2022 Graduating Senior Scholar Abstracts
Class of Summer 2022
Graduating Scholar Abstracts
Effects of Mindfulness on Posttraumatic Stress and Chronic Pain: A Scoping Review
Pablo Adrian Garcia-Tovar, M. Danet Lapiz Bluhm, PhD, RN, MSCI, ANEF, FAAN
Introduction: Post-traumatic stress disorder (PTSD) is a severe chronic disorder characterized by symptoms of re-experiencing, avoidance, negative alterations in cognition and mood, and marked alterations in arousal and reactivity following exposure to a traumatic event. It often is associated with other mental health conditions, polypharmacy, chronic pain, decreased well-being, and risk of suicide. About 15-35% of individuals with PTSD experience chronic pain. PTSD is common among veterans who have experienced significant trauma as a result of their military service. Treatments for PTSD include evidence-based non-pharmacological (i.e., prolonged exposure, cognitive processing therapy, and eye movement desensitization and reprocessing) and pharmacologic (i.e., serotonin reuptake inhibitors) interventions. However, attrition rates for these treatment interventions are reportedly high (35-40%). Mindfulness-based therapies have been shown to be as effective as traditional treatments while having significantly lower drop-out rates. The aim of this review is to synthesize current studies on the impact of mindfulness on PTSD symptoms and chronic pain, offering valuable information for healthcare providers to improve patient care.
Methods: The review is guided by the clinical question: “What are the effects of mindfulness-based treatments for PTSD and chronic pain?” A search of the PubMed and Scopus databases was conducted using the terms “mindfulness,” “post traumatic stress,” and “chronic pain,” with the time frame of 2018 to 2024. The initial search yielded 73 articles, with 15 relevant studies; 10 studies were on veterans and five (5) among civilians. Articles were assessed for risk of bias and their key data were summarized in evidence synthesis tables. Data were qualitatively synthesized according to emerging themes (i.e., measurements, symptom reduction, and populations).
Results: Studies measured pain and PTSD through self-reported data (i.e., numerical scales and checklists) or externally measured factors (i.e., cardiac data, sleep data, and brain activity, medical examinations and physiological measurements). Results support a positive correlation between mindfulness treatments and reduction of PTSD and chronic pain symptoms. All studies reported at least moderate improvements in chronic pain, stress, PTSD symptoms, and mood, with benefits such as extending quality of life, brain activity, and trauma-related symptoms. Some studies reported long lasting effects at three (3), six (6), and 12 months. The effects of mindfulness treatments were consistent across veterans and civilian populations, as well as genders.
Conclusion: There is strong evidence to support mindfulness-based therapies as an effective treatment for PTSD and chronic pain. The benefits of mindfulness have led the U.S. Department of Veteran Affairs to develop the Mindfulness Coach app, which helps the at-risk veteran populations to reduce stress, improve sleep, enhance concentration, and alleviate anxiety and depression. Further studies are needed to determine the uptake of mindfulness-based interventions in the community, especially among populations at risk.
Comparison of the Composition of the Oral and Gut Microbiomes in Hispanic Individuals with Dementia
Alvin Philip, Yannick Wadop Ngouongo, Tiffany Kautz, Bernard Fongang
Glenn Biggs Institute for Alzheimer’s & Neurodegenerative Disease, University of Texas Health Science Center, San Antonio, TX, USA
Alzheimer’s Disease (AD) poses a significant challenge due to its debilitating nature and the absence of effective treatment options. Alterations in the oral or gut microbiotas have been reported in cognitive impairment conditions, including individuals with mild cognitive impairment (MCI) or AD dementia. While research into the gastrointestinal microbiome holds promise, progress has been hindered by the limited return rate of stool samples. Thus, novel strategies for gathering data on AD patients are imperative. Salivary testing emerges as a potential approach for specimen collection, offering a greater return rate than stool samples. However, the differences between the oral and gastrointestinal microbiome in AD remains understudied. In this cross-sectional study, we compared alterations in the composition of the oral and gut microbiotas of individuals (N=32, %F = 66.7, Age = 70.2±6.4) with Normal Cognition (NC, 15), MCI (13), and AD (4) recruited in San Antonio. We used the gastrointestinal and saliva microbiome features (abundance and diversity) obtained through 16S rRNA gene sequencing of stool and saliva samples to analyze the bacterial composition of each participant. We conducted linear discriminant effect size and differential abundant analysis to identify gut and saliva features statistically different between individuals when condition status progresses from NC to Dementia (adjusted p-value < 0.005). Diversity analysis using Shannon index revealed that there was no difference in oral microbial diversity between NC and MCI individuals, as indicated by the Wilcoxon test (P=0.32). However, NC and MCI participants had a significantly richer and more diverse oral microbiome compared to those with AD (NC vs AD: P=0.027; MCI vs. AD: P=0.032). Suggesting that NC and MCI individuals had a more resilient oral microbiome with high resistance to pathogens than those in AD group. Conversely, we found that there was no difference in gut microbial diversity among the three groups (NC vs MCI: P =0.27; NC vs AD: P =0.15; MCI vs. AD: P=0.56).The beta diversity analysis showed that there was no difference of the between stool/saliva samples from NC, MCI, and AD. Key differences were identified in the microbiomes between NC, MCI, and AD groups when applying the linear discriminant analysis effect size (LEfSe) algorithm. LEfSe analysis (with LDA score cut off > 2) revealed MCI individuals had a significantly increased abundance of oral bacteria Selenomonadaceae and Saccharimonadaceae. For the gut microbiome, the bacteria Erysipelotrichales, Negativibacillus, Holdemania, Family XIII UCG-001, and Defluvitaleaceae were enriched in NC individuals. The gut bacteria Enterobacteriaceae and Enterobacterales exhibited an increase prevalence in MCI group. While the bacteria Butyricicoccus and Paludicola were higher abundant in AD group. Our results demonstrated alterations in the composition of the oral and gut microbiotas between different stages of AD. The alterations of the oral and gut microbiomes across the three groups predominantly involved anti-inflammation-promoting bacteria belonging to Firmicutes phylum and inflammation-promoting bacteria from Proteobacteria phylum. Further studies are needed to
validate these results.
