Posters

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The Postdoc Poster Session is an opportunity for postdocs to share their research in a relaxed atmosphere with fellow researchers on campus. Posters are encouraged to be accessible to a broad academic audience including graduate students, postdocs, and faculty from a wide spectrum of disciplines.  

Winners will be selected based on abstract and presentation quality by the SOPS executive committee. 

Prizes: $200 for 1st place, $150 for 2nd place, $100 for 3rd place, $50 for 4th place

Thanks to Proteintech for sponsoring the 1st place prize! 

Development of the acoel Hofstenia miamia into a model for the study of chromatin regulation of regenerative processes

John Allen | Arts and Sciences | Biology

Poster # 01

Introduction: Regeneration is a striking phenomenon in which certain animals can replace lost body parts. However, how organisms output a correct regenerative response is not completely understood. We hypothesize that the active regulation of chromatin, i.e., how the genome is organized and accessed, is critical in converting external signals into gene expression changes required for regeneration. Using planarians, we have shown that histone H3 lysine 4 (H3K4) methyltransferase complexes are functionally important in regeneration, but the mechanistic details underlying their functions remain elusive without transgenic tools. To overcome this, we are adopting a complementary model species, the acoelomate Hofstenia miamia. These acoel worms will allow us to use transgenesis to address questions of both molecular mechanism and evolutionary conservation.

Methods: Gene family phylogenic trees were constructed by aligning planarian, Hofstenia, and selected species homologs and inferring phylogenic trees. dsRNA was injected into Hofstenia to induce RNA interference (RNAi). After RNAi knockdown, worms were bisected and observed for regeneration defects.

Results:  Paralogous gene duplications were observed for the subunit dpy30 in both Hofstenia and Schmidtea, suggesting functional diversification for dpy30 in these lineages. The complement of H3K4 methyltransferases for Hofstenia was determined and revealed four putative kmt2 family genes. RNAi for Hofstenia kmt2 showed regeneration defects.

Discussion: Understanding how mechanisms of regeneration are conserved between species provides insight into how regeneration evolves, how it is regulated, and what could result in loss of regenerative ability. This work compares two regenerative species to uncover how epigenetic regulation of chromatin states regulates regeneration.

Platelet secretion in wound healing: the essence of α-granule endocytosis, biogenesis, and release kinetics

Daniëlle Coenen | Medicine | Molecular and Cellular Biochemistry

Poster # 02

Introduction: Wound healing is a multi-step process involving different cell types and growth factors. Though platelet-rich plasma has been used to accelerate wound repair, detailed functional understanding of how direct platelet functions affect wound healing is limited. We investigated the roles of platelet cargo uptake and release in a full-thickness wound model.

Methods: Two dorsal circular excisions were made on mice defective in α-granule biogenesis, endocytosis (platelet-specific), exocytosis, and wildtype mice. Wounds were measured daily to assess healing progression. On days 3 and 7, wound sites were harvested for histology. Levels of bioactive molecules were analysed from wound tissue extracts.

Results: Mice with defective α-granule biogenesis and cargo packaging showed severely impaired wound healing with a distinctive morphology and histology. Mice with improper endocytic trafficking and fibrinogen uptake/storage also showed remarkably slower wound healing. Interestingly, exocytosis-defective mice presented with faster healing kinetics, and histologically resembled healing and morphology of wildtype mice. Over the course of wound resolution, levels of various bioactive molecules changed. Most of the ones examined decreased, except for FGF2 and MMP-9, reflecting final remodelling. This was altered in α-granule biogenesis-defective mice, where wound FGF2 and MMP-9 levels decreased instead of increased as the wounds healed. Furthermore, the correlation between wound resolution and wound IL-1β, MMP-3, TIMP-1, and VEGF levels was less clear or non-existent in these mice.

Conclusions: Platelet functions have specific roles in the progression of wound healing and, potentially via extravascular migration, may impact the presence and levels of bioactive molecules in the skin microenvironment.

Immunochemical and transcriptomics evidence of Schwann cell reprogramming in an in vitro model of human peripheral nerve degeneration

Gabriela Aparicio | Medicine | Neurosurgery

Poster # 03

Introduction: Schwann cells (SCs) are activated upon nerve injury to acquire a repair phenotype able to promote axon regrowth. However, scarce information is available on the SC repair phenotype in humans. The goal of this project was to investigate injury-related responses in cells from human nerves degenerated in vitro. 

Methods: We optimized methods for the culturing of isolated fascicles and whole nerve segments to recapitulate key events associated with the process of Wallerian degeneration such as SC activation. Microscopy analysis was done to characterize the cellular composition of the human nerves as well as the injury response. 

Results: Evidence of cell proliferation was found in endoneurial cells, possibly SCs, in in vitro degenerated nerve segments without signs of apoptotic cell death. However, myelin and axon degeneration were both incomplete after 2 weeks of culturing in vitro, as determined by measuring the expression of myelin protein zero and neurofilament, respectively. Spatial transcriptomics analysis of intact and degenerated tissues revealed dramatic changes in the gene expression profiles of all cell clusters particularly those consisting of or including SCs. Curiously, cellular reprogramming was not restricted to SC-containing clusters. Cells from epineurial and perineurial areas also reprogrammed their transcriptome after culturing in vitro.

