University Of Massachusetts Amherst

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Twenty-five years ago, the University of Massachusetts launched a training program at the interface of chemistry and biology. The program built on existing strengths and harnessed our commitment to building bridges between the physical and life sciences. Since then, the University of Massachusetts has undergone a complete transformation in the life sciences. Massive investments in state-of-the-art core facilities and research buildings coupled with the hiring of over 50 new faculty has revolutionized the research capacity on campus. By establishing a collaborative community and interdisciplinary curriculum, the Chemistry-Biology Interface (CBI) training program has been at the center of this growth. The 31 CBI Training Faculty now train a total of 162 talented students, 54 of whom are eligible for support from the CBI training grant. CBI students come from five participating graduate programs: Chemistry, Chemical Engineering, Microbiology, Molecular & Cellular Biology, and Polymer Science & Engineering. The CBI curriculum is designed to complement the requirements of these programs, to achieve four objectives: 1) to provide fluency in both chemistry and biology concepts, tools, and opportunities; 2) to communicate and collaborate effectively with scientists from diverse chemistry and biology research backgrounds; 3) to follow best practices in performing rigorous and reproducible research; and 4) to increase awareness of career opportunities and build the appropriate skills to network effectively in order to take advantage of these opportunities. Robust participation of ~ 60 CBI student members who complete the curriculum, regardless of funding, demonstrates the perceived high value of this training. In this proposal, we seek to enhance the training capacity of the CBI program by requesting twelve trainee slots, which will be matched with four slots annually from UMass. Over the next five years, the cohort of CBI trainees will participate in several innovative training activities, including a revised curriculum incorporating the principles of rigor and reproducibility, specialized laboratory modules designed to introduce trainees to new concepts and techniques, and a biennial Alumni Symposium where current trainees will have the opportunity to connect with our network of 143 alumni. Equipped with this comprehensive skillset, graduates from our CBI program will be well-prepared to solve frontier problems in biology and biomedicine.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Circadian clocks are time-tracking systems that allow organisms to adapt to the time of day, and drive many cellular functions. Their alteration can lead to various diseases, including cancer, heart disease, and metabolic disorders. In this work, we propose development of a chemical biology toolbox, consisting of both imaging and protein-targeting platforms to facilitate studies of circadian rhythms at the molecular level that have otherwise not been possible. The core clock is comprised of a transcriptional-translational feedback loop with multiple protein components, including paralogs. Disparities have been observed between promoter activity and protein translation, among responses of core clock components to stimuli, and compensatory mechanisms resulting from knock-down and knock-out strategies. To address these gaps and facilitate additional studies of the molecular clock, we will use chemical biology-based strategies to: 1.) generate orthogonal chemiluminescent scaffolds to track promoter activity and protein translation of multiple circadian genes in a parallel, high-content manner; and 2.) develop small molecule and protein-based tools to directly target circadian proteins and their interactions. For studies of the molecular clock, it is essential to track circadian rhythms and target core clock proteins in a dynamic and selective manner. Firefly luciferase-based reporters have been used to assess promoter activity of individual circadian genes, and most protein-based studies involve cells derived from a single luminescent mouse model. We will use orthogonal chemiluminescent probes (Nano-lanterns) to develop a multi-signal platform to simultaneously track and associate promoter activity and protein translation relationships among multiple genes. Perturbation of circadian proteins is also essential for understanding mechanisms, including paralog roles. Currently, there are few options outside of genetic approaches, which can result in unilateral changes and activation of network compensation mechanisms. Molecular tools offer the ability to directly target the functional components of the clock – proteins, and/or their interactions. While small molecules present an attractive approach, relatively few exist that directly target core clock proteins. Hence, we propose to generate new agents for interrogating the circadian clock system: we will repurpose validated clock protein-binding small molecules by synthetically converting them into protein degraders (PROTACs), and use yeast surface-display to identify nanobodies that bind circadian proteins and prevent specific interactions. Together, these approaches present a powerful means to understand the mechanisms of the circadian clock, and can be used in a variety of models and in studies of diseases, including to uncover new therapeutic targets.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Flavivirus are major mosquito-borne pathogens infecting millions of people worldwide each year. Currently there is no antiviral therapy available for treating West Nile, Dengue and Zika viral infections. The first vaccine CYD- TDV (Dengvaxia) against DENV was approved last year but shows only 56% overall efficacy against the four dengue serotypes. The flaviviral two-component NS2B/NS3 protease is required for viral replication and thus an attractive antiviral target. However, extensive screening and rational design efforts have failed to identify any clinically viable inhibitors at this point. Two key factors have likely contributed to the challenge. First, traditional screening efforts rely primarily on binding affinity to predict the drug efficacy. Yet, increasing evidence has emerged to show that the residence time of drug-target interaction is a more reliable predictor of in vivo pharmacological activity. These kinetic rate parameters are generally not available during early stages of drug discovery. Second, NS2B/NS3 proteases display complex conformational dynamics during function and inhibition, which is still poorly understood. This project aims to develop a new label-free single molecular approach to resolve the conformational states of NS2B/NS3 proteases. Key to the approach is the use of an innovative nanopore tweezers where the protease is confined with the pore lumen, allowing dynamic structural changes during substrate or inhibitor binding to be continuously monitored by current fluctuation signals. Analysis of the current traces will provide a complete profile of binding affinity and kinetic rates as well as the distribution of conformational states. Specifically, we will first build a nanopore tweezers tool set that is readily tunable for trapping various flaviviral proteases. Secondly, we will track and analyze the functional states of the NS2B/NS3 protease in the presence of various substrates. Influence of critical residues, substrate, construct design on the dynamic equilibrium between the “open” and “closed” states will be assessed to provide insight into the mechanism of protease activity. Finally, the nanopore tweezers will be deployed to determine the structural dynamics and binding thermodynamics and kinetics profiles of NS2B/NS3 interacting with various inhibitors. Once the inhibition profiles are established, the nanopore tweezers confined NS2B/NS3 system will be tested for screening a diverse compound library to identity novel allosteric inhibitors with improved drug-like properties compared to active-site inhibitors. This work will provide unprecedented kinetic information on the function- structural dynamics relationship of NS2B/NS3 complex and mechanisms of substrate binding and inhibition, as well as establish a new paradigm for high-throughput drug screening that is independent of enzymatic activity.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Asthma is the most common chronic disease of childhood and causes more preventable hospitalizations and lost school days than any other childhood disease. Children in lower-income, urban, and racial/ethnic minority populations are more likely to have asthma, to have poorly controlled disease, and to experience preventable morbidity. Multi-level interventions that address the complex array of interrelated societal and environmental factors that contribute to these disparities are poorly supported in traditional fee-for-service payment models. In contrast, accountable care organizations (ACOs) are designed to support and sustain interventions that address social determinants of health as part of medical care. A substantial and growing number children with asthma in higher-risk populations receive care in ACOs, but their effect on asthma quality of care, outcomes and disparities is not known. This study will be the first to address this critical gap in knowledge by taking advantage of a natural experiment taking place in Massachusetts (MA), a state with high rates of childhood asthma: In 2018, MA, launched 17 new Medicaid ACOs with varied organizational features (e.g., size; age mix). We will use detailed state-level claims data to: 1) determine the association between implementation of the ACOs and changes in childhood asthma quality indicators, health outcome for Medicaid-insured children and 2) assess changes in socioeconomic and racial/ethnic disparities in these outcome measures comparing children enrolled in Medicaid ACOs to matched commercially-insured children. We then turn to understanding the influence of ACOs’ organizational features on change by: 3) using mixed-methods to generate detailed characterizations of the ACOs’ organizational features and 4) test the association between these features and childhood asthma quality indicators and outcomes. We will use innovative methods of risk adjustment that take social risk factors into account and propensity matched difference- in-difference analyses to account for factors other than ACO implementation that may affect outcomes. This study will take place at a moment when the profound inequities in health and healthcare in the U.S. are starkly illuminated by the COVID-19 pandemic. Socioeconomic and racial/ethnic disparities in childhood asthma persist despite the existence of evidence-based treatments resulting from decades of work and billions of dollars directed at improving asthma care. Structural changes in healthcare are needed for equitable delivery of evidence-based asthma care, which should reduce or even eliminate these long standing disparities. The ACO model moves healthcare payment and delivery in a direction that could facilitate this change. This study will address major gaps in knowledge as to whether the large investments being made in developing the ACO model may pay off for the millions of children at increased risk for long-term poor health due to preventable consequences of asthma.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Infertility and subfertility are critical health problems affecting about 9 % of couples worldwide. Since the first successful “Test-Tube” baby in 1978, over 5 million babies were born using Assisted Reproductive Technology (ART). ART includes such techniques as in vitro fertilization (IVF), intrauterine insemination (IUI), intracytoplasmic sperm injection (ICSI) and embryo transfer techniques. ART is used in humans, and in animals of economic relevance. In humans alone, IVF and ICSI are used ~ 800,000 times per year. In all species, the limiting factor for successful pregnancies to occur is obtaining good quality preimplantation embryos which have a direct influence in implantation and pregnancy rates. Capacitation involves crosstalk between metabolic and signaling pathways. In the previous period, we showed that a short incubation with the Ca2+ ionophore A23187[7] can induce in vitro fertilizing capacity in sperm from sterile knock-out (KO) genetic models. We hypothesized that, at least, in part, A23187's effects were due to changes in metabolism. When sperm metabolism was changed using starvation and rescue protocols, we observed that intracellular Ca2+ was elevated. In addition, we found that, similar to A23187, sperm incubated in the absence of nutrients become immotile. Once nutrients are added back to the incubation media, sperm motility is rescued and those sperm depict higher percentage of hyperactivation and enhanced in vitro fertilization (IVF) rates. Unexpectedly, eggs fertilized with sperm incubated in metabolic enhanced conditions were more efficient in producing blastocysts and those blastocysts generated more implantation sites and produced more pups when transferred to pseudo-pregnant females. This proposal has basic and applied goals. The basic science objective is to understand the molecular basis of these methods with particular emphasis on the crosstalk between calcium and metabolic pathways. At the translational level, our goal is to use novel sperm incubation conditions to improve ART.

