Rising Stars: URM 2019


Investigating pathogen-driven evolution of the human Natural Killer (NK) receptor, NKR-P1A

The innate branch of the immune system plays a critical role in initiating appropriate immune responses to invading pathogens. Natural killer (NK) cells are the first line of lymphocytes that respond to viral infections. They are tightly regulated by cell surface receptors, which can be either stimulatory or inhibitory, whose signals integrate to either activate or inhibit a given NK cell. Importantly, NK cells are key players in controlling herpesvirus infection and in recent years, many NK receptors have been found to directly interact with ligands expressed on virally-infected cells. Many of these are inhibitory ligands that serve as decoys to evade immune recognition. Recently, the NKR-P1 family of NK receptors in rodents have been identified as targets of the beta-herpesvirus, cytomegalovirus (both rat and mouse), where they encode viral ligands that engage the inhibitory NKR-P1B receptor to evade NK detection. Strikingly, these ligands do not share a genetic origin, thus have evolved independently. In light of this, we hypothesize that a similar mechanism is taking place in human herpesviruses with the human homolog, the NKR-P1A/CD161 receptor. Using biological tools, we are embarking to determine if this is indeed the case, with the hopes of revealing potential ways to counteract viral pathogenesis.


The Role of Inflammation in Prostate Fibrosis and Urinary Dysfunction

Lower Urinary Tract Symptoms (LUTS) is an umbrella term for a number of symptoms associated with bladder and/or voiding dysfunction. LUTS in men has been linked to prostate tissue remodeling and higher collagen content, in periurethral prostate tissue, during induction of fibrosis (Cantiello, et al. 2013). Fibrosis is an aberrant wound healing process that is thought to contribute to LUTS by causing stiffening of the prostate gland and resultant noncompliance of the prostatic urethra. Inflammation is known to be positively correlated with prostate fibrosis in LUTS patients and contributes to prostate fibrosis in animal models (Darnell, et al. 2015, Wong, et al. 2014). Since prostate fibrosis is linked to LUTS, prostatic inflammation could potentially promote the development of LUTS, through the generation of chronic profibrotic pathology. Utilizing a Chronic Prostatitis patient-derived uropathogenic Escherichia coli strain, named Chronic Pain 1 (CP1), known to cause inflammation in mice, the interplay of inflammation, fibrosis, and LUTS was explored. Our studies demonstrate a potential etiological role for a bacterial infection in mediating LUTS. Furthermore, these results identify a role for STAT6 signaling in mediating fibrosis in the prostate and suggest these effects could be dependent on Th2 cytokines.


Structural Basis for Transcriptional Control of HIV-1 Genome Function

Heterogeneous transcriptional start site usage by the human immunodeficiency virus (HIV-1) affords 5’-capped RNA transcripts containing a single 5’-guanosine that are selectively packaged into virions and function as genomes (gRNA) and two or three 5’-guanosines that function as mRNAs. To understand how 5’-nucleotide variability modulates transcript fate, we probed the structures of HIV-1 leader RNAs by 2H-edited NMR. 5’-capped transcripts that begin with a single guanosine preferentially adopt a branched multiple hairpin structure that sequesters the cap and inhibits interactions with the cellular translation initiation protein eIF4E. In contrast, capped transcripts that begin with two or three 5’-guanosines are extensively remodeled in a manner that exposes the Cap and downstream polyadenylation site residues and promotes eIF4E binding. Our findings reveal how as few as one or two 5’ guanosines can modulate transcript structure, function, and fate of the ~9 kilobase HIV genome


Engineering the Neural Microenvironment in Cancer and Wound Repair

The overall focus of my research has been the neural microenvironment and its role in glioblastoma (GBM) and peripheral nerve (PN) repair. GBM is a highly lethal brain tumor characterized by a diffuse border and median survival time of less than 15 months. PN injuries are very common affecting 20 million Americans, of which most do not gain functional recovery. Current gold standard therapies in GBM and PN injury are inadequate. Thus, biomedical innovations are required to improve outcomes for these patient populations.