Conclusions: Overall, we found that in vitro degeneration partially recapitulates human SC responses to injury, including myelin degradation and conversion into repair cells. This model may prove useful to investigate the complex molecular and cellular responses of human peripheral nerve cells after trauma or degenerative disease.

Utilizing a safety-by-design approach for the development of Zn-doped Hydroxyapatite nanoparticles for nitrogen delivery

Jarad Cochran | Agriculture, Food and Environment | Integrated Plant and Soil Sciences

Poster # 04

Introduction: To sustainably boost food production, researchers are developing nano-enabled fertilizers and pesticides. These materials, like Zn-doped Hydroxyapatite (ZnHAP), offer precise nutrient delivery and reduced environmental impact. 

Methods:This NP come in two forms: bare Zn-doped hydroxyapatite NP and with urea adsorbed to the NP. Additionally, surfactants, such as Silwet L-77, are incorporated to facilitate nanomaterial uptake through plant stomata by reducing surface tension. To evaluate the potential toxicity of ZnHAP, both with and without Silwet L-77 surfactant, mortality, reproduction, and growth were used as biological endpoints, with Caenorhabditis elegans used as our model species. Additionally, a Zn specific dye was used to track Zn sequestration inside the nematode after exposure. 

Results: ZnHAP exhibits moderate toxicity, with mortality rates ranging from 24% to 34% at concentrations of 100 mg/L to 275 mg/L, possibly due to Zn dissolution post-uptake. However, when urea was adsorbed to the NP, mortality decreased to zero, regardless of concentration. Reproductive capacity was unaffected without urea, but in the presence of adsorbed urea, ZnHAP showed a concentration dependent decrease in reproduction, with reproduction decreased by 60% at the highest concentration. Finally, the addition of Silwet L-77 at 0.1% and 0.2% induces significant toxicity in C. elegans alone and when combined with ZnHAP. 

Discussion: These findings suggest ZnHAP are potentially a less toxic NP for nitrogen delivery, and thus a promising option for nitrogen-enabled fertilizers. Further investigations into the toxicity and behavior of alternative surfactants in conjunction with ZnHAP with and without urea are ongoing.

Monitoring the conformational ensembles of a Bifurcating Electron Transfer Flavoprotein in solution

Anurag Priyadarshi | Arts and Sciences |Chemistry

Poster # 05

Bifurcating electron transfer flavoproteins (Bf-ETFs) perform electron transfer 'bifurcation' in a variety of microorganisms, including both bacteria and archaea. Bf-ETFs contain two flavin adenine dinucleotide (FAD) molecules. The bifurcating FAD accepts a pair of electrons from NADH and dispenses them to two separate pathways.(1) An exothermic electron transfer (ET) reaction to a high-potential acceptor, mediated by the second FAD termed ET-FAD, is used to drive endothermic transfer of the second electron to a lower potential acceptor: ferredoxin or flavodoxin semiquinone. Conformational dynamics are essential in maximizing the energy efficiency of bifurcation. The open and closed conformations of Acidaminococcus fermentans ETF (AfeETF) and Acetobacterium woodii (AwoETF) have been resolved by X-ray crystallography,(2) however our small angle neutron scattering (SANS) demonstrates that an ensemble of conformations must be invoked to explain the structure in solution. Moreover, we find that the ensemble is responsive to reduction of the flavins and binding of substrate NADH. We also demonstrate novel computational approaches integrating and extending the capabilities of methods initially designed for interpreting SAXS.

19F NMR provides high sensitivity spectra in which the observed chemical shifts are highly responsive to changes in local chemical environment.(3) Moreover, both these methods are non-perturbative of flavin oxidation states, removing the interpretational challenges that attend fluorescence and X-ray derived information as well as Cryo-EM structures of flavoenzymes. Therefore we are advancing this pair of methods to permit observation of Bf-ETF's conformational dynamics. In the current study, incorporation of 5-Fluo-Trp in wild type and mutant AfeETF was also accomplished, and 19F NMR measured with the objective of monitoring the conformational ensembles. The generation of multiple mutant AfeETF was done with the objective of inserting fluorinated Trp probe at multiple locations in the protein. Future directions include investigating the spectral responses upon partner protein binding and/or population of different redox states. Additionally, we will assign the observed signals to specific residues based on mutagenesis, to obtain insight into local structural features. 

(1) Mohamed-Raseek, N.; Miller, A. F.,  J. Biol. Chem. 2022, 298, 101733.

(2) Chowdhury, N. P.; Mowafy, A. M.; Demmer, J. K.; Upadhyay, V.; Koelzer, S.; Jayamani, E.; Kahnt, J.; Hornung, M.; Demmer, U.; Ermler, U.; Buckel, W., J. Biol. Chem. 2014, 289, 5145–5157.

