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Novel Kinase Inhibitor Screen on Estrogen Receptor (ER)-positive Breast Cancer and ER Mutants
No full textPurpose: Breast cancer is the second leading cause of death in women in the United States. Triple-negative breast cancer is named for its lack of estrogen (ER), progesterone (PR) receptors, and HER2. The lack of receptors makes these tumors increasingly more challenging to treat, resulting in worse clinical outcomes than other receptor-positive subtypes. Some ER-positive tumors mutate to express an ER-negative phenotype, effectively reducing sensitivity to common anti-estrogenic drugs and making them more triple-negative and metastatic. Most estrogen-receptor mutations (both naturally occurring and genetically engineered) in ER-positive breast cancers occur at the ligand-binding site, rendering them ineffective. This change in estrogen receptor effectiveness can alter signaling cascades and affect downstream events in the cell, thereby promoting uncontrolled cell growth and insensitivity to drugs. Kinases are responsible for signal propagation in cells. Therefore, alterations to estrogen receptor signaling could significantly affect kinase expression and activity. Little is known about the 538 kinases encoded in the human kinome; consequently, they must be studied more thoroughly. Suppose a kinase inhibitor drug effectively inhibits the growth of cells with mutated estrogen receptors. In that case, there may be an effective treatment option against triple-negative breast cancer and these naturally occurring mutations.
Methods:Wild-type ER-positive breast cancer cells (MCF-7) were treated with 1µM KCGS Drug Library kinase inhibiting compounds from Dr. David Drewry at the SGC at UNC Chapel Hill. After 72 hours, cells were stained with crystal violet and imaged for cellular viability and morphologic changes. In addition, ER-mutated ER-positive breast cancer cells (MCF-7 Y537S and MCF-7 S167A) were treated with the same compounds at 100nM for 72 hours and stained with crystal violet.
Results: The data from the screening indicate that eight kinase inhibitors affected the morphology and viability of Y537S and S167A mutants to a greater extent than the wild-type MCF-7 cells at a log-dose lower concentration. One kinase inhibitor affected the S167A mutant to a greater extent than the Y537S mutant at the same attention (100nM).
Conclusions:Mutations to the estrogen receptor of typically ER-positive breast cancers may give insight into the development of interventions for triple-negative breast cancers. In the future, additional ER mutants will be screened against the same kinase inhibitors. In addition, kinase inhibitors may also give insight into the kinases essential to cancer viability and metastasis
Investigating Associations Between Asthma and Nasal Abnormalities: A Computed Tomography (CT) Approach
No full textPurpose: Asthma affects over 300 million people worldwide and 3,500 people suffer asthma related deaths each year. Although there is no clear cause of asthma, approximately 90% of asthmatics suffer from cold/exercise induced bronchoconstriction, a symptom triggered by the inhalation of cold and/or dry air. As the nasal passages account for most of the heat and moisture transferred to inspired air during respiration, anatomical variation in nasal morphology may contribute to asthma development. While the existence of nasal anatomical variants is well documented, little is known about the prevalence of such variants among asthmatics. Methods: Accordingly, this study sought to investigate potential associations between asthma and three common nasal anatomical variants: septal deviations, concha bullosa, and paradoxical turbinate. This study analyzed Computer Topography (CT) scans of a diverse, mixed sex sample (n=242) from the New Mexico Decedent Image Database (NMDID). The asthmatic individuals (n= 120) were identified through associated medical records and compared to a control sample of non-asthmatics (n = 122). CT scans were analyzed using Avizo permitting qualitative coding of each anatomical variant for presence and type. Chi-square tests of independence were then used to test for differences in variant prevalence between the asthmatic and control samples. Results: The results of our study show significantly higher prevalence of concha bullosa in asthmatics compared to control individuals (χ2= 5.87, p=0.015), with 70.0% asthmatics exhibiting at least one pneumatized turbinate compared to only 54.9% of control individuals. Conclusions: This result suggests a potential relationship between the presence of conchae bullosa and asthma, possibly due to this variant negatively influencing intranasal air-conditioning capabilities. Future work employing computational fluid dynamics (CFD) analyses may be able to further elucidate the influence concha bullosa have on nasal passage air flow and conditioning. Such work could provide important insights into the role nasal anatomy may play in asthma prevalence and severity. This project was supported by Texas Center for Health Disparities grants RF00241 & RI40241Texas Center for Health Disparities grants RF00241 & RI4024
Role of methylation in risk for cognitive impairment in Mexican Americans
