Stanford University

NIH Research Projects · FY 2025 · 2008-12

Project Abstract Mathematical learning disabilities (MLD) impact up to 14% of school-aged children, and are linked to high rates of morbidity and poorer health outcomes making it a significant public health concern requiring extensive health resources. Designing effective interventions to remediate MLD and identifying the cognitive and neurobiological features underlying their efficacy are critical steps for addressing the public health burdens of innumeracy and learning disabilities more broadly. Leveraging a productive, innovative, and high-impact line of research, we propose to investigate neurocognitive mechanisms underlying response to intervention (RTI) aimed at remediating core and persistent cognitive impairments in children with MLD. To achieve this goal, we will use a theoretically-motivated integrated symbolic/non-symbolic (iSNS) intervention with a randomized controlled design to enhance cross-format mapping between symbolic and non-symbolic representations of quantities. We will develop innovative computational models to investigate individual differences in latent cognitive processes, including evidence accumulation, sensitivity to item difficulty, and performance monitoring, that underlie learning and brain plasticity in children with MLD. Our central hypotheses are that (1) iSNS will remediate numerical problem-solving deficits and strengthen latent cognitive processes in children with MLD, and that (2) plasticity of neural representations and reconfiguration of functional brain circuits and networks will contribute to learning, retention, and transfer in children with MLD. Crucially, building on innovative systems neuroscience approaches, we will leverage novel computational tools and quantitative network analysis of functional brain circuits linking visuospatial attention, cognitive control, and memory formation systems to advance foundational knowledge of the neurocognitive mechanisms underlying RTI in children with MLD. Findings from our novel approach and neurocognitive models will have major implications for informing the etiology of MLD, the neurobiology of learning disabilities more generally, and the neurocognitive basis of individual differences in RTI. Findings will also provide new insights into individual differences in learning, with broad consequences for optimizing learning in all children. More broadly, our proposed studies will provide a deeper understanding of dynamic neurocognitive processes underlying learning, retention and transfer (generalization) in children with learning disabilities.

NIH Research Projects · FY 2025 · 2008-09

The multidisciplinary T32 postdoctoral training program in Cardiovascular Imaging at Stanford (CVIS) is designed to train the next generation of cardiovascular imaging investigators by exposing them to three complementary areas – clinical, engineering, and molecular imaging. With the rise in the impact of cardiovascular disease on U.S. and world health and the rapid advances in imaging technologies and cardiovascular biology, it is critical that trainees be provided a broad, multidisciplinary and collaborative training program to foster their ability to translate cardiovascular imaging research into clinical applications. The program goals include rigorous training in the scientific method, critical analysis, logical reasoning, and independent thinking in a highly collaborative setting. Trainees develop a focused area of cardiovascular imaging research expertise and exposure to a wide range of complementary research techniques. Mentors model collegial and productive collaboration, provide guidance in oral and written communication, and model responsible, rigorous, and reproducible research practices. The program proposes to continue training four postdoctoral fellows in multidisciplinary cardiovascular imaging research. Fellows are appointed to the CVIS T32 annually, with a strong encouragement to seek their own funding for additional years as part of the skills imparted by the program. Thirty trainees so far have benefited from this program. Four fellows are currently in training. Continuous and consistent evaluation from the trainees report a high degree of satisfaction with the program. Fourteen of the past trainees have gone on to become independent researchers in premier academic institutions and in industry. The Program is directed by Joseph Wu, MD, PhD (Contact PI), Professor of Radiology and Cardiovascular Medicine and Director of the Stanford Cardiovascular Institute (CVI); John Pauly, PhD, Professor of Electrical Engineering; and Koen Nieman, MD, PhD, Professor of Radiology and Medicine (Cardiology) at Stanford University. Administrative and program management support is provided by a dedicated team of educators in the Stanford CVI. An Internal Advisory Board consisting of senior Stanford faculty from a broad range of disciplines and an External Advisory Board consisting of leading experts in cardiovascular imaging research in the U.S. play a vital role in monitoring the progress of this training program, providing ongoing support and advice as needed. The overarching goal of the program is to train the next generation of investigators with expertise in advanced cardiovascular imaging technology, dedicated to identifying innovative solutions, and capable of translating basic research into clinical success.

NIH Research Projects · FY 2026 · 2008-09

PROJECT SUMMARY/ABSTRACT The pursuit of tailored treatment strategies for individual patients remains crucial for enhancing cost- effectiveness in clinical practice. Despite advancements in statistical methodologies and machine learning, persistent challenges impede the progress and implementation of precision medicine in clinical practice. Limited sample sizes pose a significant hurdle in estimating individualized treatment effects on clinical outcomes, necessitating the utilization of information from multiple data sources. However, effective integration of such data requires appropriately addressing population heterogeneity, privacy constraint, and features alignment across datasets. Furthermore, even with a group of well-developed prediction models of different complexity in place, there is still a need to devise smart strategies for adaptively employing them in practice. Lastly, addressing treatment effect heterogeneity in clinical trials remains challenging, particularly in efficiently synthesizing information from both discovery and validation stages without introducing bias. Our proposal aims to develop innovative solutions to aforementioned problems. First, we will introduce a novel transfer learning approach to accommodate overlapping but non-identical prediction feature sets in source and target populations. Second, we will develop a latent class model leveraging knowledge graph information from multiple sources for flexible feature alignment. Third, an innovative dynamic prediction strategy will be created to optimize the sequence of acquiring prediction features, thereby enhancing prediction accuracy while minimizing measurement cost. Fourth, we will extend reinforcement learning at a single site to federated learning setting under privacy constraints so that adaptive strategy such as personalized dynamic treatment regimen can be better developed. Lastly, we will propose a comprehensive framework for integrating information from both discovery and validation stages in studying the treatment effect heterogeneity, enabling unbiased inference of treatment effects among a selected subgroup of responders. All methodological developments will undergo rigorous numerical studies and real-data applications, ensuring their effectiveness, and will be disseminated widely to benefit the clinical community.

