Myosin modulators are a novel class of pharmaceutical agents designed to treat patients with cardiomyopathies by directly modulating cardiac myosin function in the sarcomere. Compounds including omecamtiv mecarbil (OM), danicamtiv (Dani), and deoxy-ATP (dATP) have previously been shown to increase myofibril ATPase activity while mavacamten (Mava) reduced ATPase activity. In the absence of actin, ATPase activity is a combination of the direct effects of nucleotide binding and the equilibrium between the high activity (DRX) and low activity (SRX) states of myosin. In this study, I investigate how these small molecules affect the single turnover kinetics of pig cardiac heavy meromyosin (pcHMM) in the absence of actin. pcHMM bound to fluorescent mant.ATP or mant.dATP is rapidly mixed with a high concentration of unlabeled ATP. The rate constant for replacement of mant.ADP by ATP defines the turnover of mant.ATP. Preliminary titration experiments demonstrate that OM, Dani,and Mava all inhibit ATP turnover with an IC50 of 0.59 µM, 3.5 µM, and 0.276 µM, respectively. 100% dATP increases the ATP turnover by 100%. These Experiments indicate that each myosin modulator differentially alters cardiac HMM activity. I will discuss how each modulator affects ATP turnover by a direct effect on catalytic activity vs an effect on the amount of HMM in the super-relaxed population. Heart failure is the leading cause of death in America, and can result from diseases such as dilated cardiomyopathy and hypertrophic cardiomyopathy (DCM and HCM, respectively). Current therapies focus on treating the symptoms of the disease and do not directly treat the underlying functional changes. These myosin modulators directly treat the underlying alterations in contractility caused by DCM and HCM, making them a powerful tool in combating heart failure.

Trauma-induced coagulopathy is a severe complication of trauma that alters the normal mechanism of blood clotting through a number of complex factors. If clots are hypercoagulable, there is risk for dangerous vascular blockages. Conversely, if the clotting is hypocoagulable, it can lead to fatal hemorrhaging. Prior research indicates that actin has a major impact on platelet activity and blood clot formation. Actin is a highly abundant cytoskeletal protein that forms long, insoluble filaments. When released into the blood during cellular death, these filaments have complex effects on blood clot formation. Actin filaments can be integrated into the scaffolding of the clot, increasing strength. My experiment aims to investigate the roles of actin and on human blood clotting. Healthy donor whole blood in 3.2% sodium citrate was spiked with either a saline control or recombinant human skeletal muscle-derived actin (final concentration 200 nM) and allowed to incubate for 5 min. Samples were then activated with either 10 mM adenosine diphosphate (ADP) or 2 mg/mL collagen. The platelet aggregation response was then measured by impedance aggregometry. Each pair of control and actin conditions was run simultaneously. The impedance area under the curve (AUC) was compared between control and actin groups under each activation condition using a paired t-test with significance at p<0.05. Preliminary results show ADP was no different between the control and actin groups (p=0.400, n=5). The AUC in response to collagen was significantly higher in the presence of actin compared to control (p=0.005, n=7). Exogenous muscle actin appears to increase platelet aggregation through the collagen but not the ADP activation pathway. Further investigation is required to better characterize this interaction. A better understanding of the mechanisms of actin on hemostasis could direct research into pharmaceuticals and therapies that could yield better outcomes for trauma patients.
 

Alzheimer’s disease (AD) is characterized by aberrant cleavage of Amyloid Precursor Protein (APP) leading to toxic β-Amyloid (Aβ) peptide aggregates. Presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are intimately involved in this process. We found that alternative splicing of PSEN2 leads to the increased inclusion of a cryptic exon (part of the RNA that codes for a protein), in the typically non-coding intron 9 of PSEN2, titled exon 9B (PS2x9B), which has elevated abundance in individuals with sporadic AD. My aim is to uncover the link between PS2x9B and sporadic AD. I designed a PCR approach to measure the ratio of PS2x9B inclusion compared to non-mutated Wild Type (WT) in cDNA generated from parietal and temporal lobe tissue samples. I distinguished PS2x9B inclusion by amplicon size on a gel and measured the ratio to WT via band intensity using ImageJ. I found that the ratio of PS2x9B inclusion was significantly elevated in sporadic AD cases in the parietal lobe (p = 0.02) and the temporal gyrus (p = <0.001). Next I transfected SH-Sy5y cells with plasmids containing either WT or PS2x9B PSEN2 joined to a Flag-tag. I extracted proteins from these cells and control empty plasmids, and used Western blot to examine size differences between each plasmid’s protein products to research the impact of PS2x9B inclusion on PSEN2 protein levels. I hypothesize that inclusion of PS2x9B will lead to truncated proteins due to an early stop codon. Additionally, I am working to establish the consequences of PS2x9B on APP processing and Aβ cleavage. I expect that PS2x9B could cause either an increase in Aβ production or modify its length. These findings will allow us to expand our understanding of PSEN2 and alternative splicing in sporadic AD which could guide development of novel gene therapy treatments for AD.