No full textPurpose: Mexican Americans (MAs) are the largest aging (›65 years old) and growing US ethnic minority group, with a unique risk for cognitive impairment (CI) in comparison to non-Hispanic whites (NHWs). MAs have an earlier age of onset and a risk for CI that is largely metabolism related in contrast to NHWs who have a more inflammation-based risk for CI. CI is defined in this study as individuals diagnosed with either Alzheimer’s disease (AD), or mild cognitive impairment (MCI) (a likely precursor to AD). Risk for CI is multifactorial and involves an epigenetic form of gene regulation called DNA methylation, which involves the addition of a methyl group to the cytosine base of DNA. DNA methylation patterns can be altered or possibly reversed through changes in environmental factors such as diet and lifestyle. Our aim was to identify differentially methylated sites of the genome associated with CI and determine DNA methylation profiles that are specific to MAs and NHWs. Methods: Peripheral blood was drawn from 551 Texas Alzheimer's Research and Care Consortium participants (299 MAs and 252 NHWs) and DNA was typed on the Illumina Infinium MethylationEPIC chip array, assessing >850,000 CpG genomic sites. Participants were compared according to cognitive status (control versus CI(AD/MCI)) among each ethnic group. Beta values that represent relative degree of methylation were normalized using the Beta MIxture Quantile dilation (BMIQ) method. Differential methylation between control and CI was assessed using the Chip Analysis Methylation Pipeline (ChAMP), limma and cate packages within R studio. Results: Two significant differentially methylated sites were associated with CI at an FDR-adjusted p-value threshold <0.05: cg13135255 in MAs and cg27002303 in NHWs. Three differentially methylated sites were suggestively associated with CI at an FDR-adjusted p-value threshold <0.1: cg01887506 and cg10607142 in MAs, and cg13529380 in NHWs. Four of the five significant and suggestively differentially methylated sites were hypermethylated in CI compared to normal controls, except for hypomethylated site cg13529380. The site most significantly associated with CI was cg13135255 within the CREBBP gene in MAs (FDR-adjusted p-value = 0.029). The CREBBP protein is a histone acetyltransferase, involved in epigenetic regulation, and plays a role in memory formation. Conclusion: This is the first study to report these specific CpG sites as either significantly or suggestively associated with CI among MAs and NHWs. These sites may be used in addition to other methylated sites to develop risk assessments that are ethnicity specific for CI. Following further validation and replication in other cohorts these sites may aid development of ethnic specific therapeutics that could deter or delay CI in the future.This research project was supported (in part) by funding provided to the Texas Alzheimer's Research and Care Consortium by the Darrell K Royal Texas Alzheimer's Initiative, directed by the Texas Council on Alzheimer's Disease and Related Disorders and R01AG07086
Evaluating the Effectiveness and Ethical Implications of Disclosures in Conflict of Interest Management within Research Academic Institutions
Get PDFThis capstone project delved into the efficacy and ethical dynamics of conflict of interest (COI) disclosure policies within eight Texan academic research institutions. Comprehensive analysis of the policies covered readability, ethical and practical considerations, and content with regards to the 42 CFR Part 50 Subpart F Federal regulation. Results unveiled significant inconsistencies in the readability and depth of the policies. Texas A&M University’s COI policy was a standout in readability, while many institutions' policies demonstrated higher complexity. A detailed content analysis exposed most policies adopting a legalistic tone, veering towards risk minimization. Overall, the findings advocate for more comprehensible, ethically grounded, and regularly updated COI policies in the academic realm. In evaluating the policies against the 42 CFR Part 50 Subpart F Federal regulation requirements, it was observed that while most policies emphasized educational and legal facets, only a few consistently included crucial elements like contact details and repercussions for non-compliance. Meanwhile, the examination of informed consent forms reflected the disparities in rate of COI disclosure inclusion among the institutions, indicative of divergent institutional guidelines and definitions
A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy
No full textPURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.Funded by National Institutes of Health/National Eye Institute grants (EY012231, EY028773, EY028949, EY032930, EY032931, EY033330, and EY033477)
Long-term HIF-1alpha stabilization reduces respiration, promotes mitophagy, and results in retinal cell death
Get PDFOcular hypertension during glaucoma can lead to hypoxia, activation of the HIF transcription factors, and a metabolic shift toward glycolysis. This study aims to test whether chronic HIF activation and the attendant metabolic reprogramming can initiate glaucoma-associated pathology independently of ocular hypertension. HIF-1alpha stabilization was induced in mice for 2 and 4 weeks by inhibiting prolyl hydroxylases using the small molecule Roxadustat. HIF-1alpha stabilization and the expression of its downstream bioenergetic targets were investigated in the retina by immunofluorescence, capillary electrophoresis, and biochemical enzyme activity assays. Roxadustat dosing resulted in significant stabilization of HIF-1alpha in the retina by 4 weeks, and upregulation in glycolysis-associated proteins (GLUT3, PDK-1) and enzyme activity in both neurons and glia. Accordingly, succinate dehydrogenase, mitochondrial marker MTCO1, and citrate synthase activity were significantly decreased at 4 weeks, while mitophagy was significantly increased. TUNEL assay showed significant apoptosis of cells in the retina, and PERG amplitude was significantly decreased with 4 weeks of HIF-1alpha stabilization. A significant increase in AMPK activation and glial hypertrophy, concomitant with decreases in retinal ganglion cell function and inner retina cell death suggests that chronic HIF-1alpha stabilization alone is detrimental to retina metabolic homeostasis and cellular survival
