Below are summaries of the projects of NIH intramural scholars funded as part of the Maximizing Opportunities for Scientific and Academic Independent Careers (MOSAIC) Program.
Project Title: The role of host mRNA cleavage by RNase L in viral infectionsInstitution: National Institute of Diabetes and Digestive and Kidney DiseasesGrant ID: K99GM143484
Abstract Text: Viral infections remain a challenging public health issue worldwide. The presence of many viruses, such as Influenza A and Hepatitis C, activate a latent Ribonuclease (RNase L) in human cells. Activated RNase L cleaves the single stranded regions of viral and host mRNAs. Cleavage of these RNAs leads to physiological changes in the cell, such as autophagy, senescence, decreased cell motility, interferon production and cell death. However, many aspects of RNase L activation in cells are not yet understood. Recent in vivo and in vitro kinetic studies suggested that RNA cleavage by RNase L is modulated in the cell by an unidentified factor. RNase L can directly interact with the ribosome and with several translation factors involved in different steps of protein synthesis. Due to these interactions it was proposed that RNase L’s cleavage activity is modulated by the translation of the host messenger RNAs (mRNA). Therefore, in specific aim 1, I will investigate this relationship between host mRNA cleavage by RNase L and translation at the global and individual gene level by combining two high-throughput sequencing methods, ribosome footprint profiling and RNA sequencing in RNase L activated human cells. In addition, ribosome-mediated RNase L cleavage activity will be also directly observed at the individual gene level by a novel technique, the real-time fluorescent single molecule detection of translating nascent peptides (SINAPS) in living cells. In aim 2, I will also explore the potential involvement of translation factors in translationmediated RNA cleavage by RNase L by first mapping the details of interactions of RNase L and translation factors by mutagenesis studies and Cryo-Electron Microscopy. Then these interactions will be disrupted in living cells to probe their potential involvement in mediating RNA cleavage by RNase L. Furthermore, RNase L activation can lead to many physiological changes in the cell, such as autophagy and apoptosis. It is plausible that the level of active RNase L is the determinant of which physiological processes will occur. To investigate the correlation between active RNase L levels and RNase L mediated changes in the cell, I will develop a fluorescent RNase L activation indicator that will allow us to sort cells into homogenously activated populations in aim 3. Subsequently, in these sorted cells I will detect changes in the transcriptome and translatome and assess signatures of physiological changes of autophagy and apoptosis in the context of RNase L activation levels. In addition, I will also study how RNase L cleaved host RNA fragments contribute to activation of innate immune response pathways in cells by using Cross-linking Co-Immunoprecipitation and sequencing (CLIP-seq). In summary, the proposed project will investigate unexplored aspects and outcomes of RNase L activation. Uncovering new aspects of the defense against viral infections will enable further studies and potentially contribute to the development of new therapeutic strategies.
Public Health Relevance Statement: The persistent presence and continuing emergence of viral infections is one of the major obstacles to improved human health. The majority of viruses, such as human immunodeficiency virus 1 and potentially SARS-CoV2, activate an endoribonuclease, RNase L, that consequently induces several physiological changes in the cell from autophagy to apoptosis. In the present proposal I will investigate how the activity of RNase L is modulated and its role in determining cell fate by using a combination of new innovative and classical molecular biology techniques.
Project Title: Multiplex Imaging in Therapy Refractory Tumors: Understanding the Spatiotemporal Facets of an Immunosuppressive EnvironmentInstitution: National Institute of Allergy and Infectious DiseasesGrant ID: GM147841
Abstract Text: Background/Significance. In recent years, it appeared that the cancer treatment revolution had arrived: immunomodulatory therapies. Although these breakthroughs unleash the immune system and can be deadly to tumors, there are a high percentage of cancers that fail to respond. One complicating aspect is tumor heterogeneity, both within the same cancer type and within a single patient. This aspect is especially prominent in pancreatic adenocarcinoma (PDA), which is notoriously resistant to many frontline immunotherapies and has a low 5-year survival rate. Although mouse models have made research into PDA more accessible, these models frequently fail to capture the spectrum of tumor heterogeneity in human PDA. Innovation. To address this limitation, I will use a unique collection of mouse pancreatic tumor clones with distinct and reproducible responses to treatment, mimicking the tumor heterogeneity seen in the clinic. This will provide me with unparalleled comparative and combinatorial power to interrogate the tumor microenvironment. Additionally, my proposed experimental approach will leverage emerging high-content imaging methods that are able to reveal the phenotype, activity, and spatial organization of the tumor microenvironment in situ.
