Cystic fibrosis (CF) is a genetic disorder affecting the lungs, and chronic polymicrobial lung infections are responsible for decreased life expectancy and poor quality of life of CF patients. Staphylococcus aureus (SA) is a microbe commonly found in the respiratory tract of CF patients, and this organism adapts within the lung environment to establish chronic infections. Among the most common bacterial adaptations is the emergence of mutants known as small colony variants (SCVs). There are multiple subtypes of SCVs that arise from mutations in different metabolic pathways. Recent studies have demonstrated that SCVs are prevalent in the CF respiratory tract, are more difficult to treat with antibiotics, and are associated with worse lung health. SCVs are very difficult to detect in clinical laboratories, thus complicating the selection of appropriate treatment by physicians to improve the health of CF patients. The goal of this study is to determine if SCVs can be more readily detected than with standard culture by using mass spectrometry to identify proteins that distinguish these variants from normal colony S. aureus. Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) will be used to identify proteins unique to specific SCV subtypes by separating those proteins using ionization and Tandem Mass Spectrometry. This analysis will generate isolate-specific spectra of peaks which will subsequently be compared to each other using Principal Coordinate Analysis (PCoA). We hypothesize this technique will identify differences between proteins produced by each SCV type, which can then be distinguished from normal colony S. aureus, allowing the rapid identification of these variants. As a result, we anticipate the detection of SCV’s will improve, which will help inform physicians to select appropriate treatments to target SCVs.

Atherosclerosis, a cardiovascular disease that results from fat (cholesterol) accumulation in the walls of large arteries, is the major cause of heart attacks and strokes. Gene therapy, delivered directly to the artery wall, has the potential to prevent and reverse atherosclerosis by removing cholesterol from the artery wall. Our group has used gene therapy to prevent and reverse atherosclerosis in a rabbit model; however, the effects are partial. Reasons for partial success likely include expression of inadequate levels of the therapeutic transgene product (apolipoprotein AI; a protein that removes cellular cholesterol and transports it to the liver for excretion). To address this limitation and achieve more-effective gene therapy, we hypothesized that incorporation within our gene-transfer vectors of DNA sequences that activate transcription from nearby gene promoters (sequences known as “cis-regulatory modules or “CRMs”) would increase the amount of apolipoprotein AI mRNA transcribed from our vectors. We used a bioinformatics approach and in vitro measurements of transcript levels in cultured vascular cells, to identify 11 potential CRMs within the sequences of 4 highly expressed vascular cell genes. Some of these CRMs increased apolipoprotein A1 mRNA levels, but not above levels we had achieved using other strategies. We are now repeating this approach, using newly available in vivo data generated with single-cell RNA sequencing of vascular tissue and with a separate technique that identifies highly expressed vascular cell-specific genes. We will use the sequences of highly expressed genes that we identify, along with bioinformatics tools, to identify new CRMs that are more likely—than the CRMs identified previously, using in vitro data—to increase apolipoprotein AI transgene expression above previously attained levels. We anticipate that incorporation of these CRMs into our gene-therapy vectors will increase the efficacy of our vectors in preventing and reversing atherosclerosis.

Traumatic spinal cord injury (tSCI) often leads to a debilitating loss of sensory, motor, and autonomic function. Currently there are no treatment options available for patients with tSCI. Immediately following the initial trauma, microvessels in the spinal cord rupture, leading to hemorrhage within the spinal cord. Bleeding is a major contributor to a cascade of subsequent injuries, defined as secondary injury, such as swelling, inflammation, and oxidative stress, which results in the expansion of the initial injury. We hypothesize that enhancing blood clotting would limit secondary injury, and subsequently lead to better functional outcomes. To test this, we employed newly developed hemostatic nanoparticles (hNPs), which are designed to localize to the injury site and reduce bleeding in a contusion tSCI model in rodents. The hNPs or control nanoparticles were introduced intravenously within 3 minutes after the injury, and tomato lectin was injected at the end of the experiment to label all patent blood vessels. Clusters of hNPs were found within areas of hemorrhage and blood clot within the injury epicenter, and never seen co-labeled with tomato lectin, suggesting that hNPs were only within parenchyma in areas of active bleeding. Our unique ultrafast contrast enhanced ultrasound (CEUS) imaging was used to visualize hematoma size, local spinal blood perfusion and swelling in real-time. CEUS imaging data showed there was 50% reduction in hematoma size in hNPs treated animals compared to control. We also found significant reductions in hypoperfused volume (50%, p<0.05) as well as spinal cord swelling (30%, p<0.01) in hNPs treated animals compared to controls. Current studies are underway to 1) analyze real-time hemodynamic data obtained from ultrafast CEUS imaging, 2) evaluate chronic 3D blood flow imaging, and 3) quantify functional and histological outcomes from hNP treatment after tSCI.

