Vector-borne parasites are emerging globally due to climate change and increased animal movement. Among these are filarial nematodes, which are of veterinary and medical importance. Onchocerca lupi is an emerging, zoonotic filarial nematode that infects dogs, cats, coyotes, and humans in the southwestern United States. Animal infections are associated with ocular disease; however, many animals remain asymptomatic and likely go undiagnosed. In humans, O. lupi infection is often associated with aberrant locations, including the cervical spine, requiring invasive surgical intervention. There have been 8 confirmed human cases within the USA, coincidentally from areas where animal cases have been reported. Many knowledge gaps remain around O. lupi including details of its life-cycle, vector species, and prevalence in companion animals and wildlife across its range. Another gap is the need for accurate, rapid diagnostic tests for detecting infection in animals and humans. Currently, microscopy and molecular diagnostic methods that require subsequent DNA sequencing are used for confirming clinical suspected onchocerciasis cases (i.e., conventional polymerase chain reaction, cPCR). This creates the need for the development of diagnostic tests for clinical diagnostic purposes and active epidemiological surveillance of O. lupi across endemic areas.

The objectives of my doctoral research are: I) to develop and optimize a novel qPCR assay for detection of O. lupi DNA in biological samples and II) to assess the prevalence of O. lupi in shelter dogs from Albuquerque, New Mexico. For objective I, DNA was extracted using an automated nucleic acid purification platform (Maxwell, Promega) and subjected to cPCR followed by Sanger sequencing. Then, our lab designed and optimized a novel probe-based qPCR that is sensitive and specific to detect only O. lupi. Both PCRs targeted a fragment of the cytochrome oxidase c subunit. The probe-based qPCR was designed and optimized with a detection threshold (0.3285 pg). We further optimized the assay by performing a dynamic range test to find the ideal dilution factor and inclusion of an internal positive control to ensure that inhibition levels were not causing false negatives. Suspected clinical samples of dogs and cats (n=126) from 8 states were then tested for comparison. For objective II, we collected skin samples of 404 dogs (females n=194, (48%); males n=210, (52%)) from January to September 2023. Dogs were separated into age groups: juveniles, ≥1 year-old, 121 (29.9%); adults, 1-7 years-old, 259 (64.1%); and seniors, <7 years-old, 24 (5.9%). Skin samples were processed for DNA extraction as previously described and subjected to the novel qPCR assay. Overall, 8 (1.98%) dogs, including 6 adults and 2 juveniles tested qPCR positive. This is the first study assessing the O. lupi prevalence from an urban environment in North America. My research will contribute to the knowledge of the biology and epidemiology of O. lupi. It also will advance veterinary medicine and public health by providing novel diagnostic methods for detection of O. lupi infection. Altogether, these studies will inform the veterinary, medical, and public health communities, and enable the implementation of strategies for disease control and prevention through a One Health approach.

Acute myeloid leukemia (AML) is a cancer involving white blood cells and it represents ~20% of pediatric leukemias but accounts for most of the disease-related mortality in children. Pediatric AML is a genetically heterogeneous disease, where the specific genetic alterations present impact patient survival rate and relapse. Because of this, a single treatment strategy is not feasible for all AML subtypes, underlining the need for new targeted therapies. To address this need, our lab leveraged unique local resources and expertise to conduct a high-throughput chemical screen, using ~11k compounds and 37 AML samples and normal cord blood stem cells (CB CD34+), to identify novel anti-AML compounds. Given the mixture of FDA approved and novel compounds used, the anti-AML compounds we identified fell into three general categories.

In the first group, we found compounds with known targets, as the molecule A, which has been implicated in inhibition of the proteasome. In collaboration with the drug discovery unit at Institute for Research in Immunology and Cancer (IRIC), we have generated and tested more than 60 analogs of this compound and characterized their impact on cell viability. Several of these analogs have very low (nM) IC50 values and appear to be active against other blood disorders. Based on these exciting results, we are now completing pharmacokinetic studies to understand how these molecules behave in vivo, so that we can assess their activity in tumor challenge experiments, in preparation for eventual clinical studies.

