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Molecular and Ocular Characterization of Novel Fibulin-3 Variants Involved in Retinal Degeneration
No full textPages xi-xxiv are misnumbered as pages x-xxiii.Distinct mutations in fibulin-3 (F3), a secreted extracellular matrix glycoprotein, have been associated with various ocular diseases including Malattia Leventinese (ML) and the most common macular degenerative disease, age-related macular degeneration (AMD), which ultimately lead to vision loss. AMD is a late onset disease characterized by the progressive loss of photoreceptors and retinal pigment epithelial (RPE) cells that result in irreversible blindness. Although AMD is an etiologically complex disease due to a variety of genetic and environmental risk factors, insight into its pathogenesis can be gained by studying phenotypically similar early-onset monogenic macular diseases. One such disease is ML, a rare macular dystrophy caused by an autosomal dominant Arg345Trp (R345W) mutation in the F3 protein. Previous research has demonstrated that the R345W mutation leads to protein misfolding, inefficient secretion, and accumulation at higher intracellular steady state levels in cultured cells. However, it remained unclear whether other potentially pathogenic or clinically-identified F3 variants recently reported in the human population also share features similar to that of R345W. We hypothesized that secretion defects in one or more F3 mutants may be a shared mechanism that ultimately contributes to ocular disease. First, I characterized 15 clinically-identified F3 mutations, some of which were identified in patients with AMD, primary open-angle glaucoma (POAG), or had non-discript retinal abnormalities. I found that of the mutants tested, only a single F3 variant, L451F, presented with a significant secretion defect as well as similarities in its biochemical and molecular properties to that of R345W. Subsequently, I generated a retinal disease mouse model of the L451F mutant utilizing recombinant adeno-associated virus (rAAV) in order to robustly evaluate disease phenotypes and uncover how L451F and other F3 mutations (i.e. R345W) are involved in retinal degeneration
Validation of AXL as a Therapeutic Target in Pancreatic Cancer
No full textPancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the US, has a high metastatic rate that is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, has been identified as a critical factor that drives metastasis and immune suppression in many cancer types. Here we demonstrate that AXL-deficient PDA bearing mice have longer median survival, smaller tumors, fewer metastases and higher sensitivity to gemcitabine treatment compared to AXL wild-type (WT) PDA mice. AXL-deficient PDA displays a more differentiated histology, lower equilibrative nucleoside transporter 1 expression and has a more inflammatory and active immune microenvironment, all of which likely contribute to improved survival. As a result, AXL-deficient PDA treated with gemcitabine show more DNA damage (γH2AX) compared to WT PDA treated with gemcitabine. Single cell RNA sequencing of PDA genetically engineered mouse models reveals that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and AXL expression correlates with classic markers of mesenchymal tumor cells. This AXL-positive mesenchymal PDA cell population is critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target for pancreatic cancer patients. Multiple pharmacological strategies to inhibit AXL have been explored in pre-clinical models of PDA with promising therapeutic efficacy and similar phenotypes as genetically manipulation of AXL. These results provide a robust rationale for clinical studies aimed at investigating the effect of AXL inhibition in conjunction with standard therapy in pancreatic cancer patients
Assess Effectiveness of Opioid Prescription Policies for Acute Pain Management
Get PDFBACKGROUND: In 2017, the Department of Health and Human Services (HHS) declared the Opioid Crisis a public health emergency. Regulatory agencies and institutions have adopted several guidelines to ensure opioids are prescribed appropriately. In October 2014, the DEA changed the schedule of hydrocodone combination products (HCPs) from schedule III to schedule II narcotics. This led to a substantial rise in Tylenol 3 prescriptions at the University of Texas at Southwestern Medical Center (UTSW) due to the institutional guideline that prevents residents from prescribing schedule II narcotics without documented approval from an attending physician.