The long-term goal of my work is to utilize differential signaling induced by the respective microenvironments to improve GBM and PN injury patient outcomes. We have engineered models to recapitulate important physical stimuli in the GBM microenvironment in vitro. These physical stimuli are altered in GBM and include interstitial fluid pressure, compressive solid stress, and substrate stiffness. We have previously demonstrated their effects in altering cell motility and survival. In a selected study, we demonstrated how compressive solid stress increases cell migration and induces a morphological subtype. Further pathway analysis of the transcriptome predicted decreased proliferation and identified miR548 as a candidate molecule for pharmaceutical intervention. In the context of PN repair, we are elucidating how simultaneously applied AC/DC electric fields (EF) affect Schwann cell phenotype. Schwann cells (SC) are major players in PNI, especially those involving a gap. They often undergo growth-induced senescence in acellular grafts, which diminishes the overall repair permissivity. Increased SC directional migration and neurotrophin production under EF stimulation in vitro preliminarily suggests this novel waveform application will improve PN repair in vivo. Further, an effect is also observed on senescent SCs, suggesting this approach is particularly applicable to healing long gap nerve injuries. Elderly patients typically have a higher proportion of senescent cells and could potentially benefit from this treatment approach as well. By curating a toolbox of extracellular stimuli in vitro, cell culture techniques, and transcriptome analysis methods, my work has expanded upon our understanding of how cells interact with their environment.

GBM and PNR exist in a dichotomy. Cell migration, proliferation, survival, and associated growth factor expression are perilous in GBM, however they are compulsory in PNR. This balance represents a useful platform for my independent research moving forward. The potential for application of similar methodology across diverse neuropathologies bodes well for research productivity. For example, we studied the effect of interstitial fluid pressure in GBM. It can also be studied in the context of diabetic neuropathy to identify potential pharmaceutical targets that would subvert the need for decompressive surgery. My future work will continue to elucidate how neural microenvironmental cues can be harnessed to achieve desirable cell phenotypes, either through pharmaceutical intervention or through exogenous supplementation.


Transgender women (TGW) are a vulnerable marginalized population who are at risk for several co-morbid conditions, of which HIV may be considered one of the most formidable threats with some HIV prevalence estimates ranging 19% – 27%.

Prevention (PreP): In addition to increasing risk behavior, substance use can increase HIV risk and disease progression through physiological mechanisms as well as curtail adherence to treatment regimens, but these issues are understudied among TGW. In response to these scientific gaps, the primary objective of the ongoing TRUST – To Reach Unrestricted services for transgender women – study, uses a two-phase mixed method approach to examine the relationship between substance use, daily use of preexposure prophylaxis (PrEP), and HIV transmission in this population.

Engagement in care (Exogenous hormone use and cardiovascular disease CVD risk): Simultaneously, TGW routinely engage in exogenous hormone use to aid with feminization. Exogenous hormone use and HIV separately, are factors found to increase CVD risk, however this is understudied in transgender populations for whom hormone use and HIV can be prevalent. Therefore, the TRUST ancillary study, which is a retrospective chart review of a clinic-based sample of transitioning adolescents, aims to determine if exogenous hormone use increases CVD risk in TGW over time.

Findings from both studies will help to provide information for patients and providers to consider when selecting and determining preventive HIV biomedical options (i.e. PrEP), and, therapeutic treatment with exogenous hormone use that can exacerbate CVD risk for those living with HIV and among those who are uninfected but at high-risk for HIV transmission.


Deciphering the embryonic origins and the genetic regulation of skeletal stem cells in the vertebrate skull

In the vertebrate skull, skeletal stem cells reside in fibrous joints called sutures and ensure the long-term growth and separation of skull bones. Humans with Saethre-Chotzen syndrome lose these skeletal stem cells at the coronal suture, leading to a malformed skull and a heightened risk of impaired brain development. Genetics studies have demonstrated that mutations in two bHLH transcription factors, TWIST1 and TCF12, cause Saethre-Chotzen syndrome, but the developmental origins of coronal synostosis has remained elusive. We utilize the unique genetic and imaging strengths of zebrafish to determine the embryonic origins and genetic regulation of long-term stem cells that grow and maintain the vertebrate skull. Simultaneous deletion of twist1b and tcf12 in zebrafish leads to the same coronal synostosis seen in humans. Sequential live bone staining in mutant zebrafish reveals an initial increase in the growth of all skull bones, which correlates with increased number of proliferative osteoblasts. Intriguingly, stalled bone growth arises only at the coronal suture, and the severity of bone growth stalling predicts coronal synostosis. RNAscope in situ for skeletal stem cell markers uncovers a depletion of the number of progenitor cells that separate neighboring skull bones at the coronal suture of mutant zebrafish. Altogether, these data suggest that alterations in progenitor dynamics cause the coronal synostosis observed in Saethre-Chotzen syndrome. Current work focuses on integrating complex genetics tools, live imaging, and genomic technologies to uncover the precise progenitor dysfunction that cause coronal synostosis and the twist1b/tcf12 dependent regulatory networks that control coronal suture development.