 

The Lieber lab is exploring chemotherapy-drug-free in vivo expansion systems that involve knock-out a gene in hematopoietic stem cells (HSCs) by precision genome editors. Part of this system is an immunotoxin that targets non-transduced HSCs and eliminates them, thereby allowing for the selective expansion of edited HSCs. The goal of my project is to test one of our target proteins CD49f, integrin α6, which has been reported to be expressed on primitive HSCs. In my studies, using flow cytometry with a PE-conjugated anti-CD49f antibody, I have confirmed that CD49f is present on 63% of human CD34+ cells, a cell fraction that is enriched for HSCs. PAI Life Sciences then set out to generate a CD49f-targeted immunotoxin. If the immunotoxin works, CD34+ cells should be highly susceptible to the conjugated immunotoxin. I then tested the immunotoxin on tumor cell lines and primary human CD34+ cells. Measured by flow cytometry, nearly 100% of HEK293T, HeLa and MDA-MB231 cells expressed CD49f. Next, I incubated three cell lines with increasing concentrations of the CD49f-peptide-saponin immunotoxin, ranging from 0.5pg/mL to 10µg/mL. The cell viability and total cell number was measured three days later by two methods: i) flow cytometric quantification, using DAPI staining and ii) counting cells in a hemocytometer after Trypan-blue staining. In tumor cell lines, MDA-MB-231 and HeLa cells, only at the highest immunotoxin concentration a ~58% decrease in total cell numbers was found. The number of viable cells was ~ 4.1% lower than that of untreated controls. Then, I performed cytotoxicity studies with primary CD34+ cells. At the highest concentration tested (10µg/mL), around 6.7% cells were killed by the immunotoxin. Overall, the data indicates that the cytotoxic activity of the new CD49f-targeted immunotoxin is suboptimal. Future steps will include the design and testing of new immunotoxin conjugates.

Despite heavy exposure to Mycobacterium tuberculosis (Mtb), the bacteria that causes Tuberculosis (TB), some individuals show no evidence of infection and by defining these resistance mechanisms, we may identify novel treatment strategies. Among Mtb resistant individuals, our lab identified the Rab5a protein as differentially expressed as compared to controls with Mtb infection. By regulating vesicle trafficking, Rab proteins modulate a variety of cellular pathways including inflammatory signaling, antigen presentation, and autophagy, likely playing a role in Mtb clearance. We hypothesized that loss of Rab5a would alter IFN-êžµ gene expression. Monocyte-like THP-1 cells were electroporated with siRNA targetting Rab5a and yielded 70-90% knockdown at 24 hours versus scrambled siRNA control. Cells were then stimulated with DNA ligands for four hours before RNA analysis. Loss of Rab5a resulted in lower levels of IFN-êžµ gene expression after stimulation with Sheared Calf Thymus DNA (p=0.002, 53.9% reduction), Poly(I:C) (p=0.01, 42.8% reduction), supercoiled plasmid (p=0.03, 45.3% reduction), and cGAMP (p=0.008, 45.7% reduction). We conclude that Rab5a expression is required for Type I IFN production through the DNA-sensing pathway. By characterizing the pathways by which Rab5a modulates the macrophage Mtb response, we may identify host targets to augment protective responses that may serve as adjuncts to current TB treatments and vaccines.

Prior research suggests that people with obesity commonly have diets with more added sugar and saturated fat, worse sleep quality, emotional eating, and lower physical activity time than people with healthy weight. However, do these lifestyle characteristics predict one’s response to a behavioral weight loss (BWL) intervention? We will explore, in people with obesity, if these lifestyle characteristics, individually and/or jointly, are related to the change in adiposity in response to a 6-month BWL intervention. The methodology consists of data from self-report questionnaires administered to participants (N=63) at baseline in the BWL intervention group in the Weight Effects on Brain Health Study. Adiposity measurements were obtained by dual-energy X-ray absorptiometry. Diet characteristics were determined and Healthy Eating Index was calculated by analyzing 3 unannounced dietary recalls from an automated self-administered 24-hour recall system. Eating behavior was measured through the Three Factor Eating Questionnaire-R18. Sleep quality was determined by The Pittsburgh Sleep Quality Index. Physical activity assessed regular activity during the previous 4 weeks and was calculated as MET-h/wk. A composite score will be generated to include the 4 lifestyle factors described. Statistical analyses are undergoing. Anticipated results are associations between poor diet quality, poor sleep quality, less physical activity, and higher scores for uncontrolled and emotional eating and a greater reduction of adiposity over 6 months, as determined by multiple linear regression. Confounding by age and sex will be explored and models adjusted as needed. Additionally, we anticipate improved predictability of lifestyle characteristics, when considered jointly, to the change in adiposity. Participants were 69.8% female with mean age of 42.2±10.9 years, BMI of 37.2±5.3 kg/m2, and visceral adipose tissue of 1875.6±1027.7 grams. This research may help illuminate if lifestyle measures are able to predict one’s response to a BWL intervention and who could benefit most from it.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage, characterized by neoplastic, monoclonal expansion of plasma cells in the bone marrow. Remarkable progress has been made in the treatment of MM with the anti-CD38 monoclonal antibodies such as Daratumumab and Isatuximab, which can kill MM cells through the induction of complement-dependent cytotoxicity (CDC). The CDC efficacy of Daratumumab and Isatuximab is however limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein (Ad35K++) capable of blocking CD46 and sensitizing tumor cells to anti-CD20 mAb triggered CDC (e.g Rituximab and Ofatumumab). Here we tested Ad35K++ in combination with Daratumumab and Isatuximab. We show that Ad35K++ increases the CDC efficacy of Daratumumab and Isatuximab on Burkitt’s lymphoma and MM cell lines (MOLP8 and SUDHL-8). Ad35K++ salvaged the efficacy of Daratumumab and Isatuximab at subtherapeutic (“low”) doses in MM lines. Daratumumab and Isatuximab treatment of MM lines (without Ad35K++) resulted in the upregulation of CD46/survival of CD46 high MM cells which escaped a second round of Daratumumab and Isatuximab treatment. Escape was reduced by combining Daratumumab and Isatuximab with Ad35K++. We also tested a small recombinant protein that targets CD59 (rIYD4) in combination with Daratumumab and Isatuximab on MM cells. rIYD4 also increased CDC killing of MM cells by Daratumumab and Isatuximab. The combination of Ad35K++ and rILYD4 additively enhanced the CDC effect of Daratumumab and Isatuximab. Studies with patient MM cells will be reported. Overall, our data demonstrate that Ad35K++ and rILYD4 are efficient co-therapeutics of Daratumumab and Isatuximab and could be used to improve the treatment of multiple myeloma.