OBJECTIVE: We sought to evaluate whether the UTSW guideline preventing residents from prescribing schedule II narcotics serves to improve patient safety and pain management.
METHODS: Prescription data and associated patient demographic data was pulled directly from the UTSW electronic medical record (EMR) for one year prior to and following the rescheduling of HCPs. Additional data was pulled for the 2019 and 2020 calendar years. The proportion of T3 and schedule II narcotic prescriptions was calculated for all time periods and stratified for age, race, provider type, and department.
RESULTS: One year before the rescheduling of hydrocodone, the vast majority of prescriptions were schedule II narcotics at 98.92% and T3 was very rarely prescribed at 1.08%. In 2014 - 2015 following the rescheduling of HCPs, there was an overall decrease in opioid prescriptions and the proportion of T3 prescriptions rose to 49.94%. In 2019 and 2020, the overall number of opioid prescriptions increased to 17,297 in 2019 and 15,395 in 2020 and the proportion of T3 prescriptions decreased to 37.12% and 33.89% respectively.
CONCLUSION: The rescheduling of HCPs led to the dramatic shift in Tylenol 3 prescriptions, indicating that regulatory agencies and institutional guidelines are driving prescribing habits. Tylenol 3 is being prescribed at a significant rate however, information regarding its addictive potential, metabolic effects, and potential adverse effects remains relatively unknown. The drug policies and institutional guidelines discussed disproportionately affect people of color and lower socioeconomic class
Identification of an Interferon Signaling Pathway Linking Membrane Cholesterol Accessibility to Host Defense Against Pathogens
Get PDFInterferon-γ (IFN-γ) is a multipotent cytokine that is critical to the host innate immune defense against bacterial infection, and functions through transcriptional induction of hundreds of IFN-γ stimulated genes (γ-ISGs). However, the antibacterial roles of many γ-ISGs remain poorly defined. Here, I describe my efforts to characterize mechanisms by which specific γ-ISGs confer cell-intrinsic immunity against bacterial pathogens. Unexpectedly, I found that IFN-γ-activated macrophages secreted a soluble product that potently inhibited infection of the Gram-positive intracellular pathogen Listeria monocytogenes. To identify this factor, I first created a cDNA lentiviral library of more than 400 highly representative γ-ISGs and carried out a gain-of-function flow cytometry screen to determine the effect of each gene on infection. The results of this screen identified eight genes that potently reduce infection. Next, I determined whether these γ-ISGs produced a soluble molecule that could suppress L. monocytogenes infection in trans. Notably, conditioned media from Cholesterol 25-Hydroxylase (CH25H)-expressing cells potently enhanced bacterial resistance of naive cells, suggesting it may be responsible for the previously observed activity in macrophages. Indeed, Ch25h-/- macrophages failed to produce a soluble antibacterial metabolite, while the enzymatic product of CH25H, 25-Hydroxycholesterol (25HC), potently inhibited L. monocytogenes infection. I demonstrated that 25HC inhibits infection of non-phagocytic cells from diverse tissue lineages, and that administration of 25HC to mice significantly reduced bacterial burden in an oral gavage model. Furthermore, I found that 25HC blocks L. monocytogenes cell-to-cell spread by attenuating the formation of plasma membrane protrusions. In collaboration with the post-doctoral fellow Kristen Johnson, I have used toxin-based biosensors to determine that activation of acyl-CoA: cholesterol acyltransferase (ACAT) rapidly mobilized a specific pool of cholesterol termed “accessible cholesterol” from the plasma membrane (PM) to the ER. Importantly, the antibacterial function of 25HC was dependent on this ability to deplete PM accessible cholesterol. Together, these studies have uncovered a heretofore unknown mechanism by which IFN-mediated reorganization of the PM restricts dissemination of intracellular pathogens
Effects of Aerobic Exercise Training on Carotid Arterial Stiffness and Brain Health in Traumatic Brain Injury
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.BACKGROUND: Vascular dysfunction and hypoperfusion in the brain are common after traumatic brain injury (TBI). The brain is perfused by elastic central arteries that decrease in compliance with age, leading to decreased ability to dampen hemodynamic pulsatility and decreased continuous blood flow in cerebral vasculature. Aerobic exercise improves cardiorespiratory fitness, health status, quality of life, and cognitive function as well as decrease mortality in normal adults. In this regard, physical activity, particularly aerobic exercise training (AET), may have favorable effects on TBI-related vascular and cerebral blood flow (CBF) changes. However, knowledge gaps regarding the effects of chronic TBI on vascular function still exist. Furthermore, little is known about the effect of AET on carotid arterial stiffness and CBF in patients with TBI with pre-existing brain injury.