NIH Research Projects · FY 2025 · 2007-06

The Stanford Cancer Institute (SCI) is an NCI-Designated Comprehensive Cancer Center that integrates key strengths in cancer biology, immunology, genetics, population sciences and clinical oncology with expertise in imaging, engineering and technology development. The translation of basic science discoveries through clinical trials and collaborative team science is a distinguishing feature of the SCI. The SCI has 300 members and includes faculty from 32 Departments and from three Schools (Medicine, Engineering, and Humanities and Sciences) within Stanford University. Seven programs (Cancer Biology and Cancer Stem Cells, Cancer Imaging and Early Detection, Cancer Immunotherapy, Cancer Therapeutics, Hematologic Malignancies, Population Sciences, and Radiation Biology) address basic, translational, clinical, and population-based components of cancer research and are supported by 11 shared resources (Animal Tumor Models, Biomedical Informatics, Bioscience Screening, Biostatistics, Cancer Imaging, Cell Science Imaging, Flow Cytometry, Genomics, Human Immune Monitoring, Proteomics, and Tissue Procurement). With a current NCI funding base of $41.9 million, $33 million in other cancer-related NIH support, $16.4 million in other peer-reviewed cancer-relevant funding and $91.3 million in total peer reviewed funds, the SCI is fulfilling its mission to translate Stanford discoveries into improving the diagnosis, treatment, and outcomes of cancer patients and to better understand cancer etiologies among various populations in the Bay Area and beyond. SCI has made major investments in Population Sciences, including the recruitment of a new Associate Director for Population Sciences. We have brought focus to our catchment area through the recruitment of a new Associate Director for Community Outreach and Engagement. SCI has developed a highly successful clinical and translational effort in CAR-T therapies bringing new treatments to our patients. Finally, SCI has begun a major initiative in cancer drug discovery uniting cancer scientists, cancer physicians, chemists and engineers across the Stanford campus in their efforts to develop new cancer therapeutics. The SCI is requesting CCSG support from the NCI for its critical activities, including in Community Outreach and Engagement, Shared Resources, Cancer Research Training and Education Coordination, Clinical Protocol and Data Management, Protocol Review and Monitoring, Program Leadership, Administration, and Developmental Funds. With new additions to the senior leadership, many new recruitments, and highly effective collaborative interactions, the SCI is poised to further accelerate its contributions to cancer research in the next funding period.

NIH Research Projects · FY 2026 · 2007-05

Project Summary Basal cell carcinoma (BCC), caused by the inappropriate activation of the hedgehog (Hh) pathway, represents a common skin cancer that, while treated surgically, can also exhibit rapid invasion and metastasis. While Smoothened inhibitors (Smoi) show efficacy, the majority of advanced BCCs acquire resistance. 5ARO54780 showed that increased expression in BCCs of the polarity kinase atypical protein kinase C (PRKCi) amplifies Gli activity and confers resistance, although the mechanism remains unknown. PRKCi cooperates with histone deacetylase 1 (HDAC1) to promote Gli deacetylation and chromatin association. A vicinal proteomics approach led to the surprising identification that isoforms of the Lamina-Associated Polypeptide 2 (LAP2) bind to the Gli zinc finger domain. Nuclear envelope tethered LAP2b and an associated acetyl-lysine reader promote Gli accumulation at a site termed the paused nuclear complex that protects Gli from degradation or nuclear export. By contrast, nucleoplasmic LAP2a binds both HDAC1 and Gli allowing it to stably accumulate on chromatin. In turn, PRKCi prevents LAP2a-HDAC1-GLI degradation while promoting switching from LAP2b to LAP2a. Now in its 10th year, 5ARO54780 will test the overarching hypothesis that PRKCi-dependent Gli deacetylation directs association with distinct LAP2 isoforms that amplify pathway activity in resistant BCCs. We will: 1) Elucidate the structure and function of the Gli-LAP2b paused nuclear complex as we identify the LAP2b functional domains required for Gli activity, identify components of the Gli-LAP2b acetyl-lysine reader, and dissect how LAP2 regulates Gli localization and mobility; 2) Elucidate the structure and function of the GLI-LAP2a activation complex as we determine whether LAP2a requires Gli for colocalization, define the non-base contact surface of LAP2-Gli1, and elucidate how PRKCi regulates LAP2a-HDAC1-Gli stability; 3) Establish resistance pathway efficacy and epistasis as we determine the prevalence of the BCC resistance pathways and determine efficacy of the LAP2-LLD in human and mouse BCC explants. Completion of the project will deepen our mechanistic insights into transcription factor trafficking and regulation in the nucleus, help optimize rational BCC therapy, and identify new therapeutic targets.

NIH Research Projects · FY 2025 · 2007-04

Anti-VEGF therapies, standard care in neovascular age-related macular degeneration (nvAMD), improve outcomes in less than 50% of patients and do not prevent vision-loss progression due to fibrosis or macular atrophy. We used human physiologically relevant models to gain understanding into the coordination of signaling pathways and cross-talk involved in the activation of choroidal endothelial cells (CECs) to migrate and form macular neovascularization in nvAMD. The scaffolding protein, IQGAP1, sustains activation of the GTPase Rac1, which is necessary for CEC migration. Rac1 is activated by AMD-related stresses involving inflammatory, oxidative and angiogenic factors, as well as by the oxysterol, 7-ketocholesterol (7KC), which accumulates in blood and Bruch’s membrane with increased age and in AMD. 7KC causes CECs to change expression of cell markers from endothelial to mesenchymal ones, suggesting endothelial-mesenchymal transition (EndMT). IQGAP1 appears involved. 7KC also causes fibrosis in models of laser induced injury. Our data support the hypothetical framework that will be tested in the next funding period: that (1) IQGAP1 is critical to EndMT induced by the oxysterol 7KC; and that (2) 7KC triggers transcriptional events that render CECs unable to maintain expression of endothelial markers but to develop into a new phenotype of migratory, mesenchymal cells that develop into fibrosis. We will also test two potential therapies to reduce fibrosis: (a) to inhibit TGFβ signaling in combination with anti-VEGF and (b) to target phosphorylation of IQGAP1 in a novel mutant IQGAP1 mouse that we created by CRISPR-Cas9-induced gene mutation. Specific Aim 1 is to test the prediction that IQGAP1 mediates EndMT-induced migration in CECs exposed to 7KC. Specific Aim 2 is to test the prediction that 7KC, mediated through IQGAP1, decreases the proportion of labeled endothelial positive to mesenchymal positive cells after laser in endothelial specific yellow-fluorescent protein reporter mice. Specific Aim 3 is to test predictions that increased age, TGFβ-signaling, or IQGAP1 serine phosphorylation will increase αSMA-labeled lesions after laser in 7KC-treated eyes and to test strategies as possible future treatments. We will also evaluate the involvement of Müller cells, pericyes, and RPE. Tools include isolated human CECs; high throughput RNA sequencing; flow cytometry; spectral domain optical coherence tomography (sdOCT); 7KC-induced models of EndMT and fibrosis; yellow-fluorescent protein endothelial reporter mice; conditional inducible endothelial Iqgap1 knockout mice; a mutant IQGAP1 mouse through CRISPR-Cas9-technology; intravitreal injections of pharmacologic agents; Micron IV laser induced injury to test 7KC-induced EndMT and lesion formation. These studies will test the role of IQGAP1 in 7KC-induced EndMT as a potential cause of fibrosis, which is poorly responsive to anti-VEGF in nvAMD, and will test two novel treatments to reduce nvAMD and EndMT.