(3) Khan, S.; Ansari, A.; Brachi, M.; Das, D.; El Housseini, W.; Minteer, S.; Miller, A.-F., J. Biol. Chem. 2024, 300, 107122.

Distinct Aortic Pathological Features in Mice with Smooth Muscle Cell-specific Deletion of Fibrillin-1

Bowen Li | Medicine | Saha Cardiovascular Research Center

Poster # 06

Background: Fibrillin-1(FBN1) is a crucial structural protein of elastic fibers; global mutations of this protein lead to aortic root and proximal ascending aortic aneurysms, exhibiting significant sexual dimorphism. FBN1 is abundant in smooth muscle cells (SMCs), the predominant cell type in the aortic wall. This study determined the effect of SMC-specific deletion of FBN1 on aortic pathologies in mice.

Methods: SMC-specific FBN1 deleted (SMC-FBN1-/-) mice were generated, and their aortic phenotypes were compared to their wild-type littermates (SMC-FBN1+/+) using ultrasonography, in situ and ex vivo imaging, micro-CT, proteomics, and histological analysis.

Results: Quantitative PCR and immunostaining confirmed the absence of Fbn1 mRNA and FBN1 protein in the aortic media, while it remained abundant in the endothelia and adventitia of SMC-FBN1-/- mice. Ultrasound monitoring observed luminal dilations in the ascending aortic region at 4 weeks of age, which progressively expanded with age in SMC-FBN1-/- mice compared to their SMC-FBN1+/+ littermates. micro-CT scanning confirmed expansions from the distal ascending region to the descending thoracic aortic region at 12 weeks of age, while no apparent dilations were observed in the aortic root, proximal ascending region, and abdominal aortic region of SMC-FBN1-/- mice. The aortic phenotype was consistent in both male and female mice. Proteomics analysis in mice at 4 weeks of age revealed a reduced abundance of extracellular matrix organization-related proteins in SMC-FBN1-/- mice, compared to their SMC-FBN1+/+ littermates. Histologically, elastic fibers with comparable laminar layers were formed in SMC-FBN1+/+ and -/- mice; however, SMC-FBN1 deletion led to thinner and fragmented elastic fibers.

Conclusions: SMC-specific deletion of FBN1 induced distinct aortic pathologies in the distal ascending aorta through the descending thoracic aortic region, irrespective of sex. Although SMC-specific FBN1 deletion did not prevent elastic fiber formation, it impaired aortic wall integrity.

Role of hypothalamic glucose receptor - Adgrl1 - in mediating leptin function

Nazmul Hasan | Medicine | Pharmacology and Nutritional Sciences

Poster # 07

Introduction: Adhesion G-Protein Coupled Receptor 1 (Adgrl1) is a hypothalamic glucose receptor involved in glucose and energy homeostasis. Mice lacking Adgrl1 develop insulin resistance and obesity. Because obesity contributes to leptin resistance, here we determined whether Adgrl1 signaling influences leptin function.

Methods: We measured the effects of leptin on food intake and body weight in mice lacking Adgrl1, specifically in the ventromedial nucleus of the hypothalamus (VMH). In addition, we selectively activated Adgrl1-expressing neurons in the VMH in Adgrl1Cre mice using chemogenetics involving designer receptors exclusively activated by designer drugs. Briefly, we injected viral vectors into the VMH of 6-8 weeks-old mice. Three weeks later, we administered clozapine N-oxide (CNO) to activate Adgrl1VMH neurons. We also measured hypothalamic Adgrl1 expression in high-fat diet (HFD)-fed and leptin-deficient (ob/ob) mice and in leptin-injected wild-type mice.

Results: Surprisingly, Adgrl1VMH-deficient mice showed reduced food intake in response to exogenous leptin despite obesity and hyperleptinemia. Conversely, chronic activation of the Adgrl1VMH neurons induced leptin resistance even without obesity. This activation also elevated baseline blood glucose levels and improved insulin sensitivity, highlighting the differential effects of Adgrl1 on leptin sensitivity and glucose homeostasis. Furthermore, leptin administration increased hypothalamic Adgrl1 expression. Interestingly, leptin-deficient ob/ob and leptin-resistant HFD-fed mice also showed upregulation of hypothalamic Adgrl1 expression.

Discussion: These findings reveal that hypothalamic Adgrl1 signaling interacts with leptin function to influence energy balance. This insight may help in understanding the molecular basis of leptin resistance in obesity, and in designing more efficient drugs to treat obesity.