Stem cell reprogramming during oncogenesis

Tissue stem cells are critical for the replenishment of dying cells and for wound-repair. Previous studies have demonstrated that many cancers can arise from the dysfunction of stem cells, either through accumulation of mutations, or more recently, aberrancies in the epigenetic landscape. SOX9, a transcription factor, plays an important role in the development and maintenance of many stem cell compartments and its expression is linked with poorer prognosis in many cancers. In the skin, SOX9 is essential for hair follicle stem cells, while epidermal stem cells are characterized by lack of SOX9 expression. Interestingly, basal cell carcinomas (BCC) overexpress SOX9, yet have been demonstrated to arise from epidermal and not hair follicle stem cells. Moreover, SOX9 is critical for BCC as genetic loss of Sox9 completely abolishes tumor formation in vivo. The mechanisms by which ectopic SOX9 contributes to tumor formation for BCC and other cancers remain to be elucidated. Utilizing a newly developed transgenic mouse that expresses an inducible SOX9 in the epidermal stem cells, I have begun annotate the chromatin and transcriptional changes that occur after induction of SOX9. Interestingly, SOX9 expression in the epidermis is sufficient for BCC-like lesions to form allowing for a tractable model to study oncogenic reprogramming in vivo. My preliminary studies indicate that ectopic expression of SOX9 in the adult epidermis shifts the chromatin landscape by activating previously silenced genes that may be important for BCC transformation. SOX9 induces chromatin accessibility primarily at distal regions suggesting a remodeling of the enhancer landscape. Exploiting a combination of immunoprecipitations and mass spectrometry, my results have implicated that SOX9 interacts with major chromatin modifiers (both active and repressive) in order to remodel chromatin. My studies have begun to directly address our knowledge regarding SOX9 mediated chromatin remodeling and subsequent activation of oncogenic and stem cell transcriptional pathways. Identifying the key SOX9 target genes as well as the proteins involved in epidermal to BCC reprogramming could lead to the development of novel therapeutics used to “reset” the tumor epigenetic landscape into a non-malignant form.


Expanding the toolbox of NMR in living mammalian cells

Advancements in in-cell NMR have permitted structural and biophysical characterization of proteins in near native environments. Originally, experiments were performed in cells that permit easy expression (E. coli) or delivery (X. Laevis) of isotopically enriched proteins. As a result, these early experiments revealed transient interactions between test proteins and the cytoplasm that influence the structure and stability of the test proteins. While these experiments were simple yet elegant, the desire to study disease relevant proteins in mammalian cells persists. From this, two main methods evolved to study proteins in mammalian cells. The Banci group, used transient transfection to deliver DNA and overexpress specific proteins in HEK293 cells. While effective at generating signals, overexpression produces significantly more protein than present endogenously and therefore alters the physiology of the cell. Selenko and colleagues began to address this problem by using electroporation to deliver low µM concentrations of isotopically labeled α-synuclein to the human ovarian cancer cell line A2780. Here we look to expand upon these methodologies to deliver disease relevant globular proteins and detect them using a combination of 19F NMR and traditional 1H-15N HSQC experiments. Successful delivery of globular proteins will allow for characterization of native interactions in the cytoplasm. Additionally, utilization of 1D 19F NMR will permit rapid detection of interactions in living cells.


Defining viral mechanisms of Dengue virus immune evasion

Dengue virus (DENV) is one of the most significant arthropod-borne flaviviruses, currently leading to >390 million human infections worldwide. DENV can cause severe disease and death in children and the elderly in endemic regions such as Asia and Latin America. DENV is genetically and serotypically divided into four serotypes (1-4) and each serotype can be further subdivided into distinct genotypes. The only licensed DENV vaccine does not perform equally well against the existing four DENV serotypes. The vaccine efficacy against DENV serotype 2 (DENV2) is remarkably low (39% efficacy), underlining the need to improve DENV vaccine design and strategies. While it is known that DENV genetic diversity exists among the four serotypes, the role of DENV intraserotypic diversity within the distinct genotypes in modulating neutralization resistance to vaccine-elicited antibodies is not well understood. To this end, we are interested in defining the role of naturally occurring DENV2 genetic variation on neutralizing antibody evasion. We are also interested in defining how antibody Fc characteristics, such as IgG subclass and binding activity to Fcγ receptors, which engage the immune system, contribute to antiviral immune responses against DENV. Our work is beginning to uncover that DENV2 genotypic variants exhibit considerable amino acid residue variability within the main target of neutralizing antibodies: the viral envelope protein that is required for cell entry. We have shown that the envelope genotypic genetic diversity observed in DENV2 modulates differential neutralization sensitivity to neutralizing monoclonal antibodies, polyclonal antibodies from DENV2-infected individuals, and polyclonal antibodies from DENV vaccinated individuals. A better understanding of the mechanism(s) by which DENV evades neutralizing antibodies will be critical to rationally design “universal” DENV vaccines with improved vaccine efficacy, and will also be important to devise immune-based strategies against other related emerging flaviviruses such as Zika virus.