Transcriptomic profiling of cardiovascular and dementia risk factors
Amely Chaj Ulloa, Yannick Wadop Ngouongo, Tiffany Kautz, Jayandra Himali, Bernard Fongang
Glenn Biggs Institute for Alzheimer’s & Neurodegenerative Disease, University of Texas Health Science Center, San Antonio, TX, USA
The American Heart Association has introduced the Life Essential 8 (LE8) as a set of cardiovascular risk factors that can be used to promote and maintain cardiovascular health (CVH). Better CVH is associated with reduced risk for heart disease, stroke, cancer, and dementia, a term for the impaired ability to remember, think, or make decisions that interfere with daily life. However, the molecular mechanisms linking cardiovascular risk and dementia biomarkers are still to be determined. In this pilot study, we selected two LE8 components, smoking (SM) and blood pressure (BP), to assess at the transcriptomic level, their association with imaging and cognitive markers of dementia. We used LE8, imaging, and cognitive assessment data from 545 individuals of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. LE8 metrics were stratified as Healthy (BP (<120/80 mm Hg, Non-smoking status)) and Poor (for all others). We selected the Hippocampal volume and MoCA as dementia markers. Gene expression data was normalized using the cumulative sum scaling (CSS) method, and differential expression analysis was conducted using MaAslin2 implemented in R software, resulting in significant differential expressed genes (DEGs) that identify overlapping genetic functions associated with cognitive function (normal cognition, mild cognitive impairment, dementia). Metascape software was utilized to explore the functional role GO/KEGG of enriched genes in each of the groups. We determined several differential expressed genes among BP groups, SM groups, as well as cognitive status. Our results indicated there were fifteenth differential expressed genes that overlapped between SM, BP, and both Normal and Dementia groups. These genes include ARHGAP9, BEST1, C1orf56, C3orf62, ERGIC1, FCHSD2, KLF7, MGAM, PGM1, PPM1F, PSEN1, PTPRE, RAD23B, RBMS2, and SLC44A2. Our results demonstrated that several key genes overlap among the three markers, highlighting the connection between BP, SM, and cognition function. Pathway enrichment analysis via Metascape also revealed significant biological processes and biomarkers that overlap among BP, SM, and cognitive status, including neutrophil degranulation, regulation of the MAPK cascade, vesicle-mediated transport, myeloid leukocyte activation, and the urotensin II signaling pathway. These overlapping functions demonstrate the role of immune response, inflammation, cellular signaling, and vesicular transport in cognitive decline. These findings suggest a linkage between cardiovascular health and cognitive function through pathways involving immune and inflammatory. Overall, our results might contribute to the broader understanding of LE8 influence on cardiovascular and cognitive health. This could shed the light on research forwards into developing diagnostic markers, preventive measures into identifying early biomarkers and therapeutic strategies by targeting overlapping connections. Further studies are needed to extend these findings into the remaining LE8 metrics.
Linking Obesity to Pancreatic Cancer: Insights from Serum Metabolite Analysis and Lipid Metabolism Alterations
Audrie Torres1; Sarahi Fernandez1; Rita Ghosh1,2,3; Deepa Ajit4, Danielle Fritze5; Jillian Woodwork5, Francisco G Cigarroa5; Gleen A Halff5; Addanki P Kumar1,2,3,6
Departments of Molecular Medicine1, Urology2, Pharmacology3, Transplant Center2, Mays Cancer Center at UT Health6, Long School of Medicine,
The University of Texas Health at San Antonio, TX, Metabolon, Morrisville, NC4
Pancreatic cancer currently ranks fourth in the United States for cancer-related deaths, with pancreatic ductal adenocarcinoma (PDAC) accounting for 90% of pancreatic cancer diagnoses. Patients with PDAC cannot benefit from cancer screening because there are no viable markers. Identifying alterations in populations at significant risk, such as obesity, could be a way to find potential indicators that are subsequently utilized as a PDAC screening method. The relationship between metabolites, obesity, and pancreatic cancer is still largely unknown, despite the fact that phospholipid and lysophospholipid levels have been linked to treatment results in pancreatic cancer patients’ metabolomic investigations. This study aimed to compare the serum metabolites associated with obesity in healthy subjects (n=11; BMI range from 18-25), overweight (n=9;
25-30) and obese (n=7; >30) with pancreatic cancer. Males (n=12) and females (n=15) were represented in the study. The study used Ultrahigh performance liquid chromatography-tandem mass spectroscopy to determine global metabolic profiles. Raw data was extracted, peaks identified, and processed using Metabolon’s Laboratory Information Management System.
Random forest analysis was used to identify biomarkers. Metabolomics analysis was performed using over 3300 commercially available compounds, with curation procedures for accurate identification. ANOVA contrasts were used for statistical analysis. The study reveals significant metabolic differences between obese and non-obese patients, as well as between overweight and non-obese patients. It also reveals altered metabolic pathways, including changes in lipid metabolism. Obese patients showed elevated levels of triphosphate, arginine, and primary bile acids. The study also reveals differential expression of 13 biochemicals involved in lipid metabolism and 8 biochemicals involved in amino acid metabolism. Fatigue and obesity resulted in altered fatty acid metabolism, energy metabolism, and bile acid metabolism. The study also found significant changes in inflammation markers, hemoglobin, and bilirubin metabolism and suggests that deregulated fatty acid synthesis may be a potential druggable target for high-risk pancreatic cancer patients. The research identifies biochemical differences between healthy, overweight, and obese individuals, suggesting metabolic reprogramming supports increased fatty acid oxidation, potentially aiding in early detection and treatment decisions.
Pancreatic Cancer, Metabolism, Pancreatic Ductal Adenocarcinoma, Obesity, Body Mass Index, Lipid Metabolism, Polyunstaratted Fatty Acids
Assessing Berberine’s Therapeutic Potential for Treating Autism-Like Behaviors in Female Mice
Chase Miller, Karina Cantu, Daniela Sanchez P. Bridgette Stewart, Georgianna G. Gould
1Voelcker Biomedical Research Academy and 2Department of Physiology, The University of Texas Health Science Center at San Antonio
Social interaction deficits and restrictive-repetitive behaviors aretreatment-resistant core symptoms of autism. Deficient serotonin (5-HT) signaling may be a contributing factor, and selective 5-HT reuptake inhibitors (SSRIs) can improve symptoms for somepatients. However, many patients with autism do not respond well to SSRIs, which block the 5-HT transporter from removing 5-HT from the synapses and extracellular fluid. This may be due to several common and rare gene polymorphisms that reduce expression or impair function of the 5-HT transporter. Given this, we characterized acute effects of the herbal remedy berberine, which inhibits auxiliary ‘uptake 2’ transporters of 5-HT such as organic cation transporters (OCTs), on social and repetitive behaviors to explore its potential as an autism treatment in 5-HT transporter knock-out, heterozygous and wild-type female mice. While data collection is still underway, we have completed it for over half of the planned experiments. To assess the impact on social preferences, we used three-chamber sociability tests and found that acute berberine treatment at 5 mg/kg i.p. had no obvious effects on social interaction or novelty preferences. We also found in tube tests for social dominance that berberine treatment enhanced social dominance in by 50% in 5-HT transporter knockout mice (from 33% to 69%). To assess the effects of berberine on restrictive repetitive behaviors, we examined its ability to reduce marble burying and discovered that 5-HT transporter heterozygotes buried more than both wild types and knock-out mice. We also observed that berberine treatment reduced burying (F (2, 41) = 8.405, p< 0.001). Finally, berberine treatment reduced serum corticosterone levels. Our findings indicate berberine treatment may be a promising strategy for reducing restrictive-repetitive behaviors in females.
Anxiety-Like Behaviors and Locomotor Activity in Aged 3-Hydroxyanthranilate
3,4-Dioxygenase Knockout Mice is Not Different Compared to Aged C57BL/6N Mice the Open Field and Elevated Plus Maze
Gabriella Gonzalez,Alexis Holmes, Miguel A. de la Flor, Jason C. O’Connor Department of Pharmacology, UT Health San Antonio,
Audie L Murphy VA Hospital, South Texas Veterans Health System, San Antonio
Levels of oxidative metabolites in the kynurenine pathway (KP) may increase due to neuroinflammation. Kynurenine 3-monooxygenase (KMO) acts as a key enzyme limiting the pathway, while 3-hydroxyanthranilic acid oxidase (HAO) functions as an intermediate enzyme downstream of KMO. In inflammatory conditions, the KP tends to favor oxidative metabolism, potentially resulting in higher levels of neurotoxic KP metabolites like quinolinic acid. These metabolites have been linked to mood disorders, mental illness, and cognitive decline. Previous studies in our lab have shown that KMO-/- mice exhibit increased anxiety-like behaviors and reduced locomotor activity compared to wild-type (WT) mice. We hypothesized that HOA-/- mice might also demonstrate differences in anxiety-like behaviors and locomotor activity compared to WT mice. To test this hypothesis, we utilized the open field (OF) and elevated plus maze (EPM) tests to evaluate anxiety-like behaviors and locomotor activity using various methods. Our findings indicate no significant differences in anxiety-like behaviors and locomotor activity between HAO-/- mice and WT mice in the OF and EPM tests. These results differ from our observations in KMO-/- mice. The behavioral disparities observed may be attributed to the distinct roles and substrates of KMO and HAO within the KP, influencing the levels of neurotoxic metabolites.
