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Integrated Multimodal Genomic Analyses Reveal Novel Mechanisms of Glucocorticoid Resistance in Acute Lymphoblastic Leukemia
Get PDFoai:dc.uthsc.edu:dissertations-1516Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Much has been discovered in recent decades regarding ALL biology, and the outcome of patients with ALL has vastly improved, especially in pediatric ALL patients. Despite very promising overall cure rates, patients who relapse have a greatly decreased prognosis with survival rates ranging from 30-60%. These numbers stand to improve even further with new targeted therapies that seek to improve or maintain cure rates while reducing treatment related toxicities which affect patients both acutely and chronically. Glucocorticoids (GCs) are essential components of modern chemotherapeutic intervention for ALL. Resistance to glucocorticoids is an important factor in determining early treatment response and overall patient survival. Reduction of glucocorticoid induced toxicities, such as osteonecrosis, can significantly affect patient quality of life and are associated with high dose glucocorticoid treatment in pediatric patients. Both endogenous and exogenous glucocorticoids exert their mechanism of action through various pleiotropic effects that regulate numerous cellular functions and can cause selective cytotoxicity in lymphoid malignancies. The complex mechanism of action of glucocorticoids is evident in the number of diverse clinically relevant molecular pathways that have been previously associated with resistance to glucocorticoids in ALL.The identification of genomic and epigenomic mechanisms of glucocorticoid resistance are important for improving ALL treatment outcomes. We used an agnostic genome-wide method to interrogate multiple types of genomic information (mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels) in primary human acute lymphoblastic leuk
Validation and Application of a Novel Target-Based Whole-Cell Screen to Identify Antifungal Compounds
Get PDFTraditional approaches to drug discovery are inefficient and have several key limitations that constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically-active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein were constructed, tagged with spectrally distinct fluorescent proteins (FPs), and mixed together. The pooled strains were then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture was compared by measuring the intensity of the corresponding FP tags. Chemically-induced population shifts indicated that the bioactivity of a small molecule was dependent upon the target protein’s abundance and thus established a functional interaction. Here, we described the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validated this approach using dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to an extensive array of molecular targets and in any genetically tractable microbe.
While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase, the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1), we were able to demonstrate that Dfr1p is essential for growth in vitro. Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when DFR1 expression is suppressed, or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase, and by inference other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.
Both the fungal fatty acid synthase and desaturase have been established as essential for fungal growth, virulence in a systemic infection, and are structurally distinct from their mammalian counterparts. However, there has been little success in developing clinically relevant inhibitors that target the fungal fatty acid biosynthetic pathway, even though there are agents that inhibit the bacterial orthologs. The purpose of this study was to identify physiologically-active compounds that interact with the fungal fatty acid biosynthetic pathway. Given the challenging nature of targeting these enzymes using traditional approaches, we attempted to apply the newly validated TAFiS method, a target-based whole-cell screen. To accomplish this, Candida albicans strains with altered expression of the fungal fatty acid synthase or the desaturase were used to screen a total of 41,261 small molecules. The primary hits were confirmed through dose responses, and 16 compounds had differential activity on the strains with altered desaturase abundance, indicating a functional chemical-target interaction. Additionally, the growth inhibition exerted by the most potent hits was restored in the presence of exogenous fatty acids, further supporting their inhibition of the fatty acid desaturase. Of the 16 confirmed hits, 11 were structurally related, containing a hydrazide core that was essential for interaction with the desaturase. Collectively, these results have identified a novel scaffold that interacts with the fungal fatty acid desaturase and can be further developed into experimental therapeutics. It also confirms that the TAFiS methodology is highly effective in identifying small molecules that functionally interact with targets that are poorly suited to traditional chemical screening approaches
Model-Based Dose-Exposure-Response Assessment for Lead and Backup Spectinamide in a Mouse Model of Tuberculosis
Get PDFDespite decades of research, tuberculosis remains the oldest pathogen-based disease that is the leading cause of death from a single infectious agent. Among many anti-tubercular therapies under investigation, the semisynthetic compounds spectinamides are a promising novel class of anti-tuberculosis agents. One such lead candidate, spectinamide 1810, and backup spectinamide 1599 have demonstrated excellent efficacy, safety, and drug-like properties in various in vitro and in vivo assessments. The dose-ranging and dose fractionation studies were designed to characterize the dose-exposure-response relationship for lead and backup spectinamide in a mouse model of Mycobacterium tuberculosis infection. In this current study, we used 26 and 23 combinations of dose level and dosing frequency for the lead and backup spectinamide, respectively. The dedicated pharmacokinetic studies with a collection of series of blood samples were conducted in healthy animals. Population pharmacokinetic analysis was performed using non-linear mixed effect modeling to estimate pharmacokinetic parameters in healthy animals. The Bayesian principles were applied for reliable pharmacokinetic estimation in infected animals by using informed priors obtained from healthy animals. The individual pharmacokinetic parameters were obtained for infected animals through post-hoc estimation and subsequently used for pharmacokinetic/-pharmacodynamic (PK/PD) indices and mechanism-based PK/PD modeling.
