Fall 2019: A total of $94K in awards was distributed to 7 recipients across 5 departments representing Storrs and UCH!
Andrew Arnold, Center for Molecular Oncology
Abstract: Primary hyperparathyroidism (PHPT) is a common endocrine disorder that affects up to 36 people per 1000 population, disproportionately affecting women such that about 2% of post-menopausal women will eventually develop PHPT. Approximately 85% of all PHPT cases are caused by parathyroid adenomas (PTAd), which can cause hypercalcemia with neurocognitive symptoms, osteoporosis, and kidney stones. Effective non-surgical therapies do not yet exist, leading to an ongoing effort to identify potential therapeutic targets based on oncogenic pathways active in this tumor type. Recently, novel somatic mutations were identified in the gene encoding stemness/self-renewal transcription factor Zinc Finger X-linked, ZFX1,2, in a subset of PTAd3,4. Sequence data from human tumors strongly suggests a direct contributory role for mutant ZFX and its downstream effector pathways in PTAd tumorigenesis because the reported mutations 1) recur in 4.5% of tested PTAd cases3, a significant proportion for this tumor type; 2) are strictly somatic/cancer associated (see COSMIC5, TCGA) and are not to be found in germline data from over 140,000 individual genomes/exomes (see gnomAD, ExAC6); 3) specifically affect residues in the critical motif-determining domain of ZFX; and 4) substitute out positively charged, basic amino acids that are likely essential to wild-type ZFX interaction with negatively charged, acidic DNA. We hypothesize that these acquired mutations effect a shift in the parathyroid transcriptional program, yielding a selective growth advantage. In this proposal, we aim to investigate the as-of-yet unstudied effects of tumor-specific mutant ZFX, which may lead to the identification of genes and/or pathways that could serve as therapeutic targets for PTAD treatment.
ISG Cores Used: CBC and CGI
Leighton Core, Department of Molecular and Cell Biology
Abstract: Regulation of RNA splicing is critical for proper gene expression levels and generation of transcript diversity, and its misregulation is the basis for numerous diseases. It is now well established that a majority of RNA splicing takes place as the RNA is still being transcribed by RNA polymerase II. Consequentially, regulation of transcription and splicing are intertwined which presents a novel framework for the study of disease-associated mutations in either process. However, connecting splicing rate and with transcription regulation is difficult due to numerous technical challenges. In addition, splicing efficiency is difficult to measure since mis-spliced transcripts are often undetectable due to their rapid degradation. Here, we propose to address these issues by combining precision run-on sequencing (PRO-seq) with full length RNA sequencing on oxford nanopore’s technology. The adaptations allow us to map the position of RNA polymerase II when co-transcriptional splicing occurs. Our approach achieves near base pair resolution and initial results from Drosophila S2 cells suggest that splicing occurs within seconds of the downstream splice sites becoming accessible to the spliceosome.
ISG Core Used: CGI
Sarah Knutie, Department of Ecology and Evolutionary Biology
"Does urbanization increase host resistance to invasive parasites in Darwin’s finches of the Galapagos Islands?"
Abstract: Human population size is increasing exponentially and, in turn, the urban environment is one of the few ecosystems that is rapidly expanding. A recent example of urbanization is in the Galapagos Islands, which is home to the endemic Darwin’s finches. The Galapagos currently hosts 225,000 tourists each year and is home to over 21,300 permanent residents. Consequently, humans have altered the natural habitat and introduced parasites to the Galapagos, such as the parasitic fly Philornis downsi, which causes up to 100% mortality in nestling Darwin’s finches. Adult flies are non-parasitic but the larval stage feeds on the blood of nestlings and brooding females. However, our recent work shows that small ground finches (Geospiza fortis, a species of Darwin’s finch) in urban areas are healthier and less affected by P. downsi compared to finches in non-urban areas, which suggests that birds are better defended against the parasite in urban areas. Specifically, my team found that small ground finches are more resistant to P. downsi in urban areas compared to non-urban areas on the island of San Cristobal, Galapagos. The aim of the proposed research is to compare overall gene expression in the blood of parasitized and nonparasitized finches in urban and non-urban areas and determine whether any immune genes (i.e. potential resistance mechanisms) differ in finches from each treatment and location. In 2019, we conducted the field work in which we experimentally manipulated P. downsi abundance in the nests of finches in an urban and non-urban area. We then collected and preserved 95 blood samples from nestling and female finches across treatments and locations. We have also successfully extracted RNA from the blood samples. Therefore, the proposed funding would cover the RNA library preparation and sequencing at CGI with Dr. Bo Reese and the RNA-Seq model and non-model systems bioinformatics workshop withDr. Jill Wegrzyn. Overall, the proposed work will increase our understanding of the benefits of urban living for endemic island species, especially in response to novel parasites, and provides preliminary data for an NSF proposal.