A thoracic aortic aneurysm (TAA) is an expansion of the aorta within the chest. TAA can rupture, often causing sudden death. Mechanisms of TAA formation and growth are incompletely understood, but likely include dysfunction/loss of aortic smooth muscle cells and inflammatory cell accumulation. We hypothesized that quantitative analyses of cell types, transcriptomes, and cell-cell communications in experimental TAA tissue would provide insights into pathogenesis. Because many TAA are caused by heritable mutations, we investigated TAA pathogenesis in a mouse model of TAA caused by a mutation in the type 2 transforming growth factor beta receptor (Tgfbr2^G357W/+ mice). We used single-cell RNA sequencing (scRNA-seq) to quantify cell types and transcriptomes in proximal aortas of Tgfbr2^G357W/+ and wild-type mice. I analyzed transcriptomes with CellChat, a tool that uses scRNA-seq data to quantitatively infer and analyze intercellular communication networks. Tgfbr2^G357W/+ aortas had fewer smooth muscle cells and more macrophages than control aortas. Transcriptome analysis revealed that Tgfbr2^G357W/+ aortas also contained a new subpopulation of fibroblasts (“new fibroblasts”) that was absent in controls. Among all cell types, CellChat identified the new fibroblasts as the strongest source of outgoing cell-cell signals, and smooth muscle cells and macrophages as the major recipients of signals emanating from both the new fibroblasts and from other fibroblast populations. Outgoing signals predicted to emerge from the new fibroblasts are mediated by matrix components (e.g., collagen, laminin), cytokines (e.g., CSF), and other ligands (e.g., angiopoietin). We conclude that TAA in Tgfbr2^G357W/+ mice have fewer smooth muscle cells, more inflammatory cells, and a new population of fibroblasts. These new fibroblasts appear to signal to other aortic cells and may play important roles in inflammation and smooth muscle cell phenotypic alteration/loss. Further characterization of the new fibroblasts and their signaling pathways may reveal new targets for therapies that prevent or stabilize TAA.

Thoracic aortic aneurysms (TAA) are excessive dilations of the aorta, inside the chest. TAA can rupture, causing sudden death. Human TAA are attributed to both decreased and increased TGF-β signaling in aortic smooth muscle cells (SMC). Experimental data from mice clearly shows that decreased SMC TGF-β signaling causes TAA; however, the connection of increased SMC TGF-β signaling with TAA is largely based on correlational data. Some of these data implicate excessive TGF-β signaling in only one of the two SMC embryonic lineages that populate the proximal thoracic aorta: the cardiac neural crest (CNC) lineage. To directly test whether increased SMC TGF-β signaling in either of the two lineages causes TAA, I am generating mice with increased SMC TGF-β signaling in either CNC-derived or second heart field (SHF)-derived SMC. I accomplish this by using a transgene that expresses a constitutively active TGF-β receptor (TBRI-CA) after activation by Cre recombinase. To express TBRI-CA in CNC-derived SMC, I mate mice with a Wnt1-Cre transgene to mice with the TBRI-CA transgene. To express TBRI-CA in SHF-derived SMC, I mate mice with an Mef2c-Cre transgene to mice with the TBRI-CA transgene. I hope to determine whether increased TGF-β signaling in SMC of either lineage causes TAA. However, because SMC TGF-β signaling plays critical roles in embryonic vascular development, I will begin by determining whether activation of TGF-β signaling in either lineage is embryonically lethal. I am analyzing genotypes of pups from each mating, and using Chi Square or Fisher Exact Test to test the null hypothesis that increased TGF-β signaling in embryonic SMC is not lethal. If my hypothesis is supported, I will then be able to determine whether SMC-targeted activation of TGF-β signaling causes TAA. My findings could provide support for development of human therapies that prevent TAA by blocking SMC TGF-β signaling.

Myotonic dystrophy type 1 (DM1) is a genetic disease that causes many serious health conditions in a variety of tissues including skeletal muscle stiffening, weakness, and degeneration. DM1 is caused by a CTG repeat expansion mutation in the myotonic dystrophy protein kinase gene, DMPK. Expression of the mutated DMPK allele binds with the splicing regulator muscle-blind-like 1 (MBNL1), causing DM1 by sequestering and limiting its critical role in splicing mRNA. A main focus of the Chamberlain lab is the development of gene therapy to treat DM1, including increasing protein expression of MBNL1 to reduce the disease effects in muscle. Overexpression of MBNL1 in skeletal muscle could be beneficial but may have negative effects on cardiac tissue. The lab discovered that high, unregulated MBNL1 expression from gene therapy vectors in cardiac tissue can result in cardiac damage. In my study, I will focus on cardiac function when testing adeno-associated viral vector (AAV)-mediated systemic delivery of the MBNL1 gene to increase MBNL1 protein expression in muscle. Using analytical methods such as echocardiography and tissue histological techniques, I will determine whether it is possible to prevent MBNL1 protein production and its damaging effects in the heart while still expressing MBNL1 protein in skeletal muscle for therapeutic disease benefits.