NIH Research Projects · FY 2025 · 2021-03

Project Summary: Arsenic contamination in the food chain is a global health problem and causes damage to most human organs. A significant need exists to develop approaches for addressing environmental arsenic. The long term goal is to develop a plant-based phytoremediation approach for contaminated land that is cost-effective and ecologically friendly as an alternative to conventional remediation methods. The objective of this study is to develop a genetics-based phytoremediation strategy for arsenic uptake, translocation, detoxification, and hyperaccumulation into the fast-growing, high biomass, non-food crop Crambe abyssinica. Nanosulfur will be utilized to modulate the bioavailability and phytoextraction of As from soil and to increase the storage capacity via enhanced sulfur assimilation. The engineered Crambe will be evaluated for removing arsenic from the soil in laboratory, greenhouse, and field conditions. Our central hypothesis is that organ-specific expression of genes, which control the transport, oxidation state, and binding of As, can be tuned to yield efficient extraction and hyperaccumulation into above-ground plant tissues. To test our hypothesis, we propose the following specific aims. 1) Genetically engineer Crambe abyssinica lines for co-expressing bacterial ArsC, gECS, and AtABCC1 and RNAi suppression of endogenous arsenate reductase CaACR2; 2) Evaluate the engineered Crambe lines for metal(loids) tolerance and accumulation; 3) Synthesize and apply nanosulfur to modulate the bioavailability, phytoextraction, and accumulation of toxic metal(loids); and 4) Conduct a pilot field study of engineered Crambe lines for phytoextraction on a contaminated site. After initial screening in tissue culture media supplemented with metals, the best performing quadruple gene stacked (ArcS+gECS+AtABCC1+CaACR2Ri) Crambe lines with wild type controls will be tested using contaminated soils with arsenic as well as co-contaminants in greenhouse. A pilot field-scale study will then be carried out at a site contaminated with arsenic. The soil will be extensively characterized, and analysis for metal content and arsenic speciation will be determined using ICP/MS, HPLC- ICP/MS as well as XANES (X-ray Absorption Near-Edge Spectroscopy). Last, soil amendments with engineered nanosulfur will be used to evaluate the impacts on soil structure and contaminant availability and phytoextraction. Nanosulfur will also be foliarly applied to plants to increase the metal storage capacity via enhanced sulfur assimilation. The expected outcome of this project is a mechanistic understanding of the biogeochemical and plant processes of arsenic remediation that connects key soil characteristics with the efficiency of phytoextraction and hyperaccumulation of arsenic. The results will have an immediate and important positive impact because the knowledge generated from this study will enable efficient and effective phytoremediation approaches to minimize or remove arsenic contamination in the food chain and enhance public health.