NIH Research Projects · FY 2025 · 2006-08

Delivered RNAi products have now been FDA approved for treating two genetic disorders resulting from mutations affecting genes expressed in the liver. Gene vector delivered cassettes that produce siRNAs have an advantage for genetic disorders because of the potential for a one-shot cure. We solved one of the mysteries of how over expression of therapeutic RNAi (transcriptional RNAi) from an AAV-U6 polII promoter driven shRNA (AAV-shRNA) caused acute liver toxicity and continue to study this in more detail. When siRNAs from such a source reached 12% or more of the total miRNA reads there was a 10% reduction in the first synthesized miR122 isoform (but not the other miRNAs) and this induced acute liver toxicity exemplified by elevated liver enzymes and in some cases liver failure and death. Because germline knockout of miR122 has a much lesser phenotype, we hypothesize the discordance in these outcomes is related to the differential expression of the miR122 precursor RNA transcript known as long-non-coding RNA 122 (lnc122) and that these two RNAs have separate but coordinated functions. We propose to elucidate the molecular function of nuclear localized lnc122 RNA and by removing lnc122 and miR122 RNAs and then reintroducing the different individual RNA components in cells, mouse liver and hepatocellular carcinoma models. This will allow us to separate the individual functions of the RNA products. We will also map the lnc122 chromatin interactions. These studies are important because not only does the miR122/lnc122 gene have a tumor suppressor function, but it is also known to have effects on normal liver regeneration, formation of hepatocellular carcinoma, and liver fibrosis associated with various liver diseases such as NASH, lipid metabolism, and viral hepatitis infection. Newer strategies to target gene transfer/expression outside the liver contain transgenes with miR122 targets in the 3'UTR to exclude leaky expression in hepatocytes. This like some of the antisense miR122 products tested in clinical trials, and hepatitis virus B and infections result in the sponging of miR122 and the long-term effects of this are unclear. At the end of the granting period, we will have a better understanding of the function of the various RNA products produced from the lnc122-miR122 locus and their role in cellular homeostasis and how this may effectively limit RNAi based therapeutics. Moreover, as we learn more about the function of this genetic locus it will provide more insights into how it participates in the disease processes noted. This may provide new insights into more optimal means to treat patients with a variety of genetic and acquired diseases.

NIH Research Projects · FY 2025 · 2006-02

Project Summary/Abstract Powerful direct-acting antivirals against hepatitis C virus (HCV) are available, but no vaccine is available. There are no effective antiviral compounds available to treat enterovirus 71 (RV71) that can cause severe neurological complications in children. In the case of SARS-CoV-2, powerful vaccines are available, but the rapid emergence viral variants warrant the continued development of novel antiviral approaches. Thus, it is important to continue to search for novel vulnerabilities in these RNA viruses. We have made the astonishing discovery that HCV-, EV71- and SARS-CoV-2-infected cells produce hundreds of virus-derived circRNAs (vcircRNAs) species of different sizes that are derived from the viral RNAs. Some of these vcircRNAs, such as the HCV internal ribosome binding site (IRES)-containing cluster I vcircRNA of HCV, are being translated to yield novel viral proteins with pro-viral functions. We also made the puzzling observation that the 3' end of the minus-strand of HCV contains an IRES that mediates the translation of a novel minus-strand protein (MSP) that is unique to the JFH1 type 2a genotype. Our hypothesis is that vcircRNAs modulate viral and cellular gene expression and function in infected cells to elicit novel pro-viral or anti-viral responses. The rationale for this proposal is that modulation of vcircRNA abundances affect the pathogenesis of HCV, EV71 and SARS-CoV-2. Our goals are to examine the functional roles for the vcircRNAs in human cultured liver cells, human liver organoids and lung A549-ACE2 cells, using bioinformatic, genetic, biochemical and cell biological approaches. Two specific aims are proposed to accomplish these goals. First, functional roles for abundant vcircRNAs and IRES-containing vcircRNAs in viral gene expression will be examined after depletion of vcircRNAs, using the Cas13-gRNA system, and after ectopic expression of in vitro-synthesized vcircRNAs. Analyses of intracellular vcircRNA localization and protein-vcircRNA interactions will be performed to gain mechanistic insights into the actions of vcircRNAs. The second aim we will test the hypothesis that the vcircRNAs are generated by cytoplasmic RNA ligases, such as the RTCB/archease ligase complex, that is involved in tRNA ligation and XBP1 mRNA splicing during the unfolded protein stress response, or the newly identified C12ORF29 ligase are involved in the formation of vcircRNAs. The expected outcomes from this application will address fundamental aspects about the functions of novel vcircRNAs and novel viral proteins in the viral infectious cycle. We hypothesize that other RNA virus families generate vcircRNAs as well, suggesting novel Achilles’ heels in viruses that can be targeted in novel antiviral approaches.

NIH Research Projects · FY 2026 · 2005-09

Project Summary The process of cell division requires that each cell receive an identical complement of the genome. Errors in chromosome segregation give rise to chromosomal aneuploidies that are causative for human genetic disease such as Down syndrome and promote the progression of most human cancers. The work in this proposal is focused on understanding the functions of the centromere and kinetochore, the primary site on the chromosome that attaches each chromosome to the mitotic spindle for proper segregation during division. The centromere and kinetochore serve as the primary control center for chromosome segregation through their roles in microtubule attachment, force coupling for chromosome movement, and error correction when chromosome attachment or alignment on microtubules is perturbed. Underlying the centromere is a specialized chromatin domain that is delineated by replacement of histone H3 in nucleosomes with a centromere specific histone variant termed CENP-A. The position of CENP-A defines where the centromere and kinetochore will form and defects in CENP-A assembly or maintenance cause centromere and kinetochore loss which result in chromosome missegregation. In this proposal we study three different aspects of CENP-A chromatin. In our first Aim we work to understand how the assembly of CENP-A chromatin is controlled so that the centromere is replenished during each cell cycle and maintained through replication and cell division. We focus on three important factors for CENP-A assembly, CENP-C, HJURP and the Mis18 complex that are essential for building new CENP-A nucleosomes at centromeres. Our second aim studies the problem of how the presence of CENP-A in chromatin dictates the sites of new CENP-A assembly. We apply a new method we have developed called DiMeLo-seq that overcomes existing limitations in studying the complex repetitive centromeres of vertebrates. This approach uses directed methylation to mark the sites where proteins are bound to chromatin and then uses long read sequencing to map the binding sites through the repetitive centromere sequences. We use this approach to study the density, distribution, and sites of regeneration of CENP-A through the cell cycle. In our third aim we study how centromeres are condensed and organized by the activities of CENP-A nucleosome binding proteins. We discovered that two centromere proteins (CENP-C and CENP-N) can bridge CENP-A nucleosomes and condense centromeric chromatin. We use human cells to test the role of this condensation activity in centromere condensation, resistance to force, and chromosome segregation. Overall, our proposal will elucidate the regulatory mechanisms and structural organization of vertebrate centromeres and how those properties ensure accurate chromosome segregation.

