2023 Graduating Senior Scholar Abstracts
Class of Summer 2023
Graduating Scholar Abstracts
New Treatment Option for Pancreatic Cancer: A Novel Approach to Overcoming
Desmoplasia and Enhancing Chemotherapy
Siri Borra, Meagan M. Ybarra, Addanki P. Kumar, PhD.
Department of Molecular Medicine, The University of Texas Health Science Center, San
Antonio, TX
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with only an estimated 6% of patients surviving past 5 years after diagnosis. A phenomenon contributing to PDAC’s therapeutic resistance is desmoplasia, initiated by the activation of pancreatic stellate cells (PSCs). Activated PSCs secrete excessive extracellular matrix components, leading to tumor stroma expansion which hinders drug penetration. Previous work from the Kumar laboratory has shown that a natural compound called palmatine with druglike properties has the potential to disrupt interaction between pancreatic cancer and stellate cell interactions (PCC-PSC) and improve the growth inhibitory activities of chemotherapeutic agent, gemcitabine. Gemcitabine- based therapy is the first-line option for treatment of PDAC. I hypothesized that “palmatine enhances therapeutic response to gemcitabine by disrupting PSC-PCC communication mediated by secreted factor(s)”. I have identified that relative to PANC-1 cells (cancer cells), PSCs secrete elevated levels of IL-1 into the extracellular space and show increased levels of transcription factor HOXA10. I further found that treatment with combination of palmatine plus gemcitabine reduced protein levels of HOXA10 and pSTAT3 in PSCs. Additionally, the observed increased STAT3 signaling in PSCs under co-culture conditions was decreased following treatment with palmatine. Taken together, these observations suggest palmatine enhances therapeutic potential of gemcitabine inhibiting PSC-PCC communication in part through IL-1-HOXA10-STAT3 signaling axis.
Mapping and Morphological Characterization of GABA-Releasing Retinal Ganglion Cells in the mouse Retina
Anthony Castaneda, Ching-Kang Jason Chen, PhD
Retinal ganglion cells (RGCs) are the sole output neurons in the retina responsible for transmitting visual information to the brain. Rod and cone photoreceptors convert photons into neural signals, which pass through retinal interneurons to drive RGCs’ signaling to the brain. Along with this signaling axis, the major neurotransmitter used in this process is glutamate, which is released when information being transmitted between neurons. The primary focus is to characterize RGCs that release not only the excitatory neurotransmitter glutamate, but also an inhibitory neurotransmitter called GABA. Mouse RGCs are diverse, with a current tally of 46 types based on the gene expression patterns therein. The diversity of the RGC types based on this newer classification method is expected to increase. However, very little effort has been reported to link these 46 types to the traditional RGC types that were characterized by morphology or by light response properties. Organizing and classifying RGCs is a complex task with vast amounts of neurons. In the lab we used mouse genetics to label specific types of RGCs and by imaging these genetically marked RGCs and virtually reconstructing the dendritic morphology of these retinal neurons using Neurolucidia360 software, hoping to bridge the knowledge gap of RGCs classification using gene expression pattern and morphological features. With successfully mapped out the location of the RGCs that release GABA in the retina and have reconstructed the dendritic structure of some of them. Finally, the findings can be linked through the publicly available single-cell RNA sequencing database to assign dendritic morphology to candidate RGC types expressing the machinery of GABA production and release. Research exploring retinal RGC diversity and function is vast and complex, and by chipping it away slowly but surely, it can help advance our current understanding of how these cells play a crucial role in vision and how their dysfunction causes dreadful diseases such as glaucoma and optic neuropathy.
Leptin Neutralizing Antibodies: A Novel and Safe Way of Treating Obesity, Reversing Liver Fibrosis, and Improving Associated Metabolic Disorders in Mus musculus
Ariana Chaudhary, Shangang Zhao, PhD
As one of the largest public health issues, obesity impacts nearly 1 billion individuals worldwide, its ramifications extending beyond heightened mortality rates and increased morbidity. Obesity contributes to afflictions such as hypertension, hyperlipidemia, diabetes, cardiovascular diseases, liver fibrosis, cancer, and mental health disorders; however, it is a treatable condition. Once thought to be a lifestyle choice, obesity is now recognized as a disease, in part caused by an imbalance of hormones, such as leptin. This study evaluates the impact of reducing leptin levels to achieve weight loss in diet-induced obese mice. Leptin reduction was achieved through leptin-neutralizing antibodies (LepAB), glucagon-like peptide receptor agonist liraglutide, and a combination of both. The findings reveal that LepAB not only induces significant weight loss as a standalone intervention (p < 0.01) but also markedly amplifies the weight loss effects of liraglutide (p < 0.0001) compared to the control and liraglutide alone. Furthermore, the improved oral glucose tolerance tests indicated increased insulin sensitivity, the reduction of trichrome staining exhibits decreased liver fibrosis, and Eco-MRI measurements confirmed that weight loss ensued from fat breakdown. These overwhelmingly positive results guide us toward the future development of combination therapy as a promising strategy for sustainable weight loss, significantly reducing the risk of preventable disease and premature death. Overall, this study offers meaningful insights into combating the pervasive epidemic of obesity, thereby uplifting societal welfare through economic and mental enhancements, as well as presenting a transformative pathway for patients to overcome obesity-related challenges.
ALKBH5 is a novel therapeutic target to treat brain metastatic breast cancer
Mannat Dhillon, Manjeet Rao PhD, Panneerdoss Subbarayalu PhD.
Greehey Children’s Cancer Research Institute, Department of Cell Systems & Anatomy, UT Health San Antonio
Introduction: Breast cancer remains one of the most frequently diagnosed malignancies and is a leading cause of cancer-related death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC) defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) accounts for approximately 10–20% of breast cancer cases. TNBC is more likely to occur in younger women and is responsible for a disproportionate number of breast cancer deaths. A major contributor to this mortality is the high incidence of relapse and distant metastases, particularly to the brain, which affect 25–50% of TNBC patients. The poor prognosis associated with TNBC is largely driven by the presence of cancer stem cells (CSCs), which contribute to tumor aggressiveness, therapy resistance, recurrence, and metastasis. Despite the growing understanding of TNBC biology, current treatment strategies remain limited and are largely reliant on chemotherapy, which is often ineffective against metastatic disease and associated with substantial toxicity. There is therefore a critical need to identify novel, targetable mechanisms that drive TNBC stemness and progression.