Effects of a Novel Antimicrobial Nanofiller G50 on Composite Properties
Jessica Malakhova, Kyumin Whang, PhD
Introduction: There is a need for improved filler systems in dental composites which can improve composite esthetics, mechanical properties, longevity, and provide additional benefits, such as antimicrobial activity. OX50 is a nanoparticle that is widely used in conventional dental composites to improve esthetics and increase mechanical loading via increasing overall filler loading. G50 is a novel antimicrobial nanoparticle made by functionalizing OX50 with a quaternary ammonium methacryloxy silane (K18).
Overall Objective: To determine the effects of using G50 on composite cure, hydrophilicity, translucency, mechanical, polymerization shrinkage, and antimicrobial properties.
Methods: The maximum loading of G50 and OX50 in a 70:30 Bis-GMA:TEGDMA dental resin system was determined. The effects of different concentrations of G50 or 0.7μm glass filler (60%, 65%, 70%w/w) on composite degree of cure (near Infrared FTIR and Shore D durometer hardness measurements), transparency parameter (TP) and hydrophilicity (contact angle measurements) were determined. ANOVA with Tukey HSD post-hoc test was used to determine statistically significant differences between groups at a p<0.05 level.
Results: The maximum loading of OX50 in the 70:30 Bis-GMA:TEGDMA resin system was 50% w/w. However, significantly more G50 could be incorporated into the resin system (70% w/w). Thus, OX50 and G50 were not directly compared, and instead G50 was compared to 0.7μm barium glass filler. Also, OX50 and G50 composites were significantly opaquer than the glass-filled composites. The use of OX50 or G50 significantly decreased the TP as compared to those of the glass filled groups (range ~7.8-9.6 vs. ~28.6-33.5, respectively, p<0.0005), increasing glass filler loading increased TP, but OX50 and G50 groups had comparable TP. This interfered with the near infrared FTIR readings and degree of monomer-to-polymer conversion (DoC) could not be determined. Thus, Shore D durometer hardness was used as the main measure of composite cure. For hardness, 50% OX50 composites had significantly higher hardness than 50% glass-filled composites (91.7±0.6 DHN vs. 88.2±0.7 DHN, respectively, p=0.0024) and were comparable to those of 60%, 65% and 70% glass filled groups (range ~90.3-93.4 DHN), even though the OX50 composites had lower DoC than the 50% glass composites (64.5% ±5.7% vs. 72.8% ±9.3%, respectively). Increase in glass filler loading increased hardness, as expected, however increase in G50 loading did not significantly increase hardness. All G50-loaded composites (range ~81.0-83.3 DHN) had significantly lower hardness than all glass-loaded (range ~88.2-93.4 DHN) and OX50-loaded composites (p<0.0001). Since the opacity of the G50 groups raised concerns of the effectiveness of light-curing, they were further heat-cured at 120˚C for 1 hour to determine the effects of G50, if fully cured. Heat-curing G50 composites significantly increased hardnesses to a range of ~90.4-91.6 DHN, comparable to those of the 60%, 65% and 70% glass-loaded and OX50-loaded composites. For surface hydrophilicity, the 50% glass-filled composites had significantly lower contact angles (81.8˚±4.4˚) than all other groups (range ~102.5˚-109.3˚).
Significance: Use of smaller nanoparticles in dental composites is crucial in providing a smooth and esthetic dental restoration. However, the smaller the particle size, the lower the possible particle loading into the composite and the weaker the composite. OX50 has a particle size of 40 nm and the maximum loading was only 50% w/w as compared to >70% for 0.7 μm glass filler. However, it was possible to load composites with 70% G50. This is significant because it would reduce the amount of larger filler needed to fill the composite and provide a more esthetic restoration. However, G50 composites were opaquer than glass-loaded composites, which makes them less esthetic, and may have scattered more light and reduced the degree of light-cure. As such, while glass-filled composites became harder with an increase in filler loading, G50 composites did not get harder and they had lower Shore D hardnesses than all glass and OX50-loaded composites. However, when fully cured using heat, they exhibited similar degrees of cure as glass-filled composites. G50 also did not affect surface hydrophilicity. If fully curable, G50 seems to be a promising nanofiller material for dental composites. Mechanical, polymerization shrinkage, and antimicrobial properties are currently being determined.
Enhancing Hispanic/Latino Cancer Survivor Participation in a Clinical Study by Addressing Recruitment and Retention Challenges
Jesus G Lopez, Amelia G Ramirez, DrPH Patricia Chalela, DrPH Derek Rodriguez, PhD Ysabel Rose Lew, BS Victoria Garza, BA Natalie Rodriguez, MS Britney Ortiz, BS Juan C. Nevarez Ramos, BA Jacqueline Cardenas, BS Janette Ramirez, MS Jason Osonma, BS Daniel Ruben Flores, BS Stephanie Rowan, PhD Dorothy Long-Parma, MD Yidong Chen, PhD Chen-Pin Wang, PhD
Introduction and Background
Cancer is the leading cause of death among Hispanic/Latino (H/L) individuals, underscoring the urgent need for their participation in clinical studies to reduce cancer-related mortality. The H/L community in South Texas comprises 4.9 million residents, nearly two-thirds of the state’s H/L population. In 2018, Texas had 1.8 million cancer survivors, with 12.1% being H/L and 77.1% being non-Hispanic whites. Despite this, H/L participation in cancer clinical trials is under 7%, compared to 75% for non-Hispanic whites. Clinical trials are vital for discovering new treatments and advancing medical knowledge, particularly for minority groups. Although community-based approaches have been shown to be effective in increasing minority representation in clinical studies, these strategies have posed difficulty in the recruitment and retention of H/L patients.
Hypothesis
We hypothesize that low participation of Hispanic/Latino individuals in clinical studies is primarily due to challenges in obtaining complete electronic medical records (EMRs) during recruitment. Furthermore, retention issues are likely to stem from difficulties in successfully contacting participants to follow up on their visits.
Methods
The Avanzando Caminos (Leading Pathways) study focuses on post-cancer survivors within six years, aiming to understand the social, cultural, behavioral, mental, biological, and medical influences on cancer survivorship. This six-year prospective observational study. Data from recruitment calls and EMR source documentation were analyzed to identify the frequency of H/L patient recruitment and retention over six months (January 2024-June 2024). Weekly reports from study research assistants (RAs) on their recruitment and retention activities were used to determine factors affecting successes.