The obtained data on spectinamides’ plasma concentrations and counts of colony-forming units were analyzed using a PK/PD approach as well as classical anti-infective PK/PD indices. The population pharmacokinetic analysis results suggest that there is no difference in the pharmacokinetic parameters of lead and backup spectinamide in infected animals as compared to healthy animals. The PK/PD index analysis showed that the efficacy of spectinamide 1810 is largely driven by concentration (Cmax/MIC) and exposure (AUC/MIC) rather than a threshold minimum inhibitory level (T\u3eMIC). Although similar results were obtained for spectinamide 1599 in previously performed in vitro experiments, in the present in vivo studies, spectinamide 1599 did not demonstrate the expected correlation between efficacy and PK/PD indices. Therefore, we could not identify major drivers for the efficacy of this compound. Additionally, a novel mechanism-based PK/PD model with consideration to post-antibiotic effect could adequately describe the exposure-response relationship for lead and backup spectinamide. This supports the idea that the in vitro observed post-antibiotic effect of these spectinamides can translate to the in vivo situation, as well. Altogether we suggest, the obtained results and pharmacometric model for the exposure-response relationship of lead and backup spectinamides provide a rational basis for dose selection for future efficacy studies of these compounds against Mycobacterium tuberculosis in mice and other animal species
Eukaryotic Initiation Factor 5A2 (EIF5A2) Contributes to Ovarian Tumor Growth and Metastasis
Get PDFOvarian cancer has the highest mortality rate among all gynecological malignancies due to lack of effective biomarkers for early diagnosis. The majority of ovarian cancer patients are already at an advanced stage when diagnosed. In addition, ovarian cancers often become chemoresistant and metastatic, and recur following initial chemotherapy
Infant Massage and Bonding
Get PDFThis project aims to appraise evidence of the effectiveness of infant massage for increasing social-emotional bonding. In our research, we focused on two studies considered Level I evidence and three studies considered Level II evidence. Study designs include quasi-experimental and randomized controlled trials. Strong evidence supports that infant massage increases social-emotional bonding and attachment. Based on the evidence, implementation varied from once per day for 10 minutes to 2-3 times per day for 15 minutes. It is recommended to use infant massage in an acute care setting and in the home to increase social-emotional bonding and attachment
Effectiveness of Aerobic Exercise for Adults with Leukemia
Get PDFWe wanted to look at the most effective interventions for increasing strength and endurance in clients with Leukemia experiencing functional decline
Computer-Assisted Coding: Post ICD-10 Implementation
Get PDFComputer-assisted coding (CAC) has been around since the 1950s and is projecting to reach $4.75 Billion by 2022. However, it has not been on the hospitals’ priority list until 2014 before the implementation of ICD-10 in 2015. Computer-assisted coding is a technology software that helps streamline the coding workflow, reduce backlogs by increasing productivity, and help coders navigate through more extended, more complex charts more quickly. The technology is a type of artificial intelligence. The idea of computer-assisted became more front-line with the implementation of electronic health records (EHRs) and the demands of a more restrictive reimbursement from payers. Accuracy, consistency, and, most assuredly, productivity has been of great importance to all organizations. Due to the increase in advanced technologies, computer-assisted coding has advanced in its performance. However, the question remains as to if it has lived up to the recent hype before the implementation of ICD-10 to increase productivity, accuracy, consistency, improve clinical documentation, etc. This study was conducted using a questionnaire to survey the Tennessee Health Information Management (THIMA) community members as to the effectiveness of computer-assisted coding five years after the implementation of ICD-10. The results of the survey show that there are organizations that are still not using CAC. The overall perception of the respondents feel CAC is not a must-have technology to code efficiently but, with the CAC, the overall coding process is satisfactory but still needs improvement
Considerations for small libraries in adopting virtual reality technology for medical education
Get PDFObjective: Describe considerations for small libraries considering the adoption of virtual reality technology as a resource for health sciences education.
Methods: A small academic medical library obtained grant funding to implement a virtual reality pilot project to support anatomy coursework.