The second group includes compounds like shikonin, which have published mechanisms of actions that are inconsistent. For instance, shikonin’s anti-cancer activity is reportedly due to inhibition of pyruvate kinase muscle isoenzyme 2 (PKM2) but also to inosine 5- monophosphate dehydrogenase 2 (IMPDH2). We tested shikonin’s efficacy against AML cells, where it was more effective than several known IMPDH inhibitors and similar to a commercial PKM2 inhibitor, but critically, less toxic to healthy CB CD34+ cells. We further assessed the metabolic and transcriptomic profiles of AML cells after shikonin treatment and observed metabolic and transcriptional changes related to shikonin’s reported binding to electron transport chain 2 (ETCII); and a rapid downregulation of MYB expression, crucial for AML cell survival. Our data show that even well characterized compounds with known targets, exhibit complex polypharmacology and that for shikonin, its anti-leukemic activity appears to result from a complex combination of inhibitory effects.

Finally, the third group includes compounds with unknown targets or mechanisms of action. We have selected one of these molecules to conduct a CRISPR chemogenomic screen to gain insight into its activity. The data showed a strong genetic signature for microtubule inhibition but also show that genes involved in regulating cell differentiation can enhance or reduce the effect of the drug. We also found that loss of another gene, TP53, rescues cells from the effects of the drug, suggesting a possible link to DHODH inhibition based on the structure of this compound. We have performed in vitro assays to measure tubulin polymerization, DHODH inhibition, and used flow cytometry to verify the expression of established cell surface markers for differentiated cells.

By understanding how these compounds affect AML growth, they can be used as targeted therapies tailored to specific genetic alterations present in different AML subtypes, ultimately improving treatment outcomes and reducing mortality in pediatric AML.

I am currently involved in a research project investigating O-linked N-acetyl glucosamine (O-GlcNAc) and its implication in neurodegenerative diseases including Alzheimer's and Parkinson's. O-GlcNAc is a dynamic post-translational modification that attaches to serine and threonine residues on proteins, and its levels fluctuate in response to cellular stress and nutrient availability. We aim to understand how dysregulation of O-GlcNAc cycling contributes to neurodegeneration and develop therapeutic strategies targeting specific O-GlcNAc pathways. A specific contribution I have made to the research team includes designing and conducting experiments to investigate how O-GlcNAc affects proteostasis at the proteome level, a mechanism hypothesized to play a crucial role in cell signaling and protein regulation. To accomplish our goals, we use molecular biology, biochemistry, and proteomics techniques.

The following are key lab techniques utilized in this project:

1. Mass spectrometry: We employ advanced high-resolution mass spectrometry to analyze peptides with and without O-GlcNAc labels. By using this method, we can identify specific sites of O-GlcNAcylation and observe how they respond to different treatments.
2. Immunoprecipitation and Western Blotting: These techniques are used to detect and isolate O-GlcNAcylated proteins. Using specific antibodies, we can monitor the abundance and interaction of these modified proteins.
3. CRISPR-Cas9 gene editing: We use CRISPR-Cas9 to generate cell lines with targeted mutations to study the functional effects of O-GlcNAc on specific proteins. Using this method, we can determine the impact of specific O-GlcNAc sites on protein function and stability.
4. Cell culture and treatment: We are cultivating neuronal cell lines and treating them with various O-GlcNAc-modulating compounds. Thus, we can better understand how changes in O-GlcNAc levels affect cell viability, proteostasis, and stress responses.
5. Fluorescence Microscopy: We can visualize how O-GlcNAc-modified high-interest proteins are distributed within cells. Observing O-GlcNAc spatial dynamics may provide valuable insights into cell function and structure.

Based on preliminary findings, O-GlcNAc modification significantly affects protein stability and function. As an example, increasing O-GlcNAc levels in neuronal cells increases the stability of critical cellular proteins, which contribute to cellular viability and structural integrity. Furthermore, our proteomics analysis revealed that specific O-GlcNAc-modified sites influence protein stability, suggesting a regulatory function for O-GlcNAc. Furthermore, I intend to validate the O-GlcNAc effect on the high-interest target proteins with biochemical assays in-vitro and in-vivo using various techniques such as CRISPR-Cas9 and mass spectrometry. The findings suggest that modulating O-GlcNAc levels may be a therapeutic target for neurodegenerative diseases. In conclusion, this project aims to explore O-GlcNAc's complex role in maintaining proteostasis as well as its implications for neurodegeneration. We are uncovering novel insights into how O-GlcNAc influences protein function and stability by using advanced lab techniques and collaborative efforts. In the future, we hope to develop treatments for neurodegenerative diseases utilizing O-GlcNAc pathways.