University Of California, Merced

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY The mortality rates for patients with critical limb ischemia (CLI) are 15-40%, depending on the treatment. Moreover, the quality of life for surviving patients remains severely reduced. Limb revascularization focused on limb rescue is critical to saving lives and life quality. Cell therapy strategies currently under investigation inject a variety of progenitor cells into ischemic tissue with varying levels of success. However, no one has examined the ability of angiogenic tip-specific endothelial cells (ECs) to promote revascularization in an ischemic limb. We hypothesize that angiogenic ECs that contain tip ECs will more robustly revascularize ischemic limbs compared with nonangiogenic ECs. The proposed studies will use highly expandable pluripotent stem cells as the starting cell source and derive tip and non-tip cell-containing ECs from human induced pluripotent stem (iPS) cells. We will then compare the ability of tip ECs versus non-tip ECs to rescue the muscle in a hind-limb ischemic mouse model. For delivery, we employ spatially nanopatterned collagen biomaterial scaffolds to enhance cell survival after transplantation into the ischemic limb. Lastly, we will incorporate placental growth factor (PIGF) mRNA delivery from aligned nanopatterned collagen biomaterials to support the release of PlGF protein for maintaining the angiogenic phenotype of tip ECs, and/or directing in vivo the differentiation of stalk-to-tip ECs. We expect these studies to show that angiogenic tip-specific ECs, not only integrate into and support current vasculature in the ischemic limb, but also initiate the growth of new blood vessels, faster re-establishment of tissue perfusion, and reproducibly rescue the ischemic limb.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY_____________________________________________________________________ Parasitic diseases remain a problem worldwide largely due to the lack of vaccines to effectively interrupt transmission or infection, as parasites often develop resistance to drugs. A combination of different factors principally drives the lack of vaccines against parasitic diseases, including a limited understanding of the nature of the humoral immune response and difficulties in proper expression of the candidate immunogen. Our current work suggests that the GPI anchor has a vital role in humoral immune response to GPI-anchored proteins (GPI- APs) and most vaccine efforts have utilized GPI-APs - which constitute the bulk of surface proteins in parasites, as immunogens. However, in these vaccine formulations, the GPI-APs are without the GPI anchor to enable soluble expression of such proteins. In this grant submission, we have shown that de-lipidation of the GPI anchor significantly reduces antibody reactivity to GPI-APs of Toxoplasma gondii, a widespread Apicomplexa parasite of animals. We hypothesize a role of the GPI anchor in humoral immune response and propose using mRNA technology as an efficient method for accurate delivery of GPI-APs to the immune system to understand its role following vaccination. We also propose the use of mRNA vaccines for the efficient delivery of highly conserved antigens of the parasite's invasion machinery, a handful of which we propose as excellent targets to halt invasion of apicomplexans. Thus, we will employ tools in immunology, molecular parasitology, vaccinology, and biochemistry to dissect the aims of the study. In Aim 1, the role of the GPI anchor in humoral immunity to T. gondii will be deciphered by comparing vaccine efficacies of parasite GPI-APs with and without their GPI anchorage following immunization. In Aim 2, the invasion machinery of T. gondii will be targeted by vaccine- elicited antibodies against conserved apical proteins of the plasma membrane that confer fitness to the parasite. With the overall goal of the project being to block infection of T. gondii and apicomplexans in general, this R21 proposal will provide new insights in vaccine development against parasitic diseases.

