• Identifying transglutaminase 2 (TG2) downstream mediators that are required to maintain an aggressive epidermal squamous cell carcinoma cancer phenotype

  Chen, Xi; Eckert, Richard (Richard L.) (2022)

  Epidermal squamous cell carcinoma is among the most common cancers in humans. It is typically treated by surgical resection and chemotherapy. However, 5 – 10% of resected cases relapse as aggressive cancer. We attribute the reason as the existence of epidermal cancer stem-like cells (ECS cells). Transglutaminase 2 (TG2) is a key ECS cell survival protein. However, how TG2 maintains the aggressive ECS cell cancer phenotype is not well understood. Thus, our goal is to identify TG2-stimulated downstream mediators that are important to maintain the ECS cell cancer phenotype. In the present studies, we show that inhibition of TG2 reduces MET tyrosine kinase receptor expression and activity, and that this is associated with attenuated cancer phenotype. Inhibition of TG2 or HGF/MET function reduces downstream MEK1/2-ERK1/2 activity, and this is associated with reduced cancer cell spheroid formation, invasion, migration, and reduced EMT and stem cell marker expression. HGF partially restores the aggressive cancer phenotype, confirming that MET signaling is downstream of TG2. MET knockdown reduces ERK1/2 signaling, doubles the time to initial tumor appearance and reduces overall tumor growth. In addition, we show that TG2 knockdown or inactivation results in a reduction in mTOR level and activity in ECS cells which are associated with reduced spheroid formation, invasion, migration, and reduced stem cell and EMT marker expression. mTOR knockdown or treatment with the mTOR inhibitor rapamycin phenocopies the reductions in cancer phenotype and stem cell and EMT marker expression. Moreover, forced expression of constitutively active mTOR in TG2 knockdown cells partially restores the aggressive cancer phenotype and stem cell and EMT marker levels, suggesting that mTOR is a necessary mediator of TG2 action. Together, the present studies suggest that TG2 maintains HGF/MET/ERK1/2 signaling and mTOR signaling to maintain the aggressive ECS cell cancer phenotype and drive aggressive tumor formation, and that TG2-dependent MET or mTOR signaling may be useful anti-cancer targets.
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  Relationship between kidney function and cognitive decline and moderating effects of race and SES

  Yang, Ruowei; Gruber-Baldini, Ann L.; Waldstein, Shari R. (2022)

  There is limited research as to whether pre-clinical chronic kidney disease (CKD) measured by a combination of kidney function markers such as the estimated glomerular filtration rate (eGFR), albuminuria, and Cystatin-C have an impact on cognition. Understanding kidney function decline as a potential early marker for cognitive decline has important public health implications given the high societal burden of clinically diagnosed kidney disease and cognitive disorders. The Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study wave 1 and 3 datasets were used for this analysis. HANDLS Wave 1 data were collected from year 2004 to 2009, and wave 3 from year 2009 to 2013. In Aim 1, mixed effects models with interaction terms were constructed to examine the effects of time-varying eGFR on several cognitive outcomes. A backward elimination method was used to trim the models of non-significant four-way interactions of eGFR*Age*Race*Poverty and all lower-order interactions. In Aim 2, mixed effects models with interaction terms similar to those used in Aim 1 were constructed to examine the effects of baseline eGFR, urine albumin and Cystatin-C on the same set of cognitive outcomes. All significant interactions were examined by interaction plots for racial and SES subgroups. The analytical cohorts included 1,206 participants for aim 1 and 638 participants for aim 2 at baseline. The main hypothesis that African Americans living below poverty would have the worst impact of lower kidney function on cognition was not supported. Over about 5 years, eGFR over time was associated with decline in verbal learning and memory among Whites below poverty. In addition, the associations between baseline kidney function markers and cognitive decline were highly equivocal. Higher baseline Cystatin-C were found to be associated with cognitive decline in attention, and this association was no longer significant after adjusting for cardiovascular factors and inflammation. It is possible that kidney function is broadly related to performance in the domains of attention, working memory and executive function. These findings suggest that functional changes in the kidney precedes cognitive decline among Whites, especially those living below poverty.
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  Regulation of retinoid homeostasis by cellular retinol-binding protein, type 1

  Zalesak-Kravec, Stephanie; Kane, Maureen A. (2022)