NIH Research Projects · FY 2025 · 2005-07

Project Summary/Abstract Our program provides veterinarians with rigorous research training leading to the PhD degree at Stanford University. Appointees join top-ranked graduate programs in the biosciences where they are mentored by outstanding researchers. They develop stronger ties to their veterinary profession through the Department of Comparative Medicine. The rationale for this program is that intense research training will enable more veterinarians to compete effectively for research grant support and become independent principal investigators, which will address a national need. Trainees choose one of 14 graduate programs in the Biosciences: biochemistry, biology, biomedical informatics, biophysics, cancer biology, chemical & systems biology, developmental biology, genetics, immunology, microbiology & immunology, molecular & cellular physiology, neurosciences, stem cell biology & regenerative medicine, and structural biology. Program faculty include five comparative medicine mentors (who are veterinarians and faculty in the Department of Comparative Medicine), and 21 research mentors. During the next funding cycle, funds are requested to support three trainees at any one time. Overall, we seek to produce highly trained veterinarian-researchers that will assume leadership roles and exert a sustained, powerful influence in their research field and in the veterinary profession.

NIH Research Projects · FY 2026 · 2005-05

PROJECT SUMMARY/ABSTRACT Deep sequencing technologies have revolutionized our understanding of gene expression in the brain. In particular, single cell RNA sequencing of many brain regions has revealed an incredible diversity of transcriptomically-defined neuronal cell types. This is true not only for anatomically defined brain regions such as the frontal cortex but also for more discrete neuronal populations such as those confined to a particular cortical layer. This diversity of transcriptomically specified neuronal cell types is a major step forward for a central goal of contemporary neuroscience: understanding how neural circuits composed of specific neuronal cell types regulate behavior. Nevertheless, this diversity in neuronal cell types immediately raises the question as to how the functional output of a brain region (or discrete neuronal population) is parcellated among the many transcriptomically-defined neuronal cell types that comprise that region. Despite the enormous work that has gone into defining these neuronal cell types, their functional relevance remains unclear in most, if not all, instances. Our proposed project seeks to fill this knowledge gap by focusing on the neurons that comprise the principal component of the bed nucleus of the stria terminalis (BNSTpr). Our genetically-targeted single nucleus RNA sequencing of BNSTpr neurons has identified many transcriptomically-defined neuronal cell types, one of which is defined by its unique expression of the neuropeptide tachykinin 1 (Tac1). We have developed intersectional genetic strategies to interrogate functionally and anatomically the relevance of the Tac1-expressing BNSTpr neuronal cell type in generating behavioral output of the BNSTpr. In parallel, our genetic strategies enable us to interrogate the contribution of the complementary Tac1 non-expressing BNSTpr neuronal cell types to behavioral output of this region. In Aim 1, we will determine the activity of Tac1-expressing and non-expressing BNSTpr neuronal cell types in freely moving mice using a genetically encoded calcium indicator in engineered mouse strains. In Aim 2, we will use intersectional optogenetic actuators to determine the necessity and sufficiency of Tac1-expressing and non-expressing BNSTpr neuronal cell types in behavior. In Aim 3, we will match the connectivity of the Tac1-expressing neuronal cell type with functional output of its projection targets; in addition, we will engineer a targeted deletion of Tacr1, the cognate receptor for Tac1, in these projection targets and test whether Tac1 signaling through Tacr1 in these projection targets is essential for behavioral output. Together, our project will determine the specific contribution of a particular transcriptomically-defined neuronal cell type to behavior in contrast to the behavioral output of the region within which it resides. Health relatedness: The BNSTpr is an integrative center linking sensory input to motor output for social behaviors, and it is critical for emotional and reproductive health. Many neuro-psychiatric conditions manifest with severe disruptions to social interactions, but how this occurs is unknown. Our work on a molecularly-specified neuronal cell type within this complex region has the potential to identify neural circuits whose disruption in disease states alters social behaviors. It may therefore suggest potential targeted therapeutic or diagnostic applications for neuro-psychiatric illnesses.

NIH Research Projects · FY 2025 · 2005-05

ABSTRACT This renewal proposal for the Adult and Pediatric Rheumatology Training Program provides exceptional basic, translational and clinical research training to post-doctoral fellows interested in research careers studying rheumatic diseases and autoimmunity. Now in its 15th year, the training program leverages Stanford’s rich research and training culture to provide the environment to train the next generation of academic independent investigators in rheumatology and Immunology. The program focuses on training rheumatology and immunology MD/MD PhD fellows, as well as MD/MD PhD or PhD fellows from other departments who have strong commitments to these research areas. Physician fellows are eligible at the end of their clinical training and must commit to 2 years of training with >80% protected research time. Fellows can select a training track in either Basic and Translational Research (Track 1) or Clinical Investigation (Track 2). Training in each track is comprised of a hypothesis-driven research project, required and recommended course work, and skill development (e.g., grant application and manuscript preparation) required for a successful independent research career. Trainees in Clinical Investigation (Track 2) are eligible for obtaining a Master of Science (MS) in Epidemiology or in Health Services Policy Research. Other trainees in Track 2 have selected course work from the MS curriculum. Trainees in either track also have the option of pursuing a MS in Biomedical Informatics. In addition to course work, all trainees in both Tracks are required to attend weekly lectures in rheumatic diseases, journal clubs, the interdepartmental Stanford Immunology Program annual retreat, semiannual T32 fellow specific retreats, the five-day Intensive Course in Clinical Research, and Responsible Conduct of Research courses. Multiple other offerings specific to Track 1 include advanced immunology seminars in molecular, cellular, and translational immunology or in computational and systems immunology. For Track 2, elective/advanced courses are offered through the Departments of Epidemiology and Health Research Policy that include study design, epidemiologic methods, statistical analysis, clinical trial methodology, outcomes research, health economics, and database design and management. Cross- fertilization between the two tracks is fostered by all trainees having a primary mentor in one of the two tracks and a secondary mentor in the other track. These various courses, conferences, retreats, and venues for presentation will provide opportunities for interactions between the rheumatology and immunology T32 trainees and the many other immunology fellows, faculty and students involved in the broad base of research at Stanford. Faculty mentors are selected carefully for their research expertise and mentoring competencies. Rigorous evaluation of the program identifies opportunities for improvement and progress toward goals.