Results/Findings
Analysis of the weekly reports revealed significant decreases in follow-up appointments (V3 and V4 visits; p < 0.05) and an increase in unsuccessful contact attempts over the past six months (p < 0.05). Common barriers included patients being unable to leave a voicemail (n=73), requesting callbacks due to busy schedules (n=70), and disconnected or discontinued phone numbers (n=59). Additionally, retrieving patient information from primary care physicians posed a major challenge, often taking up to three months for new recruitment of patients.
Conclusion and Impact
Our study highlights the primary barrier to H/L participation in clinical studies: the difficulty in obtaining complete EMRs to recruitment. To improve recruitment and retention, we identified implementation strategies such as sending birthday cards, holiday mail, and promotional merchandise have been suggested. Understanding and addressing these barriers is crucial for increasing H/L participation in clinical studies, which is essential for reducing cancer-related mortality and improving health outcomes within this community. Identifying and addressing these barriers will help increase H/L participation in future studies, uncovering previously inaccessible insights and improving overall health equity.
John Wells
Introduction: One of the most common neurodegenerative diseases is Alzheimer’s Disease (AD), which affects nearly 7 million people in the United States alone. My research aims to enhance the study of AD by supplementing the Biggs Institute Brain Bank, a resource containing hundreds of donated human brains for research and education. Brain visualizations, specifically of the interior of the brain, are one of the most crucial components of neurological analysis. This is critical in AD where various physiological markers can be clear indicators of disease presence and severity. My project focuses on developing software to process images of sliced brain tissue in order to develop enhanced cerebral visualizations.
Methods: The programming for this project was done in the Python coding language using Visual Studio Code. Initially, the images were rotated to properly orient the label. This was done by determining if the label was horizontal or vertical, then using OpenCV’s thresholding function (which generates a mask of pixels within a certain color range) to isolate the label and contour generation to generate an outline of the label. The scale of the image (pixels per real life inch) was determined by obtaining the height of the ruler (in pixels) and dividing it by the height of the ruler (in inches). Next, the position of the brain slices was identified by using OpenCV’s thresholding and contour detection. The contours were filtered to remove noise and overlapping segments. Each brain slice was then cropped out and segmented into its own image using the previously identified contours. These individual images had their backgrounds removed and were rescaled to match a desired scale and resolution. Slices were sorted in an ascending to descending order. Finally, the images were compiled into a GIF to demonstrate the coronal sections of the brain.
Results: The program successfully oriented the image in 100% of instances (n = 153), and it successfully detected and cropped the brain slices contained in an image in 98.69% of instances (n = 153). It performed accurately on a wide variety of coronal brain slices, regardless of slice orientation or anterior-posterior ordering within images.
Conclusion: This project successfully developed software to facilitate the analysis of brain slices by segmenting and resizing images of brain slices and constructing them into a GIF representation of a brain. This program will contribute to the quantitative analysis of gray matter atrophy, construction of advanced 3D models of brains, and the development of machine learning algorithms to facilitate neuroanatomical research and education.
Low-dose Eribulin Promotes NK cell-mediated Therapeutic Efficacy in Bladder Cancer
Jordan P. Svatek, Zaineb Hassouneh*, Onika D.V. Noel, Niannian Ji, Robert S. Svatek, April Risinger, and Neelam Mukherjee
Despite its immunogenic nature, bladder cancer (BCa) responds sub-optimally to FDA-approved immunotherapy. We previously showed that natural killer (NK) cells in bladder tumors are associated with patient survival, identifying these cells as important targets for BCa treatment. To develop clinically approved agents that enhance NK cell activation we identified eribulin, a microtubule destabilizer primarily used in breast cancer. Ongoing clinical trials are investigating the effectiveness of eribulin in BCa; however, the mechanism of eribulin remains unclear. Here, we explore the effects of low-dose eribulin on direct NK cell activation in vitro, including on primary patient bladder tumor cells, and in vivo utilizing multiple murine models. We found that eribulin instillation into the bladder significantly reduces BCa burden and improves mouse survival in an NK cell-dependent manner. Flow cytometry and RNA sequencing were employed to identify the mechanism of NK cell activation by eribulin, which was associated with increased migration and cytotoxicity of NK cells against BCa cells. Importantly, eribulin promoted the shift of patient-derived intratumoral NK cells towards an anti-tumor CD49a+ CD103+ NK subset (i.e., ILC1-like) while diminishing the dysfunctional NR4A2-expressing CD49a- NK subset. Moreover, eribulin decreased the overall expression of exhaustion markers on NK cells, a pattern replicated in our murine models. These findings are paradigm-shifting because chemotherapy, like eribulin, is traditionally considered immunosuppressive and our data suggests that eribulin activates NK cells. Our study reveals the novel effect of low-dose eribulin chemotherapy in inhibiting BCa growth by enhancing anti-tumor NK cell immunity, challenging previous assumptions, and opening new therapeutic approaches to improve antitumor immunity (Figure).
*First author for manuscript submission
A Stem-cell Based Approach to Deliver Neurotrophic Factors to Treat Alzheimer’s Disease
Julia Kazunas, Qing Zhou, PhD, Shujie Zhao, MD, Senlin Li, MD
Alzheimer’s Disease (AD) is one of the most common forms of neurodegenerative diseases, in which an aggregation of amyloid persecutor and/or tau proteins interfere with the brain’s activity as the disease progresses and cholinergic neurons as well as other nerve cells die out. In this regard, Brain-derived Neurotrophic Factor (BDNF), one member of the neurotrophic factor (NTF) family, is a compelling therapeutic agent to treat AD, since it interacts with receptors that support the overall function and survival of neurons affected by this disease, primarily in the hippocampus and cortex. The current investigating delivery method of BDNF to the diseased brain is through direct stereotactic injections using an adeno-associated virus serotype 2 (AAV2), since NTFs are unable to pass the blood-brain barrier (BBB). This method, however, is shown to be insufficient in clinical trials due to the limited spread of NTFs in the brain tissue. Our lab proposes a different method, a hematopoietic stem cell (HSC) transplantation-based macrophage/microglia mediated central nervous system (CNS) delivery of BDNF. Previously, our lab conducted this transplant methodology in a Parkinson’s Disease model using the Glial cell-line Derived Neurotrophic Factor (GDNF) targeting the dopaminergic neurons in the substantia nigra and striatum. Syngeneic mouse bone marrow HSCs, collected and transduced using a lentiviral vector carrying the NTF gene, were transplanted after pre-conditioning with irradiation into the recipient’s bone marrow. The engrafted cells were able to proliferate and differentiate into monocytes/macrophages, upon entering brain tissue, which further differentiated into microglia. Since this transplant method has led to high microglia turnover rates and results in an amelioration of neurodegeneration in PD, we propose expanding it on an AD model.
To evaluate the turnover rate of transplanted cells on endogenous microglia, we took full brain samples of wild-type mice that underwent whole body irradiation followed by our HSC-transplant and CSF1R inhibitor treatment. Brain samples were sectioned and stained with antibodies against the Iba1 microglia marker. We then used GPT-4 Open AI software to quantify microglia identified on the slides. Granted, since the use of AI software is still being introduced into laboratory practice, we believe that evaluation in cross referencing machine learning models that systemically count cells and manual quantification using programs such as ImageJ, should be further compared for efficacy. The results using GPT-4 showed a 76.33% infiltration of transplant-derived cells within the cortex, where out of 3418 total microglial cells, 2609 were transplant-derived. It is indicated that microglia have high turnover rates in filling the microglial niche throughout the entirety of the brain through our replacement strategy, and thus can be used to deliver BDNF for treatment of AD. It is conceivable that in the future our HSC transplantation-based macrophage/microglia mediated brain delivery of BDNF may be used for novel treatment of AD either as a monotherapy or combination therapy with monoclonal antibody (such as Kisunla) against beta-amyloid.