TDP is one of the proteins believed to cause ALS, a neurodegenerative disease leading to gradual loss of muscle control and function. Although there are treatments which mitigate symptoms of ALS, this fatal disease remains uncurable. In addition, ALS also contributes to high healthcare costs and pressure on families caring for the afflicted. TDP-43 is a protein with notable aggregation in the motor neurons of 95% of those with ALS. Research has found TDP-43 mutations leading to familial ALS, further implicating its involvement in the development of the disease. There has also been some research suggesting a relationship between TDP-43 and tau, a protein linked to Alzheimer’s Disease and other neurodegenerative diseases. It may be possible for certain genes to control both tau and TDP-43 toxicity. Mutagenesis of C. elegans identified a mutation in suppressor-of-tau (sut) gene sut-6 that caused the amino acid change of W292X. Deletion of sut-6, or sut-6(null), also inhibited tau, but W292X showed greater suppression. Furthermore, a TDP-43 transgenic model expressing mutant TDP-43 protein in C. elegans neurons indicated suppression of TDP-43 by sut-6(null). The goal of this study was to learn whether sut-6(W292X) and sut-6(null) reduce the toxicity of high wildtype human TDP-43 aggregation in C. elegans neurons. I crossed TDP-43 transgenic C. elegans with sut-6 mutation-carrying C. elegans to generate TDP-43 transgenic C. elegans with sut-6 mutations. I then conducted locomotor swimming assays to measure the swimming ability of TDP-43 transgenic C. elegans with and without sut-6 mutations, with increased swimming ability indicating reduced toxicity of wildtype human TDP-43 in C. elegans with the mutations. Investigating the relationship between TDP-43 and sut-6 mutations allows greater understanding of the mechanisms of toxicity caused by TDP-43 and tau in neurodegenerative diseases, providing greater insight into therapies aimed at targeting both proteins.

Alveolar macrophages are vital immune cells, residing in the distal lung parenchyma. They provide the first line of defense against pathogens like mycobacterium tuberculosis (Mtb). They help initiate immune response within the lung and respond to viral infection. Mtb is the pathogen causes tuberculosis. This disease is fatal, and is the leading cause of death among infectious diseases globally. TOLLIP is a ubiquitin-binding protein that is involved in regulating innate immune responses by interacting with many receptors and the transport of endosomal cargo. Currently, TOLLIP’s role in TB pathogenesis is unknown, however, mice without the TOLLIP gene (Tollip-/-) have more severe Mtb disease. The goal of my project was to measure effects of adding mycolic acid and integrated stress response inhibitor (ISRIB) to macrophages of Tollip-/- and B6 Wild Type mice in vitro. I conducted the Bronchoalveolar Lavage (BAL) technique to extract macrophages from the lung via the trachea of B6 Wild Type and Tollip-/- mice. After macrophages were harvested, I plated them in mycolic acid and ISRIB in according wells with media. The cells were then infected with tuberculosis in the animal BSL-3, and data was collected through enzyme-linked immunosorbent assay technique and critically analyzed. Results illustrate that in both B6 Wild Type and Tollip-/- macrophages, adding ISRIB and mycolic acid results in an increase in production of the cytokine TNF, but Tollip-/- had a higher increase. Furthermore, the Tollip-/- samples had high variation in amount of TNF produced while Wild Type had minor amounts, raising further questions about how TOLLIP-deficient alveolar macrophages react when mycolic acid or ISRIB is added. Ultimately, the findings from this research, which is better understanding what specifically stimulates certain cytokines to help immune response to tuberculosis, is essential, and this is a small stepping stone in the broader goal of developing therapeutics for tuberculosis.

 The primary goal of post-transplant care is to achieve the optimal balance of immunosuppression between infection and rejection. Physicians currently rely on laboratory markers such as changes in serum creatinine. Physicians use kidney allograft biopsies to detect acute rejection but biopsies are expensive, invasive, and susceptible to sampling error. This study investigates the use of donor-derived cell free DNA (dd-cfDNA) as a biomarker for kidney allograft dysfunction to allow for optimization of immunosuppression. This multicenter prospective study (Caredx) examines the correlation between dd-cfDNA levels and infection and rejection episodes in pediatric kidney transplant recipients. Dd-cfDNA is measured 3 to 12 months post-transplant and compared to the clinical outcomes of major infection events and biopsies. Data is collected from November 2019 to 2023 on 59 participants from Seattle Children’s Hospital, St. Louis Children’s Hospital, and Emory University. Using patient’s charts in EPIC I abstract patient demographics, transplant characteristics, laboratory, and kidney biopsy results. I assist with patient recruitment and retention by keeping track of when patients are within sample windows and ensuring their sample requests are sent. Preliminary data from the first 10 samples revealed that elevated dd-cfDNA levels were associated with viral infection or acute rejection episodes. We observed a decrease in the dd-cfDNA levels following treatment for BK viremia. Following data collection, we plan to submit an abstract to the American Society of Nephrology in May of 2023. Additionally, we will prepare a manuscript to submit to the Journal of Pediatric Transplantation in the Summer of 2023. Use of dd-cfDNA will enable early intervention and minimize damage to allografts leading to improved longevity and quality of life. Furthermore, the biomarker is less expensive, less invasive, and more accessible than reliance on transplant biopsies holding the potential to break down economic and physical barriers to health care.

CCR5 is a co-receptor required for HIV to infect the body. Several AIDS patients were completely cured after receiving hematopoietic stem cell (HSC) transplants from donors with a mutation in their CCR5 gene making it functionally inactive. However, HSC transplantation is a very risky and expensive procedure, making it inaccessible for most AIDS patients in developing countries. Our aim was to develop a technology that introduces CCR5 gene mutations in the HSCs of AIDS patients in vivo by a single intravenous injection of a gene transfer vector. The vector delivers a genome editing enzyme (base editor) targeted to the CCR5 gene. CCR5-gene edited HSCs will give rise to CCR5-negative HIV target cells, thereby blocking HIV infection and providing life-long protection. We tested three strategies to functionally inactivate CCR5 expression: i) creating a premature stop codon, ii) eliminating the ATG start codon and iii) mutating splice acceptor sites to skip exons. We employed an advanced adenine base editor version (ABE8e) and an early version of cytidine base editor (CBE) delivered with helper-dependent adenovirus vectors (HDAd5/35++) that efficiently infect HSCs in vivo. The base editors are directed to specific target sites by single guide RNAs (sgRNAs). We screened a small library of sgRNAs and identified two species (sgSTOP2 and sgR5-1) that mediated the highest on-target editing rates and CCR5 down-regulation. HDAdAd5/35++ vectors were produced using these sequences. In a test cell line infected with these vectors, 50% of CCR5 alleles were edited. This blocked HIV infection in 40% and 95% of HDAd-ABE8e-sgSTOP2- and HDAd-CBE-sgR5-1-infected cells, respectively. We concluded that the HDAd-CBE-sgR5-1 vector is more efficient in blocking HIV infection and will further improve this vector and test it in primary human lymphocytes and HSCs in the context of HIV infection. This approach has the potential to provide a technically simple HIV/AIDS therapy.