Research Goals and Aims. The overall theme of my research is to understand the fine-grain cellular interactions that lead to an immunosuppressive tumor microenvironment, and how to then subvert and reprogram those interactions to improve synergistic combination therapies. Specifically, I propose two conceptually related aims that explore mechanisms to overcome the immunosuppressive tumor microenvironment. I aim to dissect how the myeloid compartment (Aim 1) and immunogenic cell death (Aim 2) can be manipulated to reinvigorate anticancer immune responses. Through these aims I will develop a platform for interrogating the underlying functional immunity of the tumor site, host-pathogen interactions, and the impact of therapeutic intervention.
Career Development Plan and Environment: My mentor, Dr. Ronald Germain, a renowned expert in immunology and microscopy, is an investigator in the NIH Intramural Research Program, one of the largest research centers in the world. In this unique environment, I will directly benefit from the numerous resources in place to support my research project and career development, including microscopy and immunology core facilities, frequent seminars and opportunities to engage colleagues/mentors, and regular workshops on grantwriting, mentoring, and laboratory management. Furthermore, I have facilitated multiple collaborations that will aid in expeditious attainment of my proposed research aims and my transition to independence. By the end of my training plan, I will be well-positioned to launch a productive independent career at the intersection of microbiology and cancer immunology.
Public Health Relevance Statement: Pancreatic cancer is an aggressive and therapeutically difficult cancer to treat. Mouse models of pancreatic cancer are therefore paramount to our mechanistic understanding of tumor microenvironment dynamics and immunomodulatory interventions. The overall goal of this research is to use cutting-edge microscopy techniques to mechanistically understand the fine grain cellular interactions that dictate tumor responsiveness and leverage them to improve synergistic therapeutic strategies.
Project Title: Quantitative Characterization of the Extracellular Matrix Components of Connective Tissue: Fingerprinting Macromolecular Components through Low-Field Magnetic ResonanceInstitution: Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentGrant ID: K99GM140338
Abstract Text: Fibrotic activity, the accumulation of macromolecules, alters the composition and microstructural organization musculoskeletal connective tissue. The ability to non-invasively quantify and characterize the significant extracellular matrix components such as proteoglycan content and collagen fibrils organization is important clinically since fibrosis is a common inflammatory response that plays a role in many pathologies. Recent advances in instrumentation for low field magnetic resonance (MR) has enabled its adoption in the field of macromolecular characterization, porous media, and recently biological tissues. LF MR is advantageous as an affordable non-cryogen alternative to high-field MR imaging with a greater detectable dynamic range of quantitative MR parameters. This adaptation for this imaging modality is limited by the lack of appropriate phantoms of connective tissue and the identification of biomarkers for healthy and diseased tissue. Composite gels that replicate the salient structural and compositional features of connective tissue will be developed and used to optimize low field MR methods and identify LF biomarkers. These methods will be applied to articular cartilage and lumbodorsal fascia connective tissues. The project is anticipated to have a significant positive impact on the clinical capability and utility of LF MR as an affordable point of care diagnostic application.
Public Health Relevance Statement: Musculoskeletal (MSK) conditions are the leading contributor to disability worldwide, between one and three, and one and five people live with a musculoskeletal pain condition. Regular assessment of the state of MSK connective tissue is needed to better identify the pathophysiological origin of the inflammatory and fibrotic response. This research will advance the adaptation of low field magnetic resonance imaging (MRI) as a novel, noninvasive, and affordable point of care diagnostic imaging method capable of assessing the state of MSK connective tissue and effective of regenerative medicine.