NIH Research Projects · FY 2024 · 2005-04

ABSTRACT Protein folding in the cell is critically dependent on the assistance of molecular chaperones. The ring-shaped chaperonins are essential members of the cellular folding machinery. These large protein complexes consist of two stacked seven- to nine-membered rings. Chaperonins bind unfolded substrates in their central cavity and use binding and hydrolysis of ATP to mediate polypeptide folding. Substrate proteins are thought to fold upon encapsulation in the central cavity formed by each ring. The long term goal of this program is to understand how the chaperonin of eukaryotic cells, TRiC, mediates polypeptide folding., TRiC is hetero-oligomeric and uses ATP cycling to open and close a built-in lid over the central chamber. Intriguingly, TRiC has the ability to fold some eukaryotic proteins, such as actin, that cannot be folded by any other chaperone. Despite its essential role in cellular folding, little is known about the mechanism and substrate binding properties of TRiC. Our work in the previous funding period provided important mechanistic and structural insights into this chaperonin. We established that the conformational cycle of TRiC is significantly different from that of bacterial chaperonins and demonstrated subunit diversity confers dramatic functional asymmetry to this seemingly symmetric chaperonin. Importantly, in the previous funding period we developed a recombinant system to study this chaperonin that for the first time makes it possible to generating mutants that can be studies functionally and structurally. These breakthroughs enable us to study how TRiC facilitates folding in unprecedented detail, through the following proposed aims: 1. Characterize of the nucleotide cycle of the chaperonin TRiC: Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. We want to understand how the ATPase cycle of TRiC is coordinated among the different TRiC subunits, and how ATP cycling drives conformational changes in the chaperonin and how the "built-in" lid that opens and closes in response to ATP-binding and hydrolysis. 2. Define the molecular basis of TRiC-substrate interactions: Little is known about the molecular basis of TRiC-substrate interactions. We want to define the substrate recognition code of the binding sites for the different subunits in the chaperonin and define the motifs within substrates that are recognized by TRiC. 3. Investigate the mechanism of TRiC-assisted substrate folding: The exact role that chaperonins play with respect to the substrate is still a mystery. We will explore the effect of TRiC on substrate proteins during the different stages of the folding cycle by combining biochemical approaches together with crosslinking and fluorescence spectroscopy. Importantly, recent observations have highlighted the links between TRiC and several pathological states incuding cancer, viral infection and neurodegeneration. Thus our project deciphering the mechanism of this chaperonin in cellular folding will help develop therapies to ameliorate these human diseases.

NIH Research Projects · FY 2026 · 2005-01

PROJECT SUMMARY A key question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly in the mammalian brain may provide insights into the etiology of human brain disorders. In the mammalian brain, each neuron on average forms connection with thousands of other neurons. The assembly of these complex circuits depends on cell-cell communication during many steps of neural development. In the previous three cycles of this grant, we have developed methods such as MADM (Mosaic Analysis with Double Markers) and viral-genetic manipulations in mice that allowed us to label and genetically manipulate specific neuron cell types, down to individual neurons, and study genes that play key roles in dendrite morphogenesis and target selection of axons. Specifically, we have recently identified two cell-surface proteins, Teneurin-3 (Ten3) and Latrophilin-2 (Lphn2), that are expressed in complementary patterns in the interconnected nodes of hippocampal network, following a “Ten3→Ten3, Lphn2→Lphn2” connectivity rule . We have shown that Lphn2 acts as a heterophilic repulsive ligand and Ten3 acts as a homophilic attractive ligand to direct Ten3+ proximal CA1 axons to selectively target to distal subiculum; at the same time, Ten3 acts as a repulsive ligand to direct Lphn2+ axons to proximal subiculum. We have also developed a method that allows us to profile cell-surface proteomes with exquisite sensitivity and spatiotemporal control. In this proposal, we will expand on both of these recent advances. Specifically, we will investigate whether Ten3 and Lphn2 instruct wiring specificity in multiple nodes of the extended hippocampal network and in other brain regions, how Ten3-Lphn2 interaction leads to axon repulsion, and whether G protein signaling is essential for Lphn2’s action as a receptor or a ligand. Complementary to the in-depth studies of Ten3 and Lphn2, we will use our cell-surface proteomic profiling methods to broadly survey changes of cell- surface proteomes from developing to mature neurons, and to identify new cell-surface proteins that regulate dendrite morphogenesis and neural circuit assembly.

NIH Research Projects · FY 2025 · 2004-12

PROJECT SUMMARY/ABSTRACT Agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) offer benefits over standard care laser treatment of the peripheral avascular retina in severe retinopathy of prematurity (ROP). Our lab provided proof of concept that regulating VEGF receptor 2 (VEGFR2) signaling in retinal endothelial cells (ECs) not only inhibited disordered intravitreal neovascularization (IVNV) in severe ROP, but also facilitated ordered EC division to allow vascularization into peripheral avascular retina (VPAR). VPAR occurs after intravitreal anti-VEGF treatment in infants with ROP, but reactivation with IVNV is difficult to distinguish from VPAR. To avoid complications from reactivated IVNV, clinicians tend to treat all new angiogenesis, but laser reduces potential VPAR and associated expanded visual field. Also, some anti-VEGFs reduce systemic VEGF levels with reports of adverse events in treated infants. There is the need to safely target pathologic VEGFR2 signaling to only inhibit IVNV and not VPAR. Our lab strives to understand signaling mechanisms that permit VPAR but inhibit IVNV in ROP without damaging the retina or the infant. We found that specifically in retinal ECs, regulation at the level of VEGF receptor 2 extended vascularization into the peripheral avascular retina, whereas endothelial STAT3 or the erythropoietin receptor (EPOR) either increased or inhibited IVNV. In our novel retinal EC culture model, we distinguished VEGF-activated tyrosine sites on the C-terminal domain (CTD) of VEGFR2. Sustained VEGFR2 signaling, important in p-ERK-mediated IVNV, activated the tyrosine (Y1175) site but not the Y1214. We also found that the adaptor proteins, MEMO1 and IQGAP1, sustained signaling through VEGFR2 leading to disordered or invasive angiogenesis in disease models. In transgenic humanized mice, signaling through the EPOR improved retinal function following oxygen induced damage. Based on our findings, we developed the hypothetical framework that regulation of angiogenesis leads to IVNV or VPAR and is mediated by signaling events involving1) activation of tyrosine phosphorylation sites on VEGFR2 or 2) adaptor proteins, e.g., MEMO1, IQGAP1, and that 3) crosstalk between EPOR and VEGFR2 signaling provides retinal vascular/neural protection. We propose 3 aims to test the prediction that: 1) IVNV will be increased by OIR- induced VEGF signaling through VEGFR2 in mice expressing mutant Y1212 (analog of human Y1214); 2) to test prediction that adaptor protein, MEMO1, interferes with Y1175 activation, whereas adaptor protein, IQGAP1, leads to Y1175 activation, retinal EC proliferation and IVNV; and 3) To test the prediction that crosstalk between VEGFR2 and EPOR signaling supports polarized retinal EC migration, as in VPAR, and that recovery of retinal neural function through EPOR involves EPOR-mediated vascular protection against high oxygen-induced damage. Methods include: novel mice with CRISPR-engineered mutations combined with conditional inducible knockout; optical coherence tomography; Micron IV imaging; OIR; OptoMotry and ERG.