ISG Cores Used: CBC and CGI
Masako Nakaniski, Center for Molecular Oncology
Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) have proven to be the most effective clinical agents for suppressing inflammation and cancer, primarily by reducing the production of prostaglandin E2 (PGE2)1. Unfortunately, long-term treatment with NSAIDs is often associated with varying toxicities, including most commonly gastroduodenal ulcerations2. The likelihood of developing clinically significant NSAID-induced intestinal injury, however, is non-uniform within the general population, suggesting that there may be underlying genetic and/or environmental factors that influence susceptibility to NSAID-induced GI toxicity3.
We have recently found that genetic deletion of mPGES-1, the terminal enzyme that generates inducible PGE2, in strain A mice (A/J:KO) causes spontaneous ulcerations throughout their colons, while the same gene deletion in C57BL/6 mice (B6:KO) failed to induce a comparable phenotype. This differential response to PGE2 abrogation suggested that these mice may serve as a useful model to study the varying susceptibility to NSAIDs in human population. In initial studies to characterize this phenotype, we found that A/J mice harbor a significantly lower fecal abundance of Akkermansia muciniphila, a mucin-degrading bacterium, when compared to B6 mice, regardless of mPGES-1 genotype. A. muciniphila produces mucin-derived short-chain fatty acids (SCFAs), such as propionate and acetate, which can modulate host gene expression4. Furthermore, a membrane protein of A. muciniphila, Amuc_1100, has been shown to activate Toll-Like receptor 2 (TLR2), which enhances the gut barrier function in mice5. Given that PGE2 is also critical for maintaining mucosal homeostasis, we hypothesize that strain-specific abundance of A. muciniphila, in collaboration with mPGES-1 deficiency, may impart significant effects on host gene expression and that these epigenetic changes may ultimately affect mucosal integrity in the colon. For a future grant application, we plan to propose the following aims; 1) examine the gut microbiota in the feces and colon tissues of A/J:KO and B6:KO, 2) determine whether the a subset of bacteria affect the development of colonic ulceration in A/J:KO mice, and 3) examine the potential mechanisms by which A. muciniphila or Amuc_1100 affects epithelial barrier function in intestinal organoids generated from A/J and B6 mice. For this seed grant, we propose to perform a genome-wide expression profiling of colons from these mice to identify specific pathways associated with the sensitivity to colonic ulceration. We anticipate that the data generated by this pilot study will strengthen the future proposal by providing mechanistic links for understanding the interplay between the levels of PGE2 and the abundance of A. muciniphila in mucosal homeostasis.
ISG Cores Used: CBC and CGI
Mario Perez, Department of Internal Medicine
Abstract: The use of electronic cigarettes (e-cigarette) is increasing and has already surpassed conventional cigarette use among youth. E-cigarettes have been considered safer than conventional cigarettes under the premises that they contain lower concentrations of chemicals than cigarettes. However, recent news reports have provided evidence of the adverse effects that using e-cigarette, in particular to pulmonary health. In-vitro studies and animal studies have provided evidence of the potential harms of “vaping”, yet the mechanisms by which e-cigarettes leads to pulmonary disease remain unclear. A recent study showed that exposure to e-cigarette vapor induces gene expression changes in pathways associated with inflammation, cell metabolism, apoptosis and DNA repair in ex-vivo human bronchial epithelial cells. A different study, evaluated the gene expression from participants exposed to e-cigarettes using nasal epithelial cells, showed that e-cigarettes suppress immune and inflammatory-response genes more than conventional cigarettes and to our knowledge, the only study reported in induced sputum of e-cigarette users showed proteomic changes related to altered innate immunity. Although highly valuable, most of these reports were performed in-vitro or in animal models, which may not reflect the changes occurring in the airways and respiratory system of e-cigarette users, and no study has been reported on the transcriptome of e-cigarette users. It is also likely that e-cigarettes have effects at the systemic level on not limited to the respiratory system since a dynamic cross-talk between the lungs and circulatory system has been well described. Therefore, I hypothesize that e-cigarette use alters the airway and systemic transcriptomes involved in response to the noxious stimuli they deliver.