Sarcopenia, the age-related of loss of muscle mass and function, is associated with a decline in quality of life in the elderly and has few effective treatment options. Sarcopenia is linked to mitochondrial dysfunction and elevated mitochondrial oxidant production. We are investigating the role of elevated mitochondrial oxidative stress in sarcopenia using a mitochondrial targeted therapeutic and a mouse model of accelerated sarcopenia. SS-31 is a mitochondrial targeted peptide that associates with cardiolipin, decreases oxidant production, and increases ATP production in vivo. Superoxide dismutase 1 knockout (Sod1KO) mice lack superoxide dismutase 1 (an enzyme that converts the oxidant superoxide into hydrogen peroxide and molecular oxygen) resulting in an accelerated sarcopenia phenotype. We hypothesize that improving mitochondrial function with SS-31 treatment will delay the decline in muscle function in the Sod1KO mice. To test this, we administered SS-31 to SOD1KO mice through surgically-inserted osmotic pumps for 8 weeks between 3 and 4 months of age, the published timeframe for the onset of skeletal muscle decline in SOD1KO mice. Muscle force generation and fatigue resistance was tested in vivo in the gastrocnemius before pump insertion and monthly after pump insertion for 4 months. At the end of the treatment we used histological and biochemical analyses of mouse tissue samples to determine skeletal muscle fiber type, metabolite and protein concentrations, and muscle fiber respiration and oxidant production. We expected SOD1KO mice with SS-31 to have a lower rate of decline in muscle force production and increased fatigue resistance over time, higher max ATP production, and decreased oxidative stress. The effect of SS-31 on muscle function, mitochondrial quality, and redox homeostasis has exciting potential as a translational therapeutic treatment for human sarcopenia.

Hemophilia A is an X-linked bleeding disorder caused by the absence or defective function of plasma coagulation factor VIII (FVIII). A significant complication with hemophilia A treatment by FVIII transfusions is the development of inhibitory antibodies to the FVIII protein. This project uses a combined treatment of weekly intramuscular injections of an engineered FVIII DNA plasmid vaccine with liver-directed gene therapy using a DNA plasmid encoding the FVIII protein to treat hemophilia A in mice. This strategy was designed to suppress the immune response in order to achieve persistent expression of FVIII without inducing the formation of anti-FVIII antibodies. To test the success of this experiment, blood samples were collected at various time points throughout the project and the plasma was used in FVIII antigen and FVIII inhibitor assays. Clotting assays were performed to test the activities of FVIII and FVIII inhibitors. This project consisted of three different experimental groups: a group that started FVIII vaccines before liver-directed gene therapy, a group that started FVIII vaccines after liver-directed gene therapy, and a control group that received PBS only combined with gene therapy. We hypothesized that the groups receiving the engineered FVIII plasmid vaccine should have decreased levels of inhibitor antibodies and increased expression of the FVIII protein compared to the group receiving PBS only. If this project succeeds using hemophilia A as a model, this strategy could potentially be used in other disorders with undesired immune responses.