  Retinoic acid (RA) is the main active metabolite of Vitamin A, an essential diet-derived nutrient. RA signaling regulates cell differentiation, proliferation and apoptosis. RA levels are tightly regulated throughout the body via the expression and activity of catabolic and biosynthetic enzymes, and chaperone proteins, including cellular retinol binding protein, type 1 (CRBP1). CRBP1 binds to retinol and retinal, protecting them from non-specific oxidation, and facilitating their delivery to the appropriate enzymes for RA biosynthesis. CRBP1 has been shown to be decreased in disease states that display dysfunctional proliferation and differentiation, including cancers. Reduction of CRBP1 levels directly correlates with reduction in RA and restoration of CRBP1 expression has been shown to increase RA levels and positively impact RA-dependent outcomes. Research on the role of CRBP1 in disease has been limited because of its low abundance and poor immunogenicity. We have developed a targeted, bottom-up proteomics approach for absolute CRBP1 quantitation in complex biological matrices and have utilized this assay to answer important biological questions regarding the role of CRBP1 in regulating RA and RA-mediated signaling. While proper RA homeostasis is essential for biological processes throughout the body, the research in this thesis has focused on its role in the small intestine, heart, and lung. In the small intestine, RA plays an essential role in regulating the gut immune response. In instances of cellular stress in the intestine, RA levels are decreased. We have employed our CRBP1 quantitative assay, along with retinoid metabolite quantitation and quantitative gene expression, to systemically probe the mechanism of disrupted retinoid signaling in intestinal disease via an in vitro model of the small intestine. Proper RA levels are also necessary for growth and development, including heart and lung morphogenesis, and have also been shown to be disrupted in many diseases, such as heart failure and lung cancer. Using a global CRBP1 knock-out mouse model, we have also explored the in vivo effect of loss of CRBP1 on retinoid signaling via multi-omics analysis. Together these studies will help further our understanding of the mechanisms and impact of CRBP1 loss in diseases of the intestine, heart, and lungs.
• Skeletal muscle properties, gross motor performance, and quality of life in survivors of childhood hematologic and oncologic health conditions

  Rock, Kelly; Marchese, Victoria (2022)

  Background: Gross motor skills such as running, hopping, and jumping are important for age-appropriate activities and sports throughout the lifespan. Difficulty with gross motor skills negatively affects one’s quality of life (QoL). Gross motor skills require the activation of the large lower-extremity skeletal muscles and the muscle properties of these muscles such as strength, size, and neuromuscular activation contribute to gross motor performance. Children with chronic hematologic and oncologic health conditions, such as sickle cell disease (SCD) and musculoskeletal sarcoma (MSS), are at risk for impairments in skeletal muscle properties, limitations in gross motor performance, and reduced QoL. However, there remains a lack of knowledge of skeletal muscle properties and their relationships to gross motor performance and QoL in children with chronic hematologic and oncologic health conditions. Methods: Quadriceps skeletal muscle properties (strength, size, and neuromuscular activation), gross motor performance, and quality of life were measured in children with SCD and adolescent, young adult MSS survivors of childhood cancer (CCS), and healthy controls. The effect of functional strengthening (PT-STRONG) was assessed in a sub-population of MSS CCS. Results: Children with SCD and adolescent and young adult MSS CCS presented with impairments in muscle properties including decreased knee extension strength and lower quadriceps rate of muscle activation (RoA), poorer gross motor performance, and reduced QoL compared to controls. MSS CCS demonstrated decreased surgical limb knee extension strength, and quadriceps muscle thickness and RoA compared to their non-surgical limb, and decreased bilateral knee extension strength, gross motor performance, and physical QoL compared to normative values. In children with SCD, positive relationships between RoA, strength, gross motor performance, and quality of life were identified. In MSS CCS, positive correlations between muscle thickness and strength, and between strength and gross motor performance were identified. In response to PT-STRONG, MSS CCS participants demonstrated individual improvements in neuromuscular activation, gross motor performance, and physical QoL. Conclusions: Children with SCD and adolescent and young adult MSS CCS demonstrate changes in muscle properties that are associated with limitations of gross motor performance and reduced quality of life.
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  Care Interactions Between Staff and Nursing Home Residents with Dementia

  McPherson, Rachel; Resnick, Barbara (2022)

  Background: The quality of staff-resident care interactions is critical to residents living with dementia. Limited work has focused on understanding the quality of care interactions among nursing home (NH) residents and examining what factors are associated with the quality of staff-resident care interactions. Purpose: The purpose of this dissertation was to: (1) comprehensively describe staff-resident care interactions; (2) assess racial and gender differences in the quality of care interactions between staff and residents living with dementia; and (3) test the resident and community factors that are associated with the quality of care interactions between NH residents living with dementia and staff. Methods: Utilizing baseline data from a randomized pragmatic trial that included 553 residents from 55 NH facilities, Aim 1 used descriptive statistics to describe the characteristics of care interactions in NHs and a multiple linear regression to determine differences in the quality of care interactions between actively engaged and passively engaged residents, Aim 2 used analyses of covariance to examine racial and gender differences in the quality of care interactions, and Aim 3 used structural equation modeling to test the resident and community factors that were associated with quality of care interactions and test for invariance between model fit based on resident race and gender. Results: Although the majority of care interactions were positive, 21% of the interactions were negative and neutral. Active engagement was significantly associated with more positive care interactions than passive engagement. There was a racial difference in the quality of care interactions such that Black residents received significantly more positive care interactions than White residents. Increased pain and comorbidities were associated with more negative care interactions, while higher community star rating and for-profit communities were associated with more positive care interactions. Conclusions: Understanding the quality of staff-resident care interactions in NHs and the factors that are related to the quality of care is important to guide future interventions and training curricula for NH care staff. Using this information to improve care interactions is important so that all NH residents living with dementia experience positive interactions regardless of pain, comorbidities, race, or community characteristics.
• Telehealth Care: How reliable are our physical therapy outcome measures?