NIH Research Projects · FY 2025 · 2021-01

SUMMARY Aromatase inhibitors (AIs) are drugs that inhibit estrogen synthesis and that are prescribed to prevent the recurrence of estrogen responsive breast cancers. However, AIs, such as the commonly prescribed Letrozole (LET), are associated with severe side-effects that further burden the quality of life, including insomnia, hot flashes, depressive symptoms and cognitive deficits. The precise mechanisms by which AIs may give rise to these CNS symptoms remain unclear and difficult to study in humans, as control for individual differences in disease severity, treatment history and experienced stress is lacking. Furthermore, AI treatment is recommended for 3 to 5 years, yet little is known about the effects of long-term AI use on the brain and behavior, especially with regards to age-related cognitive decline and Alzheimer's disease (AD) risk. We propose to develop a primate model for AI-induced CNS effects to advance our knowledge in this area and facilitate the design of novel therapeutics. This application uses the marmoset (Callithrix jacchus), a small primate with a brain architecture, sleep patterns, cognitive abilities, emotional responses and thermoregulation patterns that are comparable to those of humans (1) to study the effects of chronic LET use on the brain and behavior and (2) to test whether DHED, a prodrug that delivers E2 selectively to the brain, can effectively and safely prevent LET-associated adverse effects. To achieve these aims, middle-aged male and female marmosets treated with LET, LET + DHED or Vehicle for 3 years will be studied longitudinally for changes in sleep/wake patterns, cognitive performance, emotional regulation, and thermoregulation. The monkeys will be outfitted with an activity monitor for sleep/wake patterns analysis. Cognitive function will be assessed via an automated computerized battery. Thermal imaging will be used to measure changes in facial skin temperature during a thermal challenge. Emotional regulation will be assessed by measuring heart rate variability and facial skin temperature in monkeys viewing emotional and neutral videos. Following these in vivo behavioral assessments, analyses of brain tissues from underlying brain regions (hypothalamus, hippocampus, prefrontal cortex, locus coeruleus) will be carried out to quantify gene expression of selected genes, tauopathies, β-amyloid deposition and neuronal excitability. The results will have important translational applications for AI-treated patients by (1) characterizing the effects of AIs on multiple neural and behavioral outcomes; (2) determining whether long-term estrogen suppression promotes the development of an AD-like phenotype and (3) whether providing the brain with an alternate source of estrogen can counteract the adverse effects of AIs on the brain and behavior.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY College students are at risk for engaging in heavy alcohol use, affecting academic performance and mental and physical health. Brief motivational interventions reduce alcohol use and consequences, but intervention gains decay 3-6 months post-intervention. Mandated college students, who have violated university alcohol policy, are an important intervention target; they drink more, experience more alcohol-related consequences, and are less likely to maintain reduced drinking than typical students. To promote student health, brief motivational interventions need to be improved to promote maintenance of reduced drinking. This K01 outlines the necessary research and training experiences to prepare the PI to become an independent researcher focused on developing interventions that promote not only initiation, but also maintenance of reduced drinking among young adults. Notably, the PI will learn the innovative, engineering-inspired multiphase optimization strategy (MOST), which uses the factorial experimental design to develop highly efficacious interventions. The proposed research consists of three stages. First, a systematic review will examine theories of and empirical research on maintenance of behavior change. This will yield recommendations for mechanisms that are likely to improve maintenance of reduced alcohol use among young adults. In the subsequent stages, all participants will be mandated students who receive a brief motivational intervention, providing a context for studying maintenance-enhancing constructs and intervention strategies. In the second stage, the utility of constructs for predicting maintenance will be examined in 475 mandated students, followed 1-, 3-, and 6-months post- intervention. Constructs proposed by two prominent models of maintenance—coping motives, parent-student communication, maintenance self-efficacy, and recovery self-efficacy—in addition to promising constructs identified in the systematic review, will be examined as predictors of maintenance trajectories. Third, intervention strategies targeting identified maintenance enhancement constructs will be developed and pilot tested for acceptability among 60 mandated students; a mandated-specific parent handbook will be piloted among 20 of their parents. A pilot optimization trial will also be conducted with 80 mandated students and their parents. Students will be randomly assigned to one of up to 16 conditions in a factorial experimental design. Results of the proposed research will yield important information on the factors that promote maintenance of reduced alcohol use, supporting subsequent grant applications to further develop a maintenance-enhancement intervention. The PI will work with a highly skilled mentoring team (Drs. Kate Carey, Katie Witkiewitz, Linda Collins, Rebecca Spencer) to gain expertise in: 1) maintenance of behavior change theories; 2) intervention development; 3) MOST; 4) management and analysis of longitudinal research; 5) grant writing. The research and training activities proposed in this K01 will produce both an independent research scientist and important scientific knowledge on maintenance of health behavior change, an understudied topic.

NIH Research Projects · FY 2024 · 2020-08

Project Summary / Abstract Upper-limb paresis is the most common impairment following a stroke affecting 75% of stroke survivors, which can be more prominent in one of the two limbs. Most recovery of functional impairments occurs within the first few weeks after stroke and plateaus thereafter. Unfortunately, even after patients reach a stable phase of recovery, their functional level of the stroke-affected limb may decline. Therefore, it is clinically important to maintain the regained functional level beyond the first couple weeks of spontaneous recovery by continuing to practice the use of the affected limb during daily living. Wearable technologies have emerged as a low-cost, objective tool to monitor the performance of the upper limbs during activities of daily living (ADLs). However, to date, there exists no study that has investigated the effectiveness of a mobile-health (mHealth) system aiming to enable high-dosage motor performance in chronic stroke survivors in the real-world setting. Specifically, the optimal configuration of the goal setting, feedback mechanism and ways to share data among the stakeholders (patients and clinicians) remains unknown. This proposal aims to develop and validate an mHealth technology that aims to encourage affected limb use during the performance of ADLs in chronic stroke survivors. To accomplish this goal, we will employ the unique finger-worn ring sensor (accelerometer), developed by our academic-industry partnership, that can capture both gross-arm and fine-hand use of the limbs that are essential in the performance of ADLs. We will study important aspects of making positive behavior changes to encourage the affected limb use by fully leveraging the computational insights drawn from sensor data combined with clinical insights from providers. To accomplish this research goal, Aim 1 will focus on the development of an mHealth platform, composed of body-networked sensors and cloud-based systems, to monitor the real-world use of the limbs in chronic stroke survivors. In Aim 2, we will develop machine-learning based algorithms to extract clinically meaningful information regarding real-world upper limb use from sensor data. Aim 3 will investigate the optimal design of our mHealth system – such as individual tailoring of the goal, design of the feedback, medium and timing to deliver feedback, and ways to share data among the stakeholders (patients and clinicians) – via human-centered design approaches. Finally, in Aim 4, we will validate the short-term (8 weeks) effectiveness of the mHealth system in improving the use of the affected limb through a field deployment study. We believe that outcomes of this project will open a new door leading to previously unexplored datasets and understanding of patient-technology interactions to promote positive behavior changes to enable a high dosage of physical and occupational therapy, which can form the basis of a wide range of future investigations of hemiparesis rehabilitation and personalized disease management.

