters underlying reactivity. Ultimately, this understanding can be harnessed in the development of new catalysts, bioimaging agents, and systems for targeted drug delivery. Our work is poised for the exploration of key unanswered questions in enzyme catalysis, such as how allosteric binding events are converted into metallocofactor activation, or how entropic factors regulate radical chemistry, or how energy conversion occurs in mitochondrial respiratory proteins. Using a wide range of biophysical and spectroscopic methods, swArMs provide a platform with which to explore all of these questions, and to examine the mechanisms of regulation underlying function and dysfunction in metalloenzyme reactivity that are critical to human health.

NIH Research Projects · FY 2024 · 2020-08

Summary Chromatin folding is a key step to pack DNA molecules 10,000-fold into a germ cell, but exactly how the meiotic chromatin is folded and how spatiotemporal folding impacts transcription, chromosome pairing, and recombination remains largely mysterious. Homologous chromosome pairing and recombination are required for accurate chromosome segregation. Mis-segregation of homologous chromosomes is a major cause of miscarriage and birth defects (e.g., Down Syndrome). Three papers have recently reported that high-order chromatin organizations/domains dynamically shape recombination landscape and germline transcriptomes. These dramatic chromatin reorganizations depend on chromatin axes because the domain boundary proteins (e.g., cohesins) are located in the axes. What remains unknown is 1) how meiotic chromosome axis contributes to meiotic gene transcription, homologous chromosome pairing, and chromosome stiffness via chromatin reorganization; and 2) whether the meiotic chromosome structure dynamics are sex- and stage- specific. Our long-term goal is to decipher the meiotic genome organization and its roles in transcription, homologous pairing, and recombination. The proposed work here will specifically test the overall hypothesis that meiotic chromosome axes regulate transcriptome and homologous pairing via controlling local and global chromatin folding in a stage- and sex- dependent manner. To test this hypothesis, we will pursue three specific aims: Determine whether meiotic chromosome axis regulates 1) transcription and homologous pairing via reorganizing local chromatin loops 2) chromosome stiffness and pairing by mediating global chromatin folding. 3) Uncover the temporal and sexual differences of meiotic transcriptomes and chromatin organization. Method: In aim 1, local chromatin reorganization in spatial domains will be detected by chromosome conformation capture (Hi-C) contact map and the corresponding changes of transcriptional levels within these domains can be measured via RNAseq. Chromosome interactions will be examined locally by Hi- C analysis (aim 1) and stiffness will be measured globally by micromanipulation (aim 2). Fluorescent in situ hybridization will be used to verify the intra- and inter-chromosome interaction found in Hi-C map. Hi-C, single- cell RNAseq, and micromanipulation will be introduced to reveal the 4D meiotic genome reorganization and sexual dimorphism of transcriptome and chromatin folding in aim 3. The approach is innovative because multiple advanced methods including Hi-C, single-cell RNAseq, micromanipulation, nano-newton force measurement, and quantitative immunocytology will be integrated to generate a more complete picture of meiotic chromosomes. The proposed research is significant because it is expected to provide a deeper understanding of chromosome structure and how the structure varies in different stages and genders. Ultimately, insights from these studies will help us develop diagnosis and treatment for infertility, miscarriage, and birth defects.

NIH Research Projects · FY 2025 · 2020-08

PROJECT ABSTRACT This application is to evaluate the potential of a super-resolution microscopy system to image disrupted nanoscale chromatin folding as an early event in carcinogenesis and explore its potential to improve cancer risk stratification. Abnormal chromatin structure is among the most universal characteristics of tumor cells and has been used for clinical cancer diagnosis for two centuries. However, due to the diffraction-limited resolution of conventional light microscopy, only microscale structural abnormalities can be observed. As a result, cells undergoing early stages of malignant transformation often appear normal. Such limitation in image resolution has compromised our ability to accurately risk-stratify precursor lesions or distinguish aggressive from indolent forms. Recent advances in super-resolution fluorescence nanoscopy now enable us to image molecular-level chromatin structure down to a resolution of ~20-30 nm. Our group recently improved the throughput and robustness in stochastic optical reconstruction microscopy (STORM)-based super-resolution microscopy and enabled robust imaging of chromatin folding on the most widely used clinical samples. Built upon our preliminary studies that revealed a significant and gradual disruption of nanoscale chromatin folding in early carcinogenesis, this project will first further confirm the disrupted chromatin folding that accompanies carcinogenesis and identify their molecular characteristics and functional consequences. Second, we will optimize the workflow of super-resolution imaging system, sample preparation and image analysis to enable efficient and reproducible analysis of nanoscale chromatin folding in clinical tissue samples. We will also validate our finding of disrupted chromatin folding in patients with various colorectal precursor lesions and cancer. Third, we will evaluate the potential of imaging nanoscale chromatin folding to in patients with colorectal adenomatous polyps. This study will establish the scientific basis and underlying molecular profile of disrupted nanoscale chromatin folding in early carcinogenesis, opening a new avenue for risk stratification, facilitating the development and evaluation of new preventive strategies.

NIH Research Projects · FY 2025 · 2020-07

Project Summary/Abstract A fuller understanding of living systems at the molecular level has led to revolutionary changes in the fields of chemistry and biology and has made the boundary between these traditional disciplines less defined. To leverage the exciting scientific advances at the chemistry-biology interface and to be poised to make major contributions to biology and medicine, chemists and biologists must speak a common scientific language. The Chemistry-Biology Interface Training Program (CBI-TP) at the University of Illinois, Urbana-Champaign (UIUC) will offer an environment in which chemists and biologists can acquire interdisciplinary training that enables them to learn a common language and to acquire a shared set of research skills without compromising their ability to acquire deep discipline-specific knowledge in specialized areas of chemistry or biology. Through shared experiences in the classroom and in the laboratory, the CBI-TP is intended to enhance the studies of graduate students enrolled in six departments at UIUC: Chemistry, Chemical and Biomolecular Engineering, Biochemistry, Microbiology, Cell and Developmental Biology, and Molecular and Integrative Physiology. Each year, 14 trainees will be supported by the NIH, in addition to 7 matching positions supported by UIUC. Highlights of the program include courses specifically developed for the CBI-TP, first year laboratory rotations, strong training in rigor and reproducibility, an opportunity for internships, a highly diverse student body, and extremely strong institutional support. The program also offers unique opportunities for professional and career development including (i) a student-run seminar program during which trainees invite, host, and discuss their research with outside seminar speakers, (ii) creation of Individual Development Plans (IDPs) for which trainees are required to research at least three career options, join a professional organization, and attend at least one career-related workshop, (iii) options to earn relevant certifications such as the Certificate in Entrepreneurship and Management, and/or take part in internships, and (iv) participation in multiple career development activities, intended to better prepare graduate students at the chemistry-biology interface for interesting and challenging career paths and opportunities. No matter their ultimate career goals, the CBI-TP platform at UIUC, running for 19 years now, has no equivalent elsewhere on our campus. The goal of the CBI-TP is to arm trainees with the breadth of education necessary to bridge the gap between the chemical and biological sciences. The trainees have been, and will continue to be, among those scientists making significant contributions to biomedicine as participants in and leaders of multidisciplinary teams in industry, government, or academia.