SARS-CoV-2 induced exacerbation of HbA1c in Type 2 Diabetics
No full textBackground: The Covid-19 pandemic started when severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was discovered in Wuhan, China. Although the majority of those infected suffer mild symptoms and recover, it is estimated that about 20% of infected patients can develop pneumonia, and some of these patients can develop acute respiratory distress syndrome (ARDS). Diabetes mellitus is a complex disease that affects millions of people worldwide. When it comes to type 2 diabetes mellitus the CDC reports that about 11.3% of the United States population is affected by diabetes. Case Presentation: A 56-year-old Hispanic male presented to the clinic for his routine 6-month diabetes follow-up. He had a medical history of type 2 diabetes mellitus, morbid obesity, benign hypertension, BPH without urinary obstruction, and unspecified hyperlipidemia. For his diabetes, the patient took 500 mg metformin, 4 mg glimepiride, and 2 mg/dose semaglutide. The patient had been compliant with his medication. His only relevant family history included his mother with a diagnosis of diabetes mellitus. The patient denied constitutional, cardiovascular, respiratory, and neurological ROS questions. There was no change in the patient’s medical history except that he had contracted SARS-CoV-2 three months before the visit. The patient described his symptoms, and his infection was classified as a mild version of the disease. The patient’s vitals were within normal limits, and he had a BMI of 37.3. His general, cardio, respiratory, and skin PE findings were all normal as well. HbA1c was recorded at 10.2% and his estimated average blood glucose was 242 mg/dl. Both values had increased from 7.1% and 157 mg/dl respectively since his previous visit on July 22, 2022. At this current visit, his (nonfasting) glucose was 324 mg/dl. The patient’s semaglutide was stopped and replaced with tirzepatide in hopes of reducing his HbA1c along with helping him lose weight. Unfortunately, the patient could not tolerate a higher dose of metformin. Discussion: The question remains whether this patient’s sudden increase in HbA1c of 3.1% from 7.1% to 10.2% could be attributed to the patient’s mild infection of COVID-19. A study published by Joshi & Pozzilli in 2022 in the Diabetes Research and Clinical Practice journal found that SARS-CoV-2 can dysregulate glucose homeostasis even in patients with no previous risk factors for diabetes mellitus. One report that studied the relationship between these two variables found that there was an association between severe COVID-19 and increased blood glucose. They also found that HbA1c was slightly elevated in those with severe COVID-19 compared to mild COVID-19 however, this finding did not reach significance. Physicians taking care of type 2 diabetic patients can caution their patients on the possibility of being infected with COVID-19 and worsening their A1c levels. For those patients battling a severe form of COVID-19, their A1c levels could also be measured after the infection to rule out COVID-19-induced diabetes mellitus. This case report also expands the list of long-term complications from COVID-19
Factors Influencing Parents' Decision to Use Complementary and Alternative (CAM) Therapies in Children with Chronic Conditions
No full textIntroduction/Background: Complementary and Alternative Medicine, or CAM, therapies are defined as treatments, or therapies, outside of conventional medical practices, some of which have a basis outside of traditional Western practices and are often used in children with chronic conditions. Studies have found that patients, or caregivers of patients, do not always inform their doctors when they are using a CAM therapy, with this disconnect sometimes leading to interference in their treatment plan and possibly becoming a complicating factor. Therefore, it is important to understand the factors that influence a caregiver to use CAM therapies in order to bridge the gap in knowledge linking various factors to CAM therapy usage. Methods: Participants were recruited from the UNTHSC Health Pavilion Pediatrics and Family Medicine Clinics, as well as from various social media platforms. Data was collected through a survey questionnaire that 50 total eligible participants filled out. Participants answered questions regarding their CAM therapy usage, or their willingness to. They additionally answered questions about sociodemographic factors, their child's condition, and possible physician discussion of CAM therapies. Data was analyzed using descriptive statistics comparing CAM therapy usage to the variables mentioned previously. Results: Type of chronic condition was not associated with an increase in CAM therapy usage. 