NIH Research Projects · FY 2025 · 2020-07

Project Summary The past decade has been truly transformative for the Applied Life Sciences at the University of Massachusetts, Amherst (UMass). During this period a $150M investment in state-of-the art equipment housed in 30 core facilities operated by PhD-level center directors and over $400M in new research buildings has revolutionized the research capacity at our institution. This massive infrastructure growth has been matched with the hiring of over 50 new faculty in the life sciences. During this tremendous expansion we have developed the Biotech Training Program (BTP) in Applied Life Sciences, which leverages campus investments to provide outstanding training to a talented group of graduate students to prepare them for careers in the Biotech workforce and related areas. This training is guided by these objectives: 1) create a scholarly and social environment to facilitate new and strengthen existing interdisciplinary networks at UMass, particularly those at the interface of engineering and the life sciences; 2) educate students in the fundamentals of quantitative biotechnology through lecture and laboratory courses; 3) train students in the commercial impact of biotechnology through a tailored industrial internship and regular interactions with industrial personnel; 4) provide students with opportunities to improve interdisciplinary communication, expand career opportunities, and sharpen professional skills; and 5) increase the number of students, particularly those from underrepresented groups, who pursue careers in biotechnology. BTP faculty are recruited not by departmental affiliation, but by membership in the Institute for Applied Life Sciences (IALS), their research in biotechnology and their commitment to student training. For this reason, the UMass BTP recruits students from twelve PhD programs: Biomedical/Chemical/Civil/Mechanical Engineering, Chemistry, Microbiology, Molecular & Cell Biology, Neuroscience & Behavior, Plant Biology, Polymer Science & Engineering and Veterinary & Animal Science. We request twelve Trainee slots (matched with three and a half slots annually from UMass). Traineeships are awarded to students for the 2nd and 3rd years of study during which students complete the BTP curriculum. Innovative features include the Frontiers in Biotechnology course that is co-taught by industry personnel; specialized Laboratory Modules in biotechnology- techniques modeled after industry workshops; student-run Journal Club to break down discipline barriers and promote modern data analysis fluency; leadership in campus recruiting efforts for a diverse student population and accessibility to disabled students; an annual Symposium in Biotechnology (years 1 and 2) or BTP retreat (every 3rd year) that each offer a Biotech Battles experience where students solve real-world problems guided by industry experts; and targeted partnering with the Office of Professional Development for career exploration and planning. We have established industrial partnerships to support the hallmark of our BTP, a formal internship for all Trainees. Together, this comprehensive program prepares students well for careers in biotechnology.

NIH Research Projects · FY 2026 · 2020-04

PROJECT SUMMARY / ABSTRACT Our research program is focused on developing robust platforms that enable protein-based biologics as therapeutics for intracellular targets. Biologics offer distinct advantages over small molecule drugs, because of their high specificity, including against targets that are considered undruggable with small molecules. However, large size and hydrophilic nature of proteins present the formidable challenge of transporting them to intracellular locations. Protein-based therapeutics have been, thus, largely limited to extracellular targets. Recognizing this gap, we have focused on developing a platform with the combination of characteristics, critical for transporting proteins inside the cells. Accordingly, we arrived at a new protein-encapsulating nanogel system that effectively and tracelessly delivers the proteins inside the cells in its functional form. Over the next five years, we seek to build on these accomplishments to explore new scientific directions. We will create a new antibody-directed antibody conjugates (ADACs) platform with the goal of engaging currently undruggable intracellular targets in specific cells. ADACs contain two distinct antibodies – one for targeting extracellular epitopes for cellular specificity and another for engaging a specific intracellular target. We leverage the combined capabilities of the protein-polymer self-assembly to generate nanogels with the bioorthogonal chemistries developed for decorating the nanogel surfaces with antibodies, to create ADACs with tunable physiochemical characteristics. We will investigate the potential for ADACs in targeted protein degradation (TPD) of model intracellular targets that are considered undruggable, because of a single missense mutation or single site post-translational modification. In this context, we will also fully test the potential of the TRIM21 E3 ligase pathway for proteasomal TPD using antibodies and compare its efficacy with the more classical inhibition pathway in eliciting downstream responses. Concurrently, we will also develop new strategies for increasing the efficacy and specificity of antibody-based biologics using the ADACs platform. Endosomal escape has been identified as the major bottleneck in the efficacy of intracellular biologics. We propose two new, mechanistically distinct strategies for functionalizing nanogel surfaces that promote endosomal escape. Similarly, to substantially enhance cellular specificities of therapeutics, we propose a high-risk, high-payoff strategy for in-cell assembly of antibody-like molecules using Boolean logic based convergence. We have chosen the components of our approaches to be independent, such that each of our scientific goals independently inform new strategies for solving critical and unsolved issues in the field of intracellular biologics. Simultaneously, these goals are also designed to be convergent such that when put together, the impact of our overall approach will be exponentially higher. Collectively, our program will inform and develop strategies for enabling biologics as the next generation therapeutics for intracellular targets.

NIH Research Projects · FY 2025 · 2020-02

Project Summary/Abstract Point of Care Diagnostics for Liver Disease using Fluorescent Nanosensors Liver fibrosis/cirrhosis is a major driver of mortality and morbidity worldwide. Rapid diagnosis of liver fibrosis is crucial to optimizing patient outcome and minimizing economic impact of this disease. Current strategies for detecting liver damage (fibrosis/cirrhosis) use biomarker and mechanical strategies that are expensive and difficult to translate into Point of Care (PoC) platforms. Robust PoC liver diagnostics would enable regular monitoring of chemotherapy patients and game-changing diagnostics for the developing world. In preliminary studies we have demonstrated that polymer-based sensor arrays on paper substrates can generate serum ‘signatures’ that can diagnose liver fibrosis with clinical relevance. In our proposed research we will build on this foundation to create effective lateral flow device (LFD) diagnostics for liver fibrosis. In our multi-pronged strategy, we will: Aim 1: Synthesize engineered polymer conjugates (Rotello) and use these as sensor elements to provide multi-channel outputs serum sensing. Protein/serum selectivity will be guided by integration of synthesis (Rotello) with computational/machine learning tools (Van Lehn) in a feedback-driven cycle. Thesse studies will be performed in solution to facilitate sensor optimization. Aim 2: Fabricate LFD devices and immobilize polymers downselected from Aim 1 onto surfaces to provide prototype sensing systems suitable for PoC use. (Rotello) These sensors will be tested and optimized using model sera generated by spiking healthy serum. Aim 3: Apply LFDs downselected from Aim 2 to profile liver fibrosis using pathological samples and liver diagnostics insight provided by Rosenberg and Peveler. These studies will focus on detection and staging of liver fibrosis, using statistical methods developed by C. Rotello. Aim 4: Effective sensor systems identified in Aim 3 will be explored using proteomics by Vachet. These studies characterize protein binding to the polymer sensors, providing insight into how the sensor works and potentially new biomarkers for fibrosis. The key driver of the proposed research is the development of PoC systems for diagnosis of liver fibrosis; effective achievement of this goal would provide strategies that could be translated to numerous disease states. The focus on polymer-protein affinity and selectivity will provide new insight into fundamental aspects of these interactions, and the integration of machine learning into this process will develop new polymer design strategies biomedical applications.