NIH Research Projects · FY 2024 · 2004-09

A fundamental fact of vision is that our perception of the external world is shaped by a number of behavioral and contextual factors. These factors include visual selective attention, in which sensory information is filtered in favor of items that are behaviorally and contextually relevant. In addition, it includes the modulation of visual processing during saccadic eye movements which occur several times each second. These factors are known to modulate the processing of visual information and to contribute to adaptive visually guided behavior. In the primate brain, the visual and oculomotor systems are highly interconnected, and past work has shown that movement-related signals exert robust influences on visual processing in visual cortex. The current proposal focuses on addressing key questions concerning the role of gaze control mechanisms in visual selection and visual stability, two ways in which those mechanisms clearly influence visual perception and cognition. These questions will be addressed using a broad set of innovative approaches and tools including newly developed, large-scale, high-density Neuropixels (NP) probes made specifically for use in nonhuman primates. In this first aim, we will test the role of persistent activity in the selection of visual signals and in visually guided saccades in a set of key, complementary experiments that include large-scale neurophysiological recordings with primate NP probes. Our hypothesis is that persistent activity in the frontal eye field (FEF) serves primarily to select the visual information required to guide saccadic eye movements, and that this function is mediated by dopamine D1Rs. In the second aim, we will address a major open question regarding the basis of stimulus-driven attention by testing the contribution of posterior parietal cortex (PPC) to the representation of visual salience in the brain, and to saliency-driven behavior. Experiments in this aim combine the use of reversible inactivation of PPC with large- scale neurophysiological recordings and behavior. In the third aim, we will address another major open question regarding the basis of the distortions in vision that occur during saccadic eye movements. We will leverage the use of large-scale recordings, and the use of reversible parietal inactivation to test the role of PPC in perisaccadic changes in visual processing within extrastriate visual cortex and the FEF. Overall, our focus on the influence of gaze control mechanisms on visual processing, combined with our use of state-of-the-art neurophysiological approaches and causal methods, are likely to produce results that exert a large and sustained impact on our understanding of the neural mechanisms of visual perception and cognition.

NIH Research Projects · FY 2026 · 2004-04

Project Summary/Abstract The Candida Genome Database (CGD) is considered THE resource for comprehensive information about the human fungal pathogen Candida albicans and related Candida species, and is widely used by the Candida research community, who rely upon CGD in their everyday work. C. albicans is the third or fourth most common nosocomial bloodstream isolate; mortality rates are high (35% or greater) and treatment is costly. It is thus vital that there is a comprehensive and up-to-date resource for researchers investigating the biology and pathogenesis of C. albicans and related species, as such a resource accelerates their research. The central challenge for any community database is to turn data into knowledge, which the community can access, use and build upon. This is especially important in this era of high throughput technologies, which produce a flood of such data. A research community is clearly best served by the collection of all relevant data in a single location, followed by manual, expert curation of those data, coupled with tools to allow users to search and navigate the data in an intuitive fashion. Most of the data available in CGD are not available from any other site, and no other site performs curation of the C. albicans literature. We re-use software wherever possible, writing our own only when necessary. This philosophy has served us well, in that we have built CGD into an indispensable resource with modest staff, and we will continue to apply this model going forward. In this renewal for CGD we propose to build on our previous successes. We will use high-throughput data to improve the sequences and primary annotations for Candida genomes—reference genomes provide the fundamental platform upon which a community’s research builds, and it is vital that they be accurate and correctly and comprehensively annotated. We will perform real-time curation of the experimental literature, capturing gene names, mutant phenotypes, Gene Ontology Terms, etc., from papers as they are published, allowing a bench biologist to, at a glance, find the salient, up-to-date information about any gene to which their research leads them. We will modernize CGD— the code upon which it is built was written over a quarter of a century ago—providing both additional functionality as well as improved and more responsive navigation. Finally, we will provide support to the Candida scientific community, ensuring that we are continuing to serve their needs as the indispensable resource that we have become. Together, successful completion of these aims will support and accelerate research into fungal pathogenesis, and thus have a positive impact on human health.

NIH Research Projects · FY 2025 · 2004-03

PROJECT SUMMARY Signaling proteins, including G-protein-coupled receptors (GPCRs), impact genome modulators, including ATP-dependent chromatin remodelers (ADCRs), to control gene expression in homeostasis and disease. The signaling and chromatin regulators operative in epidermis, however, have not been fully characterized, in part due to the hundreds of genes that comprise each of these classes of proteins. This cycle, AR049737 knocked out all 101 GPCR and 116 ADCR genes expressed in epidermis to identify essential roles for the ADGRL2 adhesion GPCR as well as for ncBAF and SRCAP ADCRs in controlling epidermal differentiation. This revised competing renewal will define the mechanistic basis for these new essential actions, sustaining the long-term focus of AR049737 in characterizing signaling and genome regulators of epidermal homeostasis. First, we will test a working model in which the ADGRL2 GPCR is activated by FLRT3 and/or TENM2 from adjacent cells to induce pro-differentiation signals, which are modulated by specific proteins that AR049737 recently found associated with the ADGRL2 cytoplasmic domain in differentiating keratinocytes. These proteins include the RapGEF2 guanine nucleotide exchange factor and the PPP2R2A phosphatase, which are posited to enable ADGRL2-pro-differentiation signaling, and the RASAL2 GTPase-activating protein, which is postulated to oppose it. Aim I will test key features of this model using genetic approaches in tissue to illuminate the mechanistic basis for the actions of the ADGRL2 GPCR in epidermal differentiation. Second, we will study the premise that the diverse impacts of ADCRs are enabled by the specific ADCR-associated proteins that target their actions across the genome. These efforts focus on ncBAF, which AR049737 found to be essential for induction of epidermal differentiation genes and SRCAP, which AR049737 found to be essential to suppress ectopic differentiation in keratinocyte progenitors. We will define the impact of DNA sequence-specific transcription factors (TFs) on ncBAF and SRCAP targeting and activity in epidermal homeostasis, including IRF6, which co- regulates nBAF differentiation genes, and SNAI2, which co-suppresses SRCAP differentiation targets. Aim II will thus use genomics approaches to define the molecular mechanisms responsible for the opposite impacts of ncBAF and SRCAP ADCRs in epidermal differentiation. At the end of proposed funding, this effort will have defined mechanistic features of newly identified essential roles for GPCRs and ADCRs in the control of epidermal homeostasis.

NIH Research Projects · FY 2026 · 2003-09

PROJECT SUMMARY Knee osteoarthritis (OA) is a major cause of disability without a cure that affects 20+ million US adults and leads to large financial burdens. While the etiology of OA is unknown, it is thought to be related to altered biochemical or biomechanical function of the joint. OA is slow to develop; the current treatment strategies for OA are pain control using lifestyle or therapeutic interventions, until the patient requires a total knee replacement. The progressive loss of periarticular muscle mass and function reduces joint stability and health. Loss of muscle mass and function is associated with aging, as is OA development and progression. While many OA studies examine the bone, cartilage, synovium, and meniscus in the knee, the interplay between muscle function and structure, and the initiation and progression of OA is unclear, despite recommendations of exercise as a conservative treatment. In this study, we seek to build new imaging tools to sensitively measure the effects of muscle strength, quality, and function on OA initiation and progression. This is an appealing area to study because disease- modifying treatments such as exercise, physical therapy, and muscle-building drugs could both manage pain and change disease progression. Toward this goal, we will build imaging tools to provide unparalleled insights into exercise-induced microstructural changes in the muscle, subtle compositional changes in OA, and biomechanically inspired biomarkers of how joint loads affect cartilage pressure. In Aim 1, we will build and validate a multi-vendor, highly accelerated, multi-parametric whole-bilateral- limb magnetic resonance imaging (MRI) protocol that will enable acquiring high-resolution quantitative imaging of both knees, thighs, and calves in under 20 minutes of scan time. In Aim 2, we will compute fully automated and novel biomechanics-inspired imaging biomarkers for the cartilage, bone, and muscle. These will include cartilage pressure maps to assess spatial joint loading profile, as well as neural shape models to model the complex shapes of bones and muscles. In Aim 3, we will assess the responsiveness of these imaging measures in a two-year longitudinal study in patients with early OA undergoing a walking-based muscle strengthening intervention. We will quantify the impact of exercise on muscle structure, tissues in the knee, pain relief and OA progression. We hypothesize that our novel imaging biomarkers have stronger association than current functional tests on pain relief and OA progression, and that our baseline imaging can predict subject-specific pain relief. Overall, our novel tools will elucidate the mechanistic interplay between exercise and OA physiology, towards developing precision muscle strengthening programs. These insights will help accelerate the elusive quest towards developing disease-modifying strategies for OA.