NIH Research Projects · FY 2025 · 2023-08

Project Summary We propose to study the relationship between the structure, dynamics, and function of enzymes by examining how changes to their conformational ensembles regulate their catalytic functions. Understanding this relationship is critical for understanding macromolecular phenomena such as allosteric regulation, yet it remains difficult, because the relevant conformational changes involve a hierarchy of motions that occur across broad lengthscales (sub-Å to multi-nm) and timescales (ps-s). Our lab is developing a new generation of structural measurements that combine temperature perturbations with static and time-resolved X-ray crystallography, allowing us to explore the conformational landscapes of protein molecules in detail. We aim to apply these methods to temperature-sensitive orthologs from key enzyme families, including kinases, proteases, and ATP-dependent chaperones, to understand how changes to their conformational ensembles modulate their biological functions. The specific goals of our work are: (1) Use multi- temperature X-ray crystallography, combined with traditional biochemical and biophysical assays, to quantify the relationship between conformational states and catalytic activity. (2) Characterize previously invisible conformational states of enzymes, including cryptic pockets that can be targeted for drug discovery, using time-resolved temperature-jump crystallography. (3) Continue developing new hardware and software to improve the collection and analysis of data from multi-temperature and temperature-jump crystallography. Our research represents a novel approach to understanding how the balance of active and inactive conformations drives the regulation of protein function. Successful completion will yield new information about the structure-function relationships of biologically and clinically important enzymes and provide new opportunities for targeting them with therapeutics. We expect that similar changes to protein conformational ensembles underlie thermal regulation and other types of allosteric regulation in these enzyme families, and therefore we expect our results to be generally useful in understanding allosteric regulation more broadly. Finally, our work will develop a framework for studying the relationship between protein structure, dynamics, and function that exploits the response of protein conformational ensembles to temperature, and we aim to democratize the use of multi-temperature and temperature-jump crystallography as a general tool for the structural biology community.