NIH Research Projects · FY 2025 · 2020-01

Project Summary The overarching goal of this R25 application is to provide training to diverse high school and undergraduate students who will work with supportive faculty mentors to understand how environmental chemical exposures contribute to non-communicable diseases, and how hazard and/or exposure mitigation can improve health. Program faculty come from a diverse range of disciplines: toxicology, endocrinology, cell and molecular biology, veterinary and animal sciences, chemistry, epidemiology, chemical engineering, civil and environmental engineering, and science communication. We will focus on recruiting women and individuals from underrepresented groups to participate in this summer program, which includes three aims: 1) To involve high school age young women in introductory summer research experiences and recruit those with an interest in participating in research projects or science communication projects. By partnering with the local Girls Inc. organization, we will offer week-long introductory training programs for 9th grade girls from diverse but underserved communities in Western MA. With 24-26 girls participating every year, we will use this program to recruit 10th and 11th grade girls to participate in Aims 2 and 3. 2) To create a learning community focused on understanding how environmental chemical exposures contribute to non-communicable diseases and develop scientific strategies to create solutions to these challenging problems. High school and undergraduate students will work with UMass faculty to understand how environmental chemical exposures contribute to non-communicable diseases, and how hazards and/or exposures can be mitigated. Research will focus on one of these areas: • molecular and cellular models of diseases relevant to environmental chemical exposures; • characterizing chemical exposures and their association with human diseases; • understanding the effects of environmental chemicals on conditions such as cancer and metabolic diseases; • creating solutions for exposures to hazardous environmental chemicals including use of green chemistry principles to avoid hazards entirely and pollution remediation when exposures cannot be avoided. 3) To train diverse participants in methods to communicate complex environmental health concepts in the context of our learning community and in broader communities. High school and undergraduate students will work to create science communication tools, aimed at educating a lay audience about one or more topic addressed in the laboratories in Aim 2. Communication tools could include static or animated infographics, informational videos, podcasts, or other social media tools.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY/ABSTRACT The long-term goal of my research group is to develop “next-generation” nucleic acid-based platform for understanding how living organisms function and for disease diagnostics. In particular, we are creating a series of DNA probes for measuring cell membrane biophysical interactions, especially the biomechanical features at cell-cell junctions, as well as developing fluorogenic RNA aptamer-based sensors for targeted imaging inside living organisms. Our immediate goal for the next five years is to make precise intercellular force measurement and regulation readily available for implementation in life science laboratories. We will demonstrate how these novel DNA-based tools can be broadly used to understand the basic mechanical principles of development, physiology, and disease. These DNA mechanical probes and actuators will also serve as the critical foundation for developing novel strategies in tissue engineering, regenerative medicine, and cell therapy. Mechanical forces play fundamental roles in many intrinsic and collective cellular processes, such as tissue regeneration, morphogenesis, and tumor metastasis. While extensive studies have focused on the forces between cells and extracellular matrices, mechanical interactions among individual cells appear to be important yet poorly characterized. These intercellular forces are known to be critical during wound healing, cancer cell invasion, and other developmental and homeostatic processes. However, the molecular principles that govern these finely balanced mechanotransduction events are still poorly understood. To depict the mechanisms of these collective cellular processes, it is essential to measure and fine-tune intercellular forces at the molecular level, and then correlate the patterns of mechanical landscapes with the specific molecular machineries that regulate cellular signaling. In the past project period, we have developed precise and easy-to-use DNA-based fluorescent probes to visualize and quantify forces at cell-cell junctions. These synthetic DNA probes can spontaneously anchor onto the external surfaces of live cell membranes and allow sensitive imaging of a broad range of intercellular molecular forces simply after a brief incubation. During the next 5-year project period, we plan to further develop highly robust, versatile, and high-throughput DNA-based mechanoprobes and novel mechanical actuators. These tools will be further used to provide unique insights for elucidating the mechanical mechanisms of several key collective mechanosensitive cellular events in neural development, cancer metastasis, and immunology. High-throughput screening platforms will also be validated and applied for the identification of novel modulators of intercellular forces and potential drug candidates. In contrast to many current mechanobiology studies that are based on techniques typically performed in only a few specialized laboratories, the DNA probes, actuators, and screening platforms developed in this project can be potentially widely adopted for measuring and regulating molecular forces at cell-cell junctions.

NIH Research Projects · FY 2023 · 2019-09

PROJECT SUMMARY/ABSTRACT The central goal of this project is to serve as a strong, interdisciplinary center of forecasting research and science. We will achieve this through innovation in development of forecasting methodologies and systematic research into optimal the communication and visualization of forecasts. By working with the US Centers for Disease Control and Prevention in this cooperative agreement, we aim to extend existing methodologies to incorporate new data sources and model structures, thereby improving influenza forecast accuracy in the US. We will review and revise existing FluSight forecasting guidance, targets, and accuracy evaluation at the national, regional, and state levels. This will include conducting stakeholder interviews with federal, state, and local epidemiologists to track uses of forecasts and outcomes and identifying unmet needs from current forecasts. We will develop and refine methods to create forecast ensembles, with a specific focus on developing ensemble weighting schemes using robust, penalized methods to estimate model weights. We will identify methodologies and data sources that increase forecast accuracy for start and peak week forecasts, peak intensity, and short-term forecasts at the national, regional, and state level. Our work here will focus on developing multi-scale spatial models that leverage state and zip-code level data on influenza infections from public and private sources. We will develop communication products and methods to describe forecast results and uncertainty for federal and state public health officials and the public. We will achieve this by incorporating new visualizations into our existing interactive data visualization product for influenza forecasts in the US and studying systematically the end-user perception of various visualization and data presentation layouts. Finally, we will develop and adapt successful seasonal methodologies, data sources, and communication approaches for forecasting the timing, intensity, and short-term trajectory of an emerging influenza pandemic. Specifically, we will create, test, and disseminate weekly data summaries and visualizations from the most up-to-date sources of reported influenza cases in the US (including data from our real-time point-of-care data sources), and validate our new spatial models against simulated pandemic data.