Quantifying Alzheimer’s Pathology using Qupath Machine Learning Analyses
Kelsey Hernandez, Morgan M. Smith M.S., Elizabeth Ochoa Ph.D., and Kevin Bieniek Ph.D.
Background: According to a recent Alzheimer’s Association report, an estimated 6.9 million Americans age 65 and older currently live with Alzheimer’s disease, the most common form of dementia. Alzheimer’s disease is characterized by impaired memory, thinking, social, and behavioral skills. Underlying these clinical symptoms, Alzheimer’s disease is also clinically diagnosed according to three primary biomarkers: amyloid beta aggregation (A), tau neurofibrillary tangles formation (T), and neuronal loss (N), collectively known as A/T/N. While amyloid beta and tau biomarkers can be detected histologically after autopsy, quantifying neuronal loss is more challenging. Therefore, we developed a QuPath algorithm to accurately identify and quantify Nissl bodies as an estimate of neuronal loss and amyloid beta plaques to quantify disease progression.
Methods: To detect Nissl-stained neurons as well as amyloid beta pathology via QuPath, we utilized hierarchical structuring to establish a parent-child relationship between the annotated and predicted objects, using the annotated objects as reference points for the algorithm. The hierarchical structuring was based on a random trees model at high resolution, with a smoothing value of 0.5 and 1.0. During the training period, approximately four regions of interest were identified on five training slides to ensure the algorithm’s accuracy and variability. These regions were combined to create a more specific training image, allowing the algorithm to develop on a whole-image scale rather than isolated annotations.
Results: Training was considered complete when the manual identification and quantification of Nissl bodies within neurons was congruent with the prediction proposed by the algorithm. This accuracy confirmation method was repeated on 29 additional, randomly identified, testing regions and six overall testing slides. Based on this review, the Nissl classifying algorithm performs with an average error rate of 2.204% (95% confidence interval of 1.294 to 2.974) in an approximate 8.96 x 105 μm region.
Conclusion: Overall, we provide a set of classifiers for both Nissl positive neurons and demonstrate that QuPath has the capacity to accurately analyze both traditional histologic stains (Nissl) as well as immunohistochemical stains (amyloid). Future work will focus on validating the amyloid beta classifying algorithm in a testing data set via a verifying metric. After validating we plan to further develop the amyloid beta algorithm to differentiate specific amyloid pathologies, for example cerebral amyloid angiopathy, diffuse amyloid plaques, and cored amyloid plaques. Additionally, future research may also include improving the diagnostic accuracy of neuropathologies in a clinical setting.
Hematopoietic Stem Cell Transplantation-Mediated GDNF Delivery as a Potential Therapy for ALS: Enhancing Microglial Infiltration
Luke Cyrus, Qing Zhou, PhD, Shujie Zhao, MD, Barath Prashanth Sivasubramanian, MD, Senlin Li, MD
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of motor neurons in the motor cortex and spinal cord, leading to muscle weakness, paralysis, and ultimately, fatal respiratory failure. Current therapies for ALS are palliative, highlighting the urgent need for novel treatment strategies aimed at slowing disease progression and preserving motor function. In our laboratory, we have been exploring innovative approaches initially developed for Parkinson’s disease (PD) research to potentially treat ALS. Hematopoietic stem cell transplantation (HSCT) was utilized as a platform for the delivery of Glial Cell-Derived Neurotrophic Factor (GDNF), a potent neuroprotective agent. Donor HSCs were collected from adult mice and transduced ex vivo with lentivirus expressing the GDNF gene under the control of a highly active macrophage synthetic promoter (MSP). A genetic mouse model (MitoPark) was used in this study. Over 4 months and 7 cycles of mobilization-assisted transplantation, GFP+ blood cell proliferation stabilized at approximately 77%. Our findings demonstrated that HSCT-based GDNF delivery effectively mitigated progressive motor and non-motor dysfunction in PD mice. Specifically, treated mice showed significant improvements with a 46.3% increase in horizontal and a 54.9% increase in vertical activities compared to baseline measures. Restoration of striatal dopaminergic levels reached 84% of those measured in normal mice. Importantly, our non-toxic HSCT-based GDNF gene therapy approach was well-tolerated, with no observed adverse effects throughout the experiment. In the MitoPark mice, the disease pathogenesis sufficiently induced macrophage infiltration and differentiation into microglia-like cells over time. However, in the SOD1G93A mouse model of ALS, the disease pathology does not allow extensive infiltration of macrophages by our HSC therapeutic approach. Therefore, we used a novel modified methodology to experimentally achieve increased infiltration. Before HSC transplantation, we depleted the brain and spinal cord of microglia by administration of CSFR1 (a crucial protein for microglia survival) inhibitors, allowing high rates of macrophage infiltration. After CSFR1 systemic administration, we performed our HSC therapy in the ALS mice models. Subsequently, we harvested brain and spinal cord tissue and utilized immunohistochemistry staining techniques and Image J Particle Analysis software to observe levels of macrophage infiltration (displaying green fluorescence from the GFP biomarker found in the transplantation-derived macrophages/microglia-like cells) relative to the total microglia (displaying red fluorescence from the Iba1 biomarker common to all microglia, including both endogenous and transplanted microglia-like cells). We obtained 76% transplanted microglia (610) among the total microglial cells (805), indicating that our new HSCT approach was effective. In future studies, we will test our HSCT-based GDNF gene therapy approach in the treatment of ALS by examining the mitigation of neurodegeneration and motor function recovery. We will also develop a novel AI video-recognition model using hundreds of labeled video samples to train the model to accurately predict the mouse model’s ALS neuro score, so as to eliminate the margin of error that comes from human analysis, as well as to optimize the delivery of our treatment by determining disease onset as early as possible.
Effects of the Anti-Diabetic Pharmaceutical, Metformin, on Freshwater Algae and Zebrafish Behavior
Lyla Arnold, Georgianna Gould, PhD
Metformin, an anti-diabetic drug, is increasingly found in freshwater ecosystems due to its high excretion rate and incomplete degradation in wastewater treatment. This study examines Metformin’s effects on Chlorella pyrenoidosa algae and Danio rerio (zebrafish). The first part of the research assesses Metformin’s impact on algal growth and dissolved oxygen levels. The hypothesis suggests that higher Metformin concentrations will decrease algal growth and increase dissolved oxygen. Initial trials used 5µg, 10µg, and 50µg doses, followed by higher concentrations of 100µg, 150µg, and 200µg. Results showed reduced algal growth with higher Metformin levels, but no significant change in dissolved oxygen, indicating a direct mitochondrial impact without affecting photosynthesis. These results highlight C. pyrenoidosa’s sensitivity to Metformin and the need for more research on pharmaceutical pollution in aquatic ecosystems. The second part investigates Metformin’s behavioral effects on zebrafish, hypothesizing that higher concentrations reduce anxiety and alter social behaviors. Behavioral changes were measured using light-dark maze and social preference tests. Zebrafish exposed to higher Metformin levels spent more time in lighted areas, indicating reduced anxiety, and showed decreased social interaction, supporting the hypothesis of behavioral changes due to prolonged exposure. In conclusion, Metformin pollution significantly affects algal growth and zebrafish behavior, with higher concentrations reducing algal growth and altering zebrafish behavior. These findings underscore the environmental consequences of pharmaceutical pollution and the importance of preventive measures in pharmaceutical production to protect freshwater ecosystems.