  Reoli, Rachel; Whitall, Jill; Bastian, Amy (2022)

  Background: Changes in global health have prompted a transition from the in-person healthcare model towards the telehealth care model for patients with a variety of health conditions, including patients with cerebellar impairments and patients with Parkinson’s disease (PD). While it is known that patient satisfaction is high in the telehealth care setting, it is unknown whether the tools that clinicians utilize in the in-person setting are reliable and valid in the virtual setting. Thus, the goal of this project is to evaluate the psychometric properties of two physical therapy (PT) outcome measures, that have been previously established in the clinical in-person setting, in the telehealth setting. Methods: Nineteen individuals with cerebellar impairments and nineteen individuals with PD were recruited on a voluntary basis. The Scale for Assessment and Rating of Ataxia (SARA) was used to assess the degree of cerebellar damage and the Berg Balance Scale (BBS) was used to assess the impact of PD. Participants completed two testing sessions (one virtual and one in-person) during which the outcome measure of choice was performed. Outcome measure performance was video recorded in both environments. Videos were independently scored by four raters with varying levels of PT experience. Concurrent validity was assessed via Spearman’s Rank Order Correlation Coefficient, α<0.05, comparing the virtual SARA and BBS scores to their “gold standard” in-person scores. Inter-rater reliability for four raters was evaluated with an Intraclass Correlation Coefficient (ICC) (2,4), α<0.05. Results: The SARA and the BBS were found to have large concurrent validity with Spearman’s rho significant at the two tailed, alpha <0.01, 0.90, n=14; 0.87, n=18, respectively. Similarly, the SARA and the BBS had excellent inter-rater reliability in the clinic ICC (2,4) 0.97 and 0.90, n=19 for both measures. Virtually, the BSS had moderate inter-rater reliability (ICC (2,4) 0.72, n=18) and the SARA had excellent inter-rater reliability (ICC (2,4) 0.98, n=14). Conclusion: Our study shows that both the SARA and the BBS can be used in the virtual telehealth setting. Additionally, clinicians with varying years of PT experience can accurately score the SARA and the BBS for patients with cerebellar impairments and PD, respectively.
• Healthcare Epidemiology of Emerging Infectious Diseases

  Nadimpalli, Gita; Harris, Anthony D.; O'Hara, Lyndsay M. (2022)

  Background: Readmission within 30-day in coronavirus disease 2019 (COVID-19) is an important indicator of patient safety and quality of care. Conflicting evidence exists regarding in-hospital mortality in solid organ transplant (SOT) recipients with COVID-19. Furthermore, during the pandemic, an increase in hospital-acquired infections (HAI) potentially partially due to environmental contamination was observed. Objective: This thesis evaluated i) the impact of comorbidities on the odds of 30-day readmission following COVID-19 hospitalization, ii) the risk of in-hospital mortality among SOT recipients with COVID-19, and iii) the efficacy of a novel continuously active disinfectant (CAD) to reduce bioburden and infectious pathogens on environmental surfaces in a critical-care setting. Methods: A retrospective cohort study was conducted using the Premier database of COVID-19 in-patients in the United States. The association between comorbidities and readmission, and the relationship between SOT and in-hospital mortality were examined using logistic regression and log-binomial models respectively. General linear models and logistic regression were used to evaluate the efficacy of the CAD in a randomized controlled trial. Results: Among 331,136 COVID-19 patients, 36,827 (11.1%) were readmitted within 30 days. Each additional comorbidity category was associated with increased odds of all-cause readmission (adjusted odds ratio [aOR], 1.18; 95% confidence interval (CI):1.17–1.19) and readmission for COVID-19 (aOR, 1.10; 95% CI:1.09–1.11). Among 378,111 COVID-19 patients, 3,527 (0.9%) SOT recipients had an increased risk of in-hospital mortality (adjusted relative risk, 1.26; 95% CI, 1.17–1.35). The mean difference in environmental bioburden between the new CAD and the standard disinfectant was -0.59 (95% CI: -1.45 , 0.27) and the odds of detection of epidemiologically important pathogens was 14% lower in rooms cleaned with the CAD compared to the standard disinfectant (OR 0.86, 95% CI: 0.31 -2.32). Conclusions: In COVID-19 patients, readmission was common within 30-days. SOT recipients with COVID-19 are at an increased risk of mortality. Larger trials may be warranted to further evaluate CAD to limit the transmission of infectious pathogens. A better understanding of these risk factors will facilitate hospital epidemiologists to better manage emerging infectious diseases by improving bed-flow planning, resource allocation, and implementing appropriate measures to deliver high-quality patient care.
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  Moving from Moral Distress to Moral Emotion: A Socioecological Model