OBJECTIVE: We hypothesized that TBI patients may continue to suffer from vascular impairments at the chronic stage and may benefit from aerobic exercise with improvements in cardiorespiratory fitness, decrease in carotid arterial stiffness, and improved brain health.
METHODS: Twenty-three participants with a history of mild to severe TBI and twenty-five age- gender-fitness level-matched participants with no history of TBI were recruited for the normal control group (non-TBI). All participants were 18-65 years old and have a sedentary lifestyle. The groups were divided into young (19-44 years old) and middle-aged (45-63 years old) for further analysis. Carotid arterial compliance was measured using common carotid artery echography and applanation tonometry. Transcranial Doppler was used to measure the cerebral blood flow velocities. Carotid arterial compliance was calculated from the carotid diameters and blood pressures.
Seventeen of the TBI survivors (age: 48±13 years, 10 women) with persistent neurological symptoms 6-60 months after initial injury were randomized to 3-month moderate-intensity AET or control stretching program (SAT) and completed the interventions. Among them, 10 sustained mild TBI and 7 had moderate to severe TBI. Cardiorespiratory fitness was assessed by peak oxygen uptake (VO2peak) using a modified Astrand-Saltin treadmill protocol. Carotid arterial compliance was measured as measured. Neuropsychological function was assessed using the NIH Toolbox cognition battery and the PROMIS assessment.
RESULTS: In the cross-sectional portion of the study, hemodynamic parameters indicate that the TBI group had higher brachial blood pressure (116.4 ± 10.4 vs. 109.8 ± 9.3 mmHg, p < 0.05). and carotid systolic blood pressure (113.0 ± 11.0 vs. 102.8 ± 10.6 mmHg, p < 0.05) than the non-TBI groups at rest. Arterial compliance was significantly lower in the TBI vs. non-TBI group (0.101 + 0.025 vs. 0.120 + 0.029 mm2/mmHg, p < 0.05). Additionally, cerebrovascular resistance was significantly higher in the TBI vs. non-TBI group (0.168 + 0.0332 vs. 0.145 + 0.0290 mmHg/mL/min, p < 0.05).
Ten participants were randomized to AET group and seven to stretching group. No age, gender, or VO2peak differences were noted at baseline between AET and stretching groups. The duration of the intervention was twelve weeks. Although no statistically significant changes were observed following the intervention, different trends were observed. VO2peak increased by 7% in AET yet decreased by 4% in stretching; arterial compliance increased by 12 % in AET and decreased by 2% in stretching; NIH Toolbox fluid composite score, which assessed adaptability to new experience, improved by 15% in AET and 9% in stretching; and the NIH Toolbox total composite score, which involves adaptability to new experiences as well as past knowledge and skills, improved by 7% in AET versus 4% in stretching.
CONCLUSION: The results suggest that TBI is associated with increased blood pressure, which is consistent with existing literature. The elevated blood pressure potentially leads is associated with decreased arterial compliance and increased resistance in the cerebral vasculature. Previous literature suggests that decreased cerebral blood flow may be associated with the cognitive impairment in TBI patients.