NIH Research Projects · FY 2026 · 2023-07

Project Summary/Abstract There are persistent mental health and health care access disparities between rural and urban communities. Within rural communities, foreign- and US- born Latinos experience worse mental health and access to care than rural Whites or urban Latinos. While the social determinants of rural health have been recognized as a major underlying cause of health, there is limited investigation of and data on the structural and community factors specific to improving mental health and health care access of rural Latinos. The overarching goal of the proposed study is to investigate the association between mental health and health care access and immigrant policies among Latinos in rural regions by conducting a multilevel, cross-sectional study of the impact of policy contexts, social climates, and Latinos’ direct encounters with institutions that implement policy. The specific aims of this study are to: 1) investigate the impact of exclusionary county policy contexts and social climates on rural Latino mental health and health care access; 2) investigate the impact of rural Latinos’ encounters with exclusionary immigrant policy on their mental health and health care access; and 3) investigate the extent to which county policy contexts, social climates, and policy encounters jointly influence rural Latino mental health and health care access. To achieve these aims we will, first, collect county-level data on local policy contexts and social climates and will implement hierarchical regression models to test their associations with mental health and health care access, net of covariates. We will also conduct a population-based survey of foreign- and US-born Latinos in rural counties in California and Arizona. We will use survey data to construct a measure of respondents’ level of exclusionary encounters with policies and will implement hierarchical regression models to test their association with mental health and health care access. Finally, we will conduct analyses of the correlations between county policy contexts, social climates, and policy encounters and conduct tests of mediation and moderation to assess how their relationships contribute to mental health and health care access. The study’s multi-level approach will contribute to knowledge on the mechanisms that influence Latino and immigrant health. The study will produce new data to inform health policy and Latino-focused interventions in rural communities aimed at promoting mental health and increasing access to health care and safety net services.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Multiprotein complexes known as inflammasomes form in innate immune cells to trigger inflammation upon detection of pathogens or tissue damage. Abnormal inflammasome activation leads to chronic inflammation, which is the culprit of numerous life-threatening diseases such as cancer, diabetes, cardiovascular disorders, and the cytokine storm in SARS-CoV-2 infection. Inflammasome assembly is controlled by protein-protein interactions as it requires the self-association and oligomerization of multiple copies of three proteins: sensors to detect danger signals, a protease to activate inflammatory factors, and the adaptor protein ASC to connect sensor and protease. Inflammasome formation leads to plasma membrane rupture and concomitant cell death, thus resulting in the release of proinflammatory cytokines and inflammasome particles to the extracellular environment. These extracellular inflammasomes are internalized by nearby cells to perpetuate and amplify the inflammatory response. Removing or sequestering extracellular inflammasomes will likely inhibit or reduce inflammation. Therefore, extracellular inflammasomes are potential therapeutic targets. Our laboratory’s extensive experience on the function and structure of the adaptor ASC, and its interactions with other inflammasome proteins, has led us to create hydrogels designed to form specific protein-protein interactions with inflammasomes; thus, they have the potential to broadly inhibit inflammation by effectively capturing and removing extracellular inflammasomes. This project focuses on identifying the hydrogelation factors leading to optimum biding of inflammasome particles in cell-free systems (Aim 1); and determining the anti-inflammatory efficiency of the hydrogels in the presence of activated innate immune cells (Aim 2). Our experimental plan will combine cell biology and biochemical approaches, including live/dead cell imaging, flow cytometry, immunoblotting, enzyme-linked immunosorbent assays and fluorescence spectroscopy. Overall, we expect to develop a hydrogel technology of broad applicability to reduce inflammation in the absence of drug loading by targeting the inflammasome, which is implicated in many inflammatory diseases.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY ABCB10 is a human mitochondrial inner membrane ATP binding cassette (ABC) transporter that uses energy from ATP hydrolysis to export a substrate out of the matrix. This transporter is essential for protection against oxidative stress during erythropoiesis (abcb10 knock-out mice die in uterus due to anemia and oxidative damage) and protecting the heart (ABCB10 protein level is upregulated in human ischemic myocardium). Despite its potential clinical relevance for treating anemia and protecting the heart against oxidation, the identity of ABCB10’s substrate was unknown until our group recently identified biliverdin, a heme degradation product with antioxidant properties, as the physiological substrate for this transporter. We have also found that zinc mesoporphyrin, a heme analog, increases the basal ATPase activity of the transporter like substrates do. Identification of these substrates has opened the door to the biochemical and structural studies proposed in this project, which will contribute to a better understanding of the molecular mechanisms by which this important transporter works. Our experimental approach involves the use of functional (ATPase assays), spectroscopic (Luminescence Resonance Energy Transfer, LRET), and mutational analysis of ABCB10 reconstituted in lipid nanodiscs. This experimental system has many advantages for the in vitro study of ABC transporters in a “native- like” lipid bilayer and at physiological temperature. We can produce functional human ABCB10 in bacteria, facilitating the production of the numerous mutants needed for this research. Aim 1 will determine the conformational changes that ABCB10 undergoes during its basal ATP hydrolysis cycle and how those molecular movements are modified during activation by substrate. According to our preliminary data, this aim is expected to prove that ABCB10 functions through small conformational changes. If our hypothesis is correct, our findings will challenge the generally accepted idea that all related ABC exporters follow a similar molecular mechanism. Aim 2 will determine substrate-transporter interactions that are critical for ABCB10’s stimulation. We will study the protein’s ATPase activity and associated conformational changes in response to a) variations in the chemical groups of the substrates and b) mutagenesis of residues in a putative substrate binding pocket. Our preliminary results suggest that the substrate’s carboxyl groups and two arginines in the binding pocket are critical for ABCB10’s stimulation. Mutagenesis of these arginines cause constitutive ABCB10 activation (gain-of-function). Here, we expect to gain information about substrate specificity, find putative inhibitors, identify essential residues in the binding pocket, and define conformational changes that accompany alterations in protein’s function. In general, this project will provide molecular information that can validate current structural models in the ABC transporters field and provide ideas to modulate ABCB10’s activity for therapeutic purposes.