Materials & Methods: Breast cancer cells lines (231Br met, MDA-MB-231, MDA-MB-468,) were cultured in standard medium. Breast cancer cells were treated with vehicle control or mefloquine. After 48h of drug treatment BC including metastatic BC cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays and immunofluorescence analysis of DNA damage proteins including 53BP1 and gH2Ax.
Results: Our analysis reveals that m6A demethylase ALKBH5 is amplified and is highly overexpressed in BC patients. Using in vitro and in vivo orthotopic mouse models, we discovered that ALKBH5 supports breast cancer growth and metastasis. Notably, we show that ALKBH5 supports TNBC stem cell self-renewal. We discovered that ALKBH5 induces the expression of several genes involved in stem cell function, cell cycle and DNA repair. Consistent with that we demonstrate that depletion of ALKBH5 blocks cell cycle progression, leading to termination/stalling of replication fork and impaired DNA repair, and apoptosis of TNBC cells.
Conclusion: These findings indicate that Mefloquine has significantly reduced Brain metastasis TNBC and TNBC proliferation in various aspects. Our data suggest that Mefloquine is a potential therapeutic agent to inhibit ALKBH5 activity.
Acknowledgements: Special thanks to Prof. Manjeet Rao, Rao lab current & past members, and collaborators and Voelcker Biomedical Research Academy (VBRA). Funding Sources: NIH-NCI R21, NIGMS R01GM113245-01, CPRIT
Investigating Imitative Deficits in Fragile X Syndrome Through Eating Behaviors in Mice Models
Paulina Garcia, Eric Oh, Rodrigo Gonzales-Rojas, and Hye Young Lee, Ph.D.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in imitative behaviors, which are essential for social learning and interaction. Fragile X syndrome (FXS), recognized as a leading monogenic cause of ASD, caused by a mutation in the Fmr1 gene has similarly been linked to impairments in imitation mouse models. Previous studies have demonstrated the fact that Fmr1 knockout (KO) mice exhibited reduced imitation of scratching behaviors observed in demonstrator videos (Gonzales-Rojas et al., 2020, Scientific Reports). A recent study further suggests that feeding behavior in mice may also involve an imitative component (Xie et al., 2022, Neuroscience Letters).
Before evaluating this behavior in Fmr1 KO mice, it is critical to first establish whether wild-type (WT) strains exhibit imitative eating behaviors as a baseline. To this end, we examined two widely used strains—FVB and C57BL/6J (BL6)—across both male and female cohorts, to determine baseline levels of imitative eating. Over the course of three weeks, four cohorts (male BL6, male FVB, female BL6, and female FVB) were exposed to video demonstrators of a continuously performing eating behavior or ambulatory behavior, for 60 minutes across six experimental sessions. Behavioral responses were recorded to assess the presence of imitative eating patterns across strain and sex. This initial control phase aimed to identify a reliable baseline strain suitable for subsequent KO testing.
Preliminary analyses were inconclusive, leading us to refine the protocol: we extended the study to five weeks (ten experimental sessions), increased the number of subjects, and removed a ten-minute beaker habituation period to reduce sleep duration that had previously limited observable eating behaviors. The revised design included all four original WT cohorts and introduced Fmr1 KO mice. The present study investigates whether Fmr1 KO mice display deficits in imitative eating behavior. By comparing the eating responses of Fmr1 KO and WT mice following exposure to demonstrator videos, we aim to determine whether Fragile X-associated imitation deficits extend beyond previously documented motor patterns to encompass socially mediated feeding behaviors.
Effect of estrogen/progesterone transcription program on FOXO1 dysregulation in endometriosis
Zulema Naomi Garcia, Virgil Bideau Sheralyn, Bijaya Kumar Nayak MD, Nameer B Kirma, PhD
Introduction: Endometriosis is a chronic, estrogen-dependent inflammatory disorder characterized by the presence of endometrial-like tissue outside the uterine cavity. It affects approximately 10% of women of reproductive age worldwide, leading to symptoms such as chronic pelvic pain, infertility, and dysmenorrhea. One key process disrupted in endometriosis is decidualization, a differentiation event initiated by progesterone (P4) in which endometrial stromal cells (ESCs) acquire a specialized, secretory phenotype in response to estrogen, (E2) progesterone, (P4) and cyclic adenosine monophosphate (cAMP). This process is important for embryo implantation and normal reproductive function. In women with endometriosis, impaired decidualization may contribute to infertility and altered endometrial receptivity. Fork head Box 1 (FOXO1) is a major transcription factor that regulates P4 receptor binding to genomic targets in ESCs during decidualization, but less is known about its role in endometriosis and the exact mechanisms require investigation. Our hypothesis states that FOXO1 dysregulation caused by an altered hormonal response in ESCs contributes to endometriosis development.
Methods: To further investigate the effects of progesterone on FOXO1 expression and its hormonal influence on endometrial cells, we observed the cell morphology over a time course, using primary endometrial stromal cells from patients with and without endometriosis. We conducted further studies using an established cell line (tHESC). With the following treatments: Control, E2 (10nM), P4 (10µM), cAMP (500µM) [collectively called, EPC] and an additional treatment with P4 alone. (10µM). We observed the decidualization in cell culture for 9 days of EPC treatment. We then tested the levels of the decidualization marker prolactin (PRL) using ELISA and qRT-PCR. We also examined RNA levels using qRT-PCR, visualized proteins using Immunofluorescence and western blotting,
Results: We showed impaired decidualization in ESCs from endometriosis, which exhibited lower PRL expression with EPC treatment. Further, our initial findings show that FOXO1 protein is suppressed by P4 in eutopic ESCs from women without endometriosis, whereas this response was refractory in ESCs from subjects with endometriosis. Little effect was observed on FOXO1 with E2 alone. We also assessed whether this differential response on FOXO1 in endometriosis contributes to ESC survival and found that endometriosis ESCs undergo less apoptosis than non-endometriosis ESCs. Protein expression levels of FOXO1 and P4 receptor were significantly lowered when subjected to high levels of P4, while lower levels of P4 treatment resulted in higher protein expression. Immunofluorescence microscopy of FOXO1 during decidualization in endometriosis ESCs showed more nuclear localization of FOXO1 compared to the normal.