Role of cellular cross-talk during early-stage lung cancer

Lung cancer remains a major health problem, accounting for millions of deaths worldwide. Recent data has demonstrated that signaling between supporting microenvironment cells and lung stem cells is important for lung repair. Cells that make up the lung microenvironment can drive stem cell functions such as self-renewal, differentiation, and cell-fate decisions. It is plausible that communication between cancer cells and supporting cells is also important during early-stage lung cancer development. However, our understanding of signaling between tumor and supporting cells, especially during the earliest stages of cancer development remains poor. To address these important questions, I will use single-cell RNA-sequencing to analyze gene expression changes in cancer cells and non-cancerous supporting cells populations during early-stage lung cancer development in autochthonous mouse models and identify receptor-ligand relationships for functional validation. These studies will elucidate how the microenvironment supports early-stage lung cancer development and identify new diagnostic markers and novel druggable targets for early-stage disease.


Engineering an aortic valve with cellular and mechanical functionality

Heart valve disease is an increasing clinical burden associated with high morbidity and mortality, and patients are limited to valve replacements that lack the ability to grow and remodel. This presents a major challenge for pediatric patients who require a valve capable of somatic growth and at smaller sizes. A tissue engineered heart valve (TEHV) capable of growth and remodeling while maintaining proper valve function would address this major need. In this study, we create a TEHV by mimicking the structural components of the valve leaflet layers using 3D bioprinting and incorporating valvular interstitial cell (VIC)-like cells to actively regenerate and remodel. First, we generated a potential suitable cell source of human iPSC-derived mesenchymal stem cells (iMSCs) that mature into VIC-like cells. Next, we used 3D printing and a combination of poly-ε-caprolactone (PCL) and hydrogel blend of natural and synthetic polymers to create a cell-laden multilayered leaflet that recapitulates the layers of the valve leaflet. Lastly, the PCL-component of the valve leaflet was mounted onto a valve stent and feasibility studies were conducted using a left-ventricle flow simulator to evaluate the hemodynamic performance of the PCL-TEHV under aortic valve conditions. We demonstrated a cell source can be derived from autologous iPSCs, generated a multilayered leaflet scaffold using a combination of natural and synthetic biomaterials, and verified the hemodynamic performance of the leaflet under aortic flow conditions. These promising findings are the first steps to a pre-clinical TEHV with the ability to regenerate and remodel with the patient.


Pulmonary Drug Delivery using Water-in-Perfluorocarbon Reverse Emulsions

Improved treatments are needed for lung diseases like Cystic Fibrosis and Acute Respiratory Distress Syndrome. Each involves mucous or fluid build-up in the lung that limits ventilation and, thus, delivery of inhaled drugs. Delivery is most needed within the damaged regions of the lung, but if an area is not ventilated, inhaled drug will simply not reach it. My thesis proposes using antibacterial perfluorocarbon ventilation (APV): the lungs are filled with an emulsion containing a disperse phase of water-soluble antibiotics and/or other drugs within perfluorocarbon liquids. An emulsion is formed by mixing drug solutions with perfluorocarbons and fluorosurfactant, for water droplet stability. We deliver antibiotics for lung infections and growth factors for lung repair. Objectives: (1) determine optimum bactericidal emulsion formulation in vitro, (2) optimize emulsion for lung repair in vitro, and (3) determine antibacterial emulsion treatment efficacy in an animal infection. Approach: (1) build a two-chamber bioreactor to mimic antibiotic diffusion from emulsion, through bacterial layer, and into bloodstream, (2) determine growth factor delivery to epithelial cells via migration, proliferation, barrier function responses, (3) instill emulsion into infected animals and examine bacterial killing. Outcomes: (1) achieved more bactericidal emulsions at higher total antibiotic concentrations and low fluorosurfactant concentrations—droplet stability limits drug release, (2) achieved comparable response in cell migration from emulsions, but delivery was insufficient to evoke similar proliferation and barrier function responses, and (3) APV treatment with higher antibiotic concentrations is comparable to inhaled treatments for bacterial killing.