NIH Research Projects · FY 2025 · 2003-09

SUMMARY Celiac disease (CeD) is a gluten-induced inflammatory disorder of the small intestine for which no non-dietary therapy has been approved for clinical use. In this ongoing collaborative project, we study the pathogenic role of transglutaminase 2 (TG2), a validated drug target for CeD, through a synergistic combination of tools and concepts from chemical biology and mucosal immunology. During the past grant cycle, we have uncovered a novel pathway by which antigenic gluten peptides are potently escorted into the endosomes of antigen presenting cells through the formation of complexes involving TG2, 2-macroglobulin (2M), and low density lipoprotein receptor-related protein 1 (LRP1). In the next cycle, we will characterize this mechanism for TG2- dependent inflammatory T cell activation through the following Specific Aims: Aim 1: Structure-function analysis of molecular interactions involved in LRP1-mediated gluten antigen presentation: We will biochemically characterize how a representative intermediate formed between TG2 and a gluten peptide is recognized by 2M. Our goal is to define the molecular features facilitating 2M-TG2 recognition and the role of the peptide in this three-component interaction. Separately, we will also study how the resulting peptide-TG2-2M complex is recognized and endocytosed by the LRP1 receptor. Aim 2: Defining the pathogenic role of the LRP1 pathway for gluten peptide uptake in CeD pathogenesis: While LRP1-mediated gluten antigen endocytosis has been studied in cultured cells, we do not know the identities of small intestinal cells that deamidate and take up gluten peptides and present them to CeD-specific T cells. Using fluorogenic peptides and peptidomimetics that undergo LRP1-mediated endocytosis, we will identify intestinal cells that exhibit this property in selected strains of mice. In turn, we will leverage mouse models developed through the support of this grant to examine the role of the TG2-2M-LRP1 pathway in the loss of oral tolerance to dietary gluten as well as the onset of gluten-induced villous atrophy. Not only does our proposed research offer the promise of breaking fundamentally new ground in our understanding of CeD pathogenesis, but the resulting tools and methods could be leveraged to investigate the relevance of the TG2-2M-LRP1 endocytic pathway in other patho-physiological situations.

NIH Research Projects · FY 2026 · 2003-09

PROJECT SUMMARY Influenza and SARS-CoV-2 are respiratory viruses that represent a continual threat to the health of many Americans, and will likely account for 100,000 deaths and many times that number of hospitalizations in the US alone each year. The available vaccines are inadequate, with the recent flu vaccine estimated to be only 40% effective and while the RNA vaccines for SARS-CoV-2 have been remarkable at preventing severe illness or death, they do not prevent reinfection in many people. Many with pre-existing conditions or immune deficiencies (e.g., obesity, diabetes) are vulnerable, which leads to suppressed response to vaccine or increased risk of severe outcome upon infection. Thus, the need to understand and improve both vaccines and the response to them is urgent. The Stanford CCHI has been a leader in understanding influenza vaccination and infection, and very quickly developed complementary expertise during the pandemic, making great strides in both understanding SARS-CoV- 2 infection and vaccine responses, and also in developing new technologies that promise even greater advances. These range from Dr. Wang’s insights in the effects of antibody glycosylation and lung inflammation, to Dr. Barnes seminal work on the structures of antibodies to SARS-CoV-2 antigens. Work that Dr. Barnes will carry further by designing and testing novel flu and SARS-Cov-2 antigen constructs. Dr. Khatri has also developed bioinformatic methods that have revealed conserved gene signatures regarding the immune response to viruses, and which he will use to define the cells and mechanisms that underlie these signatures. The clinical core under Dr. Chinthrajah will recruit and vaccinate obese and diabetic patients at risk for severe COVID and Influenza illness, and the results will be analyzed by Dr. Wang and also by the Human Immune Monitoring core under Dr. Maecker. Dr. Davis will head the Technology Development project which will continue his development of immune organoids and the use of spleen organoids particularly to characterize both existing and novel vaccine responses, and also attempt to reconstruct immune responses to vaccination and infection using skin and lung organoids from the same donors. Organoid responses to vaccination or infection will be validated against data in human subjects using our collection of influenza and SARS-CoV-2 specific T and B cell probes, as well as through TCR and BCR sequence analysis. Organoid processing and banking will be handled by the clinical core and all projects will make use of this resource, which will be especially useful in testing particular immunogens for their ability to make the desired antibodies, or the use of gene editing to identify key loci mediating particular effects. Immune organoids and the networks we propose to create will give us the ability to test hypotheses and define mechanisms in an entirely human system. The overall theme of this submission is to continue our efforts to understand Influenza and SARS-CoV-2 vaccination and infection using a broad range of approaches, integrating molecular, structural and bioinformatic methods, in vivo with unique cohorts and in vitro with organoids.

NIH Research Projects · FY 2026 · 2003-04

PROJECT SUMMARY A central question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly may provide insights into the etiology of human brain disorders. The fruit fly olfactory circuit has been an excellent model to investigate the mechanisms by which wiring specificity of neural circuits arises during development. In this circuit, axons of 50 types of olfactory receptor neurons (ORNs) match precisely with dendrites of 50 corresponding types of second-order olfactory projection neurons (PNs), forming 50 discrete glomeruli in the antennal lobe. This allows olfactory information to be faithfully delivered from peripheral sensory organs to higher brain centers, enabling innate and learned olfactory behavior. Thanks to the continual support of this grant since 2003, our studies have made the Drosophila antennal lobe one of the best-understood circuits in the molecular, cellular, and developmental underpinnings of wiring specificity. During the last grant cycle, we made three key advances: 1) we determined single-cell transcriptomes of PNs and ORNs across development, which have produced differentially expressed genes as candidate wiring molecules and cell-type-specific drivers for labelling and genetically manipulating individual PN and ORN types; 2) we developed a cell-surface proteomic profiling method that allowed us to identify new wiring molecules; 3) we established an explant culture that recapitulates wiring specificity in vivo, allowing us to examine the dynamic process of circuit assembly in wild-type and mutants using time-lapse imaging. In this proposal, we will take advantage of these advances to further investigate the cellular and molecular mechanisms by which the olfactory circuit is assembled and wiring specificity is achieved. Specifically, we will utilize new genetic tools and time-lapse imaging to determine the cellular events that lead to PN dendrite patterning, segregation, and ORN-PN synaptic partner matching. We will investigate the mechanisms by which cell surface receptor teneurins work with putative ligands and downstream signaling molecules to instruct synaptic partner matching. We will also identify additional instructive wiring molecules and study their mechanisms of actions using data from single-cell transcriptomes and in vivo assays that can distinguish attraction vs. repulsion in ligand-receptor interactions.