NIH Research Projects · FY 2023 · 2019-07

A major driver of the U.S opioid crisis is limited access to effective medications for opioid use disorder (MOUD) that reduce overdose. Traditionally, jails and prisons in the U.S. do not initiate or maintain MOUD for inmates with OUD prior to their return to the community, which places them at high risk for fatal overdose. A 2018 law (“Chapter 208”) made Massachusetts (MA) the first state to mandate that five county jails deliver all FDA-approved MOUDs (extended-release naltrexone [XR-NTX] buprenorphine-naloxone [BUP-NX], and methadone). Chapter 208 establishes a 4-year pilot program to expand all FDA-approved forms of MOUD at five county jails; two more county jails in MA voluntarily joined this initiative. The law stipulates that MOUD be maintained in individuals receiving it prior to detention, and initiated prior to release among sentenced inmates where appropriate. The seven jails must also facilitate continuation of the medication in the community on release. The Massachusetts Justice Community Opioid Innovation Network proposes to partner with these seven diverse jails and community treatment providers to conduct a Type 1 hybrid effectiveness- implementation study of Chapter 208. We will: (1) Perform a longitudinal treatment outcome study among inmates with OUD who receive XR-NTX, BUP-NX, methadone, or no MOUD in jail utilizing MA’s powerful and innovative Public Health Data Warehouse, a collection of over two dozen linked state administrative data sets, to examine post-release MOUD initiation, engagement and retention, as well as fatal and non-fatal overdose and recidivism. Propensity score methods will adjust for selection effects. (2) Conduct an implementation study to understand contextual factors that facilitate and impede delivery of MOUDs in jail and community care coordination, and best practice strategies that optimize MOUD delivery in jail and coordinated care with community partners. (3) Perform a pilot randomized comparative effectiveness trial of antagonist (XR-NTX) versus agonist treatment (BUP-NX or methadone) initiated prior to release among 100 sentenced and follow- them 12-months post-release to examine outcomes that cannot be assessed in administrative data and determine procedures, acceptability, feasibility and effect sizes to support planning for a future randomized comparative effectiveness trial. (4) Calculate the cost to the correctional system of implementing MOUD in jail, and conduct an economic evaluation from state-policymaker and societal perspectives to compare the value of MOUD prior to release from jail to no MOUD among matched controls. The Mass JCOIN team, in collaboration with the MA Department of Public Health, seven MA county jails and community treatment partners, has the experience and expertise to fulfill the study aims. The Chapter 208 initiative has important implications for future policy and practice in the justice and OUD treatment systems at the local, state, and national levels. This study’s insights into Chapter 208’s implementation will inform the efficient development of future strategies to address OUDs in jail populations nationwide.

NIH Research Projects · FY 2026 · 2019-01

Project Summary Regulated proteolysis controls the quality and quantity of proteins. In bacteria, energy dependent proteases eliminate aberrant proteins by recognizing distinctive marks arising from failed quality control, such as incorrectly exposed hydrophobic regions of proteins or specific tags attached upon prolonged translational arrest. These same machines control levels and dynamics of native folded proteins often through recruiting auxiliary factors to increase selectivity. In this proposed work, we focus on two major energy dependent proteases systems that are conserved in bacteria where understanding of their mechanistic regulation and scope is incomplete. The ClpXP protease selectively degrades major regulators during the Caulobacter cell cycle driven by a dedicated hierarchy adaptors and processes replication factors. Illuminating the molecular details of how adaptors deliver these substrates will reveal rules of specificity crucial for understanding programmed degradation. Similarly, the allosteric regulation of the Lon quality control protease by ligands or substrates is important for its function as a quality control protease, with emerging models suggesting that dynamic conformational changes can modulate specificity. In addition, findings that Lon can selectively remove DNA bound proteins support new regulatory roles for this highly conserved protease. By using proteomics, transposon-sequencing, biochemistry, and genetics to understand how bacteria govern the specificity and activity of these proteases at a mechanistic and cellular level, we will gain critical insight into pathways that control normal growth and stress responses in all bacteria.

NIH Research Projects · FY 2025 · 2018-07

Project Summary Engineered Polymer Nanoemulsions for Treatment of Wound Biofilm Infections The goal of the proposed research to create new therapeutics targeting multidrug-resistant biofilm infections. These infections are difficult to treat. The refractory nature of biofilm infections make them non-responsive to standard antibiotics, a situation exacerbated by acquired antibacterial resistance. In our research, we have integrated the nanomedicine capabilities of Rotello with wound biofilm expertise of Patel to develop crosslinked nanoemulsions (XNEs) that use a crosslinked polymer network to stabilize nanodroplets of essential oils. These XNEs kill biofilm-based bacteria with minimal effects on host cells and can eradicate biofilms through incorporation of antimicrobials into the oil component of the XNE. XNEs have good efficacy (killing ≥99% of bacteria in biofilms) using the in vivo wound biofilm model developed by Patel. Consistent with other antimicrobial nanomaterials, however, killing is less effective in vivo than in vitro. In our proposed research, Rotello will develop new block copolymers to generate block copolymer XNE (B-XNE) therapeutics. The B-XNEs will then be incorporated into hydrogel wound dressings to provide controlled release of B-XNEs to treat wound infections. B-XNEs and B-XNEs in wound dressings will be tested in vitro and in vivo using realistic and challenging wound biofilm models. Aim 1: Rotello will synthesize block copolymers and use these to parametrically vary size and charge of B-XNEs. These B-XNEs will then be used to carry antibiotics, providing synergistic activity with essential oils. B-XNEs will be screened for activity using luminescent methicillin-resistant Staphylococcus aureus (MRSA) biofilms, and then tested against other bacterial species by Rotello and Patel. Co-culture models employing mammalian cells will be used to downselect agents that maximize antibacterial activity and minimize mammalian cell toxicity. Aim 2: Rotello will incorporate B-XNEs into hydrogels to provide antimicrobial wound dressings. B-XNEs and hydrogels will be co-engineered to provide controlled release of B-XNEs. These B-XNE will be screened using luminescent MRSA to identify promising B-XNE-hydrogel combinations, and further tested as in Aim 1 by Rotello and Patel. Aim 3: Rotello and Patel will use murine wound biofilm models to test B-XNEs and B-XNE wound dressings. These studies will combine parametric pilot experiments using luminescent MRSA by Rotello with full pre-clinical evaluation by Patel with MRSA and Acinetobacter baumannii wound biofilms. Efficacy in these models will be quantified by decreased bacterial counts, enhanced wound healing, and diminished purulence as outcomes.