GelBox: Open-source software to improve rigor and reproducibility when analyzing gels and immunoblots

Utku Gulbulak | Medicine | Division of Cardiovascular Medicine

Poster # 08

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/ 

Insulin Resistance and Alzheimer's Disease: Targeting Astrocyte Insulin Receptors to Address Gait Disturbances 

Ting-Hsuan Lu | Medicine | Pharmacology and Nutritional Sciences

Poster # 09

INTRODUCTION - Alzheimer’s disease, a leading cause of dementia, leads to memory loss, confusion and mobility issues. Currently an estimated 5 to 7 million older Americans are affected, with this number potentially rising to 13.8 million by 2060, highlighting the urgent need for better treatments. Besides cognitive decline, Alzheimer’s patients often suffer from central insulin resistance. Insulin is vital for brain function, regulating energy use and sharing processes for the clearance of beta-amyloid, a protein that forms harmful plaques. Astrocytes, a type of glial cell in the brain, contain insulin receptors that help manage cellular energetics. Disrupted insulin signaling in these cells impairs glucose metabolism and may contribute to Alzheimer’s progression. Here we overexpressed a truncated human insulin receptor beta-subunit (hIR-beta) with constitutive activity in somatosensory astrocytes in the 5XFAD model (mouse model of Alzheimer’s disease) and tested for functional amelioration on gait outcomes and multiple central processes.  

METHODS – Using two-photon imaging combined with a GCaMP Ca2+ sensor, we examined astrocytic network performance in somatosensory cortex in the aging 5xFAD and wildtype mice. We also investigated neurovascular coupling, and astrocytic network at rest and during ambulation.

RESULTS – Overexpression of hIR-beta in astrocytes improved network connectivity and neurovascular coupling. This improvement could help recover impaired brain function and slow disease progression.

DISCUSSION - This approach appears valuable for addressing dysregulated gait and may also benefit other brain regions, potentially improving overall cognitive function.

The regulatory role of asprosin in hypertension

Rubab Akbar | Medicine | Internal Medicine-Endocrinology

Poster # 10

Introduction: Asprosin is a novel adipokine, identified through the study of genetic disease called neonatal progeroid syndrome (NPS). So far, two spatio-temporally distinct functions of asprosin have been discovered. Asprosin cell-autonomously induces hepatic glucose release and stimulates appetite via the activation of agouti- related protein (AgRP) neurons of the hypothalamus. Asprosin performs these two spatio-temporally distinct functions via two different receptors. Ptprd (Protein Tyrosine Phosphatase type δ), a membrane bound phosphatase receptor mediates asprosin’s orexigenic function, while a G-protein coupled receptor, Olfr734 (mouse ortholog of OR4M1), acts as the hepatic receptor for its glucogenic function. 

Methods: Blood Pressure measurement was done by tail cuff method and Telemetry.

Results and Discussion: In this ongoing study we are assessing the role of asprosin in regulation of blood pressure (BP). Our preliminary results show that asprosin deficient female mice (NPS) present with significantly lower BP, which can be completely rescued with intra-nasal treatment of recombinant asprosin. Further, at the mechanism level, our preliminary data shows that asprosin’s hypertensive effects are mediated by Ptprd signaling in the oxytocin neurons. Both male and female mice, with genetic loss of Ptprd from oxytocin+ neurons (Oxy-cre+;Ptprdflox/flox) had significantly lower MAP (mean arterial pressure) when compared to wild type littermate controls (Oxy-cre+;Ptprd+/+). 

This study identifies a novel function of asprosin and represents a new avenue for therapeutic development for treatment of hypertension.

Exploring the Microbial Diversity and Composition of Three Cigar Product Categories

Sanjay Joshi | Agriculture, Food and Environment | Kentucky Tobacco Research & Development Center

Poster # 11

Introduction: Cigars and cigarillos are emerging as popular tobacco alternatives to cigarettes. However, these products may be equally harmful to human health than cigarettes and are associated with similar adverse health effects. 

Methods: We used 16S rRNA gene amplicon sequencing to extensively characterize the microbial diversity and investigate differences in microbial composition across 23 different products representing three different cigar product categories: filtered cigar, cigarillo, and large cigar. High throughput sequencing of the V4 hypervariable region of 16s rRNA gene revealed 2124 Operational Taxonomic Units (OTUs). 

Results: Our findings showed the three categories of cigars differed significantly in observed richness and Shannon diversity, with filtered cigars exhibiting lower diversity compared to large cigars and cigarillos. We also found a shared and unique microbiota among different product types. Firmicutes was the most abundant phylum in all product categories, followed by Actinobacteria. Among the 16 genera shared across all product types were Bacillus, Staphylococcus, Pseudomonas, and Pantoea. Nine genera were exclusively shared by large cigars and cigarillos and an additional thirteen genera were exclusive to filtered cigars. 

Discussion: Analysis of individual cigar products showed consistent microbial composition across replicates for most large cigars and cigarillos while filtered cigars showed more inter-product variability. These findings provide important insights into the microbial diversity of the different cigar product types.

Loss of Glucose in Urine Reprograms Mitochondrial Function and Increases Mannose Production in the Kidneys to Maintain Adequate Supply of Energy in Renal GLUT2 Knockout Mice

Nadia Rashid | Medicine | Pharmacology and Nutritional Sciences

Poster # 12

INTRODUCTION: Glucose transporters such as GLUT2 and SGLT1/2 are responsible for reabsorption of glucose in kidneys. Lack of renal Glut2 induces massive glycosuria in both humans and rodents, however, both are able to maintain normal supply of energy even when they are fasted. We therefore aimed to determine how renal Glut2 KO mice maintain normal energy and glucose homeostasis at baseline despite massive loss of glucose in urine.  We hypothesized that quantifying mitochondrial proteome and metabolome in the kidneys would identify novel adaptive pathways to explain this phenotype. 