NIH Research Projects · FY 2025 · 2002-08

PROJECT SUMMARY Mucins are densely O-glycosylated proteins with extended regions of clustered Ser/Thr-linked O-glycans, a structural feature that imparts a rigid and extended conformation. Their range of biological functions include physical stiffening of the glycocalyx to modulate cell survival in low adhesion settings, and biochemical interactions with glycan-binding receptors on other cells. Altered mucin expression and glycosylation patterns have been strongly linked to cancer progression. Crude measurements of these changes are currently used for cancer diagnosis but are imperfect due to their lack of molecular-level detail. A detailed map of mucin O-glycan structures and sites has been impossible to obtain, as mucins are recalcitrant to conventional mass spectrometry-based glycoproteomics methods. As a consequence, the cellular pathways underlying aberrant mucin structures are not well defined. We are pursuing these questions with the long-term goal of identifying more accurate cancer biomarkers and new therapeutic targets. During the previous funding period, we developed new mass spectrometry-based glycoproteomics methods and used them in fundamental studies of the enzymes that initiate mucin-type O-glycosylation, the polypeptide GalNAc transferases. Examples of our accomplishments include (i) development of the IsoTaG method for intact glycoproteomics via isotopic recoding and mass-independent glycopeptide discovery; (ii) identification of an optimal tandem mass spectrometry method for O-glycosite discovery; and (iii) development of a bump/hole strategy to identify biological substrates of polypeptide GalNAc transferases that initiate mucin-type O- glycosylation. In preliminary work for this application, we repurposed mucin-specific proteases (“mucinases”) from gut-resident microbes as tools for mapping O-glycosites on mucin domains. In the next funding period, we plan to develop a comprehensive “mucinomics” platform. We will use engineered mucinases as glycoform-sensitive probes of mucin expression on cells and tissues. We will also develop a mucinase-based enrichment strategy for mass spectrometry-based discovery of new mucin domain molecules as well as O-glycosite mapping. Integrated into this workflow will be newly developed ionization methods and search algorithms for O-glycosite identification. Finally, we will use the mucinomics platform to define pathways by which prevalent oncogenes drive altered mucin expression and glycosylation in cancer.

NIH Research Projects · FY 2025 · 2002-07

Project Summary/Abstract Our training program recruits diverse postdoctoral fellows, predominately physicians, and develops their skills to conduct infectious disease epidemiology research. The program stresses epidemiology as a key tool to face growing infectious disease threats. It highlights the global epidemiology of infectious disease, because the increased connectivity of human populations means that the epidemiology and control of infectious diseases requires a global perspective. The program will be interdisciplinary, engaging 29 faculty research mentors from seven Departments (Medicine, Pediatrics, Epidemiology, Biology, Pathology and Microbiology, Immunology and Civil and Environmental Engineering). Three trainees per year will be drawn from candidates for the Infectious Diseases Fellowship Programs in Pediatrics and Internal Medicine; exceptional applicants from other postdoctoral programs at Stanford may be considered if they have demonstrated prior commitment to Infectious Diseases epidemiologic research. Trainees will conduct research under the mentorship of a program faculty member committed to trainee success. Research is expected to be cross-disciplinary to take advantage of the breadth of campus faculty and the varied exceptional research expertise available at Stanford. To promote interaction among trainees and faculty we will convene research seminars conducted by trainees; interdisciplinary seminars in infectious diseases epidemiology; weekly infectious diseases grand rounds; lectures on infectious diseases, ecology and risks; and an annual one-day research retreat. Each trainee will be counseled by a Research Committee comprised of their mentors and others with expertise relevant to the research and to individual career goals. At least twice a year, trainees will meet with their mentors and the program directors to discuss their Professional Development Plan. In addition, as part of this T32 renewal, we propose to increase our emphasis on epidemiologic methods including requiring that all fellows supported by the training grant complete four courses taught by the Department of Epidemiology in global infectious disease epidemiology and modeling. This classroom instruction will complement the in-depth mentored research experience so that trainees will develop both the content expertise and applied skills critical to become independent researchers. All trainees will be strongly encouraged to apply for independent support. Trainees will be appointed to the program for a year at a time. They will spend between 1 and 3 years in the program. We envision that graduates of this program will assume independent research or public health leadership positions and engage in careers that address the great infectious disease threats of their generation.

NIH Research Projects · FY 2024 · 2002-05

PROJECT SUMMARY Numerous environmental and cellular factors can induce DNA lesions or create other types of challenges that can slow DNA replication, leading to replication stress. Failure to alleviate this stress and restart stalled replication forks can cause genome instability, which can drive cancer initiation and progression and affect the cellular response to chemotherapy. Hence, deciphering the cellular response to replication stress––the long- term goal of this application––is imperative for understanding fundamental aspects of tumorigenesis. One crucial aspect of the replication stress response involves replication fork reversal, a process that leads to the formation of a four-way junction structure by the remodeling of both nascent and parental DNA strands. There are a family of ATP-dependent translocases that promote fork reversal in vitro and in cells, but why so many enzymes are involved in this process is not understood. We showed that one of these translocases, HLTF, prevents a stress-resistant mode of replication by promoting fork reversal, and we elucidated the basic mechanism by which cells continue replication upon HLTF loss. The object of this application is to further elucidate the mechanisms by which HLTF mediates the replication stress response, characterizing its unique role in this process. HLTF is a multi-functional protein and recent studies using a panel of HLTF mutants that are each deficient for one of its activities have revealed unexpected roles for these activities in HLTF’s known functions, raising a number of new and exciting questions. This application will address questions about the molecular mechanisms by which HLTF promotes fork slowing and fork reversal in cells and determine the impact of its loss on replisome stability, genome stability, cell proliferation. Aim 1 will investigate which domains of HLTF are needed for fork slowing, fork reversal, and resistance to replication stress, and HLTF’s association with the replication fork. HLTF’s dynamic interaction with the fork will also be probed. Aim 2 will investigate a newly discovered function for HLTF in stabilizing the replication fork. These experiments will employ a combination of molecular, cellular, genetic and proteomic approaches as well as single-molecule imaging and tracking methods in Xenopus extracts to solve fundamental questions about how cells respond to replication stress.