ISG Cores Used: CBC and CGI
Paola Vera-Licona: Center for Quantitative Medicine
Abstract: The concept of differentiation therapy emerged from the fact that hormones or cytokines may promote differentiation ex vivo, thereby changing the phenotype of cancer cells. The differentiation therapy hallmark of success has been the treatment of acute promyelocytic leukemia (APL), an Acute Myeloid Leukemia subtype that is now highly curable by the combination of retinoic acid (RA) and arsenic. Based on this success,
several differentiation therapies that induce maturation of diverse AML subtypes are in development [1-3]. However, studies of AML have shown a hierarchical model of cancer where tumorigenic cancer stem cells (CSCs) differentiate into relatively mature nontumorigenic progeny [4; 5]. In this hierarchical model, tumors are composed of phenotypically heterogeneous cancer cells that resemble various differentiation states of their lineage of origin. It is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that eradicate CSCs or induce their differentiation. The success of these therapies depends on CSC differentiation being unidirectional rather than reversible. As such, computational methods that help to quantify the reversibility potential of differentiation therapy targets to select the most optimal ones, would greatly aid the study and development of differentiation therapies. In this pilot project, parting from a dynamical systems and control theory framework and with the use multi-omics data, we will develop a computational systems biology pipeline to quantitatively characterize AML maturation state interconversion in clinically relevant models of ATRA-based APL differentiation therapy.
ISG Core Used: CBC
Spring 2019: A total of $96K in awards was distributed to 6 recipients in 5 departments representing Storrs and UCH!
Sarah Hird, Department of Molecular and Cell Biology
"Antibiotic resistance in the mallard microbiome"
Abstract: Pharmaceutical antibiotics (hereafter "antibiotics") are one of the most profound medical advancements of the 20th century, but their widespread use has led to antibiotics being deposited across many natural systems. For some organisms, like waterfowl, antibiotics in water may exert selective pressure on the microbial members of the microbiome to become resistant to the antibiotics. Given that the microbiome can be important in many axes of host biology, selecting for antibiotic resistance in the microbes could have cascading effects on the host and microbiome. Here, we propose to use shotgun metagenomics sequencing on oral and feather microbiomes from wild mallards collected at three locations in Connecticut (with associated soil samples) to identify and quantify antibiotic resistance genes in the mallard microbiome. The data will also be used to identify genes of functional importance to the avian oral and feather microbiomes, two largely unexplored environments. The metagenomic data will be analyzed in conjunction with data collected for and funded by an ongoing study in the Hird lab: we will have empirical estimates of environmental antibiotics at each sampling locality (using HPLC-ms/ms analysis of water) and 16S rRNA amplicon libraries. Six genomes from feather microbiome bacteria that our lab has collected will aid in metagenomic assembly. Identifying abundant genes in the oral and feather microbiomes of waterfowl and quantifying the amount and specificity of antibiotic resistance genes in our samples will provide the necessary preliminary data for an extramural grant from the National Science Foundation's Division of Integrative Organismal Systems program. Specific Aims: Collect 36 shotgun metagenomic datasets, for duck oral, duck feather and soil microbiomes from three locations in Connecticut. Hypothesis: Antibiotic pollution will be reflected in the mallard microbiome through a correlation between environmental antibiotics and antibiotic resistance genes.
