Poster Session E (+Lunch and Office Hours)

Fibrosis affects almost every tissue in the body, including pulmonary, dermal, and cardiac tissues, and is the pathological outcome of misregulated wound healing or chronic inflammation. For example, idiopathic pulmonary fibrosis (IPF) is hypothesized to be initiated, in part, by repeated micro-injuries to the alveolar epithelium, resulting in deposition and accumulation of scar tissue, increased tissue stiffness, and ultimately loss of lung function. Currently FDA approved therapeutics for IPF (e.g., pirfenidone and nintedanib) only slow the progression of fibrosis and cannot reverse disease pathology. Development of new therapeutics often is challenged by poor in vivo efficacy despite promising preclinical findings. Improved human model systems are needed to better understand the pathobiology of IPF and improve therapeutic strategies. In this work, we are establishing human, multidimensional culture models that allow probing of dynamic cell-microenvironment interactions that regulate activation and persistence of wound healing cells. A lentiviral reporter system of alpha smooth muscle actin (αSMA) expression has been established for stably transducing a range of human cells, including lung epithelial and fibroblast cell lines and primary cells, and monitoring changes in phenotype in situ and in real-time in response to microenvironment changes upon injury. Hierarchically-structured and photoresponsive soft materials that mimic healthy to injured and diseased states have been created, and on-going work is utilizing these systems for studying cell activation and persistence to identify targets for mitigating maladaptive wound healing responses and developing improved strategies treating fibrosis.

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Multiple sclerosis, the most common of these diseases affecting over two million people worldwide and approximately 400,000 people in the United States, is a chronic inflammatory disease, causing lesions and plaques of demyelination in the brain and spinal cord. However, none of the current clinical tests can confidently predict clinical multiple sclerosis progression or treatment efficacy due to a lack of sufficiently sensitive and effective biomarkers. T cells in multiple sclerosis patients display an activated phenotype with an increased avidity to myelin protein. As a result, we hypothesize that the avidity of autoreactive T cells is a disease marker that can be used to monitor disease progress, judge therapeutic response, or discover new biochemical disease markers for multiple sclerosis. However, none of the current technologies have achieved high-sensitivity and high-throughput measurement of cell avidity, in clinical samples, at the single-cell level. Our engineering advances in 'Acoustofluidics,' allowing for precise manipulation of single cells and liquids at the unexpected resolution, shows promising potential for measuring the avidity of highly heterogeneous, clinical autoreactive T cells. Here, I report our progress on the development of a novel 'Acoustofluidic Avidity Cytometer' to overcome the barriers in detecting clinical multiple sclerosis.

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Gene regulation is chiefly determined at the level of individual chromatin fibers. However, our current understanding of the cis-regulatory architecture of humans is almost entirely derived from the fragmented sampling of large numbers of disparate chromatin fibers across individual cells or bulk tissue. To develop a single-molecule understanding of human gene regulation, we have pioneered an approach for precisely stenciling the structure of individual chromatin fibers onto their underlying DNA templates using non-specific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of these chromatin stencils enables the nucleotide-precise readout of the primary architecture of multi-kilobase chromatin fibers (Fiber-seq). Fiber-seq exposes widespread plasticity in the linear organization of individual chromatin fibers, and illuminates principles guiding regulatory DNA actuation, single-molecule transcription factor occupancy, and the coordinated actuation of neighboring regulatory elements along individual single-molecule chromatin fibers. Finally, application of Fiber-seq to human primary cells enables the simultaneous mapping of both the primary genetic and epigenetic states of individual regulatory alleles – directly exposing the functional impact of both rare and common regulatory DNA variation. Overall, single-molecule chromatin fiber sequencing opens new vistas on the primary architecture of human gene regulation.

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Our understandings of biological rhythms have recently expanded beyond the well-characterized ~24h circadian rhythms through our group's recent discovery of a mammalian 12h-clock regulating 12h rhythms of gene expression and metabolism. The mammalian 12h-clock is evolutionarily conserved (likely evolved from the circatidal clock of marine animals), cell-autonomous, and established independently from the circadian clock. However, the exact prevalence, function and regulation of the 12h-clock are still poorly defined. I recently uncovered wide-spread 12h transcriptome in mouse BAT, WAT, liver, adrenal gland, skeletal muscle, lung and aorta, with distinct phase and amplitudes signatures observed in these tissues. I further proposed a tripartite regulatory network comprising of E26 transformation-specific (ETS), Basic Leucine Zipper Domain (bZIP)-containing and Nuclear transcription factor Y (NFY) family of transcription factors (TF) that are responsible for the transcriptional regulation of mammalian 12h-clock, which mainly functions as a vehicle regulating the capacity of genetic information flow from DNA to proteins. Lending further evidence to the vehicle-cargo hypothesis is our recent findings that 12h-clock controls 12h rhythms of nuclear speckle liquid-liquid phase separation dynamics, which in turns leads to 12h rhythmic nuclear speckle spatial chromatin recruitment and gene expression control. Owing to the early stage of the 12h rhythm field, little is currently known about the definitive functions of 12h rhythms in mammals. However, new evidence supports the notion that the endogenous mRNA surveillance and protein quality control systems cycle with a 12h period. I hypothesize that multiple stress conditions can perturb this system from its normal 12h cycle, and if uncorrected, will contribute to increased disease susceptibility. Thus, the identification of novel regulators of the mammalian 12h-clock will most likely uncover new players implicated in stress responses and stress-associated pathologies.

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Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year in endemic areas and threatens to spread into non-endemic areas as climate change intensifies. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains poorly understood. As hepatocytes are a major target of yellow fever virus (YFV) infection, coagulopathy in severe YF has been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, which suggests that a consumptive process also contributes to YF coagulopathy. To define the relationship between coagulopathy, hepatocellular tropism, and tissue damage, we compared infection and disease in mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic fever and suggest directions for clinical management of severe YF cases.

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What makes each species unique? This question has fascinated philosophers and scientists alike for centuries. The diversity of life is mediated, at least in part, by protein-coding genes whose sequences are unique to a given species or lineage to the exclusion of all others. These genes are considered evolutionarily novel and often give rise to novel traits and adaptations. Novel genes were long thought to evolve exclusively by divergence from ancestral genes, just as new species descend from ancestral ones. However, it has recently become clear that novel genes can evolve de novo from ancestral sequences that previously lacked coding capacities (e.g. intergenic, regulatory or other "non-genic" sequences). Indeed, the genomic revolution revealed the existence of de novo genes in viruses, bacteria, fungi, plants and animals. In the human genome, de novo genes are highly expressed in the cerebral cortex, suggesting a contribution to improved cognitive ability. Several de novo genes have been associated with cancer (2), and ongoing studies in my laboratory have uncovered novel candidate de novo genes in the human genome located at or near disease-associated variants currently thought to land in "gene desert" regions. It is becoming increasingly clear that de novo genes mediate the molecular determinants of species-specificity, including human-specific traits and disease mechanisms. Despite far-reaching implications, a concrete biochemical understanding of the evolutionary transition from a non-genic sequence to a functional protein-coding gene is currently lacking for any species. My laboratory is working to elucidate the molecular mechanisms of the extraordinary paradigm of evolutionary innovation that is the phenomenon of de novo gene birth.

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