Conclusion: ESCs from endometriosis subjects are deficient in P4 induced decidualization, as demonstrated by lower PRL after EPC treatment and refractory response in FOXO1 expression after P4 treatmentFOXO1 dysregulation in ESCs may further contribute to endometriosis development, which will be validated by future functional studies.
Confirming the Genotypes of the Mouse Lines of FVB and C57BL/6 Strains Using the PCR Method
Bella Gonzalez, Eugenio Elizondo, Hye Young Lee PhD
Genotyping is the process of determining an individual’s specific genetic makeup by analyzing targeted DNA sequences. This technique is essential for accurate strain identification and characterization of the laboratory animals. The polymerase chain reaction (PCR) method serves as the primary tool for identifying the presence or absence of specific DNA sequences through the use of sequence-specific primers. These primers are single-stranded DNA molecules that are critical to the PCR process due to their ability to selectively amplify desired DNA sequences. Following PCR amplification, gel electrophoresis is used to visualize and analyze the amplified products. During electrophoresis, DNA fragments migrate through the gel matrix based on their size, allowing for the identification of specific base pair lengths that correspond to target sequences. The success of PCR amplification and subsequent genotyping is determined through careful gel analysis. Positive and negative controls are incorporated into each experimental run to ensure procedural integrity and validate results. In this laboratory setting, genotyping is utilized to identify genetic markers specific to relevant mouse lines in the FVB and C57BL/6 (BL6) strains. These two strains exhibit distinct phenotypic characteristics, including differences in memory performance, anxiety levels, and depression-related behaviors. Accurate mouse identification is crucial for maintaining experimental validity across laboratory projects. Given the diverse phenotypic characteristics associated with each mouse line, proper genotyping ensures that researchers can account for line-specific traits when designing experiments and interpreting results. This systematic approach to line verification supports the reproducibility and reliability of research conducted within the laboratory.
Behavioral and Neurogenic Outcomes in KMO-/- and HAO-/- Mice
Alexis Holmes1, Miguel A. de la Flor1,2, Natalia Kuhn-Sandoval1,2, Jason C. O’Connor1,2
1Department of Pharmacology, UT Health San Antonio
2Audie L Murphy VA Hospital, South Texas Veterans Health System, San Antonio
Kynurenine 3-monooxygenase (KMO) and 3-hydroxyanthranilate 3,4-dioxygenase (HAO) are key enzymes in the kynurenine pathway (KP). These enzymes are increased in response to brain injury, illness, or aging. This can lead to higher levels of 3-hydroxykynurenine (3-HK) and quinolinic acid (QA), which push the pathway toward oxidative metabolism. This shift has been linked to neurodegeneration and problems with learning and memory. Previous studies in our lab suggest that KMO knockout (KMO-/-) mice have lower levels of 3-HK and QA and higher levels of the neuroprotective metabolite kynurenic acid (KA), but show more anxiety-like behavior compared to wild-type (WT) controls. Based on this, we thought that HAO knockout (HAO-/-) mice might show similar anxiety-like behaviors. To test this, we used the open field test and elevated plus maze. We found no statistical differences in anxiety-like behavior between HAO-/- and WT mice. We also measured general movement and found no significant differences in locomotor activity between the two groups.
We also thought that KMO-/- mice might recover better after ischemic stroke because of their lower levels of 3-HK and QA and higher levels of KA. To test this, we scored the skill reach test in adult male and female WT and KMO-/- mice before and after stroke. These results are currently being analyzed. In addition, we are studying whether KMO-/- mice have more cell proliferation in the dentate gyrus of the hippocampus.
Gene Expression of Inflammation and Aging Markers in Females
Born to Obese Mothers – the Preliminary Insights
Linda Lopez, Ariana Samaniego, Meera Rath, PhD, Egle Bytautiene Prewit, MD, PhD
Introduction: Obesity poses a significant threat to long-term health, contributing to chronic disease and a reduced lifespan. Additionally, the effects of obesity extend across generations, with maternal obesity increasing the risk of adverse long-term health outcomes for offspring, such as a higher susceptibility to breast cancer in female offspring. Our laboratory’s research using a mouse model demonstrated this transgenerational vulnerability: female mice born to obese mothers and exposed to the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) showed decreased lifespan, a significantly higher incidence of tumors, and elevated circulating IL-6 compared to their counterparts born to non-obese mothers. The pro-inflammatory cytokine IL-6 is notably elevated in various cancers and tends to increase with age, indicating a potential role in aging and disease development. The Klotho gene declines with age and is regarded as a marker of aging. The objective of this project was to examine how maternal obesity and postnatal exposure to carcinogenic agents influence the expression levels of IL-6 and Klotho in female offspring during mid (30 weeks) and late (52 weeks) life.
Methods: CD-1 mice were fed either a standard fat (SF, 5.8% fat) or a high-fat (HF, 34.9% fat) diet for three months before conception, during pregnancy, and throughout lactation. After weaning, female HF and SF pups were switched to an SF diet. At six weeks, the females received 10 mg of DMBA in peanut oil via oral gavage once a week for four weeks, until they were nine weeks old. Tissues were collected at 30 and 52 weeks of age. mRNA levels of IL-6, α- and β-Klotho in liver and kidney tissues were measured using qPCR (1-2 pups per dam/per diet/per age). For data analysis, the unpaired T-test and Welch test were used as appropriate (P < 0.05). Based on the literature and our data, we expected that IL-6 would increase with age and exposure to maternal obesity, while Klotho should decrease.