METHODS: We used 8-10 weeks old male (C57BL/6) renal Glut2 KO and control mice. We adopted 2D-DIGE coupled with MALDI TOF/TOF mass spectrometry to quantify mitochondrial proteome. Untargeted metabolomics was performed to determine changes in metabolite levels in kidneys. 

RESULTS: Mitochondrial proteomics revealed significant alterations (1.5 up/down fold) in proteins involved in mitochondrial respiratory chain and energy homeostasis. Using western blot, we validated the downregulation of pyruvate carboxylase involved in gluconeogenesis, and glycine amidinotransferase involved in amine, polyamine and creatine biosynthesis in renal Glut2 KO mice compared to control. Kidney metabolomics demonstrated that mannose was 18-fold higher in the KO mice as compared to control group. Mannose enters glycolysis pathway through the enzyme phosphomannose isomerase (MPI). We further confirmed the increased levels of MPI in renal Glut2 KO mice using western blotting. 

CONCLUSION: Our data shows that reprogramming of mitochondrial function and enhanced mannose production are compensatory mechanisms that make up for the loss of glucose in urine in renal Glut2 KO mice. These pathways can be targeted to improve efficiency of SGLT2 inhibition in treatment of diabetes mellitus.

The biochemical and morphological characterization of platelet derived extracellular vesicles after the infusion of CAR-T cells in lymphoma patients

Lin Li | Medicine | Biochemistry

Poster # 13

Background: Diffuse large B cell lymphoma (DLBCL) represents a highly aggressive malignancy and stands as the most prevalent form of malignant lymphoid neoplasm. Chimeric antigen receptor T cells (CAR-T) are a new frontier of immunotherapy for fighting refractory neoplastic diseases. Up to 45 % of patients with lymphomas respond, the reasons for failure are not always clear. Blood platelets exert fundamental roles in thrombosis, inflammation and angiogenesis, contributing to different pathologies from cardiovascular disease to cancer. Extracellular vesicles (EVs) are membranous vesicles produced by all cells under physiological and pathological conditions. Platelet- and cell-derived EVs are important in cell-to-cell communication, and EVs can cross biological barriers to deliver their cargo (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells.

Aims: To explore the biochemical and morphological characterization of platelet derived EVs following CAR-T cell infusion in lymphoma patients and healthy controls.

Methods: In this study, platelet derived EVs from platelet poor plasma (PPP) of DLBCL patients (n=3) before and after CAR-T cell infusion as well as PPP and platelet rich plasma (PRP) of healthy controls (n=2), will be purified. EVs were isolated and characterized by fluorescence Nanoparticle Tracking Analysis (fNTA), ExoView and immunofluorescence microscopy to investigate their specific biochemical and morphological properties. Lipidomic and proteomic profiles will be explored on platelet-derived EVs in lymphoma patients before and after CAR-T treatment.

Results: The size distribution for EVs from PRP and PPP from healthy controls as well as PPP from DLBCL patients were characterized by fNTA and EVs particle counts on different CD9, CD63 and CD81 were measured and imaged by ExoView Tetraspanin chips. The Super-Resolution immunofluorescence microscopy for PRP and PPP were used to visualize the EV staining with CD63, platelet marker CD41 and α- granule marker P-selectin. Very few EVs in our preparations were stained with P-selectin in EVs prepared from either PRP or PPP. Comparing PRP-derived to PPP-derived EVs from healthy controls, the amounts of EVs are much more. PPP-derived EVs were smoother and discoid shape before CAR-T treatment, and they had more pseudopodia formation and aggregation after CAR-T treatment. Lipidomic and proteomic data are being acquired in subsequent phases of the study.

Conclusions: In summary, our study provides new insights into the molecular composition of human platelet derived EVs, EVs derived from PRP have higher amounts compared to PPP in healthy controls, and validate a method for isolating highly pure platelet-derived EVs as a basis for preclinical studies. EVs particle counts on CD9, CD63 and CD81 showed differences from PPP after CAR-T treatment compared to before in lymphoma patients. Future acquisition of lipidomic and proteomic EVs data promises further insights into this paradigm.

Spatially-Explicit Simulations Predict How Different Modes of MyBP-C Function Modulate Isometric Twitches

Caterina Squarci | Medicine | Internal Medicine-Cardiology

Poster # 14

Introduction: Multiple groups are trying to develop sarcomere-based therapies for Heart Failure with reduced Ejection Fraction. Trials that attempted to activate myosin with omecamtiv mecarbil showed minimal benefit, in part because the increased contraction compromised diastole. Cardiac Myosin Binding Protein-C (cMyBP-C) regulates both contraction and relaxation under physiological conditions and could be a more effective therapeutic target. However, cMyBP-C’s complex function makes it difficult to study in biological experiments.