83.3% of participants whose child's condition was more severe reported having used CAM therapies while 76.9% of participants whose child's condition was less severe reported having used CAM therapies. Sociodemographic factors such as child race/ethnicity (p= 0.412), caregiver race/ethnicity (p=0.236), caregiver education level (p=0.180), caregiver marital status (p= 1.000), caregiver employment status (p=0.575), and reliable form of transportation (unable to perform test) were not associated with CAM therapy usage. Physician discussion of the following CAM therapies were associated with an increased usage, or willingness to use those CAM therapies: Botanical drugs/natural product drugs (p=0.032), breathing and relaxation techniques (0.009), dietary supplements (0.002), and psychotherapy (0.040). Conclusion: There was a trend possibly associating CAM therapy usage and severity; however, statistical tests were unable to be performed and prevented statistically backed conclusions to be made. Physician discussion of certain CAM therapies is associated with an increase in participants using those CAM therapies, or being willing to use them. Further studies with larger sample sizes gathered from more diverse recruitment sites are needed to better explore these relationships and possible factors that may influence a caregiver to use CAM therapies for their child with a chronic condition(s)
Population-Specific mtDNA Indices of Mitochondrial Stress Associated with Alzheimer's Disease in Mexican Americans and Non-Hispanic Whites
No full textAge is known to be the biggest risk factor for Alzheimer's disease (AD), and Mexican Americans (MAs) are the fastest-aging ethnic group in America. This puts MAs at a uniquely elevated risk for AD. This study aimed to identify ethnicity-specific markers for mitochondrial dysfunction associated with cognitive decline and detectable in peripheral blood cells (PBMCs). Specifically, copy numbers of mtDNA and nuclear DNA in PBMCs and blood plasma from non-Hispanic white and Mexican American participants were assessed by qPCR as potential markers of mitochondrial dysfunction and increased risk for cognitive decline. Evidence from these experiments shows that there are ethnicity-specific markers for this blood-based phenotype of mitochondrial dysfunction, which may also be indicative of an increased risk for cognitive decline and Alzheimer's disease
Structural optimization of VU0609159 as an activator of Slack potassium channels
No full textPurpose: Slack (Slo2.2) is a sodium-activated potassium channel widely expressed throughout the brain and encoded by the KCNT1 gene. This channel modulates the firing patterns and general excitability of many types of neurons, contributing to neuronal resting membrane potential, action potential repolarization, and afterhyperpolarization. Increasing evidence suggests that channelopathies that alter Slack activity, triggers cognitive dysfunction, as has been found for Fragile X Syndrome (FXS), the most common cause of intellectual disability (ID) and inherited autism. FXS results from the absence of fragile X mental retardation protein (FMRP) which interacts directly with Slack channels to regulate outward currents termed IKNa in neuronal cells. Prior studies involving animal models of FXS demonstrated that lack of FMRP leads to reduced IKNa currents due to diminished Slack activity, affecting neuronal function. We therefore hypothesize that small molecule activators of Slack have potential utility as therapeutics for neurological disorders associated with Slack hypofunction. Thus, the objective of this study is to discover small molecule activators of Slack potassium channels that may be used as in vitro probes to investigate such a hypothesis. A high-throughput screen using a thallium (Tl+) flux assay identified the hit compound VU0609159 (VU159) as a moderately potent Slack activator. Here we report our efforts to develop structure-activity relationships (SAR) in the VU159 series through an iterative, systematic optimization plan using parallel library synthesis.
Method: Our approach involved identifying multiple regions of VU159 that could be readily diversified and using short efficient synthetic routes to produce small libraries of analogs. Systematic substitution using a variety of monomers was carried out around the western benzoxazolone ring, the central amide and linker region as well as the eastern aromatic ring. Structure and purity of all analogs were confirmed using spectra obtained from a Bruker Fourier 300HD NMR spectrometer and an Agilent 6230 time-of-flight LC/MS. Cellular activity was then evaluated using a Tl+ flux assay in HEK-293 cells that stably express wild-type (WT) Slack channels.
Results: Each region of the VU159 scaffold proved tolerant of modification to some degree. Multiple fused heterocyclic rings proved competent replacements for the benzoxazolone ring with some analogs providing superior potency. Synthesis of alkylated linkers identified preferred enantiomers. Secondary amides were preferred to tertiary amides. Likewise, cyclic linkers were generally less effective than acyclic linkers. Finally, substitution of the eastern aryl ring was allowed with some analogs demonstrating enhanced potency.
Conclusion: Our systematic optimization plan has identified multiple slack activator analogs with improved activity relative to VU159. Multiple regions of the scaffolds are amenable to SAR development, which greatly enhances the probability of reaching our goal of highly optimized in vitro probes. Additional modifications, including preparation of analogs that combine optimal features, could provide additional SAR and analogs with optimal potency for use as an in vitro probe.We gratefully acknowledge the National Institute of Mental Health (R21MH125257) for their support of our research directed toward the discovery of new small molecule activators of Slack potassium channels