  Imboden, Rachel; Hopkins, Karen M., 1954-; Shdaimah, Corey S. (2022)

  Among social workers, morally relevant distress has been associated with negative emotional and physical outcomes for the practitioner as well as increased intent-to-leave and burnout. Moral distress, a term that arose in the nursing literature, has traditionally been understood as the discomfort that arises when one knows the right thing to do, but is unable to act on that knowledge. However, social workers’ perspectives and experiences of moral distress have not been fully explored, including whether certainty and constraint are central features. A diverse sample of 20 master’s prepared social workers participated in individual semi-structured interviews exploring experiences they identified as morally or ethically troubling or distressing. Grounded theory was used to guide analysis of respondent interviews and revealed that constraint was a common, but not essential feature of morally distressing scenarios. Additionally, uncertainty was often described as an aspect of the experience. The Model of Moral Emotion emerged during the analytic process, demonstrating the layered and contextual nature of a range of morally challenging emotions (e.g., anxiety, depression, anger, traumatic stress). Within this model, moral emotion is shaped by the features of the moral scenario (i.e., constraint, conflicts, complexity, complicity, and trespass) and the individual’s capacity, via internal and external resources, to work through practice challenges. This micro level experience is nested within the mezzo level of practice (e.g., agency context, resources, and culture), the macro level of practice (e.g., sociopolitical, economic, and historical context), and the practitioner’s development as an individual and professional (chronosystem). The Model of Moral Emotion is best suited for use as an assessment tool with students and practitioners to explore how and in what contexts moral emotion develops, how it is experienced or impacts the practitioner, and what actions or responses are needed. Future research will benefit from exploration of the positive end of the moral emotion spectrum (e.g., moral courage), the nuances of diverse cultural understandings of moral emotion, and possible adaptation of the model for quantitative analysis.
• Single-Nucleus Multi-Omic Characterization of Age-Specific Effects of Traumatic Brain Injury in the Mouse Hippocampus

  Bhatia, Parth; Ament, Seth A. (2022)

  Traumatic brain injury (TBI) has been characterized as a silent epidemic with growing incidence rates worldwide. Recently, single cell genomics has revealed multiple microglial subtypes activated during inflammation, and emerging evidence suggests that distinct subtypes contribute to age-related differences in neurodegeneration and neuroprotection. In this thesis, I test the hypothesis that differential microglial activation states contribute to the worsened outcomes to TBI associated with aging. Using single-nucleus RNA and ATAC sequencing, I describe cell type specific transcriptional and epigenomic changes in microglia in the context of TBI in young vs. aged mice. I found substantial transcriptional effects of TBI in microglia in both young and aged mice. Regulatory network models predict common repression of homeostatic genes but differential activation of regulatory activity with transcription factors like Nfe2l2 and Runx1 in rescuing and repressing a neuroprotective state of microglia.
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  Method Optimization of a New Automated Platform for Proteome-Wide Structural Biology

  Johnson, Dante; Jones, Lisa M. (2022)

  Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein HOS and dynamics requires integrated approaches, including mass spectrometry (MS), which has evolved into an indispensable tool for proteomics research. One approach readily integrated with MS is protein footprinting. In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change. The method can modify thousands of proteins in a single experiment leading to structural information across the proteome. IC-FPOP modifies proteins on the microsecond timescale making the method suitable to study fast biological processes. However, the single cell flow system developed for initial IC-FPOP experiments had temporal limitations motivating the design for a higher throughput platform. My research describes the development of a new platform for IC-FPOP entitled Platform Incubator with XY Movement (PIXY). PIXY permits IC-FPOP to occur in a sterile system using a temperature-controlled stage top incubator, peristaltic pumps for chemical transport, mirrors for laser beam guidance, and a mobile stage for XY movement. Automated communication amongst the entire PIXY system was made possible using LabVIEW software which allows the analysis of one sample in only 20 seconds. Well over 2000 proteins in HEK cells can be oxidatively modified by IC-FPOP in PIXY. This allows for a greater amount of structural information to be obtained. The capabilities of this high throughput platform permit other cell based experimental applications including fluorescent imaging and time-dependent solution transfer. PIXY’s ability to accommodate automated time points and subsequent changes over time make it a powerful tool for probing protein biochemistry in the native cellular environment.
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  Mindfulness, Self-Compassion, Emotion Regulation, and Parenting Stress in Mothers of Preschoolers