NIH Research Projects · FY 2026 · 2017-09

Various forms of chronic distress have been linked with premature aging, and development of cardiometabolic diseases (CMD), which are leading causes of death for older adults. Both chronic distress and CMD conditions are strongly linked with risk of Alzheimer's Disease and Alzheimer's Disease-Related Dementias (AD/ADRD). While metabolic changes affecting vascular health are proposed as a key pathway driving the relationship between chronic distress and major diseases of aging, understanding of molecular mechanisms underlying such metabolic changes is limited. High-throughput technologies permit simultaneous measurement of hundreds of metabolites in plasma (“metabolomics”) and provide a broad picture of an individual’s metabolic profile. In our first funding cycle, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS)-based metabolomic score of chronic distress (anxiety and depression), in independent data sets of largely non-Hispanic White women; this score was associated with higher risk of incident CMD. In this renewal, using cutting edge metabolomic and biostatistical approaches along with several additional cohort studies, we propose to extend our initial findings and address the following specific aims: (1) Strengthen our existing chronic distress metabolite-score by adding novel, previously unknown metabolites strongly associated with the distress phenotype, and biochemically identify these validated but unknown metabolites to provide new mechanistic insight; (2) Assess our chronic distress metabolomic score (and its components) in key populations including African-American (AA) and Hispanic men and women and White men, and optimize the score in each population. We will also evaluate associations of the score (and components) with CMD and secondarily AD/ADRD risk in AA men and women, White men, and preliminarily in Hispanic men and women, and (3) Evaluate if chronic distress influences the distress-related metabolite score using causal methods and evaluate the distress-metabolite score as a potential mediator of the relationships of chronic distress with CMD risk and secondarily with AD/ADRD risk. We will achieve our aims by leveraging the robust data resources of five prospective studies: the Jackson Heart Study (n=5,306; 100% AA men and women), the Multi-Ethnic Study of Atherosclerosis (n=6,814; 39% White, 28% AA, 22% Hispanic, 12% Asian-American men and women), the Nurses’ Health Study (NHS; n=121,700, 98% White women), the Women’s Health Initiative (n=161,808 women; 18% non-White), PREDIMED (n=7,447; White men and women). Each cohort has similarly assessed chronic distress, blood metabolomic profiles, relevant covariates, CMD and dementia risk outcomes over up to 20 years of follow-up. NHS and MESA also have genetics data and repeated metabolomics measures. This work will extend our understanding of biologic pathways underlying chronic distress and their association with subsequent CMD and dementia risk among both women and men and across racial and ethnic groups.

NIH Research Projects · FY 2026 · 2017-08

Project Summary/Abstract This research program will focus on the development and application of computational methodologies to deter- mine in vivo RNA 3D structure as well as characterizing the structural organization of functional elements for all RNA transcripts (termed transcriptome). The fates and functions of RNAs in health and diseases are determined by their structures. However, little is know about the folding of the transcriptome in the 3D space. Recently, high- throughput sequencing couple with proximity-ligation by chemical crosslinkers have been developed to investigate spatial RNA-RNA interactions at transcriptome scale. Current computational approaches are not readily suitable for analyzing the new types of RNA proximity-ligation sequencing datasets to dissect the higher-order structures, especially the 3D structures of all RNAs (termed RNA 3D structurome). To overcome these challenges, I will build a unique research program to develop statistical and computational methodologies for analyzing proximity- ligation sequencing data to reconstruct and characterize the 3D organization of RNAs in living cells. Over the next five years, the goals of my research program are to comprehensively elucidate the in vivo RNA 3D struc- tuome in high resolution and precision and decode the organization features of RNA functional elements. We will develop a methodological framework for inferring the 3D structures of RNAs using proximity-ligation sequencing data from living cells and verify them using experimentally verified 3D structures. We will use this the framework to study the 3D organization of RNAs from diverse categories (i.e., ribosome RNAs, messenger RNAs, and long noncoding RNAs) and from various cell types. Successful development of the proposed approaches will improve the understanding of RNAs folding in 3D and their functional impact on health and disease.

NIH Research Projects · FY 2026 · 2017-02

PROJECT SUMMARY/ABSTRACT Chronic alcohol use disrupts a number of cognitive functions, including attention, memory, flexibility, and other aspects of executive control. This is exacerbated by both exogenous stress and stress associated with alcohol use and withdrawal. We hypothesize that a key factor relating widespread cognitive dysfunction to problematic alcohol use is cognitive effort, which is needed to sustain diverse cognitive functions, particularly under conditions of heavy demand. In particular, we hypothesize that a history of chronic alcohol and stress disrupts the willingness and ability to exert cognitive effort, and conversely, that pre-alcohol limitations in cognitive effort predict future problematic alcohol use. There is strong interest in the neural basis of cognitive effort, but to our knowledge, no studies have examined the relationship between cognitive effort and alcohol at either a behavioral or neural level. Here we will investigate a neural circuit critical for cognitive effort and impacted by stress and alcohol use. This circuit is formed by the locus coeruleus norepinephrine (LC-NE) system, its strong projections to the anterior cingulate cortex (ACC), and ACC projections to the dorsomedial striatum (DMS). Unique to this proposal is 1) the integration of these systems into a unified circuit and 2) the investigation of this circuit in the integrated framework of cognitive effort, alcohol, and stress. We will measure the impact of chronic intermittent ethanol (CIE) and/or forced swim stress (FSS) on willingness and ability to perform two cognitively-demanding tasks (extradimensional set shifting and cognitive effort discounting in a sustained attention task) in mice. We will identify CIE/FSS associated changes in ACC noradrenergic regulation using RNAscope and probe them with CRISPR-mediated NE receptor knockdown (Aim 1). We will record neural dynamics in this network during cognitive performance after CIE/FSS (Aim 2), and manipulate the activity of this circuit using optogenetics to reversibly disrupt or rescue cognitive effort (Aim 3). Finally, we will collaborate with INIA Neuroimmune investigators to identify the impact of the phosphodiesterase inhibitor apremilast on cognitive function after CIE/FSS and characterize its effect in this circuit (Aim 4). Studies will integrate individual and sex differences to capitalize on heterogeneity in behavior and its relationship to underlying neural function. Cognitive effort is a novel and powerful framework for investigating the impact of alcohol and stress on cognition. Our studies have the potential to unite diverse findings related to the cognition and AUD. By identifying changes in neural circuitry underlying CIE/FSS-induced changes in cognitive effort, there is significant potential for identifying broadly-influential treatments for AUD that strengthen cognitive abilities to serve as a bulwark against the combined influences of alcohol and stress.