Heightened erythrocyte disposal as a modifier of the innate immune response

Heightened or on-demand erythrocyte disposal occurs during pathologic states such as in the hemoglobinopathies or during medical interventions such as transfusion of very old or effete red blood cells (RBC). Though virtually all mammalian cell types are equipped to ingest and process circulating iron, the macrophage is the primary cell responsible for engulfing and processing damaged RBC to maintain suitable plasma iron levels. The macrophage is also the sentinel immune cell in tissue environments tasked with sensing and eliminating microbial invaders, yet how the macrophage prioritizes signal when faced with competing biological stressors is less known. We hypothesized that a competing stressor such as heightened RBC disposal compromises macrophage effector function during ongoing bacterial infection. To test this, we developed an experimental model where leukoreduced, damaged RBC are fed to macrophages infected with the prevalent opportunistic pathogen, Klebsiella pneumoniae. Our findings indicate that heightened disposal of damaged RBC suppresses Stat1 (and its downstream effectors) in macrophages and impairs host defense against Klebsiella pneumoniae intrapulmonary infection.


Use of a genetically-encoded biosensor to study striatal acetylcholine release in awake, freely-moving mice

Acetylcholine (ACh) has important roles in the induction of synaptic plasticity, as well as in action control, decision making and substance abuse. Cholinergic interneurons (CINS) are tonically-active and serve as the predominant source of striatal ACh. While a great deal about the physiology of these neurons and their roles in behavior is known, little is known about real-time ACh during behavioral changes or effects of drugs of abuse. We are using the novel, genetically-encoded ACh sensing fluorescent reporter, iAChSnFR, to address these questions in dorsal striatal slices and the in vivo striatum. In striatal slices, we found that the iAChSnFR responded dynamically to electrical stimuli and that these responses were synaptic in nature. In vivo measurements with iAChSnFR expressed in dorsal striatum indicate that ACh levels are enhanced by peripheral cocaine treatment but inhibited by peripheral injection of ethanol. We are currently examining changes in striatal ACh during behaviors that are thought to involve CIN activity.


Localized Delivery of Ibuprofen from a Bilayer Delivery System (BiLDS) in a Rat Supraspinatus Tendon (Rotator Cuff) Injury and Repair Model

The high prevalence of tendon re-tear following rotator cuff repair motivates the development of new therapeutics to promote improved tendon healing. Controlled delivery of non-steroidal anti-inflammatory drugs (NSAIDs) to the repair site via an implanted scaffold is a promising option for modulating inflammation in the healing environment. Furthermore, biodegradable nanofibrous delivery systems offer an optimized architecture and surface area for cellular attachment, proliferation, and infiltration while releasing soluble factors to promote tendon regeneration. Previous work confirmed the in vitro sustained release of ibuprofen (IBP) from Labrafil-modified poly(lactic-co-glycolic) acid (PLGA) microspheres within sintered poly(ε-caprolactone) (PCL) electrospun scaffolds. Biocompatibility of this bilayer delivery system (BiLDS) was also demonstrated with primary rat bicep and Achilles tenocytes in vitro. However, the effect of these IBP-releasing BiLDS on tendon healing in vivo was unknown. Therefore, the objective of this study was to investigate the biological and mechanical implications of localized delivery of IBP from the BiLDS implanted at the repair site in a rat rotator cuff injury and repair model. We hypothesized that the controlled release of IBP from the BiLDS would improve tendon healing by decreasing the expression of pro-inflammatory cytokines and mitigating inflammation, thus improving tendon remodeling and mechanics. This collection of work exploits the ability of a transformative technology to provide physical and chemical cues for improved tendon repair.


The genetics underlying Zika virus neuropathogenesis

Zika virus (ZIKV) is a growing and likely recurrent global health concern punctuated by recent outbreaks in French Polynesia, the Americas, and most recently India. ZIKV, which is a mosquito-borne illness in the same viral family as West Nile, Dengue, and Yellow Fever, preferentially infects proliferating neural progenitor cells (NPCs) in the developing brain. Prenatal exposure to ZIKV can result in severe outcomes such microcephaly, intracranial calcifications, and fetal demise. In fact, women infected with ZIKV in the first two trimesters are 17 times more likely to give birth to an infant with microcephaly, though it is estimated that only 1-13% of maternal infections result in this brain malformation. It remains unclear why some maternal infections result in birth defects and others do not. Using advanced stem cell-derived NPC induction methods, I performed genome-wide CRISPR/Cas9 genetic screens and “village-in-a-dish” mixed culture single-cell RNA sequencing experiments to better understand the genetics underlying differential sensitivity to ZIKV infection. I found hundreds of host factor genes that the ZIKV uses to replicate in NPCs, as well as a relatively common single nucleotide polymorphism in the antiviral IFITM3 gene that confers enhanced vulnerability to ZIKV infection. Together, this work highlights the need for improved efforts to control global mosquito populations while also identifying many targets for therapeutic intervention for ZIKV and other devastating RNA viruses.