Results: IL-6 and α-Klotho were significantly increased in kidneys at week 30 in HF females (P=0.014 and P=0.042, respectively). At week 30 of age, β-Klotho and IL-6 levels tended to be lower in the HF group in the liver. Later in life, at week 52 of age, IL-6 levels (P=0.018) were significantly lower in the kidneys of HF mice, while there was no difference in α-Klotho levels (P=0.46). In the liver, β-Klotho and IL-6 tend to be lower at 52 weeks of age.
Conclusions: Our findings indicate that exposure to maternal obesity and postnatal carcinogenic chemicals impacts the gene expression of IL-6 and Klotho in the liver and kidneys of female offspring. In HF female offspring, compared to SF, 1) IL-6 mRNA levels were higher in the kidneys at 30 weeks but lower at 52 weeks; 2) IL-6 levels in the liver remained consistently lower; 3) α-Klotho gene expression increased in the kidneys, while β-Klotho expression decreased in the liver. A limitation of this study is the small number of mice in each diet group that were not exposed to a carcinogen. Therefore, although our data show expected changes in IL-6 mRNA in the kidney during mid-life and β-Klotho in the liver at both ages, further research is needed to better understand how maternal obesity and cancer development influence females born to obese mothers, especially regarding inflammation and aging markers.
Retrospective Athlete Mental Health Study
Autumn MacPherson & Summer Rolin Psy. D
Although often overlooked, student-athletes experience increased levels of stress due to the pressure of sports, navigating new environments, and balancing their academic responsibilities. There was an added stressor due to the COVID-19 pandemic, which suggested a nationwide mental health crisis, specifically, in an NCAA student-athlete COVID-19 well-being study, students had reported mental health concerns 150 to 250 percent higher than previously reported by NCAA athletes. All these factors have led to various mental health concerns in the student population, such as insomnia, depression, anxiety, and adjustment disorders. To combat this, the UTSA student athlete population has direct access to psychology services. A Pre-Participation screener is utilized as part of a preseason physical to identify students who may benefit from services. Students complete a Sports Mental Health Assessment Tool 1, SMHAT-1, a standardized assessment tool created by the International Olympic Committee as a general gauge of students’ current mental health standings. Data was collected retrospectively from student screeners and from the UTHSCSA Electronic Medical Record, which included sex, ethnicity/race, education level, injury, medication, and mental health visits. The majority of the sample, 592 people, were male, and 325 were female, with a mean age of 20.12. Positive variables were created based on test cut-scores to identify athletes tested as positive on the screening measure. Across all sports, the rates of mental health variables were below 10%. Chi-square analysis was used to examine the relationship between gender and positive rates for depression, suicidal ideation, anxiety, ADHD, sleep problems, disordered eating, alcohol misuse, and drug misuse. The assumption of independent observations was met. Statistical significance was evaluated at the p ≤ .05 level. There was a significant difference between females testing positive for anxiety versus males (χ² (1) = 41.67, p≤ 0.00), ADHD (χ² (1) = 13.68, ps 0.00), and Disordered eating (χ² (1) = 4.97, р = 0.02). Whereas males had significantly higher positive results for alcohol (χ² (1) = 14.29, p≤ 0.00) and drug use (x? (1) = 4.75, p= 0.03). Overall, this data will provide the ability further to examine gender differences in mental health in the future. Along with this, the data provides insight on what concerns could more easily arise in future athletes, and brings attention to the risks of mental health concerns that so many athletes may be prone to in future years.
Investigating Symptom Onset and Therapeutic Strategies for Parkinson’s Disease and Amyotrophic Lateral Sclerosis
Emad Makinsi, Georgianna Gould, Senlin Li
Amyotrophic Lateral Sclerosis (ALS) and Parkinson’s Disease (PD) are neurodegenerative disorders affecting about 30,000 and 1 million Americans, respectively. Both conditions exhibit progressive motor deficits, starting with muscle twitching and weakness, and later developing into breathing difficulties and slurred speech. Research is ongoing to identify causes and treatments.
A key protein linked to PD is α-Synuclein, which enhances neurotransmitter release and aggregates with biogenic aldehydes like 3,4-dihydroxyphenylacetaldehyde (DOPAL). To explore its effects, we used mice overexpressing α-Synuclein alongside knockout mice lacking aldehyde dehydrogenase enzymes (ALDH 1a1 and ALDH 2). This setup expedited neurodegeneration and motor dysfunction, raising questions about the role of α-Synuclein and elevated DOPAL levels in social withdrawal and motor impairment.
While no significant differences were observed during the physiological measurements or behavior tests, A test in which there were prominent differences was the marble burying test, which was used as a measure anxiety-like behaviors following novel environment and social interactions. This suggests that the mice with overexpression of α-Synuclein buried fewer marbles than wildtype mice, suggesting that they may have experienced less anxiety-like behaviors after sociability tests. This may also suggest that there may be some interplay between biogenic aldehydes that we focused on in the study, and α-Synuclein.
Additionally, the glial cell-derived neurotrophic factor (GDNF) has shown promise in protecting motor neurons. Li lab studies demonstrated that SH-SY5Y cells treated with GDNF from rats and humans had increased viability by 59% and 92%, respectively. However, delivering GDNF to the brain is challenging due to the blood-brain barrier (BBB).
To overcome this, the Li lab utilized genetically engineered hematopoietic stem cells (HSCs) modified with lentiviral vectors containing GDNF. This allows HSCs to differentiate into macrophages that can cross the BBB and target ALS-affected areas. They limited differentiation to macrophages by using a specific promoter in the lentivirus. After preparing the HSCs, they were transplanted into mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which models ALS. Testing included behavioral assessments and evaluations of tyrosine hydroxylase (TH)-positive dopaminergic neurodegeneration in the substantia nigra and striatum. Results showed that using macrophage-specific promoters reduced dopaminergic neurodegeneration and improved locomotor activity. Future plans in the Li lab involve using AI for neuro-scoring to better assess neurodegeneration in mouse models, enabling earlier detection of ALS and PD.