  Endy, Katherine; Sacco, Paul; Jones Harden, Brenda (2022)

  Parenting stress is known to have adverse effects on both parents and children and is particularly salient during the early childhood years. Parenting stress can lead to harsh parenting behaviors which in turn have negative consequences for children, including internalizing and externalizing behaviors. Parental emotional dysregulation and low levels of compassion toward oneself are factors associated with elevated parenting stress. Mindfulness has been identified as a state of being negatively associated with symptoms of high stress, anxiety, and depression, and has been shown to be beneficial for parents of children with autism and those with chronic illnesses. The purpose of this dissertation study is to analyze the relationship between mindfulness and parenting stress, looking specifically at self-compassion and difficulties with emotion regulation as possible mediators. Using an electronic survey administered by Qualtrics Panels, I gathered data from mothers of 2- to 5-year-old children and analyzed the data using mediation models. I hypothesized that self-compassion and/or difficulties with emotion regulation would mediate the relationship between mindfulness and parenting stress. Results were that self-compassion partially mediated the relationship between mindfulness and parenting stress. Difficulties in emotion regulation did not mediate the relationship between mindfulness and parenting stress. This research has implications for the field of social work, and for parenting interventions in particular, as it has the potential to expand our understanding of how mindfulness works to reduce parenting stress.
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  Novel Bioactive Low-shrinkage-stress Nanocomposite with Antibacterial and Remineralization Properties and Thermal-cycling and Aging Resistance.

  FILEMBAN, HANAN; Xu, Huakun H.; Weir, Michael D. (2022)

  Dental composites continue to be the material of choice in daily dental practice for several reasons, including their good mechanical properties, conservative cavity design, and superior esthetics. However, the longevity of current resin composite restorations ranges only 5-10 years. Recurrent caries and tooth fracture are the most common types of failure during the first 6 years of clinical service. These failures are often caused by the polymerization shrinkage stress of the dental composite materials. Thus, there is an increased need to develop a new generation of bioactive dental composite with the ability to reduce polymerization shrinkage stress, long-term antibacterial, remineralization abilities, and excellent mechanical properties. Therefore, this dissertation aims to develop a new bioactive low-shrinkage-stress dental composite containing dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) which could be a promising approach to increase the chances of success of composite restorations and strengthen tooth structures. First, we found that the new bioactive low-shrinkage-stress resin composite significantly reduced the polymerization shrinkage stress, without compromising their mechanical properties. Increasing the DMAHDM mass fraction increased the antibacterial effect in a dose-dependent manner. Next, we investigated the low-shrinkage-stress composite mechanical stability and antibacterial durability in thermal cycling for 20,000 cycles, equivalent to two years of clinical life. We found that the bioactive low-shrinkage-stress composite possessed good mechanical properties that matched commercial composite both before and after thermal cycling. The new composite had potent antibacterial activity, which was maintained and did not decrease after thermal cycling. Lastly, we further examined the mechanical and antibacterial durability of a bioactive low-shrinkage-stress after 50,000 and 100,000 thermal cycles which corresponds to 5 and 10 years respectively of in vivo function. We found that the bioactive low-shrinkage-stress composite maintained its antibacterial potency after thermal cycling, indicating long-term antibacterial durability. In addition, it possessed good mechanical properties that were comparable to commercial composite both before and after thermal cycling. The triple benefits of antibacterial, remineralization, and lower shrinkage stress have a great potential to inhibit recurrent caries and increase restoration longevity.
• Investigating Fn14 as a cell surface portal for targeted nanotherapeutic delivery to triple-negative breast cancer (TNBC) cells

  Carney, Christine; Kim, Anthony J.; Winkles, Jeffrey Allan (2022)