Methods: Here we used FiberSim, a spatially-explicit model of half-sarcomeres to investigate how different potential modes of cMyBP-C function modulate contractile properties. In the model, cMyBP-C molecules are restricted to 9 stripes in the C-zone of the half-sarcomere where they have the appropriate stoichiometry (3 cMyBP-C molecules to 18 myosin molecular per 43 nm thick filament repeat). The cMyBP-C molecules can transition between a null state (no effect) and two states that respectively stabilize myosin in its suppressed super-relaxed / interacting heads motif / OFF configuration or bind to available sites on the thin filament. cMyBP-C molecules that are bound to actin increase thin filament activation via cooperative effects.

Results: The time-course of isometric twitch contractions is prolonged when cMyBP-C molecules bind to the thin filament. Peak force is reduced when cMyBP-C stabilizes the SRX state. Simulations in which some cMyBP-C molecules bind actin and some stabilize myosin in the suppressed state have smaller slower twitches. 

Discussion: Computational models could constitute a valid tool to integrate the study of cardiac pathologies.

Proteomics and Lipidomics Analysis of Mesenteric Lymph from Mice with Impaired Chylomicron Secretion

Khaga Neupane | Medicine | Saha Cardiovascular Research Center

Poster # 15

Background: Postprandial triglyceride (TAG) levels are an independent predictor of cardiovascular risk in humans. Chylomicrons (CMs) are the primary carrier of dietary TAG and they travel to circulation via intestinal lymph. The protein component of lipoproteins, including chylomicrons, can impact their metabolism in the plasma. While previous findings have discovered roles of several proteins associated with lipoproteins, the protein composition of newly secreted CM’s, prior to entry in the circulation where they are rapidly metabolized, has not been thoroughly studied using modern mass spectrometry methods. Furthermore, the lipid composition of newly released chylomicrons has not been extensively reported.

Objectives:  1) Determine the protein and lipid composition of mesenteric lymph during fasting and after a lipid bolus, 2) identify proteins in lymph that may impact the metabolism of newly secreted chylomicrons prior to entry into the circulation, and 3) determine the impact of disruption of intestinal CM secretion in the composition of mesenteric lymph.

Methods and Results: Mouse mesenteric lymph was collected under fasting conditions and after infusion of a mixed lipid bolus into the duodenum of wild type and Dennd5b-/- mice. This study revealed that, compared to wild type mice, Dennd5b-/- mice exhibit significantly reduced TAG content in the lymph (5 h, -94%, p<0.001). Electron microscopy imaging and analysis on the lymph samples revealed that the lymph of wild type mice had both greater number and larger sized lipid particles in comparison to the lymph of Dennd5b-/- mice. In addition, lipidomics analysis of fasting and postprandial lymph showed significant increases in triglycerides and free fatty acids in wildtype, but not Dennd5b-/- mice. Shotgun proteomics revealed significant changes in the protein content of lymph that was collected at fasting and 4 hours after lipid bolus administration. Gene ontology analysis for differentially expressed proteins revealed that intralipid infusion affects the expression of proteins involved in several lipoprotein metabolism pathways, only in wild type mice. 14 proteins were observed to be significantly increased in response to lipid including: Saa1 (+10%; p<0.01), Mttp (+51%; p<0.01), and Pla2g12b (+51%; p<0.01). These protein changes were confirmed by western blotting analysis of lymph. The Saa1 effect was of particular interest. Because Saa1 is known to bind to lipoproteins, we used size-exclusion chromatography to analyze the distribution of Saa1 protein across lipoprotein fractions in lymph. This demonstrated that Saa1 in lymph is associated primarily with very large size fractions that typically contain chylomicrons and to a lesser extent with HDL-containing fractions. In oil gavage studies in mice, we found that plasma Saa1 is elevated at 4 hours post-gavage and is primarily associated with HDL. To determine the source of post-gavage Saa1, mouse intestinal tissue was analyzed by western blot and immunofluorescence for Saa1. Western blotting and immunofluorescence demonstrate that intestinal Saa1 is increased in response to high fat diet.

Conclusions:  This study suggests that wildtype mice have lipid-poor ApoB-containing lipoproteins in lymph during fasting conditions. We identified several proteins that respond to dietary lipid ingestion and may contribute to postprandial lipoprotein metabolism in the lymph and periphery. In wildtype mice, a lipid bolus increases lymphatic Saa1-3 concentrations and Pla2g12b that may remodel CM’s prior to reaching the circulation. These effects were not observed in Dennd5b-/- mice, suggesting that these responses to dietary lipid are dependent on successful CM secretion from intestinal epithelium.

Aging influences color and oxidative stability of beef biceps femoris muscle

Ana Paula Pereira | Agriculture, Food and Environment | Animal & Food Sciences

Poster # 16

Introduction: Wet aging of postmortem skeletal muscles has been extensively utilized to enhance beef tenderness. On the other hand, prolonged aging could adversely affect beef color. Biceps femoris (BF) is a glycolytic muscle less susceptible to aging-induced discoloration. The impact of wet aging on the color stability of beef BF is not completely understood. Therefore, the aim of the present study was to examine the effect of postmortem wet aging (for 0, 7, 14, and 21 days) on color stability of beef BF muscles.