HIV-2 represents approximately 1-2 million of the 38.4 million people living with HIV (PLHIV) worldwide. Existing guidelines and recommendations for the treatment of HIV-2 infection are largely derived from in vitro data and small-scale clinical studies, and are often extrapolated from studies of people living with HIV-1. Furthermore, of the two most common HIV-2 groups (A and B), only a few examples of drug resistance in group B have been reported in literature. As a result, in-depth knowledge about the mutations that arise in group B HIV-2 isolates and the roles that these mutations play in antiretroviral drug resistance is lacking. The aims of my project are (1) to develop a novel approach for creating full-length, infectious HIV-2 group B clones that encode patient-derived integrase sequences, and (2) to demonstrate that this approach can be used to characterize resistance to Integrase Inhibitors (INI), which are an important class of antiretroviral drugs. Specifically, the parental plasmid for cloning and virus expression is 7312A-JK; this plasmid contains a full-length HIV-2 genome with group B gag and pol gene sequences. To facilitate downstream steps, I have replaced the integrase-encoding portion of p7312A-JK pol with a short, synthetic linker. This cassette clone will serve as the acceptor for longer, synthetic DNA fragments that encode patient-derived group B HIV-2 integrases. My ultimate goal is to generate a panel of 10 replication-competent group B clones that can be given to members of the Gottlieb lab for culture-based drug susceptibility testing. This work is an essential step toward a more comprehensive knowledge of drug resistance in HIV-2 which, in turn, is important for evidence-based treatment of all HIV-2–infected individuals, including those infected with group B strains.

The study on Digital Spacing Profiling in Antibody-Mediated rejection (AMR) looks at patients post-kidney transplant. This study involves the use of Digital Spacing technology to analyze the tissue samples of patients suffering from antibody-mediated rejection. Antibody-mediated rejection remains the most formidable cause of kidney transplant failure. Digital spatial profiling of biopsy specimens is a novel technology that provides unprecedented insight into spatially registered molecular changes to better define pathophysiology and potential therapeutic targets. We used the Nanostrong GeoMx platform to define the spatial transcriptome of AMR and compared these results with pathologic, laboratory, and clinical data. We hope that the results from this study will provide novel insight into the pathophysiology of AMR, and lead to improved diagnosis and treatment of AMR. The primary goal is to characterize patients and kidney transplant genomic profiles using digital spatial profiling in the setting of biopsy-proven antibody-mediated rejection, de novo donor-specific antibodies, and/or donor-derived cell-free DNA. Rejection is caused by the body’s response to the transplanted tissue and is typically treated with medication. This study looks at patients who may have different reasons to receive a kidney transplant and how their treatment impacts their organ rejection. The study utilizes clinical and pathological data to learn more about antibody-mediated rejection in kidney transplant recipients, specifically. With IRB approval, we identified eligible subjects through the review of UWMC electronic health records, abstracted patient data, then looked at the patterns from the profiling. Data analysis is underway. This work provides an atlas for the future prevention of AMR, research on this topic is essential to understanding and helping transplant patients live the healthy lives that transplants promise them. Along with that, this research furthers the medical field's scientific understanding of the human body and how to create more effective treatments for illnesses.

 Cancer metastasis, the spread of tumor cells to different parts of the body, significantly increases patient mortality. An early step in the metastasis process is invasion into surrounding tissues. One way that tumor invasion is studied is through tumor organoids. Tumor organoids mimic tumors in vitro through 3D cell culture. While it is generally held that invasion continues to increase monotonically over time, I recently discovered that invasion of tumor organoids has a temporal pattern, where invasion spikes up, then decreases after a certain time. This finding suggests more complex regulation of invasion dynamics than thought previously. The goal of this project is to investigate the temporal dynamics of cell-matrix associated RNAs and proteins during breast tumor organoid invasion. I can perform a qPCR time course of particular genes related to invasion to measure the levels of RNA at specific time points. In addition, I can check for protein levels using a novel method developed in the Cheung lab. This method utilizes bio-orthogonal click-chemistry to perform rapid, selective pairing of intracellular proteins with azidohomoalanine, a clickable methionine analog. Click-chemistry allows us to pick up proteins that cells are either secreted or on the surface of the cells. To define a cancer-specific invasion signature of tumor invasion, I compare RNA and protein dynamics in breast tumor organoids with normal mammary organoids (FVB) migrating in 3D collagen gels. I hypothesize that there will be specific genes associated with increased and decreased invasion levels. In future work we will target those genes by either suppressing those genes that increase with invasion or increasing the expression of invasion suppressors. We expect this work to reveal new regulators of the metastatic process.