Assessing Mitochondrial Function Differences in Naked Mole Rats and Mice Using Seahorse XF96 Profiling
Aneesa Moreno, Adam Salmon, Steve Otero, Harshita Anandabarathi, Jarett Goldblatt, Scarlette Hong, Ariv Sadh, Michael Saxton
The aging process is associated biologically with the decline in many major body functions which result in age related diseases such as cardiovascular, neurological, and more. These age-related impairments are closely associated with the decline in mitochondria function, which affects energy production, redox balance, and cellular respiration. This study aimed to compare mito-respiration between tissues from long-lived naked mole rats (NMRs) against short-lived mice, focusing on tissue specific differences using Seahorse XF96 profiling. NMRs have been shown to withhold resilience to many different biological factors within aging as well as cancer, their lifespan reaching 40 years while a mouse’s average lifetime ends at around 4 years. Frozen brain, liver, and heart tissues from both species were assayed using the Seahorse XF96 platform to measure oxygen consumption rates (OCR) driven by Complex I and Complex II substrates. Analysis in mouse and naked mole rat tissues suggest that the mouse liver exhibited the highest mitochondrial respiration within Complex I and II while NMR liver displayed greatly reduced activity. This data suggests NMR liver may rely less on Complex II-driven respiration, potentially reducing ROS generation and preserving tissue integrity during aging. Mouse liver’s high respiration could pertain to a higher chance of oxidative stress within its species, leading to greater risk of age-related diseases and mitochondrial dysfunction. Overall, these findings support the idea that reduced mitochondria activity in some NMR tissues, particularly the liver, might serve as a protective strategy to limit oxidative damage and extend lifespan. The Western Blot analysis is currently underway to assess protein-level expression of mitochondria and oxidative-stress related markers in the same tissues. Future experiments will expand the number of biological replicates and include a stratification by age and sex to improve resolution of species-specific mitochondrial adaptations.
Role of Hypothalamic Oxytocin Receptor in Mediating the Maternal Cardiovascular Health Benefits
Genna Mountcastle, Ariana Samaniego, Egle Bytautiene Prewit, MD, PhD
Epidemiological data show that women who did not lactate have a higher risk of cardiovascular disease later in life compared to those who did. We hypothesize that lactation provides long-term maternal cardiometabolic health benefits due to increased oxytocin production, which remains active throughout the lifespan. Preliminary data from the lab indicated that mice that did not lactate had lower circulating oxytocin (OXT) levels and reduced gene expression of oxytocin (Oxt), oxytocin receptor (Oxtr), and CD38 (an activator of central OXT release) in the hypothalamus (HT) than mice that breastfed their pups. The objective of this study was to investigate the role of Oxtr in the HT in mediating the cardiovascular benefits of lactation in mice.
Experimental Design and Methods: CD1 mice HT were collected at delivery for tissue culture experiments. The HT was cut in half, placed in a 6-well plate with media, and combined with either 100 µL of media, plasma collected from dams on day 1 after stopping breastfeeding, a CD38 antagonist, or a combination of CD38 antagonist and plasma. The supernatant was sampled at 4 and 24 hours of incubation. The tissue was collected 24 hours after the procedure. The levels of Oxt, Oxtr, and CD38 in HT were measured using qPCR. The OXT concentration in the supernatant and plasma was measured with an OXT immunoassay. Cre-lox Oxtr knockout in HT (KO) and Cre-lox control (CTRL) mice were bred, delivered, and lactated. Their blood pressure was measured at 6 months post-delivery using the tail-cuff method. For data analysis, the Student T-test and one-way ANOVA with post hoc tests were used as appropriate (p < 0.05).
Results: HT explants treated with a CD38 antagonist and plasma showed higher, though not statistically significant, Oxtr and CD38 gene expression compared to the other exposure groups (data not shown). Systolic, diastolic, and mean blood pressure in KO mice lacking Oxtr in HT were significantly higher than in CTRL mice (Figures 1 – 3, respectively). The plasma concentration of OXT tended to be lower in KO mice compared to the CTRL group (205 ± 24 pg/mL vs. 223 ± 33 pg/mL).
Conclusion: Our tissue culture experiments demonstrate that lactation during pregnancy in mice increases Oxt levels by acting on Oxtr in the hypothalamus, resulting in long-term benefits for maternal cardiovascular health. Data from KO mice indicate that Oxtr is crucial for the oxytocin cycle, as maternal cardiovascular health is impaired in its absence.
Discerning the molecular basis of the neoadjuvant use of Phellodendron amurense with radiation in prostate cancer
Avani Nagaragere, Sean Vargas, Joseph W Basler, Chul S Ha, Ikeno Yuji, Rita Ghosh, Addanki P Kumar
Department of Molecular Medicine, Pathology, Radiation Oncology and Urology; The University of Texas Health Science Center, San Antonio, TX
Radiation therapy is a standard treatment for prostate cancer patients diagnosed with localized disease. Although radiation is an effective treatment option for most patients, progression to recurrence and side effects that preclude optimum quality of life are critical barriers. Therefore, adjuvants that can prevent cancer relapse or lower RT-mediated side effects are an active area of cancer research. Along these lines, previous studies from the Kumar laboratory showed that a natural compound (Nexrutine) enhanced radiation-induced growth inhibitory activities of prostate cancer cells in culture and in a preclinical animal model. Additionally, in a clinical trial prostate cancer patients that received Nexrutine prior to radiation had lower levels of prostate specific antigen. Prostate specific antigen is clinically correlated with tumor volume. To uncover the molecular basis of the observed beneficial effects, we performed digital spatial profiling on formalin-fixed paraffin embedded prostate tumor specimens from pre-treatment diagnostic and post-radiation biopsy samples. Pathway enrichment analyses of these data revealed enrichment of genes involved in androgen response, mTORC1 signaling, oxidative phosphorylation, ribosome biogenesis, and inflammatory signaling. We propose that changes in cancer cell metabolism influences inflammatory signaling that impinges on androgen response leading to the development of recurrent prostate cancer following radiation treatment.