Methods: The BF muscles (IMPS #171B) were excised (24 h postmortem) from both sides of eight (n = 8) beef carcasses (USDA Select grade) obtained from the USDA-inspected meat laboratory of the University of Kentucky. The muscles were further divided into two equal-length sections, vacuum-packaged, and randomly assigned to aging at 2°C for either 0, 7, 14, or 21 days. During each aging period, muscle sections were fabricated into 2.5-cm thick steaks, individually over-wrapped, and allocated to refrigerated storage for 0, 3, or 6 days.  Instrumental lightness (L*), redness (a*), yellowness (b*), hue, chroma, and R630/580 were evaluated on each day of storage. The experimental design was a randomized complete block design, where each carcass served as a block. The main effects of muscle source and aging days, and their interactions were analyzed using the Mixed Procedure of SAS. The least square means for protected F-tests (P < 0.05) were separated by using least significant differences and were considered significant at P < 0.05. 

Results: BF steaks from 14 and 21 of days aging exhibited greater (P < 0.001) L* values on day 0 of storage, whereas non-aged steaks (0 day) exhibited greater (P < 0.001) lightness on day 6 of storage. During storage, non-aged steaks exhibited an increase (P < 0.05) in lightness, while BF steaks from 14 and 21 days of aging demonstrated a decrease (P < 0.05) in L* values from day 0 to 6. Aged BF samples (7, 14, and 21 days) exhibited greater (P < 0.05) a* values than their non-aged counterparts on storage days 0 and 3. Nevertheless, a* values decreased (P < 0.05) in all samples during storage. On days 0 and 3 of storage aged BF steaks exhibited greater (P < 0.05) b* values then compared to their non-aged counterparts. Aged BF steaks demonstrated a decrease (P < 0.05) in yellowness, whereas their non-aged counterparts exhibited no changes (P > 0.05) in b* values during 6 days of storage. Aging did not affect (P > 0.05) hue angle. During storage, an increase (P < 0.05) in hue angle was observed in all BF samples from day 0 to 6. Aged BF steaks exhibited greater (P < 0.05) chroma compared to their non-aged counterparts on storage days 0 and 3, whereas on day 6, aged and non-aged samples exhibited similar (P > 0.05) chroma. All BF steaks exhibited a decrease (P < 0.05) in chroma from day 0 to 6 of storage. Non-aged and aged samples exhibited overall similar (P > 0.05) color stability (R630/580) on days 0, 3, and 6; whereas on day 3, aged steaks exhibited greater (P < 0.05) color stability than their non-aged counterparts.

Conclusion: These findings suggested that wet aging influenced the color stability of beef BF muscle by improving surface redness and color stability. Wet aging may be a practical processing strategy in the retail beef industry to improve the color stability of BF steaks.

Cancer and Alzheimer's Disease/ Alzheimer's Disease Related Dementia: Potential for Drug repurposing

Khine Zin Aung | Public Health | Biostatistics

Poster # 17

Introduction: In recent years, an inverse association between cancer and Alzheimer's disease (AD) has been reported.  Various factors have been hypothesized as important contributing factors. However, the exact mechanisms are still unknown. Research suggests that cancer patients receiving chemotherapy are more likely to have cognitive deficits during their treatment course, and identifying underlying mechanisms is warranted. In this study, we investigated the genetic connection between various types of cancer and dementia using pathway-specific Polygenic risk scores (PRS). 

Methods: We obtained phenotype data from the National Alzheimer’s Coordinating Center (NACC), genotype data from the Alzheimer’s Disease Genetics Consortium (ADGC), and summary statistics for five cancers (liver, lung, prostate, breast, and colon cancers) from the United Kingdom (UK) Biobank. We mapped genes to each of the 18,826 Gene Ontology (GO) categories for Homo sapiens and discovered 7,300 biological processes comprised of groups of three or more genes. We removed the APOE region and analyzed the associations between AD/ADRD and each cancer by using pathway specific PRS. 

Results: Individuals having higher biological pathway-specific cancer PRS were relatively likely to have a lower risk of AD/dementia-related phenotypes. The most significant biological processes are cell cycle related pathways, angiogenesis, and neurodevelopmental pathways, Angiogenesis and atherosclerosis pathways are also significant in TDP-43 and cerebrovascular pathologies respectively. 

Conclusion: The disruptions of specific biological pathways are contributing to the inverse relationship of cancer and AD related dementia. Our results will gain insights into genetic risks for both AD and cancers and may contribute to precision medicine. 