NIH Research Projects · FY 2026 · 2022-04

U*RISE at UC Merced - Project Abstract National statistics clearly indicate a disparity in the number of underrepresented minorities obtaining higher degrees in biomedical or biobehavioral disciplines. Given that the University of California, Merced (UC Merced) is an established Hispanic-Serving Institution (HSI) with the highest percentage of Hispanic undergraduates enrolled of any of the 10 UC campuses, UC Merced is ideally situated to realize the greatest benefit from an NIH U*RISE T34 training grant. Our proposed U*RISE program will support undergraduate scholars of primarily Hispanic-descent given our demographics, but we expect to use this opportunity to attract more students from other backgrounds to enhance the diversity of our campus and ultimately of our training pool. Thus, our proposal has potential for the highest impact to help transform the diversity of our undergraduate student body and ultimately the diversity of the graduate ranks as well. Data from our campus indicate a justified need to enhance and enrich the quantitative skills of our undergraduate scholars via focused research training in computational and/or systems biology. Faculty expertise and a history of extramural funding to support UC Merced’s many computational biology and Applied Math components makes a U*RISE program focusing on quantitative skills highly pertinent and sustainable at UC Merced. Thus, U*RISE at UC Merced will emphasize providing an enriching research experience for undergraduate scholars by providing unique opportunities in computational biology and/or systems biology including bioinformatics, multiple “-omics” approaches, and big data analytics. The innovation in our program lies in the cohort approach in which cohorts of trainees will work collaboratively on projects and in our novel learning modules. The strong institutional commitment from the Executive Vice Chancellor and Provost, the Office of Undergraduate Education, and the Undergraduate Research Opportunities Center (UROC) will help ensure the successful training and placement of all our scholars. Levering other institutional resources such as UC Merced’s Health Sciences Research Institute (HSRI) will also help succor the program’s successes. In summary, given UC Merced’s HSI status, demographics, and demonstrated need for enhanced training of its undergraduate scholars with a focus on quantitative skills makes it an ideal training institution to establish a U*RISE program with the potential for very high impact.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Circadian clocks are intracellular enzymatic systems that provide a biochemical representation of local time with profound consequences to health across diverse organisms. Unlike most enzymes, biological clocks need to be insensitive to a range of physiological temperatures and cellular energy levels so that organisms can anticipate dawn and dusk reliably. However, the mechanisms by which circadian clocks in any organism achieve insensitivity to temperature (i.e., temperature compensation) and cellular energy levels (i.e., metabolic compensation) are far from understood. Therefore, the overall vision of the LiWang lab for the next five years is to elucidate the mechanisms of temperature compensation and metabolic compensation in the circadian clock of cyanobacteria. Over the past 20 years, we have made many impactful discoveries on mechanism of the cyanobacterial clock and developed innovative methodologies and tools along the way. Thus, we are very well positioned to succeed at filling critical gaps in knowledge in the field of biological timekeeping. A major expected outcome of the work proposed here is a detailed cause-and-effect model linking clock protein behavior and interactions to temperature and metabolic compensation phenotypes in vivo.

NIH Research Projects · FY 2025 · 2021-05

We propose a graduate research training program in Interdisciplinary Biomedical Science and Technology (I-BioSTeP) at the University of California Merced, a Hispanic-serving institution that opened in 2005. The major objective of this program is the establishment of the first doctoral level graduate training program at UC Merced in the biomedical sciences to train diverse cohorts of students to identify and solve pressing biological problems using quantitative interdisciplinary approaches that prepare them for competitive careers in the biomedical field. Transformative advances in the biomedical research arena increasingly require contributions from many different fields and I-BioSTeP leverages the uniquely interdisciplinary structure at UCM, to join faculty from six different departments forming a diverse, interdisciplinary research community with a common vision for research and education. Because UCM is still in its developmental stages, an initiative like I-BioSTeP can have truly transformative impact by establishing a nucleus of high quality training and research on campus. As part of the program we will establish an I- BioSTeP designated emphasis (a graduate minor) open to all graduate students in science and engineering who participate in the new coursework we are developing, thus enhancing the overall graduate training offerings on campus. Opening a training program for the highly skilled biomedical research workforce of tomorrow at UC Merced will also have an enormous impact on the region, producing a diverse cohort of trained professionals that will raise the economic and educational standards of the surrounding educationally and economically disadvantaged communities. The specific objectives of our program are (i) to increase the diversity of our trainee cohorts (ii) to ensure retention and speed up the time to degree (iii) to produce graduates with a broad training in an interdisciplinary curriculum for biomedical sciences and (iv) to inculcate a sense of belonging and teamwork skills and (v) to ensure successful transition into the biomedical research workforce. There are many unique aspects of our proposed program that specifically address these objectives including the diversity of our current graduate student body and applicant pool that we target, a core interdisciplinary curriculum of courses with lab components, a summer bridge program that addresses retention by bringing students out two months before classes and immersing them in hands-on training, mentoring and professional development such as fellowship proposal writing, an interdisciplinary multiple mentor structure and a unique career development program that focuses on specific skills that are important in the biomedical research workforce but are commonly not addressed in such training programs - business/entrepreneurial skills and science communication skills.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract The goal of this proposal is to capture and then characterize engrams for ethanol tolerance in Drosophila. Engrams are the neurons and their molecular changes that encode lasting behavioral change. Engrams described for ethanol in mammals are limited to specific brain regions, encoding factors, and behavioral states, and little is known about effectors in ethanol engrams. Drosophila offer significantly higher throughput, facilitating whole brain capture of engrams, testing of multiple types of potential engrams, and molecular discovery of the mechanisms of encoding. Further, we developed behavioral paradigms for rapid, chronic, and repeated exposure tolerance, and we discovered that they are encoded by distinct immediate early gene transcription factors. We will develop a new method to permanently tag engrams in the brain. Permanent tagging lets us define molecular changes in engram neurons, explore their function as tolerance is acquired and then expressed, and to define their function in brain circuits. Comparing the anatomy and effector molecules for different forms of tolerance will unveil the complex actions of ethanol on the brain. Our focus on evolutionarily conserved factors provides a means for future development of diagnostics and therapeutics for alcohol use disorders.