Prostate cancer is the second most prevalent cancer among men in the United States. Characteristic biochemical markers include abundant Androgen Receptor (AR) and Prostate Specific Antigen (PSA), a downstream marker for AR activity. A rare neuroendocrine prostate cancer (NEPC), however, is characterized by low AR and PSA activity and high Synaptophysin (SYP; NEPC marker) expression. AR pathway inhibitors (ARPIs), the first-line treatment for prostate cancer, have minimal therapeutic effects on NEPC. This suggests that suppressed AR activity inhibits ARPI effect, and restoring AR could induce sensitivity to ARPIs, such as Enzalutamide. This study investigates the impact of overexpressing a tRNA, Arg-TCT-1-1, in NEPC. tRNA-sequencing of NEPC cell lines identified reduced expression of the Arg-TCT-1-1 tRNA isodecoder (Arg-TCT). Stable cell lines were generated for adenocarcinoma (AD) and neuroendocrine (NE) phenotypes, with overexpression of Arg-TCT or mutant TCT (mut-TCT). Reverse Transcriptase Quantitative Polymerase Chain Reaction confirmed Arg-TCT and mut-TCT overexpression. Cell lysate was immunoblotted for AR, PSA, and SYP; cell growth assays were then performed with Enzalutamide in DMSO to assess cell proliferation and sensitivity. Enzalutamide inhibited AR activity in LNCaP (1uM) and C4-2B AD (20 uM). Immunoblot suggests that Arg-TCT-1-1 overexpression rescues AR activity in NE cells, while mut-TCT does not affect AR activity. Cell growth assays reveal Arg-TCT-1-1 upregulation induces increased cell proliferation and enzalutamide sensitivity in NE cells. These novel findings demonstrate that upregulated tRNA promotes ARPI sensitivity in NEPC cells. Further research on how Arg-TCT-1-1 regulates AR activity and its transferability to other cancers is needed. These results suggest a promising therapeutic vulnerability if confirmed in murine models.

Syphilis is a sexually transmitted infection (STI) caused by the bacterium Treponema pallidum subspecies pallidum (T. pallidum). Syphilis is still a global health concern, as its incidence is rising in high-income countries like the United States and it is still endemic in low-income countries, where it causes significant mortality due to congenital transmission. Our chances at syphilis control would improve if a vaccine against syphilis was available. The past century of research has only yielded experimental vaccines able to produce partial protection, but the use of new techniques could improve this outcome. One possibility would be to use an array of known protective epitopes from other T. pallidum antigens crafted onto a protein carrier to induce a protective immune response. Currently, we are investigating the use of the Tp17 protein of T. pallidum as a scaffolding for a future vaccine. The Tp17 protein has a β-barrel structure with loops around the edges of the barrel. To use this protein as a scaffolding for a vaccine, we will be performing epitope mapping to determine the hierarchy of immunodominant epitopes in Tp17 to find the ideal candidate regions that will be substituted with other epitopes. We are using Enzyme-Linked Immunosorbent Assay (ELISA) to attain these data in combination with human sera to define the reactivity of the different peptides. The results will determine the most immunogenic peptides, thus indicating the ideal areas to be replaced by protective epitopes in a potential vaccine. We anticipate that several of the most reactive peptides will correspond to the protein loops outside of the β-barrel, which should be easily replaceable without affecting Tp17 structure.

Syphilis is a sexually transmitted infection (STI) that is caused by the bacterium Treponema pallidum. STIs within the U.S. remain at a concerning all-time high, with syphilis incidence increasing by 52% since 2016. Syphilis remains endemic in many low- and middle-income regions such as sub-Saharan Africa. A vaccine that’s able to provide complete protection would be an important solution in controlling this infection. To date, there has been a single study producing complete protection that was conducted by immunizing rabbits a total of 60 times, which is impractical in humans patients, but proves that protection can be achieved through immunization. Partial protection has been achieved using recombinant proteins, a method that in our laboratory allowed the identification of possible protective epitopes on T. pallidum specific antigens. Though a vaccine design able to elicit complete protection remains elusive, a chimeric mRNA-based vaccine could breathe new life into the search. This project’s main purpose is to map the immunogenic epitopes of the Tp17 lipoprotein of the syphilis pathogen with the goal of using it as a scaffolding protein for a chimeric vaccine. To this end, we have broken down the Tp17 protein into 23 20-mer peptides overlapping by 10 amino acids and used infected rabbit sera to test and identify the regions of the protein that elicit the most robust humoral response using enzyme-linked immunosorbent assays (ELISA). Preliminary data has shown that the hypothesized external loops in the structure of Tp17 are the regions of highest immunoreactivity. These reactive epitopes were mapped onto the determined Tp17 structure to create a hierarchy of regions that could be replaced with B-cell epitopes protective against syphilis that our lab has previously identified with the future goal to create and test a chimeric mRNA vaccine.

In humans, the KRAS protein normally acts as a switch to regulate cell growth. Acquisition of certain mutations result in constant activation of the protein, leading to uncontrollable cell growth. The most frequent such mutations are at the glycine-12 residue of the protein, including changes to valine (G12V). Cells with this mutation can be recognized and eliminated by T cells engineered to express an antigen-specific receptor (TCR), but posttranslational modifications, such as methylation, can interfere with the ability of such engineered T cells to recognize G12V mutant KRAS. The objective of my project is to identify and eventually disrupt mechanisms that lead to methylation of KRAS mutant antigens. I hypothesize specific enzymes are responsible for methylation of mutant KRAS. To accomplish this, I will generate CRISPR-Cas9 gene knockout libraries to screen for potentially responsible enzymes. The CRISPR library will target human genes encoding methyltransferase and demethylase enzymes. I will co-culture these CRISPR-Cas9 knockout cancer cells with T cells engineered with a TCR targeting the unmethylated KRAS mutant antigen. From the tumor cells that have survived this coculture, I will sequence genomic DNA to determine which knockouts are enriched/depleted after the co-culture. Preliminary results show certain gene knockouts are significantly enriched/depleted in co-culture compared to baseline. The sgRNAs that appear from the high-throughput knockout library most likely to be involved in the KRAS methylation pathway will be individually evaluated in additional co-culture experiments. This should allow me to assess the proliferation/susceptibility of these cancer cells in more detail. For confirmation of mechanism, I will evaluate the methylation status of the KRAS mutant antigen in cancer cells rendered susceptible. My goal is to determine the process leading to methylation of KRAS antigen and then target it to allow for more effective targeting of KRAS-driven cancers using TCR-T cell immunotherapy.