NIH Research Projects · FY 2026 · 2017-01

Abstract/Summary: The vertebrate craniofacial skeleton is a dynamic organ that arises and is maintained through an intricate balance of genetic and environmental inputs. Disruptions to either can lead to deleterious health outcomes. While significant progress has been made toward understanding the genetic and cellular mechanisms that underlie early craniofacial patterning, much less is known about the basis for craniofacial variation that manifests over extended periods of development, and depends upon the environmental context in which it occurs. Whether it's the physical interactions between cells and tissues within the developing embryo, or the mechanic forces imposed on the system, these contexts will determine how genetically-encoded systems unfold over time to determine craniofacial geometry. Implicit to these ideas is feedback in the system. Feedback is how disparate developmental units come together to form integrated functional systems - e.g., reciprocal signaling between adjacent but developmentally distinct tissues. It is also necessary for normal growth and homeostasis in kinetic systems - e.g., progenitor cells must sense environmental inputs, including mechanical load, and adjust developmental processes accordingly. Broadly speaking this proposal seeks to understand how both types of feedback are regulated at the genetic level. In doing so, three specific questions will be addressed: (1) What are the genes that contribute to craniofacial shape? (2) Do they exert their effects on more than one tissue, either via pleiotropy or as part of the same signaling pathway? (3) How do these loci interact with the environment, via mechanosensing, to affect variation in facial form? Cichlid fishes will be used as the experimental model, as they have undergone extensive evolutionary modifications of their skulls and jaws in a very brief period of time, making them ideal for genetic/genomic mapping. Cichlids are also well known for their capacity to remodel their jaws under different foraging environment, but not all cichlids share this ability, and thus plasticity itself is genetically determined and has diverged in this system. Cichlids therefore represent an ideal model to identify and parse the genetic, environmental, and GxE effects that underlie craniofacial variability. This proposal leverages these experimental attributes, and integrates advanced phenotypic, genotypic and functional tools to provide a more holistic understanding of the mechanisms that underlie craniofacial shape.

NIH Research Projects · FY 2025 · 2016-09

PROJECT SUMMARY A fundamental challenge for the scientific community in the 21st century is learning how to turn this deluge of data into evidence that can inform decision-making about improving health and preventing illness at the individual and population levels. The maturing field of real-time infectious disease forecasting is a prime example of a research area with great potential for leveraging modern analytical methods to maximize the impact on public health. Infectious diseases exact an enormous toll on global health each year. Improved real- time forecasts of infectious disease outbreaks can inform targeted intervention and prevention strategies, such as planning for surge capacity, increasing healthcare staffing, and designing vaccine studies. However we currently have a limited understanding of the best ways to integrate these types of forecasts into real-time public health decision-making. The central research activities of this project are (1) to develop stand-alone and ensemble infectious disease models and methodologies that support forecasting and inference about outbreaks and (2) to expand our collaborative, online platform for collection, dissemination, evaluation, and synthesis of forecasts from different research teams. Additionally, we will continue to develop a suite of open- source educational modules to train researchers and public health officials in developing, validating, and implementing time-series forecasting, with a focus on real-time infectious disease applications.

NIH Research Projects · FY 2026 · 2016-06

Abstract Early life stage exposures to toxicants can result in islet malformations, which may predispose individuals to diabetes. The glutathione redox microenvironment plays fundamental roles in embryonic development and cell signaling, perturbation of which can result in functional or structural alterations that only become apparent with subsequent stress or age. Surprisingly little is known about how embryos respond to oxidative stress, or the impact of toxicant exposures on pancreatic β-cell development. This project takes a multi-level approach using state-of-the-art techniques to elucidate the complex pathophysiological mechanisms by which exposures to Per-and-polyfluoroalkyl substances (PFAS) that cause oxidative stress derail islet development, and the consequences for β-cell function. We test the central hypothesis that deviations from the GSH redox microenvironment and aberrant activation of the transcription factor Nrf2- at the wrong place and the wrong time- impair β-cell development and function. There are three overarching goals of this project: 1) to deepen our understanding of the role of Nrf2 activation in embryonic β-cells and islet development; 2) ascertain the impact of PFAS on insulin biosynthesis; and 3) identify β-cell fragility and bioindicators of later-life metabolic impacts that can be translated to human health. We will use transgenic zebrafish, confocal microscopy and immunofluorescence, redox proteomics and insulin misfolding assays, and cultured β-cells to investigate exposures to two common PFAS (PFOS, PFHxS), and a legacy aqueous film-forming foam (AFFF). This work will have a sustained and powerful impact on the fields of developmental toxicology, redox biology, and the developmental origins of health and disease and provides critical advances towards developing science-based PFAS guidelines, targets for clinical interventions, and public health policies.

NIH Research Projects · FY 2025 · 2016-06

Project Summary Hsp70 molecular chaperones are central players in protein homeostasis and quality control. They mediate a diverse set of cellular functions using a deceptively simple allosteric mechanism. In their ATP-bound states, they bind substrates with rapid on/off rates and relatively low affinity, and in their ADP-bound states, substrates bind more tightly with slow association/dissociation. Hsp70s bind short sequences within unfolded regions of their substrates, preferring hydrophobic residues with flanking positively charged residues. Their transition between a high substrate affinity, ADP- bound state, and a low substrate affinity, ATP-bound state, involves major conformational rearrangement of both the N-terminal nucleotide-binding domain, and the C-terminal substrate- binding domain. While recent research has shed light on this allosteric conformational change, many questions remain and are the focus of the proposed research. What are the features of their substrate-binding sites that enable binding to many but not all sequences (they are “selectively promiscuous”)? Hsp70s work with partner co-chaperones, the J-proteins that help with cellular localization and delivery of specific substrates, and the nucleotide-exchange factors that facilitate replacement of ADP by ATP in the allosteric cycle. What are the structural origins of these partnerships, and how do they affect substrate binding and release? Preliminary results and literature observations suggest that Hsp70s can bind nearly isoenergetically to their extended polypeptide substrates in either an N- to C-orientation or a C- to N-orientation. This provocative bimodal substrate binding may have functional implications. The proposed work will examine the structural origins of this capability as well as the potential impact on the functions of the chaperone. Past work shows that the allosteric properties of Hsp70 are tunable by amino acid substitutions and by post-translational modifications. The proposed work will delve into the structural origins of this tuning, and the consequences of changes in the allosteric energy landscape for specific Hsp70 functions. Past work leaned heavily on peptide models to understand how Hsp70s bind their substrates. We will characterize the binding of Hsp70s to protein substrates to learn how the affinity for short sequence motifs translates into a hierarchy of site binding: are flanking sequences involved in selection of binding sites? How pivotal is accessibility? Lastly, the role of the Hsc70 chaperone in preparing SNAP-25 to participate in pre- synaptic vesicle docking and fusion in neurons will be studied. Methods that will be deployed in the proposed work include biochemical assays, nuclear magnetic resonance, fluorescence, mass spectrometry, and computational modeling.