Biological and Health Sciences

Graeme L. Conn, PhD

PROFESSOR, SCHOOL OF MEDICINE, BIOCHEMISTRY

Antibiotic Selection and Efflux by the Pseudomonas Efflux Pump MexXY-OprM

Bacterial resistance to antibiotics is an escalating crisis in healthcare that threatens to fundamentally alter modern medicine. An important way that many disease-causing bacteria become resistant to antibiotic treatments is to pump out (“efflux”) the drug, removing it from its site of action. This proposal will determine how some clinically critical antibiotics (aminoglycosides) are effluxed by an important human pathogen (Pseudomonas aeruginosa) that is a major cause of disease in individuals with the genetic disorder cystic fibrosis (CF), serious burns, or compromised immune systems. The specific goals of the proposal are to: 1) define how antibiotics enter the pump and how substrates for efflux are specifically selected; 2) define the structure of the efflux pump for the first time using high-resolution structural studies. The dual goals of this URC application are to support obtaining long-term external funding for this new research program by both establishing my group’s expertise in this area and through generation of critical new preliminary data and reagents. In the long-term, our studies in this new area of research will have broad implications for our understanding of antibiotic resistance by drug efflux in important human pathogenic bacteria. This work may also lay the foundation for development of new inhibitors (“resistance breakers”) of drug efflux that would allow continued use of otherwise effective antibiotic treatments.

Andrew Escayg, PhD and Michael Zwick, PhD

PROFESSOR, SCHOOL OF MEDICINE, HUMAN GENETICS

Identifying Genes Responsible for Recessive Forms of Epilepsy

Epilepsy is a common neurological disorder that has a major negative impact on quality of life and imposes a tremendous burden patients and the healthcare system. Data from recent studies indicate that genetic factors are likely to account for a substantial percentage of cases. However, access to patient DNA remains limited, creating a bottleneck to efforts to identify epilepsy genes. Furthermore, most current efforts have focused on identifying mutations in genes responsible for dominant forms of epilepsy, and little progress has been made identifying recessive epilepsy genes, largely due to the absence of appropriate human DNA samples. In addition, few studies have explored the functional consequences of identified mutations, preventing progress towards understanding disease mechanisms and, by extension, the development of more efficacious precision therapies.

As part of a collaborative study, we obtained a unique collection of 223 blood samples from 25 consanguineous pedigrees from the Afridi tribe within the Federally Administered Tribal Area of the province Khyber Pakhtunkhwa, Pakistan. Consanguineous marriages result in a high prevalence of genetically recessive disorders. Therefore, these samples represent a unique and powerful resource for identifying recessive epilepsy genes. We have already isolated DNA from all samples and performed human whole genome sequencing (WGS) on two affected siblings from 10 pedigrees. The goals of this URC proposal study are to 1) conduct WGS on 4 more pedigrees, 2) perform bioinformatic analyses on generated WGS data for all 14 families, 3) identify candidate variants in each pedigree, and 4) functionally characterize high priority variants.

Jessica K Fairley, MD

ASSOCIATE PROFESSOR, SCHOOL OF MEDICINE, MEDICINE

Describing the Role of WASH (water, sanitation, and hygiene) and Potential Environmental Reservoirs in Leprosy Transmission

Leprosy continues to pose challenging questions about transmission, susceptibility and reservoirs of infection. Given the difficulties that this non-cultivable, slow-growing bacterium presents, a multidimensional approach is critical to answer these tough questions. Environmental factors, including possible reservoirs, are undoubtedly tied to leprosy transmission, but the inability to grow the bacteria in culture hampers our understanding of these potential modes of transmission(1, 2). We need to better define these transmission factors to improve control. Our main study question thus pertains to how the environment influences transmission of leprosy. With our study design, however, we will also be able to quantify the burden of disease, investigate nutritional factors related to infection, and even describe the seroprevalence of other neglected tropical diseases and COVID-19. We question whether poor water access and quality, sanitation and hygiene (WASH) are risk factors for leprosy transmission and whether we can identify environmental reservoirs of transmissible Mycobacterium leprae by testing environmental samples using state-of-the art methods that can identify genes of reproducing bacteria. North Gondar, Ethiopia, highly endemic for leprosy, presents an ideal location to use an antibody platform that can identify exposure to both leprosy using the antigen, LID-1, as well as many other neglected tropical diseases. By identifying people exposed to LID-1, and thus at higher risk to develop leprosy, we will identify potential environmental and nutritional risk factors for leprosy and create a cohort that we can eventually follow over time. This assay will also allow us to measure exposure to the novel coronavirus, SAR-CoV-2.