Microchimerism (Mc) occurs when a small amount of genetically different cells (or DNA) is acquired from another individual. Mc is acquired naturally during pregnancy due to exchange between the mother and child and can be detected decades later. Maternal microchimerism (MMc) is when a person harbors Mc from their mother. MMc is frequently detected in healthy adults but is increased in individuals with some autoimmune diseases, including scleroderma. Few studies have investigated MMc, especially whether it changes in a woman after her own pregnancies. One study tested MMc in peripheral blood of women during the time they were pregnant and occasionally detected MMc, but not if the woman had preeclampsia. No study has addressed whether MMc prevalence and quantities change in healthy women according to the time since the woman’s own childbirths or number of childbirths. This study addresses this knowledge gap. MMc was assayed using a panel of polymorphism-specific real-time polymerase chain reaction (qPCR) assays on DNA from peripheral blood. Most assays employed human leukocyte antigens (HLA)-specific primers and fluorogenic probes targeting non-inherited, non-shared HLA sequences. Each woman and her mother were HLA typed to identify an appropriate target. In total, 142 women were tested, and 266 qPCR experiments were run. Preliminary analysis found evidence of MMc in 59 of the 266 samples and a trend of MMc prevalence being highest within the first year postpartum and ten years after childbirth. Prevalence and quantities of MMc are being analyzed in collaboration with a biostatistician. Pregnancy and childbirth are known to affect some autoimmune diseases and cancer risk. Addressing the knowledge gap about MMc according to the time since birth and the number of births in women could provide further insights about some autoimmune diseases and cancers.

Aging is associated with shifts in the composition of brain extracellular matrix chondroitin sulfate glycosaminoglycans (CS-GAGs). CS-GAGs are comprised of repeating glucosamine and N-acetylgalactosamine units that are either non-sulfated (0S-CS), mono-sulfated (4S-CS, 6S-CS), or di-sulfated (2S6S-CS, 4S6S-CS, 2S4S-CS/Dermatan) and participate in the regulation of brain plasticity. The mono-sulfated 6S-CS isomer is predicted to play a key role in the induction of circuit plasticity during neurodevelopment. Therefore, we asked whether this isomer also shows consistent age-related changes between wild-type mice and humans in the regions of the hippocampus and cortex. Our preliminary data generated from cohorts of mice ranging in age from 7 days to 2 years (50%M/50% F) reveal that 6S-CS abundance is highest at 7 days of age and declines with increasing age (9-22 mice/group). We analyzed the relative abundance of the 6S-CS isomer in n=57 hippocampal and cortical human tissue samples (age: newborn - 95 years, sex: 50%M/50% F). Initially, the human samples exhibited the highest abundance of 6S-CS isomer following birth (<1 month age) that then declined at >1M to 29 years of age, phenocopying the results from mice. However, in contrast to mice in which 6S-CS abundance decreased progressively with aging, we found that in humans, 6S-CS abundance began to increase starting at 30 to 99 years of age (R² = 0.84, p-0.0001). The biphasic model of changes in 6S-CS abundance in humans throughout normal aging was previously unknown. Collectively, these findings demonstrate that age-associated changes in brain extracellular matrix 6S-CS isomer abundance in human tissue do not reflect the age-related decline of 6S-CS isomers that occur in mice. Therefore, additional research is needed to establish the utility and robustness of using rodent models to study aging and other age-related extracellular matrix diseases in humans.

Previous studies have indicated that the neurohypophyseal hormone, oxytocin (OXT), reduces body weight in high fat diet (HFD)-induced obese (DIO) rodents, nonhuman primates and obese humans through reductions in food intake and increases in energy expenditure. Findings from recent pre-clinical and clinical data indicate that the long-acting glucagon-like peptide 1-receptor (GLP-1R) agonist, semaglutide, reduces body weight, in part, through reductions of food intake. Based on these findings, we hypothesized that the combined treatment of OXT and semaglutide would produce an additive effect to evoke weight loss in DIO rats. To test this hypothesis, rats were fed a HFD for approximately 5 months prior to being implanted subcutaneously with a 28-day minipump that infused OXT (50 nmol/day) or vehicle (VEH; saline). Data from animals that received escalating doses of the incretin hormone, glucagon-like peptide-1 (GLP-1) and semaglutide was analyzed over the 32-day infusion period. Daily energy intake and body weight were tracked for 32 days. Body composition was assessed immediately prior to onset of treatment and near the end of the treatment period using Quantitative Magnetic Resonance (QMR; EchoMRI 4-in-1). OXT (50 nmol/day) alone and semaglutide (3 nmol/kg) alone reduced body weight by approximately 9.9±1.1% (0.05<P<0.1) and 4.3±1.4% (P<0.05), respectively (N=6-7/group), relative to pre-infusion baseline, while GLP-1 at either dose (1 or 3 nmol/kg, SC) was ineffective (P=NS). However, the combination of OXT and semaglutide (3 nmol/kg) produced a more pronounced reduction in body weight (14.1±1.4%; P<0.05) compared with either treatment alone (N=6-7/group). These effects were associated with reduced energy intake, adiposity and adipocyte size (P<0.05). Together, these findings support the hypothesis that the combination of OXT and the GLP-1R agonist, semaglutide, produces an additive effect to evoke weight loss and body adiposity in DIO rats by reducing energy intake. Our findings will create a preclinical infrastructure upon which future studies are undertaken to address whether oxytocin may be used as an adjunct with other treatment approaches, including low doses of the FDA-approved GLP-1R agonist, semaglutide, to evoke weight loss in humans with obesity.