Women’s Health Post Survey Responses Collected from Metro Health Classes at Center for Refugee Services to Determine Level of Physical Health Understanding Post Class Attendance and Benefits of Classes
Madilyn Del Angel, Rami Benchoui, Dr. Fozia Ali
Women’s health classes at the Center for Refugee Services taught by Metro Health began to inform immigrant women about health and how to live a healthy lifestyle. Classes include topics like exercising at home and how to eat healthier. Post surveys began weeks after the first initial class to collect feedback and insight on how the women feel about the classes. Post survey includes questions that ask: “Are these classes beneficial?”, “Did you learn something new from today’s lesson?”, “Will you apply what you learned to your lifestyle, and if so how?”, “Will you share this information with family and friends?”, “What is your biggest barrier in regards(understanding and attending wise) to the class?”, and if there are any other comments or concerns. Post surveys were created to be easy to answer with simple yes or no questions and a few short answer responses. The questions give insight on how the women feel about the classes. To see if the women are benefitting from the classes and if there are any changes or specific topics the women would like to learn about.
Survey handouts were given to the women after the meeting and the women filled the survey out with help of translators. The surveys were given back once completed and most of all the questions were answered with some exceptions of the question that asks how they plan to use what they learned to change their lifestyle. Ten surveys were collected and all the women marked that the classes are strongly beneficial. All women also marked “yes” in response to the questions: “Did you learn something new from today’s lesson?”, “Will you apply what you learned to your lifestyle?”, and “Will you share this information with family and friends?”.This proves that the classes are beneficial and should be continued, and the women who attend are actively learning and comfortable enough to teach the newfound knowledge to others. The class the women had attended the day the post surveys were handed out was over exercising. The survey asked, “How will they change their lifestyle now?” and there were mixed responses. Some women responded saying they would exercise daily, others said they would start going on walks, and half of those who answered said they would exercise after thirty minutes of sitting; which proved they learned and retained that specific piece of information which was taught in the meeting. When asked about any struggles or barriers they come across with learning or attending the meetings the two answers that were checked off the most was the time/day the meetings were hosted and the language barriers. The meeting is taught in English with a slideshow translated for the women who mostly speak Pashto, Dari, or Farsi; some understand and speak some English but there is only one translator who is also attending the meetings. For the final question which was for comments and concerns there was a variety of answers. Two asked for help finding a job, two asked for more classes/sessions, and one asked for mental health help. Feedback gathered can help figure out how else to benefit the women/keep track.
Novel Mouse Model to Study Long-Term Outcomes After Exposure to Prenatal Inflammation
Martha Chavez-Lua, Ariana Samaniego BS, Meera Rath PhD, David Schmerber BS,
Madison Lay BS, Egle Prewit MD PhD
Background: Preterm birth, defined as childbirth occurring before 37 weeks of gestation, affects approximately 12% of pregnancies in the United States. It is a significant concern due to the incomplete development of the fetus and its strong association with intrauterine inflammation. Despite being identified as a major contributing factor to preterm birth, there is currently no effective therapy to prevent intrauterine inflammation.
Objective: To develop a novel mouse model to study long-term outcomes after exposure to prenatal inflammation. This is a first step towards our overall objective to develop therapeutic measures to prevent adverse neonatal outcomes due to inflammation and preterm birth.
Materials & Methods: On day 15 of gestation, pregnant CD-1 mice were randomized to receive intraperitoneal (IP) injections of either lipopolysaccharide (LPS) or saline 2 hours prior to the administration of a second injection saline only. Blood samples were collected hourly post-injection to measure IL-6 concentrations. Six to eight months post-injection blood pressure was monitored using the tail cuff system. The mice were then euthanized, and blood tissues were collected for further analyses. These tissues were preserved to evaluate both primary and secondary outcomes. Brain cortex tissues were utilized for xMPA Luminex assays, and serum samples were taken to measure C-reactive protein (CRP) levels.
Results: Blood pressure was significantly elevated in the LPS and saline group compared to the exclusively saline group. This applied to both the mother and offspring groups (figure). Some mothers experienced pregnancy loss during the experiment and were categorized as a separate group. This group exhibited a significant increase in blood pressure compared to both the LPS+saline and saline groups. Regarding CRP levels, there was a notable increase in the LPS+saline group compared to the saline group (figure). Similarly, mothers who lost their pregnancy showed elevated CRP levels. IL-6 expression was significantly increased in response to LPS administration, as measured hourly (not shown). Both mothers and offspring exposed to LPS displayed increased chronic inflammation (determined by IL-4, IL-10, IL-6, IL-1β, not shown).
Conclusion: In subjects exposed to inflammation (LPS) there was an increase in blood pressure & pro-inflammatory cytokines, and a decrease in anti inflammatory cytokines. Although the offspring did not receive LPS directly, they still displayed chronic inflammation due to exposure to inflammation throughout gestation. The observed relationship between inflammation inducers and anti-inflammatory cytokines, such as IL-4 and IL-10, suggests that gestational inflammation may contribute to long-term effects, including chronic inflammation and elevated blood pressure. Highlighting the potential impact of prenatal inflammatory environments on future health outcomes and a specific target for the development of therapeutics.
Elucidating Imitative Eating Behaviors in Mouse Models of Fragile X Syndrome
Quin Moyer, Eric Oh, Rodrigo Gonzales-Rojas, and Hye Young Lee
Autism Spectrum Disorder (ASD) is characterized by impairments in imitative behaviors such as facial replications or body movement which leads to deficits in social learning and bonds. Fragile X Syndrome (FXS) is an intellectual disability that is prevalent in patients with ASD. In a previous study, it was determined that Fmr1 KO mice, mouse models of FXS, have a deficit in imitative scratch behaviors after viewing a video of a demonstrator mouse scratching (Gonzales-Rojas et. al. 2020 Scientific Reports). A study done in 2022 determined that another behavior mice appeared to imitate was eating(Xie et al. 2022 Neuroscience Letters).
With this information, our lab intends to explore this new imitative behavior (eating) even further. We intend to determine if Fmr1 KO mice will have a phenotype of the same imitative deficit we saw in scratch in this new eating behavior. Before we collect data on the Fmr1 KO mice we must determine if the WT strains (FVB and Bl6) of mice imitatively eat so that we may have a control group. This initial experiment will collect data from 4 cohorts (male Bl6, male FVB, female Bl6, female FVB) over 3 weeks and 6 sessions. This study is in progress, but with the information from previous studies, we hypothesize the mice will exhibit imitative phenotypes. As we continue our study, we hope to expand our groups and identify the ideal mouse strain for future studies as well as use the Fmr1 KO mice to create a new mouse model to study ASD and imitative deficits.
Understanding the Role of Matrin 3 in Glioblastoma Growth and Progression
Rachelle Barrera, Manjeet Rao, Panneerdoss Subbarayalu
Greehey Children’s Cancer Research Institute, Department of Cell Systems & Anatomy, UT Health San Antonio
Introduction: Glioblastoma multiforme (GBM) is a high grade and an aggressive brain tumor. Life expectancy for those diagnosed with late stages of glioblastoma are incredibly low.