Homa Ghalei, PhD

ASSISTANT PROFESSOR, SCHOOL OF MEDICINE, BIOCHEMISTRY

ZNHIT3-Mediated Epitranscriptomic Regulation of Ribosome Biogenesis and Translation

Ribosomes are responsible for producing all proteins in every living cell. Because of their critical role in defining the cellular proteome, defects associated with the ribosome production, function or regulation have detrimental cellular outcomes and can cause serious human diseases, termed ribosomopathies. In human cells, bulk of the ribosomal mass accounts for ribosomal RNA (rRNA) that is chemically modified at over 200 nucleotides. These modifications are required for ribosome production and function and their dysregulation affects the efficiency and accuracy of protein synthesis and can lead to human diseases. How cells control their rRNA modification pattern to ensure the quality and quantity of their proteins remains elusive. Using yeast, we recently showed that amino acid variations in the evolutionarily conserved key ribosome biogenesis protein ZNHIT3 which cause the neurodevelopmental disorder PEHO syndrome, change the rRNA modification pattern and result in translational defects. These data suggest that, at least in the yeast model, the adverse cellular effects caused by the PEHO mutations arise from translation dysregulation. In this collaborative proposal, we plan to extend our studies to human neuronal cell lines to investigate the functional importance of ZNHIT3 and the molecular outcomes of the PEHO-linked ZNHIT3 mutations by testing the hypothesis that ZNHIT3-mediated rRNA modifications regulate human neuronal ribosome biogenesis and translational control. Our studies will serve as the foundation to dissect the mechanistic underpinnings of the ZNHIT3-mediated translation dysregulation in disease and will enable us to acquire essential data to support a grant application to NIH on this research topic.

Adam Gracz, PhD

ASSISTANT PROFESSOR, SCHOOL OF MEDICINE, MEDICINE

Biliary Epithelial Heterogeneity in Cholestatic Liver Injury

The mammalian liver demonstrates a remarkable capacity for regeneration, employing a wide range of adaptive cellular responses that preserve function following physical or chemical injury. Ductular reactions are a common feature of liver injury/regeneration and are defined by proliferative expansion and remodeling of intrahepatic bile ducts, which consist of biliary epithelial cells (BECs). Our understanding of the genetic regulation of BEC responses to liver injury remain limited, due in part to a longstanding lack of biomarkers to discriminate between specific BEC subpopulations. We hypothesize that specific subpopulations of BECs, defined by unique transcriptomic and chromatin regulatory landscapes, serve as proliferative “responders” to liver injury. We propose to apply a Sox9EGFP reporter transgene, which we recently characterized as a marker of BEC heterogeneity, to understand BEC dynamics in a mouse model of cholestatic liver disease. We will apply conventional histological and flow cytometry approaches with cutting-edge single cell genomics assays to comprehensively map cell-type specific responses to injury by BECs and peribiliary hepatocytes. The results of this study will provide an “atlas” of BEC dynamics in cholestasis with foundational relevance to human liver disease.

Xiulei Mo, PhD

ASSISTANT PROFESSOR, SCHOOL OF MEDICINE, PHARMACOLOGY

Discovery of Small Molecule Mutant SMAD4-PPI Inducer

Tumor suppressors represent a major class of oncogenic “drivers” and offer robust therapeutic window. However, direct targeting “loss-of-function” tumor suppressor is challenging. Many tumor suppressors have hypomorph mutations that weaken the normal protein-protein interaction (PPI). We aim to fix such weakened PPI through discovery of hypomorph mutation-directed small molecule PPI inducers to restore their tumor suppressor functions. SMAD4 is such a tumor suppressor with hypomorph mutations that impair its normal PPI with SMAD3. In this URC proposal, we will utilize our newly developed PPI inducer screening platform to reveal novel small molecule mutant SMAD4-PPI inducer (MuSMADid) that can induce the mutant SMAD4 PPI with SMAD3 and restore the pathway and cellular response to the tumor suppressive TGF-b signaling. From a bioactive chemical library, we have identified Ro-31-8220 as potential MuSMADid that induced the mutant SMAD4-SMAD4 PPI and restored the responsiveness of SMAD4 mutant cancer cells to the TGF-b anti-proliferation signaling. These results support our central premise that novel chemical probes can be discovered as potential MuSMADid by leveraging the established high-throughput PPI inducer screening platform to screen structurally diverse chemical libraries. We will focus on Aim 1 “Primary Screen Implementation” to identify MuSMADid hits with new chemical scaffolds, and on Aim 2 “Functional Validation” to prioritize a list of MuSMADid with selective anti-tumor activity. Top ranked MuSMADid with the strongest structural and functional evidence can be used as chemical probes to study the mutant SMAD4-dependent cancer biology and for the development of novel small molecule drug for precision oncology.