RPS3 Inhibition to Target Breast Cancer Cell Growth and Metastasis
Meera Nair, Manjeet Rao PhD, Panneerdoss Subbarayalu, PhD
Greehey Children’s Cancer Research Institute, Department of Cell Systems & Anatomy, UT Health San Antonio
Introduction: Breast cancer is the most common cancer and is the primary cause of cancer-related deaths among women worldwide. As per American Cancer Society, approximately 1 out of 8 women will be diagnosed with invasive breast cancer over their lifetime. Among the different subtypes of breast cancer, triple-negative breast cancer (TNBC) occurs in only about 12% of cases but is the most aggressive subtype and has the worst prognosis because it lacks all three hormone receptors (estrogen, progesterone and HER2). As chemotherapy is the only available treatment option for TNBC in clinics, the quality of life for TNBC patients is highly compromised due to chemotherapy associated toxicity. Therefore, there is an urgent need to find alternative targeted therapy for TNBC treatment. We focused on the RPS3 gene to study its role in breast cancer. Ribosomal Protein S3 (RPS3) is a part of the 40S subunit of the ribosome which has shown to repair DNA damage and induce apoptosis (programed cell death). Our studies show that depletion of RPS3 significantly reduced TNBC cells viability, migration, and invasion and stem cell proliferation.
Methods: Triple negative Breast cancer cells lines (MDA-MB-231, MDA-MB-468) were purchased from the American Type Culture Collection and cultured in standard medium. Breast cancer cells were transfected either with siControl or siRPS3-1 and siRPS3-2. After 48-72 hour of post-transfection these TNBC cells were analyzed for cell viability, migration, invasion, colony formation, and western blotting.
Results: First we confirmed the RPS3 protein knockdown. Our results showed both siRNAs, siRPS3-1 and siRPS3-2, knocked down protein significantly. After confirming the RPS3 knockdown, we performed breast cancer cell viability, migration, invasion, colony formation assays and stem cell assays. Our preliminary results showed that siRNA knockdown of RSP3 showed significantly reduced breast cancer cell viability, migration, invasion and colony forming ability. Interestingly RSP3 knockdown showed reduced breast cancer stem cells when compared to control siRNA transfected breast cancer cells. In addition to this, the MCM2 protein (minichromosome maintenance protein), which is required for initiation and elongation of DNA replication, were significantly reduced in RPS3 knockdown breast cancer cells.
Acknowledgements: I would like to acknowledge and thank the Rao lab and all its members who made this project possible including my mentor Dr. Panneerdoss Subbarayalu. I would also like to thank the Voelcker Biomedical Research Academy for giving me this opportunity to explore my passion in research.
The Effects of Hepatic Lipoprotein Lipase Overexpression on Hepatic Lipid Accumulation and Metabolism
Gabriella F. Perdikis, Iriscilla Ayala, Ph.D., Marcel Fourcadot, Luke Norton, Ph.D.
Background:
Lipoprotein lipase (LPL) is a multifunctional endothelial enzyme whose main function is to hydrolyze triglycerides (TGs) in circulating TG-rich lipoproteins, such as chylomicrons and very-low density lipoproteins (VLDL), to release products such as fatty acid and monoacylglycerol which can then be taken up and processed by adipose and muscle tissues, islets, and macrophages¹⁻². Adipocytes rely on LPL to break down TGs, which supplies them with fatty acids, and to serve as an important marker for adipogenesis. An increase of LPL activity in this tissue causes excessive fat storage and leads to obesity. Meanwhile, in the human liver, LPL is only produced naturally at very low levels after the early postnatal stage. The same is true for mouse liver; however, the effects of transgenic overexpression of LPL in mouse liver have shown a twofold increase in both liver TG content and insulin resistance. Insulin plays a key role in signaling to hepatic cells when to suppress glucose production. However, such cell signaling is impaired in obesity models, causing a positive feedback loop in which both insulin levels and LPL activity in white adipose tissue increase that is commonly observed in many type 2 diabetes (T2D) patients. However, little is known about LPL’s effects on hepatic lipid metabolism and storage. Through this project, we aimed to analyze the effects of liver-specific LPL overexpression on lipid metabolism and storage in mice who were fed high-fat diets.
Methods:
In order to analyze the effects of overexpressing LPL in the liver, genetically modified mouse models with CRE/Lox recombinase technology were genotyped at weaning (~3 weeks) into either a control (WT/WT, n=5) or mutant (LoxP/LoxP, n=6) group. Once the mice reached 10 weeks old, they were injected with an adeno-associated virus (AAV) containing CRE recombinase – to overexpress hepatic LPL activity in mutant mice but to have no effect on the livers of control mice – then were placed on a 60% high-fat diet (HFD for 6 weeks. At the end of the 6-week period, mouse liver tissue was collected. Sample fragments, ranging in mass from 20-25 mg, each were homogenized in TRIzol. mRNA was extracted, then treated with DNase using Invitrogen’s DNase Treatment and Removal Kit. NanoDrop readings were conducted to ensure that each sample was diluted into a concentration of 200 ng/µL. mRNA samples were then read via NanoDrop to ensure an amount of 500 ng for processing into cDNA using Promega’s cDNA Kit. Standards were made containing 9 µL from all samples and dilutions ranging from 1:2 to 1:64. Each individual sample was then diluted at a 1:10 ratio using remaining template volume (10 µL) and utilized for relative gene expression via quantitative PCR (qPCR). LPL and CRE were analyzed via qPCR, along with three other genes involved heavily in fatty acid metabolism: CPT1A, PPAR⍺, and PPARγ. Four housekeeping genes — GAPDH, 18s, ActB, and B2M — were also analyzed via qPCR.