  Metastatic triple-negative breast cancer (TNBC) is associated with a dismal prognosis due to a lack of known therapeutic targets and several difficulties in treating disseminated disease. As a result, patients with this diagnosis have an average median survival time of just 13 months. Treatment challenges are further exacerbated in the 30-55% of metastatic TNBC patients that develop brain metastases (BMs), where therapeutic agent efficacy is further limited by the blood-brain barrier. Nanotherapeutics may be especially poised for overcoming such treatment barriers, particularly if specifically targeted to TNBC cells within the brain lesions. The TWEAK receptor, Fn14, is minimally expressed by healthy breast and brain tissues but overexpressed in primary TNBC tumors and TNBC BMs. We previously demonstrated that Fn14-targeted, paclitaxel-loaded polymeric nanoparticles (NPs), termed ‘DARTs’, outperform Abraxane—an FDA-approved paclitaxel nanoformulation—following intravenous delivery in mouse models of primary TNBC and TNBC BM. In Part 1 of this work, we investigated the impact of using different Fn14 targeting moieties and moiety surface densities on NP binding affinity and uptake by Fn14-expressing tumor cells. Specifically, we found that DARTs functionalized with ~13 ITEM4 monoclonal antibodies, which are specific for Fn14, exhibit significantly greater uptake by TNBC cells compared to NPs with other ITEM4 surface densities or those with ITEM4 fragment antigen binding (Fab) regions for targeting. In addition, we determined that DARTs are predominantly internalized via clathrin-mediated endocytosis and traffic via the endolysosomal pathway. In Part 2 of this project, we further examined the utility of this formulation as a drug delivery platform in complementary xenograft and syngeneic models of TNBC BM. First, we determined that tumor cells were the predominant source of Fn14 expression in the TNBC brain tumor-immune microenvironment, with minimal Fn14 expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then showed that although DARTs, non-targeted PLGA-PEG NPs and Abraxane exhibit similar accumulation in brains harboring TNBC BMs following systemic delivery, only DART NPs specifically associated with TNBC cells. Collectively, these findings deepen our understanding of the potential use of the DART NP formulation for drug delivery to TNBC patients with BMs.
• The Anti-tumor Action and Skeletal Toxicity of Palovarotene, Retinoid Nuclear Receptor Gamma Agonists in Multiple Osteochondroma Mouse Model

  Garcia, Sonia Arely; Enomoto-Iwamoto, Motomi (2022)

  Osteochondromas are cartilage-capped tumors that arise near growing physis and are the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Multiple osteochondromas (MO) can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved drug therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including osteochondromas in rodent models. A clinical trial of the systemic treatment of Palovarotene, a RARγ agonist for MO (NCT03442985) trial was terminated due to concerns of skeletal toxicity observed in pediatric patients in a different clinical trial. The purposes of this project are to determine whether refining systemic and local RARγ agonist treatment inhibits pre-existing osteochondroma growth, to minimize the adverse actions of the RARγ agonists on adjacent growth plate, and to elucidate the molecular action of RARγ agonists on osteochondromas. A mouse model involving the conditional deletion of EXT1 in cartilage was used as MO animal model. Palovarotene (1.76 mg/Kg, daily) treatment for 2 weeks, strongly suppressed osteochondroma development in the wrists and ribs. Osteochondromas became evident under the treatment with the same dose for an additional 2 weeks, however, was significantly reduced with the increasing the Palovarotene dose to 4.0 mg/Kg. However, this increase in the drug dose exhibited skeletal toxicity, including changes in trabecular bone, thinning of the cortical bone and articular cartilage deformity. Local delivery is an alternative theory to overcome systemic exposure concerns. Two-week local application of RARγ-loaded nanoparticles inhibited osteochondroma volume in the writs of our osteochondroma model without causing effects on limb lengths compared to vehicle control. Mechanistic studies demonstrated that RARγ agonist treatment of human osteochondroma explants inhibited matrix synthesis, stimulated matrix degradation and induced cell death while systemic treatment inhibited matrix synthesis and stimulated matrix degradation in our osteochondroma mouse model. These findings indicate that RARγ agonist exerts anti-tumor function, and that local drug therapy may be an alternative to avoid systemic toxicity.
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  Soluble Immune Biomarkers as a Diagnostic Tool of Head and Neck Squamous Cell Carcinoma Histological Inflammatory Subtypes

  Ghita, Ioana; Younis, Rania H. (2022)