Alberto Moreno, MD

ASSOCIATE PROFESSOR, SCHOOL OF MEDICINE, MEDICINE

A Nanotechnology Approach for Designing the Next Generation of Pre-erythrocytic Malaria Vaccines

Malaria is the most common parasitic disease in the world. Five different species can affect humans, being Plasmodium falciparum the most lethal and Plasmodium vivax the most widespread. This disease is a public health problem since methods to eliminate the mosquitoes that transmit the parasite are failing, and the parasite is developing drug resistance. Extended clinical trials of a P. falciparum subunit vaccine based on a protein expressed on the surface of the infective forms have shown limited efficacy that wanes rapidly. Recent experimental evidence has shown that a region within this protein not included in the vaccine is the main target of neutralizing antibodies. Our research team has produced monoclonal antibodies with similar specificity and functional activity, supporting the need to include this region in designing the next generation of malaria vaccines. We have developed novel technologies of protein and vaccine vector engineering, aiming to enhance immune responses. Combining these platforms in a single vaccination regimen can improve efficacy and longevity. This proposal's protein engineering approach involves using molecular shields designed to divert the antibody responses to critical but subdominant regions. This proposal's vector engineering approach is based on a modification of adenovirus, a common virus that can cause cold-like symptoms. Tools have been developed to modify the viral vector's binding to target cells critical in initiating the adaptive immune responses. We include preliminary data in the application showing that these approaches are feasible. We aim to develop a vaccination regimen that combines these approaches to improve the overall efficacy.

Stella Papa, MD

PROFESSOR, SCHOOL OF MEDICINE, NEUROLOGY

Targeting Dopamine Signal Transduction for the Therapy of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by motor abnormalities primarily caused by degeneration of midbrain dopamine (DA) cells, which modulate striatal function. DA replacement with L-Dopa initially produces significant benefits, but over time L-Dopa efficacy declines and is further complicated by the development of involuntary movements (dyskinesias). Studies have shown that non-physiologic, chronic DA replacement induces altered responses in striatal projection neurons (SPNs). DA signals in SPNs are mediated by molecular cascades that begin with DA receptor activation and its effects on the synthesis of the cyclic nucleotides cAMP and cGMP. Additionally, the levels of these key molecules are regulated by specific catabolic enzymes, the phosphodiesterases (PDEs). Therefore, cAMP/cGMP regulation by PDE catalytic activity may have a direct impact on DA signal transduction in SPNs. PDE inhibitors (PDE-Is) have shown motor effects in animal models of PD, but the mechanisms underlying such effects are not clear. The physiological effects of regulating cAMP, cGMP or both in SPN subpopulations have not been addressed. PDEs are a family of enzymes that have substrate specificity and can be targeted with selective inhibitors. This proposal will test the hypothesis that selective PDE-Is may improve particular SPN responses to DA. We plan to analyze inhibitor-induced changes in SPN activity measured by calcium indicators in parkinsonian rats following chronic L-Dopa treatment. Data generated in this initial study will support subsequent grant submission for continuation of work. Our long-term goal is to translate findings into a new therapeutic approach to improve motor responses to L-Dopa therapy in PD.

Leila Rieder, PhD

ASSISTANT PROFESSOR, ECAS, BIOLOGY

Expanding Embryonic Assays through In Vivo Engineering of Repetitive Transgenes

Animal embryos undergo rapid cellular division, a process partially controlled by the availability of histone proteins, which are necessary to organize the embryonic DNA and control gene expression. Recently, scientists have embraced a system that allows genetic engineering of the histone genes in the powerful genetic fruit fly model system, facilitating studies in the early animal embryo. In the fly genome, the five histone genes are grouped into an array that is present in ~100 back-to-back copies. Deletion of the 100 natural copies can be rescued by adding back 12 engineered copies of the histone gene array; fewer copies do not support life. Multiple groups have used this powerful system to study the effects of histone modifications on embryonic gene regulation, as well as the sequences within the histone array that lead to histone gene regulation. However, the existing engineering system is prohibitive because it requires construction of a very large and fragile piece of DNA before insertion into the fly genome. We propose to develop two strategies in which the histone arrays are tandemly engineered within the fly, rather than using traditional bacterial cloning. We will compare time, expense, and efficacy of both strategies. Our method not only simplifies the current strategy for engineering the histone genes, but opens up the door for prohibitively difficult genetic engineering, such as multiplexing unique histone arrays and other repetitive DNA elements such as the ribosomal RNA genes and non-genic repeats. This work will result in a methods paper and future specific aims.