ISG Core Used: CGI
John Malone, Department of Molecular and Cell Biology
Abstract: One of the major unanswered questions in sex chromosome evolution is why some sex chromosome pairs diverge while others do not. Study of amphibian genomes provides a transformative system for the study of heteromorphic sex chromosome evolution in vertebrates because over 95% have sex chromosomes that are hardly divergent (homomorphic), whereas 5% have highly divergent, heteromorphic sex chromosomes that have evolved 20-30 independent times. The main objectives of this proposal are to use the Center for Genome Innovation’s (CGI’s) Nanopore PromethION and to work with the CBC to generate long read data that will assemble, annotate, and distinguish the complete Z and W sex chromosomes of the African bullfrog (Pyxicephalus adspersus), an amphibian with heteromorphic sex chromosomes. The results generated from this seed grant will serve as the foundation for a proposal to NHGRI’s Comparative Genomics Research Program (PAR-17-482) to identify transcriptional changes for genes that change from autosomes to sex chromosomes and the links to sex chromosome dosage compensation that occur in some vertebrate lineages. The ultimate goal is understand how sex chromosome evolution molds gene network changes and how these changes impact genome function across vertebrate evolution.
ISG Cores Used: CBC and CGI
Beth Taylor, Department of Kinesiology
"Identifying Novel miRNAs to Investigate as Diagnostic Biomarkers in Patients with Statin-Associated Muscle Symptoms"
Abstract: Statin drugs are lifesaving drugs that reduce the incidence of cardiac events in men and women by approximately ~40%. They are among the most frequently prescribed drugs in the U.S and world. Statins have few side effects but can produce statin-associated muscle symptoms (SAMS) consisting of pain, cramps and weakness that reduce medication adherence and quality of life. SAMS are difficult to diagnose since there are no validated questionnaires or biomarkers, and at least 30-50% of patients on statins with self-reported SAMS actually have non-specific muscle complaints unrelated to statin use. Despite this, the majority (~60%) of adults who discontinue statins report SAMS as the primary reason for statin non-adherence and discontinuation. The absence of predictors and markers for SAMS makes its diagnosis and management difficult, forcing clinicians to rely entirely on clinical complaints and drug cessation. The absence of validated methods to diagnose SAMS also has major public health importance because patients with statin intolerance have an increased risk of cardiovascular disease events and incur substantially higher healthcare costs as a result of these events. Resultantly, the need for an effective biomarker of SAMS that can be used clinically to diagnose and confirm SAMS is critical for improving statin adherence and managing SAMS. We recently received American Heart Association (AHA) funding (#17GRNT33661247) to investigate the effectiveness of eight muscle-specific miRNAs, nonprotein-coding RNA molecules that regulate cellular function at the posttranscriptional level, as biomarkers for the detection of muscle damage and possibly SAMS. We seek to utilize the current funding mechanism to first perform Illumina small RNA sequencing in order to expand our investigation beyond muscle-specific miRNAs to all circulating miRNAs and ultimately refine our proposed candidate panel of miRNAs implicated in SAMS. The final candidate panel of eight miRNAs will then be examined as planned in the AHA proposal via real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis of archived samples from an ongoing randomized, cross-over clinical trial of 40 patients with a previous history of SAMS treated with 20 mg simvastatin and placebo for two, 8 week treatment periods separated by a 4 week washout. Specific Aim 1 will use Illumina small RNA sequencing to explore and identify novel circulating miRNAs related to SAMS in a small subset of patients (n=6) with confirmed SAMS. Specific Aim 2 (as funded in the AHA proposal) will determine whether a targeted panel of eight candidate miRNAs, chosen from the quantitative analyses in Specific Aim 1, can then differentiate between patients with confirmed SAMS and those with non-specific muscle complaints. The addition of Specific Aim 1 greatly improves our scientific approach over the current funded AHA proposal, which, due to funding constraints, limits us to a generalized panel of a priori muscle-specific miRNAs. We hypothesize that patients with confirmed SAMS will be distinguished from all other patients by significant alterations in one or more miRNAs during simvastatin treatment relative to placebo, demonstrating efficacy of selected miRNAs as biomarkers that may eventually be utilized by clinicians and researchers to diagnose, treat and study SAMS.
ISG Cores Used: CBC and CGI
Ted Rasmussen, Department of Pharmaceutical Sciences
"RNAseq to ascertain hepatic differentiation pathways mediated by lncRNAs and hepatic exosome responses to drug-induced toxicity."