  Head and neck squamous cell carcinoma (HNSCC) is a devastating malignancy that occurs in close proximity to vital structures. Despite all advances in diagnostic and therapeutic measures, the overall 5-year survival rate stays at ~65% and can be dismal for recurrent and advanced stages. There is accumulating evidence indicating the immune suppressive potential of HNSCC by which it can escape and/ or suppress the immune system. The recent advent of immunotherapy showed unprecedented improvement in overall response of advanced stage HNSCC. Yet, the overall response rate of HNSCC to immunotherapy remains at ~ 15%. Hence, further understanding of HNSCC tumor inflammation is warranted. The hypothesis of the current work is that the histological inflammatory subtype (HIS) of HNSCC can provide the basis for patient stratification that can be distinguished from the cytokine profile in peripheral blood, therefore better treatment strategies and patient outcomes. Paired tumor tissue and plasma of 104 HNSCC cases were collected according to UMB IRB protocol. Scoring of the HIS subtype was carried out using immunohistochemistry (IHC) of the emerging immune biomarker Semaphorin 4D (Sema4D). Our cohort showed 52% HIS inflamed (HIS-INF), 40% immune excluded (HIS-IE) and 8% deserted (HIS-ID). Differential gene expression (DGE) analysis of 10 HNSCC tumor tissues using NanoString immune-oncologic (human IO-360) 700 genes set showed that tumors with HIS-IE clustered as IFN-γ negative/ low immune signature compared to HIS-INF and were higher in hypoxia gene expression and the myeloid cellular compartment, while the HIS-INF demonstrated higher lymphoid component. DGE revealed a novel association between the high soluble Sema4D in plasma (HsS4D) and higher transcriptional level of Osteopontin (OPN) in the tumor tissue, that we also demonstrated in vitro. Furthermore, using ELISA-Luminex™ system, HIS-IE tumor tissue was significantly distinguished from the HIS-INF, in almost 40 traditional cytokines detected in the paired plasma samples. In conclusion, our work demonstrated a stratification model of HNSCC based on the underlying HIS profile that can be detected via the soluble cytokine panel in blood. These findings can open new avenues for patient stratification and enhance personalized clinical care in the field of HNSCC.
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  Characterization of a Lipid Receptor in Tick Innate Immunity

  O'Neal, Anya Josephine; Pedra, Joao H. F. (2022)

  Innate immunity in metazoans relies on an arsenal of pattern recognition receptors. These molecules activate signaling pathways that direct antimicrobial defense. The arthropod immune system, which has been largely studied in the fruit fly Drosophila melanogaster, utilizes the immune deficiency (IMD) pathway to defend against Gram-negative bacteria. Recent studies using the blacklegged tick Ixodes scapularis determined that immune signaling in chelicerates is wired differently from dipteran insects. Notably, ticks and other chelicerates do not encode several components of the canonical IMD pathway, including classical receptors. Here, we report that the I. scapularis homolog of Croquemort (Crq), a CD36-like lipid scavenger receptor, initiates immune pathways against the Lyme disease spirochete Borrelia burgdorferi. Crq binds the lipid and IMD pathway agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and exhibits plasma membrane localization. Nymphs silenced for crq display impaired fitness, an inability to reach full repletion, and delayed molting to adulthood due to deficient ecdysteroid synthesis. Importantly, Crq relays antimicrobial signals, regulates immune gene expression, and limits acquisition of B. burgdorferi through the IMD and jun N-terminal kinase (JNK) pathways. Collectively, our findings reveal an ancient antibacterial immune response in non-insect arthropods and establish a new scientific paradigm in tick-borne diseases.
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  The Allostery and Specificity of EF Hand Calcium-Binding Proteins

  Young, Brianna; Weber, David J., Ph.D. (2022)

  EF-hand Ca2+-binding proteins (CBPs), such as calmodulin (CaM) or those belonging to the S100 protein family (S100s) undergo conformational changes upon increasing intracellular Ca2+, facilitating interactions with protein targets and inducing important biological responses. In the absence of target, the Ca2+-binding affinity of CaM and most S100 proteins is weak (CaKD >1 μM). However, when bound to effectors, allosteric mechanisms increase the Ca2+ affinity of these CBPs (CaKD <1 μM) to allow for proper Ca2+ homeostasis and maintenance of Ca2+-signaling. The Ca2+-tightening of these CBPs is described here by the “binding and functional folding” framework for detailing this physiologically relevant phenomenon. This research seeks to elucidate both the mechanisms of allostery and the basis of ligand- and target- specificity for S100 proteins and CaM. First, molecular fragments were used to differentiate between two highly similar S100 proteins, S100B and S100A1, with the goal of designing S100-specific inhibitors to block these elevated S100 proteins in various disease states. This provided insight into the specificity of S100A1 versus S100B for small molecules and will enable improved S100 protein-based drug design efforts. S100A1 and S100B binding to ions was also compared and differences between ion binding sites within the two highly similar proteins were determined. Another study revealed an allosteric mechanism in which a peptide termed BP2, derived from the STRA6 vitamin A transporter, increased the Ca2+-binding affinity of CaM upon binding. CaM-STRA6 complex formation was observed at physiologically relevant free Ca2+ concentrations (<1 μM), suggesting that retinol transport by full-length CaCaM-STRA6 may be regulated by Ca2+-signaling. The effect of CaM on multiple full-length target proteins was then discussed to further describe CaM allostery. Together, these studies lead to an improved understanding of Ca2+ signaling, CBP allostery and CBP-target- and ligand- interaction specificity.
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  The feasibility of actigraphy and care journals to examine sleep-wake patterns of preterm infants in the NICU

  Hunt, Natalie; Johantgen, Mary E. (2022)