Results: Observations were made related to the technology selection and purchasing process, space and safety requirements for implementing VR, student patterns of use, supplementary VR programs of interest to medical students (including gaming and meditation programs for stress relief), and staffing/scheduling/maintenance of the system.
Conclusions: Virtual reality technology is compelling for medical education; implementation resulted in increasing the library’s engagement with teaching faculty, students, alumni relations, and the curriculum. However, implementation requires careful consideration related to staffing, technology choices, use cases, safety, and other matters. Small medical libraries should be aware of various limitations and considerations prior to making large investments of money, space, or personnel time. Examples will be provided of concrete considerations for libraries
A Comparative Study of the Distribution and Density of Retinal Ganglion Cells and Visual Acuity in Bothrops Snakes with Differences in the Habitat Use
No full textPurpose: The density and distribution of neurons in retinas of vertebrates are associated with specific behaviors and species ecology. The visual system of snakes has adaptations to the species’ habitats, circadian rhythm, and foraging strategies. A previous study revealed a ventral area centralis in a terrestrial snake and a visual streak in a close related arboreal species. In this study, we compared the distribution of ganglion cell layer (GCL) cells in retinas of Viperidae snakes from the genus Bothrops, with differences in the habitat use, namely the arboreal B. insularis, and the semi-arboreal species B. jararaca and B. sazimai.
Methods: Three individuals of each species were euthanized with Thionembutal (100 mg/kg). The eyes were enucleated and retinas were dissected and fixed in 4% paraformaldehyde. Retinas were wholemounted and Nissl stained. The density of GCL cells was estimated using a stereological approach with the Stereo Investigator software (MicroBrightField), and isodensity maps were obtained using OriginPro 8.5.1. Visual acuity values were estimated based on the peak density of GCL cells and the eyes focal length.
Results: We observed a poorly defined horizontal streak in retinas of the arboreal B. insularis, with higher density in the central retina, while in the semi-arboreal B. jararaca and B. sazimai there were no defined retinal specializations, and the peak cell densities were located in the temporal region. The mean cell density was 4,405 ± 735 cells/mm2 in B. insularis, 4,606 ± 335 cells/mm2 in B. sazimai, and 4,881 ± 746cells/mm2 in B. jararaca. The estimated spatial resolutions were similar among the species and varied from 2.0 to 2.2 cycles per degree.
Conclusions: These findings reinforce the hypothesis that retinal specializations are associated with habitat use in snakes. A visual streak may assist in locomotion and hunting among branches, as indicated by the distribution of cells in B. insularis. On the other hand, peak GCL cell density in the temporal retina may facilitate striking behavior in the species that forage on the ground
Arid1a Haploinsufficiency Initiates Neural Crest Transformation in a Mouse Model of Mycn-driven Neuroblastoma
Get PDFMouse models of cancer are critical for developing therapeutic treatments for pediatric patients. Recent sequencing studies of neuroblastoma (NBL) patient tumors have uncovered precise deletions in the chromatin remodeler and tumor suppressor gene (TSG) ARID1A. Additional causal studies supported ARID1A’s candidacy as a putative 1p36 TSG in MYCN-driven NBL. This study aimed to causally test Arid1a loss during Mycn-driven NBL initiation through the development of a mouse model of high risk NBL.In this study, we modified a Mycn-driven mouse model of NBL to incorporate Cre mediated deletion of floxed Arid1a. Briefly, in freshly isolated embryonic day 9.5 primary trunk neural crest cells (NCCs), Arid1a was heterozygously and homozygously deleted in combination with Mycn overexpression. We injected these genetically modified primary cells into mice to determine if Arid1a loss collaborates with Mycn overexpression during NCC transformation. We evaluated tumor growth kinetics, characterized the pathological features and gene expression profiles of resulting tumors, and evaluated the Arid1a-dependent differentiation traits of tumors and primary NCCs in vivo and in vitro. We found that Arid1a loss caused a gene expression and phenotypic shift to an immature cell identity. Furthermore, heterozygous loss of Arid1a during transformation of primary murine NCCs resulted in tumors that pathologically and molecularly model human high-risk, MYCN amplified NBL. Gene set enrichment analysis demonstrated that gene expression profiles of Arid1a heterozygous tumor samples significantly correlated with previously established mesenchymal gene signatures found in high-risk NBL patients. Our model causally tested the clinical observation that 70% of high-risk MYCN amplified NBL patient tumors include 1p36 LOH. Our results support the hypothesis that ARID1A is a 1p36 tumor suppressor candidate that collaborates with MYCN to transform NCCs into high-risk NBL. Last, our model suggests that a shift in cell identity may be connected to NBL initiation