Abstract: We propose to use RNAseq to generate additional preliminary data to support two related but distinct lines of research having to do with hepatocyte biology. The overall goal is to enhance my lab’s position to obtain significant extramural funds and publication. The projects are as follows: Project 1: Analysis of transcriptional regulatory pathways governed by a novel long noncoding RNA (lncRNA) required for differentiation to hepatocytes. Project 2: Identification of lncRNA and miRNA biomarkers of hepatotoxicity in exosomes and extracellular vesicles (EX/EVs) secreted by hepatocytes. For Project 1, we have completed all research needed for a significant publication now in preparation. Developmentally-primitive hepatoblasts are bi-potent and differentiate into both mature hepatocytes and biliary epithelial cells. My lab discovered a novel lncRNA (lnc76) that is required for the development of mature hepatocytes using an inducible shRNA approach. We propose here to use RNAseq to determine which mRNAs are perturbed in the human transcriptome in lnc76-deficient versus control cells. In project 2, we aim to discover biomarkers consisting of lncRNAs and microRNAs present in EX/EVs secreted from human hepatocytes exposed to acetaminophen and ethanol. There is a significant unmet medical need for human “liquid biopsies” to detect hepatocellular toxicity in response hepatotoxic levels of both acetaminophen and ethanol.
ISG Core Used: CGI
Ming Xu: Department of Genetics and Genome Sciences
Abstract: Aging is the greatest risk factor for most common chronic diseases including cancer, heart disease, osteoporosis, dementia and diabetes, conditions that are collectively responsible for most morbidity and health care costs in older adults. Growing evidence indicates that targeting fundamental aging mechanisms such as cellular senescence has the potential to slow the progression of these chronic conditions as a group. My research goal is to design novel interventions to target cellular senescence in order to enhance life quality and lifespan in older adults by slowing the onset or progression of multiple chronic diseases. To achieve this, we must have a better understanding of cellular senescence. In this proposal, we will leverage single cell transcriptomic (SCT) technology to comprehensively compare young versus aged mouse tissues at single cell level in an unbiased manner. We will specifically examine naturally occurring senescent (SEN) cells in these tissues, with the goal of finding a common transcriptomic signature for these cells in different cell types, thus allowing the identification of pathways to target precisely.
ISG Core Used: SCGC
Kepeng Wang, Department of Immunology
"Elucidating the role of IL-17 on regulatory T cells in colitis and colorectal cancer"
Abstract: IL-17 family cytokines are critical drivers of inflammatory bowel diseases (IBD) and colorectal cancer (CRC), but the underlying mechanism remains unclear. IL-17 is known to signal to epithelial cells and fibroblasts to promote the production of cytokines and chemokines. Whether IL-17 also signals to adaptive immune cells, especially effector and/or regulatory CD4+ T lymphocytes, and whether this signaling plays a role in IBD or CRC, remains largely unknown. Our preliminary studies show that targeted ablation of IL-17 signaling on T lymphocytes reduced the development of CRC in mice, demonstrating the importance of an IL-17-T cell interaction. IL-17 signaling inhibits the accumulation of regulatory T cells in mouse models of IBD and CRC, and ablation of IL-17 receptor in the whole body resulted in marked increase in the transcriptional levels of anti-inflammatory cytokines such as TGF-β and IL-10, both of which are known to inhibit the development of IBD and primary colorectal cancer. These preliminary findings support our hypothesis that IL-17 inhibits the accumulation and activation of Tregs during the development of IBD and CRC, and that this process may be through direct signaling of IL-17 to these cells. Given the critical roles of Tregs and Th17 cells in the pathogenesis of IBD and CRC, and our knowledge gap on the direct modulation of regulatory T cells by IL-17, we set to propose the following studies: 1) test the role of IL-17 signaling to Treg cell biology using mouse models of CRC and IBD; 2) test the role of IL-17 signaling to regulatory T cells in human IBD and CRC. These studies will involve high-throughput RNA sequencing experiments with the Center for Genomic Innovation (CGI). Data obtained in these studies will be used for the application of R01 grant centering the role of IL-17-Treg interaction in colitis and colorectal cancer.
ISG Core Used: CGI