For the longitudinal portion of the study, the physiological measurements (includingVO2peak, arterial compliance, and pulsatility index) and cognitive measurements suggest the potential positive effect of AET on physiological and cognitive improvement in patients with TBI. Physical activity, both SAT and AET, can improve arterial compliance in patients with chronic TBI
KAP1 Is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs
No full textPrecise control of the RNA polymerase II (Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Much previous work to understand individual transcription steps, but insight into how these steps might be integrated is lacking. Here we reveal a mechanism that integrates Pol II cycle transitions. Surprisingly, the transcription factor KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters thereby guaranteeing a continuous progression of Pol II entry to, and exit from, the pause state. Upon chromatin docking, KAP1 first associates with Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands thereby enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by colorectal cancer cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation
Dissecting the Mitotic Golgi Membranes Mediated Microtubule Polymerization
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.A properly assembled astral microtubule network is required for correct mitotic spindle orientation, which is important in multiple development processes as it determines cell fate and function. The initiation and growth of astral microtubules was previously attributed to centrosomes and microtubule stabilizing proteins. Here in my dissertation research, I demonstrate that microtubules initiated by mitotic Golgi membranes contribute to the growth of astral microtubules and the proper orientation of the spindle. In turn, the microtubule initiation activity of mitotic Golgi membranes facilitates the proper inheritance of the single copy Golgi apparatus, which is essential in polarized cellular functions, including directional cell migration and secretion. Microtubule assembly is initiated by the Golgi resident protein GM130, which locally activates the spindle assembly factor TPX2 at the mitotic Golgi membranes. GM130 relieves TPX2 from inhibition by competing for importin α binding. The mitotic phosphorylation of importin α on Serine 62 by Cdk1 switches its substrate preference towards GM130 and enables the competition-based activation. The importin α S62A mutant impedes the local TPX2 activation and compromises the astral microtubules, which ultimately leads to misoriented spindles. Blocking of the GM130-importin α-TPX2 activation pathway reduces the astral microtubule growth rate. I also identified that the human GM130 homolog GLP harbors a domain that is highly similar to the TPX2 activating domain of GM130, which could potentially initiate microtubule assembly. My research reveals the novel role of mitotic Golgi membranes in astral spindle organization and the underlying mechanism that regulates this process in a spatio-temporal manner
Identification of Smaller Noncoding RNAs Produced by Mycobacterium Tuberculosis in Infected Macrophages That Regulate Mtb Growth and Survival
No full textIt is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different proteins encoded by Mtb in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. A subset includes small RNAs (sRNAs) between the sizes of 50-350 nts. The current study focused on the identification and characterization of miRNA-like sRNAs <50 nts produced by Mtb. A sRNA-centered RNA-sequencing approach was performed and a subset of Mtb-encoded smaller noncoding RNAs (sncRNAs) were identified. Thirty-five distinct Mtb-encoded sncRNAs were discovered, with most being induced in infected eukaryotic cells. Three sncRNAs, sncRNA-1, sncRNA-6, and sncRNA-8, predominated the read counts. They were contained in longer RNA transcripts with stable secondary RNA stem loops and structures like precursor microRNAs. My work established that sncRNA-1 positively regulates two mycobacterial transcripts involved in oleic acid biosynthesis. Loss- and gain- of-function approaches reveal that sncRNA-1 enhances Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Given evidence that RNA processing enzymes were involved in the formation of the sncRNAs, different components of core RNA degradosome were characterized for their ability to process the precursor forms of the sncRNAs. My work revealed that PNPase degrades sncRNA-8 and preliminary evidence suggests that sncRNA-1 is also likely a target, which could be critical in the oleic acid deficient media. Overall, my study reveals that Mtb produces a set of sncRNAs in infected cells, with one modulating mycobacterial gene expression and mycobacterial pathogenicity coupled to oleic acid biogenesis. Future studies will address the functions of other sncRNAs and focus on the identification of sncRNA processing enzymes