Understanding the molecular mechanism of glioblastoma growth and progression will improve the patient’s survival and quality of life. Our bioinformatics analysis showed that MATR3 levels were significantly low in GBM. The objective of this study is to establish the mechanisms by which RNA binding protein MATRIN3 facilitates GBM tumor growth and progression.
Methods: In order to test the role of MATR3 on glioblastoma, the protein was both
over-expressed (OE) and knocked-down using siRNA (siMATR3) in glioblastoma cells. To establish the significance of MATR3 in glioblastoma multiforme, we performed an over expression (OE) and knockdown (siMATR3) approach. We used several GBM cell lines including U251 and T98G cells that were purchased from the American Type Culture Collection and cultured in standard medium. Mechanistic studies were conducted using MATR3 overexpression and knockdown cells and by measuring the status of cell viability, migration/invasion, colony forming assay, apoptosis, sphere formation, target gene and protein expression analysis immunofluorescence analysis of MATR3 localization. Results: Our studies discovered an RNA binding protein, MATRIN3 (MATR3), as a novel tumor suppressor in GBM.
MATR3 is expressed at a significantly reduced levels in GBM. MATR3 inhibited short and long-term viability as well as migration and invasion of GBM cells. Overexpression of MATR3 leads to increased apoptosis. MATR3 overexpression significantly reduced the FOXM1 (regulates cell proliferation, cell cycle, DNA damage repair, apoptosis) protein. Mechanistic studies identified FOXM1, a cell cycle regulating protein as a downstream effector of MATR3.
Immunofluorescence analysis showed significantly increased numbers of 53BP1 foci in MATR3 overexpressed GBM cells compared to control plasmid transfected cells. Conclusions: Our data suggest that MATR3 inhibits GBM growth and progression by inhibiting FOXM1 signaling axis in GBM. These findings show that MATR3 plays an important role in decreasing tumor growth and progression in glioblastoma. Future research targeting the over-expression of MATR3 could lead to potential treatment and therapeutic strategy for tackling glioblastoma.
Acknowledgement: Thank you to the Rao Lab and all of its members for the help in conducting research. Special thanks to my mentor Dr. Panneerdoss Subbarayalu for all of the help he’s provided over the course of this project. Finally, thank you to the Voelcker Biomedical Research Academy and the Greehey Institute for the amazing opportunity to learn about cancer research.
Novel targeted therapy for treating renal cell carcinoma
Rashmi Basu, Emily Aller BS, Megharani Mahajan PhD, Hareesh B. Nair PhD, and Suryavathi Viswanadhapalli PhD.
The American Cancer Society expects 81,000 new kidney cancer cases and 14,000 fatalities in 2024. Chemotherapy and radiation therapy are currently used for treating kidney cancer; however, severe side effects limit their use in clinic. There is an urgent need for novel and safe targeted therapies for treating kidney cancer. Renal cell carcinoma (RCC) is the most common type of kidney cancer. Midkine (MDK) is a growth-factor that is implicated in the progression of various types of malignant tumors including RCC. MDK promotes cancer progression by promoting growth, survival, metastasis, migration, and angiogenesis. MDK levels were higher in RCC and were associated with a poor prognosis for patients. In collaboration with our industry partner (HiBis Associates LLC), we have developed a novel first-in-class high affinity (KD 38.4 nM) MDK inhibitor (MDKi), HBS-101. The objective of this study is to evaluate the efficacy of HBS-101 as a targeted therapy. In vitro activity was tested using MTT, apoptosis and invasion assays. Long term effects of HBS-101 on cell survival were measured using colony formation assays. Our study results found that HBS-101 reduced cell viability of both RCC cells A-498 and 786-O in a dose-dependent manner with an IC50 of 1 µM and 4 µM respectively. Furthermore, in clonogenic assays HBS-101 treatment reduced colony formation in both cell lines. Flow cytometry analysis showed that treatment with HBS-101 increased late apoptotic activity and cell death. Treatment with MDKi led to a significant decrease in the invasive capability of RCC cells. Collectively, these results suggest that HBS-101 effectively suppresses oncogenic activity of RCC cells and HBS-101 could serve as a novel targeted therapy for RCC.
Selisistat to Overcome Prostate Cancer Relapse
Sindhu Atluri, Michelle R Villarreal, Tyler J Smith, Rita Ghosh and Addanki P Kumar*
Background: More than 1 in 4 men who have definitive local therapy with surgery or radiation develop biochemical recurrence (BCR) indicated by the increasing level of prostate specific antigen (PSA). For these men, androgen deprivation therapy (ADT; blocking testosterone production) is the next treatment step. However, a sizable proportion of men treated with ADT develop castration-resistant prostate cancer (CRPC) including metastases, despite the initial response to ADT. Therefore, it is critical to find ways to prevent disease progression (BCR) post- treatment. Published studies show association of nuclear SIRT1 (nSIRT1) with higher tumor grade and that nSIRT1 is associated with progression to BCR. We hypothesized that therapeutic SIRT1 nuclear translocation may be useful to prevent progression to castration resistance in patients diagnosed with advanced disease. A secondary hypothesis is that combination of SIRT1 inhibitors with existing standard-of-care (SOC) treatment approaches will enhance therapeutic efficacy.
Objective: To test that selisistat, a SIRT1 deacetylase inhibitor potentiates therapeutic efficacy of AR-signaling inhibitors.
Methodology: Cell proliferation, clonogenic assays, immunoblotting and enzyme-linked immunoassay (ELISA) were used.
Key Findings: Our results indicate selisistat potentiates tumor growth inhibitory effects of AR- signaling inhibitors such as abiraterone and enzalutamide; chemotherapeutic agent Taxotere and radiation albeit differently. Selisistat treatment reduced the levels of SIRT1 and increased senescence in prostate cancer cells.
Discussion and Conclusions: Taken together, these observations suggest that SIRT1 inhibition is a new intervention strategy to potentiate therapeutic efficacy of conventional therapy.
Tanisha Arya, Daisy Medina, Manjeet Rao, Ph.D., Panneerdoss Subbarayalu, Ph.D. Greehey Children’s Cancer Research Institute, Department of Cell Systems & Anatomy, UT Health San Antonio
Despite improvement in overall survival, many patients with triple-negative breast cancer (TNBC) still succumb to this disease. Identification of new biomarkers and safe therapeutic targets is urgently needed to improve the overall clinical outcome of breast cancer patients. Our previous studies showed ALKBH5 expression, an m6A RNA demethylase, to be significantly increased in
triple-negative breast cancer patients which supports TNBC growth and progression. We performed an ALKBH5 inhibitor screen and identified Mefloquine (an FDA-approved drug) as a potent inhibitor of ALKBH5.
To conduct our research, we used in vitro assays on breast cancer cell lines BT549, MDA-MB-231, and MDA-MB-468. Assays included short-term cell viability (24, 48, and 72 hours), migration, apoptosis, cell cycle, and long-term colony formation. TNBC cells were treated with Mefloquine at dosages of 20 µM, 15 µM, 10 µM, 7.5 µM, and 5 µM for short-term cell viability, while the rest of the assays used the dosages of 10 µM and 7.5 µM. The short-term cell viability was assessed using the Cell TiterGlo assay, while apoptosis was evaluated using flow cytometry with Annexin V/PI staining. Migration assay was performed using Boyden chamber/transwell. Cell cycle distribution was analyzed by flow cytometry following PI staining, and colony formation was evaluated by counting the number of colonies (stained with crystal violet) formed after drug treatment.