  Problem: Sleep disruption in preterm infants can have negative short- and long-term effects, such as behavioral changes, poor emotional regulation, and poor verbal skills. Purpose: Assess the feasibility of using actigraphy and infant care journals to examine sleep-wake patterns and care activities of premature infants in the NICU. Methods: An observational study was done in a level IV NICU with clinically stable infants 28-32 weeks gestation at DOL 2-7. Sleep-wake patterns were examined using the Actiwatch 2 and care activities were assessed using an infant care journal over 84 hours continuously. Scoring rules for actigraph data were developed to determine rest intervals. Sleep variables collected included total sleep time, percent of sleep, and number of wake bouts during sleep period. Care activities were documented in journals, including length of activities and whether infants were asleep or awake prior to and after the activity. Actigraphy data was assess using the Actiware software and, along with care activity data, were analyzed using SPSS. Feasibility is assessed through acceptability, implementation, practicality, and limited efficacy. Results: Data were analyzed on 10 infants. Rest intervals were 155 minutes ±5.3 minutes on average with infants spending an average of 67.1% ± 11.1% asleep. Average daily total sleep time was 902.5 minutes ±158.6 minutes, or 64.3% (± 10.85%) and care clustered with routine care made up 75.5% of the documented care activities. Conclusions: The feasibility of using actigraphy to examine sleep-wake patterns was supported, despite subjectivity. Sleep data were similar to previous literature, though the this sample is of younger gestation. While the amount of activities were less than previous studies, with modifications to methodology, data collection could be improved.
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  Diagnostic Stewardship to Improve the Diagnosis of Urinary Tract Infections

  Claeys, Kimberly; Morgan, Daniel J., M.D., M.S. (2022)

  Urinary tract infections (UTIs) are common infections, however diagnosis remains challenging. Asymptomatic bacteriuria (positive urine cultures in the absence of true infection) represent a major driver of inappropriate diagnosis and unnecessary antimicrobials. Curbing the impact of inappropriate diagnosis of UTIs often falls upon antimicrobial stewardship (AMS) programs, however AMS interventions occur after diagnosis. In contrast, diagnostic stewardship is an innovative approach that intervenes at across the diagnostic pathway (urine culture ordering, processing, and reporting), upstream of traditional AMS activities, and has the potential to significantly impact diagnosis and management decisions. Best practices and optimal implementation of diagnostic stewardship, however, have yet to be fully described. Specific Aim 1 evaluated the feasibility and impact of conditional urine testing. This study compared three Veterans Affairs (VA) hospitals that had implemented this diagnostic stewardship intervention compared to three control sites. Conditional urine testing, particularly with more restrictive requirements, was associated with significantly fewer urine cultures performed. Importantly, there were no serious unintended consequences, such as secondary bacteremia, related to missed diagnosis of UTI at the population level. Specific Aim 2 sought to comprehensively review available diagnostic stewardship interventions and create expert guidance for best practices to improve diagnosis of UTI. Traditionally, guidelines for diagnosis and management of infections do not address diagnostic stewardship interventions, particularly those at the institutional or health system level. This Aim distilled available literature into best practices for urine culture stewardship through expert guidance and will serve as a guide for clinicians in the future. Specific Aim 3 applied implementation science and user-centered design principles to develop urine culture diagnostic stewardship interventions for three geographically diverse VA medical centers. This included interventions focused on urine culture ordering, processing, and reporting. Prototype intervention tools were refined using qualitative methods to facilitate local implementation at each participating site and ensure long-term intervention success. Patient-centered diagnostic stewardship can lead to improved patient outcomes and less antimicrobial misuse. The results of these three aims will be used to demonstrate the impact of urine culture diagnostic stewardship best practices on these outcomes and ultimately lead to an implementation toolkit to be used nationwide.
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  The Trueness of additive and Subtractive Zirconia Crowns

  Almutairi, Eman Sager; Masri, Radi, 1975- (2021)

  Purpose: To assess the trueness of ceramic crowns fabricated using additive technology compared to crowns fabricated using subtractive technology. Material and methods: single crown was designed using Dental Designer Software. 3D design was used to fabricate milled and printed crowns. All crowns were scanned. Geomagic software was used for analysis. Best Fit and Marginal Fit Alignment used to analyze the 3D accuracy of the crowns. One Way ANOVA used to analyze the data, and p<0.05 was considered significant. Result: Significant differences found between the additively and subtractive manufactured crowns [p=0.003,F=11.172]. Additively manufactured crowns exhibited better trueness than subtractive crowns. For the additively manufactured crowns, the Best fit Alignment showed 42.81±40.8μm deviation, and the Marginal Alignment showed a 43.11 ±38.05μm deviation. For Subtractive manufactured crowns, the deviation in Best Fit Alignment was 62.67 ±50.79μm and in Marginal Alignment was 55.35±48.69μm. Conclusion: Additive technology fulfills surface trueness criteria.

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