Results:
LPL mutant mice saw a 114-fold increase in their LPL levels when compared to control mice (p < 0.0001). However, there were very few differences in the levels of CPT1A, PPAR⍺, and PPARγ; only the ratios of CPT1A and PPAR⍺ to housekeeping gene 18s saw statistical significance (p < 0.05), whereas no statistical significance was observed in any ratio of housekeeping gene to PPARγ levels (p > 0.05).
Conclusions & Future Directions:
Overall, our results proved that the CRE/Lox model is successful at overexpressing LPL in mice. However, so far, no statistically significant link at the transcriptional level has been observed between LPL levels and the levels of CPT1A, PPAR⍺, and PPARγ.
Future directions and continuations of this project will involve protein and lipid analysis on mouse liver samples to better understand any possible links at the translational or posttranslational level between the aforementioned genes, as well as to understand specifically how LPL affects fatty acid levels, storage, and metabolization within the liver. We also plan to extend the project’s database to mutant and control mice placed on a chow diet to determine how the regulation and production of LPL is affected by diet.
1. Wang, H., & Eckel, R. H. (2009). Lipoprotein lipase: from gene to obesity. American journal of physiology. Endocrinology and metabolism, 297(2), E271–E288. https://doi.org/10.1152/ajpendo.90920.2008
Shimizu, K., Nishimuta, S., Fukumura, Y., Michinaga, S., Egusa, Y., Hase, T., Terada, T., Sakurai, F., Mizuguchi, H., Tomita, K., & Nishinaka, T. (2022). Liver-specific overexpression of lipoprotein lipase improves glucose metabolism in high-fat diet-fed mice. PloS one, 17(9), e0274297. https://doi.org/10.1371/journal.pone.0274297
Investigating the Relationship Between Cellular Senescence and Circadian Rhythms
Noelia Plantenga; Alexis Garcia; Kristi Dietert, PhD; Kevin Koronowski, PhD
As improvements are made in both healthcare and medicine, more people than ever before are living to encounter the negative effects of age related diseases. This has resulted in the need for human longevity research to address this public health crisis. Numerous health disadvantages occur when the body’s circadian rhythm becomes disrupted, and circadian misalignment increases prevalence with age. Cellular senescence also contributes to aging phenotypes, as senescent cells accumulate with age and are known to drive age-related diseases. The exact relationship between the two processes remains under investigation.
In order to evaluate the extent to which cellular senescence has an impact on the circadian rhythms of surrounding non-senescent cells, a coculture experiment was performed with Per2LucMEF cells and IMR90 cells. The Per2LucMEF cell line contains a Luciferase reporter driven by the Period 2 (or Per2) promoter. Per2 is a core component of the molecular clock, enabling the cells to emit visually detectable light in a rhythmic fashion when treated with Luciferin, the substrate for Luciferase. Prior to recording, the cells were treated with Dexamethasone, a glucocorticoid receptor agonist known to synchronize cells’ circadian rhythms in vitro. These efforts combined allowed for the visualization of circadian rhythms through Period-synced light emissions with a plate reader. The Per2LucMEF cells were cocultured with IMR90 cells which do not contain a Luciferase reporter. The IMR90 cells were separated into two groups: irradiated IMR90s and control IMR90s, to show the impact that the senescence of these cells might have on the circadian rhythms of non-senescent Per2LucMEFs. This is accomplished because irradiated cells experienced irreparable cell damage, and therefore enter the state of senescence wherein they develop SASP (Senescent Associated Secretory Phenotype). Factors contained in SASP have been shown to influence the rhythms of non-senescent surrounding cells in previous experiments. Thus, we hypothesize the circadian rhythms of the Per2LucMEF cells cocultured with irradiated IMR90 cells will have a lower amplitude than those cocultured with the control IMR90s, likely due to SASP signaling.
In order to confirm the senescent state of our irradiated IMR90 cells, we are utilizing multiple methods, including senescence-associated β-galactosidase activity (SA-β-gal) staining. This stain uses the enzyme β-galactosidase, commonly used as a marker to detect senescent cells, to reveal the extent to which irradiating IMR90 cells results in cellular senescence. The quantitative analysis of this stain will conclude in the near future, along with other tests confirming senescence, such as qPCR and Western Blot to further confirm a full cellular senescence phenotype in culture.
Using data from the coculture of Per2LucMEF and IMR90 cells, as well as the SA-β-gal stain, we hope to provide evidence of the influence senescent cells have on non-senescent cells’ circadian rhythms, leading to a better understanding of both processes and contributing to future therapies to combat age-associated diseases.
Unveiling Area Deprivation: Patterns in Studyl Retention and Zip Code
Audrey Samora, Ashley LaRoche, Jeremy Tanner, MD
In South Texas, dementia poses a growing public health challenge, especially among underserved communities with limited access to specialized care and support services. This study explores the relationship between socioeconomic status, as represented by ZIP codes, and study retention in dementia-related research within the San Antonio and Rio Grande Valley regions of Texas. Motivated by the underrepresentation of socially disadvantaged populations in Alzheimer’s Disease and Related Dementias (ADRD) studies, the project aimed to determine whether income levels associated with ZIP codes influence participant retention in studies. Using data from 768 participants enrolled in a longitudinal observational study, researchers compared the enrollment status against average income data from their respective ZIP codes. Results revealed a notable disparity: a higher proportion of withdrawn participants were from lower-income areas, while higher-income ZIP codes correlated with greater retention. Specifically, 78% of withdrawn participants were from below-average income areas, supporting the hypothesis that lower socioeconomic status presents significant barriers to study retention. These findings emphasize the importance of addressing systemic inequities to improve retention and representation in dementia research, with implications for future interventions targeting community engagement and support services.
This project was supported from P30AG066546 (South Texas Alzheimer’s Disease Research Center).
Modeling Imitative Deficits in Fragile X Syndrome through Eating Behaviors in a Mouse Model
Jocelyn Tamez, Eric Oh, Rodrigo Gonzales-Rojas, and Hye Young Lee, Ph.D.