Many with clinically diagnosed Alzheimer’s disease (AD) dementia during life show comorbid neuropathologies at autopsy. We sought to develop and evaluate a composite brain pathology score (BPS) in the Adult Changes in Thought study. We derived BPS using nine standard neuropathological indicators such as Thal phase, Braak stage, and CERAD score, using confirmatory factor analyses. We compared BPS to the AD Neuropathologic Change (ADNC) score, which quantifies neuropathologic changes that underlie AD. We performed non-nested modeling to compare BPS and ADNC scores’ associations using the last cognitive score (memory, executive function, language, and visuospatial ability) prior to death, adjusting for age at death and sex. Non-nested models were compared using Adjusted-R², Davidson-MacKinnon J-test, and Cox–Pesaran tests. We focused on people whose last cognitive data were≤2 years prior to death. We compared ADNC and BPS’ associations with last known clinical diagnosis (no dementia vs. any dementia), adjusting for age at death and sex, by considering area under receiver operator characteristic (ROC) curves. Sample size was 886 with mean age of death of 89 and 57% female. A bifactor model fit best, with residual correlations for Thal phase and CERAD score, and for LATE stage and presence of hippocampal sclerosis. The BPS explained more variance for each cognitive score and was superior and statistically significant compared to the ADNC score in non-nested model comparisons. The BPS was more strongly associated (Odds Ratio=3.2) with dementia diagnosis than the ADNC score (Odds Ratio=2.0). The area under the ADNC and BPS’ ROC curves were 0.74 and 0.78, respectively. The difference between the areas was statistically significant (p-value ≤ 0.0001). We demonstrate an approach to developing a composite BPS which incorporates multiple forms of pathology. Future efforts will focus on co-calibrating and harmonizing BPS in other autopsy cohorts.

Colorectal cancer (CRC) is a prevalent and often lethal form of cancer, with complex underlying biology and a wide range of associated risk factors. CRC is a significant global public health challenge, it is the second leading cause of cancer-related deaths in the United States. A growing body of research suggests that activin can stimulate the MAPK pathway which plays a critical role in the development and progression of CRC. In the context of colorectal tumors, innate immune cells such as macrophages, dendritic cells, and natural killer (NK) cells can both promote and suppress tumor progression. Macrophages, for example, can either promote tumor growth by secreting growth factors that stimulate angiogenesis and immune suppression, or they can suppress tumor growth by producing cytokines that activate T cells and other immune cells. In this study, our aim is to examine the relationship between activin and the MAPK signaling pathway in macrophages and to determine the role of macrophages in producing and responding to activin. We will be using RAW264.7 macrophage cell line to study the role of macrophages in cancer and Western blot to detect changes in specific proteins in macrophages stimulated with activin. We predict to see an increase in MAPK activation in activin-stimulated macrophages which will enhance tumor elimination. Our findings will also improve the understanding of the innate immune response to colorectal tumors and how it can be modulated by targeting activin signaling. This research will provide new insights into the complex interplay between the immune system and colorectal tumors and may inform the development of new diagnostic methods and more effective treatments.

Colorectal cancer (CRC) is the third most common cancer globally, and the second leading cause of cancer deaths in the US due to metastasis. The five year mortality rate of Stage IV CRC patients remains around 90%, and most treatments for stage IV CRC are palliative. Activin A, a cytokine that regulates proliferation and apoptosis, is known to induce metastatic phenotypes in CRC cells primarily through the PI3K/ AKT pathway. Glycogen synthase kinase-3 beta (GSK3β) is an enzyme downstream of AKT that regulates energy metabolism and apoptosis. An inactive form, phosphorylated GSK3β (pGSK3β), is unable to inhibit β-catenin activity, which promotes proliferation and epithelial to mesenchymal transition. Very few connections between activin A signaling in colorectal adenocarcinoma cells and GSK3β phosphorylation have been established in scientific literature. Our project aims to study the correlation between activin A signaling and pGSK3β by testing the hypothesis that activin A signaling induces phosphorylation of GSK3β in CRC tumor cells, promoting the attenuation of cell cycle arrest. We will perform Western blot analyses on FET cells treated with a vehicle control, activin A, or TGF-β, a cytokine that is activin-dependent in the context of CRC, to measure the levels of pGSK3β relative to GSK3β. We expect to see the highest levels of pGSK3β relative to GSK3β in FET cells treated with activin A and the lowest relative pGSK3β levels in cells treated with the vehicle control. The findings of this project can contribute to identifying biomarkers useful for risk stratification in metastatic CRC to provide more individualized treatments for patients. 

Risk variants of apolipoprotein L1 gene (APOL1) increase the risk of chronic kidney diseases in populations of African ancestry. We seek to study this disease, which is unique to humans, in human kidney organoids derived from induced pluripotent stem (iPS) cells. However, APOL1 is not expressed in organoids at baseline. While interferon (IFN)-gamma is a potent inducer of APOL1 expression, our prior experiments suggested that IFN-gamma itself disrupts organoid structures, limiting the degree to which the specific effects of APOL1 can be assessed. To improve the kidney organoid system as a better platform to model APOL1-associated nephropathy without IFN-gamma stimulation, I am establishing an APOL1 inducible expression system in kidney organoids. I hypothesize that cell lines with the risk variants will demonstrate an accelerated rate of degradation compared to the non-risk variant, modeling risk variant-dependent cytotoxicity. The inducible expression system will be established by generating iPS cell lines encoding Tet-On system sequences, enabling both tunable and temporal control of APOL1 expression using doxycycline. Plasmids were constructed by PCR amplifying and inserting the targeted sequences into a homology-dependent repair template with a doxycycline-inducible promoter. Ligation had to be done multiple times due to the high prevalence of self-ligation of the backbone vector and backward insert orientation. However, we found that adding alkaline phosphatase to dephosphorylate 5’ ends of the backbone vector significantly improved the integration rate and led to the successful construction of plasmids. Next, CRISPR-Cas9 gene editing will be utilized to introduce the APOL1 gene variants into the AAVS1 safe harbor locus in iPS cell lines, which can be differentiated into kidney organoids. This project aids in isolating the phenotype of APOL1 on human kidney organoids with various cell types, which will be a valuable tool in developing an in-vitro pathophysiological assay such as use in therapeutic drug discovery.