We discovered that Mefloquine significantly reduced all TNBC viability, with an IC50 of approximately 8 µM. At 24, 48, and 72 hours, a dose-dependent decrease in cell viability was observed, indicating that Mefloquine effectively inhibits TNBC cell growth. Moreover, apoptosis assays revealed a marked increase in apoptotic cells, with a significant increase in both early and late apoptosis phases at higher drug concentrations. Furthermore, migration and colony formation assays demonstrated significant reductions in TNBC proliferation at Mefloquine dosages of 7.5 µM and 10 µM. Mefloquine-induced cell cycle arrest (preliminary data) suggests that it effectively halts cell proliferation by inducing cell cycle arrest.
These findings indicate that Mefloquine has significantly reduced TNBC proliferation, migration, and increased apoptosis. Our data suggest that Mefloquine is a potential therapeutic agent to inhibit ALKBH5. Further research on Mefloquine and ALKBH5 inhibition could present a promising therapeutic strategy for TNBC treatment, potentially leading to improved patient outcomes.
Acknowledgment: We would like to thank Dr. Panneerdoss Subbarayalu, Dr. Manjeet Rao, Rao Lab Members and Collaborators, and the Voelcker Biomedical Research Academy and Staff for their invaluable support and their contributions to this research.
Implementing the ECHO Model for Harm Reduction Education: A Statewide Initiative to Enhance Capacity and Confidence in Tackling the Opioid Epidemic
Victoria Rangel, Adrienne Lindsey, MA, DBH
The opioid epidemic has contributed to a high level of fatal drug overdoses, as well as HIV, AIDs, and HCV being among the many health epidemics correlated with injection drug use, where the implementation of harm reduction techniques has been underutilized. Harm reduction practices include syringe service programs, drug checking services, education in safe use practices, naloxone distribution, and other practices. Due to the lack of harm reduction practice in the healthcare field, there is an urgent need for education. The ECHO (Extension for Community Healthcare Outcomes) model provides didactic and case-based learning, where learners are exposed to a brief harm reduction topic presentation and a case presentation from a member of the learning community. Utilizing Project ECHO allows for building capacity in new practices, and in this case was used to educate a broad, interdisciplinary audience across Texas in harm reduction practices. In May 2022, the Harm Reduction ECHO program was launched, providing instruction and case consultation in topics such as naloxone administration and distribution, overdose education for patients, person-centered care, stigma reduction strategies, and drug testing/checking. After each ECHO session, learners were given a short post-session survey via REDCap. Surveys were not anonymous, as participant information was mandatory for continuing education credits for the program. The survey consisted of satisfaction with the session content, including factors such as overall satisfaction, receptivity of the speakers to comments and questions, neutrality/objectivity of the information provided, utility of the case presentation in informing daily practice, and if the participant would recommend the program to a colleague. Additionally, the post-session survey collected learners’ confidence in their ability to apply material to their daily work. Approximately 739 unique learners participated in the program across 62 cities, representing 232 organizations. Learners were highly satisfied with the programming, with 76.88% reporting they were “very satisfied.” Learners were also very confident in their ability to apply the practice, with 88.57% reporting they were “confident” or “very confident.” Forty-four percent (44%) of learners reported adding one or more new harm reduction services since their participation in ECHO such as naloxone, HIV testing, Hepatitis C testing, education about overdose prevention, information about accessing sterile syringes, or drug testing/checking services. The ECHO model demonstrates promising preliminary results for the dissemination of harm reduction information for various providers across the state. This was seen in participants who reported being highly satisfied with the model and high confidence in applying harm reduction strategies. Also, about half of the participants (44%) reported the implementation of newly learned harm reduction services. Further research could focus on conducting similar studies in various regions to explore the possibility of implementing ECHO on a broader regional or national scale to support the adoption of harm reduction strategies aimed at tackling the drug overdose crisis.
NAD[P]H Quinone Oxidoreductase 1 (NQO1) depletion is associated with resistance to Histone Deacetylase (HDAC) inhibitors
Zachary N Hardeman, Michelle R Villarreal, Tyler J Smith, Subhash S Karki, Addanki P Kumar and Rita Ghosh*
Background: NQO1 is a detoxification enzyme. Previous work from the Ghosh laboratory has shown that NQO1 depleted prostate cancer (PCa) cells rapidly form poorly differentiated tumors. Low NQO1expression correlates with advanced PCa, disease aggressiveness and increased risk of PCa recurrence. HDACs remove acetyl groups from histones, leading to chromatin condensation and gene repression. In PCa the dysregulation of HDAC activity modulates the interaction with androgen receptor (AR), influencing AR-driven transcription. High HDAC levels correlate with poor prognosis and advanced cancer stages. However, current HDAC inhibitors have not shown clinical efficacy in PCa patients underscoring the need to understand the mechanism that underpins the lack of clinical effectiveness. In melanoma and glioblastoma cells acquired resistance to HDAC inhibitor has been linked to low NQO1 levels but whether NQO1 may be involved in HDAC inhibitor effectiveness in PCa remains unknown. Given the significant decrease in NQO1 levels during PCa progression, we hypothesized that low NQO1 activity may contribute to lack of HDAC inhibitor cytotoxic efficacy.
Objective: To determine viable cells with active metabolism following treatment with a series of HDAC inhibitors.
Methodology: I used dimethyl thiazolyl diphenyl tetrazolium salt (MTT) to determine cytotoxicity of the HDAC inhibitors. The drugs were code named as SSD01, SSD02, SSD05, SSD06, SSD07, SSD08, SSD09, and SSD10. I used isogenic hormone sensitive PCa cells (LNCaP) that either expressed NQO1 and those that were genetically knocked down for NQO1 expression. The knockdown mimicked the levels of NQO1 in PCa patient samples with ~90% inhibition in NQO1 expression. 4,000 cells were plated per well in 96 well plates and allowed to attach to the wells overnight. Cells were treated in triplicate with solvent or increasing doses (2.5 µM, 5 µM, 10 µM, and 25 µM) of HDAC inhibitors. The experiment was terminated at 48h. The Cell Titer Non-Radioactive Active kit was used. 4h after the MTT dye was added, the stop solution was added. The next morning, the absorbance was recorded at 570 nm on a plate reader and the percentage of surviving cells relative to the solvent control was calculated.
Key Findings: Preliminary observations show that 4 of the 10 novel HDAC inhibitors tested have potential cytotoxic effects on NQO1 expressing PCa cells. In contrast, none of the tested HDAC inhibitors were cytotoxic in isogenic cells depleted for NQO1.
Discussion and conclusion: The preliminary results obtained here indicate the importance of NQO1 enzymatic activity in the cytotoxicity of HDAC inhibitors in PCa cells. If these results are replicated in other PCa cells, then activation of NQO1 in combination with these drugs may enhance cytotoxicity. Although, not conclusive, this study also identifies several promising HDAC inhibitor candidates. These results suggest that during PCa progression low NQO1 activity may determine sensitivity to targeted use of HDAC inhibitors.