Human cerebral organoids (HCOs, also known as mini brains) generated from induced pluripotent stem cells (iPSCs) provide exciting opportunities to study neurological disorders in ways not previously possible from traditional animal, brain slice, or 2D cell culture models commonly used in research. Recent studies have demonstrated that HCOs can reproduce complex neural development pathways and mimic several biomarkers of neurodegenerative disorders. However, it remains uncertain if these HCOs can model the characteristic electrophysiological activities of epilepsy. We have developed a technique to record electrocorticography (ECoG) directly from these HCOs to first characterize their electrographic behavior as a step toward evaluating their potential as a clinically relevant model of epilepsy. We aimed to determine the baseline ECoG characteristics of HCOs and the changes in ECoG characteristics after exposure to common proconvulsants with different modes of action in order to assess their ability to model seizure activity. We recorded ECoG while the HCOs were bathed in normal artificial cerebrospinal fluid (ACSF) and while they were in ACSF solutions containing pentylenetetrazol (PTZ, a GABA A receptor antagonist), kainic acid (KA, a glutamate receptor agonist), or an elevated bath potassium concentration, which induces neuronal hyperexcitability. We observed an increase in spikes and seizure-like activity after high potassium and KA exposure. In contrast, there was no change in activity associated with PTZ exposure. These findings are consistent with the fact that our HCOs are primarily composed of excitatory neurons, with only a minimal number of inhibitory interneurons. Developing reliable brain organoid models of epilepsy is an important next step in the field that will revolutionize studies of precision therapy for epilepsy.

DNA polymerase (Pol) proofreading and mismatch repair (MMR) cooperatively guard against DNA replication errors and cancer. Defects in these activities produce “mutator” phenotypes characterized by elevated levels of base-substitutions and frameshifts. The clinical significance of these mutator mutations is noted in how POLE exonuclease domain mutations have been found in both colorectal and endometrial cancers. Haploid yeast with combined defects in Pol and MMR rapidly go extinct, in a process termed error-induced extinction (EEX). Organisms adapt by duplicating their genome without dividing (polyploidization) or by lowering their mutation rate via antimutator mutations. In evolution experiments with haploid mutators that display a synthetic-sick phenotype due to Pol ε proofreading and MMR defects we found that polyploids routinely beat out antimutator mutants. Although whether these results would hold true for diploids was unknown, so to investigate whether polyploids would arise in diploid strains, we did similar evolution experiments with Pol ε proofreading and MMR deficient diploids. After passaging about 200 generations, we found the vast majority of cultures had remained diploid. We then measured mutation rates of isolates from these cultures and repeatedly found antimutator phenotypes. Whole genome sequencing of independent isolates from each culture revealed various POL2 mutations in many of those that had suppressed mutation rates. Thus, spontaneous antimutator alleles and polyploidization rescues haploid and diploid cells from EEX with markedly different efficiencies. Differences in the relative frequency of each escape mechanism may reflect the nature of the mutator alleles, the starting ploidy of the cells, or the magnitude of the initial mutation rate. Our findings in diploids suggest that mutator cancer cells near the edge of error-induced extinction may similarly be under selection for spontaneous antimutator mutations and polyploidization.

Prostate cancer remains the second leading cause of cancer-related deaths of men in the US. Currently, the major challenge is to prevent the tumor cell from gaining the resistance to androgen deprivation therapy which almost inevitably leads to lethal castration-resistant prostate cancer. We are repositioning antiparasitic agents to develop a novel therapy to target androgen receptor (AR) mediated metastatic castration-resistant prostate cancer (mCRPC). Among them, bumped kinase inhibitor (BKI) 1553, which was originally designed to inhibit Toxoplasma calcium-dependent protein kinase 1, has shown to efficiently inhibit AR-dependent prostate cancer growth and AR signaling. Upon binding to its cognate ligand androgen, AR undergoes a conformational change to translocate from the cytoplasm into the nucleus to act as a transcriptional factor. The aim of my project is to identify the target site of BKI 1553 on AR transcription activation pathway. I examined the subcellular localization of AR by using biochemical subcellular fractionation and immunofluorescence assay. I also observed the phosphorylation status of AR via Western blot, and evaluated the level of AR signaling through a luciferase-based reporter assay and quantitative PCR assay of several AR target genes, including PSA, FKBP5, and Nkx3.1. The results of the study reveal the potential site of action of BKI-1553 in AR signaling. This study will provide a better understanding of the mechanism of BKI-1553 on prostate cancer and contribute to the development of a new therapy to mCRPC.