NIH Research Projects · FY 2024 · 2016-08

Project Summary/Abstract The goal of this renewal application is to determine the impact of the lipid membrane enclosing cellular cargos on the function of the major microtubule-based motor protein kinesin-1. Motor protein-based transport underlies all eukaryotic cell function and survival; understanding the mechanistic basis of motor protein regulation is critical for understanding this fundamental process of intracellular transport. Our central hypothesis is that the fluid nature of the cargo membrane is a key determinant of motor protein function. In cells, motor proteins are typically attached to cargos via a fluid lipid membrane. This membrane is “fluid” in that its constituents, including associated motor proteins, are mobile and diffuse on the cargo surface. Alterations in membrane fluidity are increasingly linked to aging and neurodegeneration, in which dysfunction in motor-based transport is a common early hallmark. Quantitative investigations of cargo membrane effects on motor function have remained limited, with most cargos in current in vitro assays lacking a physiological lipid membrane. Closing this major gap, in prior funding periods, the research team developed a robust optical-trapping assay to directly measure the transport of membrane-enclosed cargos in vitro. Using this assay, the research team demonstrated the first direct support for the central hypothesis of this project. Specifically, a fluid membrane enhances the productive binding of kinesin to microtubules; this enhancement is countered by cholesterol, which reduces membrane fluidity. These membrane effects were established in the presence of tau, an in vivo factor critical for microtubule polymerization and stabilization but that occludes kinesin binding sites on microtubules. Building on this recent work, in the next funding period the research team will test the central hypothesis by pursuing three independent but related aims. Aim 1 will leverage the recently developed assay to test the hypothesis that a reduction in membrane fluidity underlies the inhibitory effect of membrane cholesterol on kinesin binding in the presence of tau. Aim 2 will extend the assay and implement high temporal resolution detection to test the hypothesized membrane effect on kinesin binding in the absence of tau. Aim 3 will develop a stochastic simulation model to quantitatively test the hypothesis that cargo membrane fluidity determines kinesin-microtubule binding by impacting the diffusive search time of kinesin for open binding sites on the microtubule. Accomplishing the proposed project has the potential to establish cargo membrane fluidity as an unexplored physiological determinant of kinesin-microtubule binding, advancing scientific knowledge in the mechanistic basis of motor protein regulation, and providing a controlled experimental and computational platform for quantitative investigations of physiological determinants of motor protein function. Findings could pave the way for future therapeutics that target membrane properties to mitigate dysfunctions in motor protein-based transport early in the progression of diseases including neurodegeneration.