Autism spectrum disorder (ASD) is characterized by impairments in imitative behaviors, which are essential for social learning and interaction. Fragile X syndrome (FXS), a leading genetic cause of ASD, has previously been associated with imitative behavior deficits in animal models. Notably, earlier studies demonstrated that Fmr1 knockout (KO) mice showed reduced imitation of scratching behaviors observed in demonstrator videos (Gonzalez-Rojas et al. 2020 Scientific Reports). A recent research study suggested that eating behavior may also be imitative in mice (Xie et al. 2022 Neuroscience Letters).
Before evaluating this behavior in Fmr1 KO mice, it is essential to determine whether wild-type (WT) strains consistently exhibit imitative eating behaviors. In this study, we examined two commonly used strains, FVB and C57BL/6J (BL6), in both male and female cohorts to establish their baseline levels of imitative eating. Across three weeks, four cohorts (male BL6, male FVB, female BL6, and female FVB) were exposed to one of two video demonstrations for 60 minutes across six experimental sessions. The eating demonstrator video portrayed a mouse continuously performing an eating behavior, defined by biting or chewing a food pellet, while the ambulatory demonstrator video depicted a mouse walking. Behavioral responses were assessed in two ways: initially by measuring the change in pellet weight before and after each session and later through direct scoring of eating behaviors, such as biting and chewing, from video recordings. We transitioned to behavioral scoring after observing inconsistencies in the pellet weight data, including instances where pellets appeared to gain weight post-session, likely due to moisture accumulation. This transition also aimed to determine whether the alternative method would produce clearer trends.
This initial control phase was designed to identify a reliable baseline strain before proceeding to Fmr1 KO testing. Preliminary analysis of the data was inconclusive, prompting us to extend the experiment over a longer timeframe with an increased number of subjects. Additionally, we modified several experimental parameters, most notably removing a ten-minute beaker habituation period. Although initially implemented to acclimate the mice, the habituation phase was eliminated after we observed that mice often consumed the food pellet during this time, before the video demonstration began, compromising data integrity. The revised study is currently in progress, spans five weeks, and includes all four original WT cohorts while also introducing Fmr1 KO mice for subsequent testing.
The Effects of Ceramide-Lowering Therapy on Lifespan in Western-Diet Fed Female Inbred Mice
Klarisse Echevarria1, Eduardo Gutierrez1, Adolfo J. Garcia1,2, Juan Pablo Palavicini1,3, PhD
1Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health SA, 2Northwest Vista College, 3Department of Medicine, Diabetes Division, UT Health SA
The Geroscience hypothesis proposes that several chronic diseases, including Alzheimer’s disease, diabetes, and heart disease, share one underlying risk factor: aging. By modulating the biological mechanisms of aging, we can delay or prevent the onset of age-related diseases, ultimately, leading to a longer and healthier life. Ceramides, complex lipids that accumulate with age, have been linked to longevity and contribute to various age-related consequences, including cellular senescence, telomerase activity, autophagy, oxidative stress, mitochondrial dysfunction, insulin resistance, and inflammation associated with aging. Notably, the first longevity assurance gene discovered in yeast, LAG1, has been shown to encode proteins associated with the de novo ceramide synthesis. The deletion of this specific gene has been shown to extend lifespan in yeast and worms, suggesting that ceramides have a significant role in aging. This study investigated the effects of pharmacological inhibition of ceramide synthesis on lifespan using myriocin, a potent inhibitor of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step in ceramide biosynthesis. We hypothesized that myriocin would inhibit the overaccumulation of ceramides by reducing ceramide production. To conduct this experiment, 60 BALB/cByJNIA female mice were split into two arms: a control group and an experimental group (n=30/group). Both groups received a Western diet (WD); however, the experimental group received myriocin administered in their food every other week (WD+myriocin). The Western diet is a high-carb and high-fat diet that best emulates our modern American society’s eating habits and thus, has a strong human relevance. Our results have shown that at 30% mortality, there was a significant difference between the two, with myriocin showing an extension in lifespan (p=0.0163). However, maximal lifespan (90%) was not extended by the drug. As expected, WD showed unregulated weight gain, while myriocin prevented WD-induced weight gain. In conclusion, preventing ceramide accumulation proved to be beneficial for survival, particularly at early stages. The preclinical data generated in this study supports ceramide-lowering therapy as an effective treatment to extend lifespan.
Enhancing Autism Therapeutics: The Influence of Acute Cannabidiol treatment and the Estrous Cycle in Social Behavior in Female Mice
Abigail Windscheffel, Evan Zhang, Georgiana Gould
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by persistent social interaction deficits and repetitive behaviors. Current FDA-approved drugs reduce irritability and aggression but fail to effectively address core symptoms. Additionally, limited research on sex-based differences and hormonal changes complicates treatment in females. Therefore, this study aims to enhance pharmaceutical options that alleviate core ASD symptoms while investigating sex-specific treatment complexities. Cannabidiol (CBD), a non-intoxicating constituent of Cannabis sativa, has shown promise in alleviating ASD social deficits. The estrous cycle, a four-stage reproductive cycle in female mice, causes hormonal fluctuations that introduce behavioral complexities. We hypothesized that an acute 15 mg/kg CBD dose improves social behavior in adult BTBR and C57BL/6 mice, and that hormone changes during the estrous cycle influence social behavior. CBD’s impacts on autism-relevant behaviors were evaluated using social interaction sniffing, social novelty sniffing, marble burying, and a social dominance tube test. Additionally, fecal boli were counted to assess anxiety. Macroscopic and microscopic analyses were performed to determine estrous cycle stages, and hormone assays quantified estradiol and progesterone concentrations in blood samples. Significant behavioral effects were observed in marble burying, number of fecal boli, tube test wins, and tube test duration. In female mice, higher estradiol levels correlated with increased social novelty, and higher progesterone levels correlated with reduced repetitive behavior. These findings demonstrate that CBD has potential to improve ASD core symptoms and that reproductive hormone fluctuations influence behavioral outcomes.