Development of Cyanobacterial natural Products as SOCE Modulators for Neuropathic Pain

Chhabi Chaudhary | Pharmacy | Pharmaceutical Sciences

Poster # 18

Introduction: Cyanobacteria are a source of bioactive compounds and have been studied for over 50 years in the search for new antibiotics, antiviral, and anticancer drugs. In our current projects, we are investigating marine cyanobacterial compounds and their synthetic analogs for Ca2+ modulation via interactions with the sigma-2 receptor. We have identified several cyanobacterial natural products such as veraguamide which has sigma-2 receptor affinity and alter store operated calcium entry (SOCE). Recent research has shown SOCE and the sigma-2 receptor to be involved in neuropathic pain. 

Methods: Cyanobacterial samples were collected from the Las Perlas Archipelago in Panama, extracted, and the crude extract was fractionated over silica gel using a stepwise gradient solvent system. The fractions were then evaluated in a psychoactive drug screening program, and those with sigma-2 affinity were further purified to isolate veraguamide compounds. The structural modification and optimization of these cyanobacterial compounds using medicinal chemistry tools are ongoing to develop antinociceptive agents and to better understand the interaction of sigma-2 and SOCE.

Results and Discussion: Veraguamides and their analogs have shown to alter SOCE in a sigma-2-dependent manner in both mouse and human-derived neurons, and this effect can be modulated through simple structural modifications. Furthermore, the reduction of SOCE caused by these analogs has demonstrated in vivo antinociceptive effects. The sigma-2/TMEM97 receptor provides a promising target for alteration of the perception of noxious stimuli and could potentially lead to a new option for neuropathic and chronic pain management in the future.

Klotho enhances diastolic function in aged hearts through Sirt1-mediated pathways

Nastaran Daneshgar | Medicine | Internal Medicine-Cardiology

Poster # 19

Aging leads to a progressive decline in cardiac function, increasing the risk of heart failure with preserved ejection fraction (HFpEF). This study elucidates the impact of α-Klotho, an anti-aging hormone, on cardiac diastolic dysfunction and explore its downstream mechanisms. Aged wildtype and heterozygous Klotho-deficient mice received daily injection of soluble α-Klotho (sKL) for 10 weeks, followed by a comprehensive assessment of heart function by echocardiography, intracardiac pressure catheter, exercise tolerance and cardiac pathology. Our findings show that klotho deficiency accentuated cardiac hypertrophy, diastolic dysfunction and exercise intolerance, while sKL treatment ameliorates these abnormalities and improves cardiac capillary densities. Downstream of klotho, we focused on the Sirtuin1 (Sirt1)-Checkpoint kinase 2 (CHK2) signaling pathway to elucidate the potential underlying mechanism by which Klotho improves diastolic function. We found that decreased Klotho levels were linked with Sirt1 deficiency, whereas sKL treatment restored Sirt1 expression in aged hearts and mitigated the DNA damage response pathway activation. Through tandem mass tag proteomics and unbiased acetylomics analysis, we identified 220 significantly hyperacetylated lysine sites in critical cardiac proteins of aged hearts. We found that sKL supplement attenuated age-dependent DNA damage and cardiac diastolic dysfunction. In contrast, Klotho deficiency significantly increased hyperacetylation of several crucial cardiac contractile proteins, potentially impairing ventricular relaxation and diastolic function, thus predisposing to HFpEF. These results suggest the potential benefit of sKL supplementation as a promising therapeutic strategy for combating HFpEF in aging.

Pancreatic cancer-associated fibroblast supernatant suppresses T-cell function

Hoda Saghaeiannejad | Medicine | Surgical Oncology 

Poster # 20

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and therapy-resistant cancers. Despite the transformative impact of immunotherapy on various other cancers, it has largely failed to show efficacy in PDAC. A hallmark of PDAC is its extensive fibrosis which has been hypothesized to contribute to immunosuppression, thereby supporting tumor progression. Given these challenges, there is an urgent need for further research into the immunosuppressive components of the PDAC microenvironment to develop more effective therapeutic strategies.

Methods: Human PDAC surgical specimens were wax embedded for histology and mechanically and chemically digested to a single cell suspension.  Cancer-associated fibroblasts (CAFs) were isolated from PDAC tissue digest, subcultured, and supernatant from CAF culture was saved.  PBMCs were harvested from healthy donor and CD8+ cells were bead isolated.  CAF supernatant was added to CD8+ cell culture for 48 hours, and subsequent gamma interferon ELISA and flow cytometry for immune markers was conducted.  

Results: T-cells co-cultured with PDAC CAF supernatant isolated from 5 unique patient samples had lower levels of CD69 and higher levels of PD-1 when compared to control (P<0.05).  Gamma interferon secretion was also significantly lower (P<0.05) in T-cells co-cultured with PDAC CAF supernatant.

Conclusion: Supernatant from patient derived CAFs is capable of suppressing the effector function of CD8+ T-cells and reducing their capacity to initiate an immune response.  Our results indicate a clear need to investigate the human patient derived CAF secretome for possible clinical targets to reduce immunosuppression in the PDAC microenvironment.

Environmental pollutant PFOS disrupts intestinal tissue homeostasis to promote gastrointestinal carcinogenesis

Josiane Tessmann | Medicine | Toxicology and Cancer Biology

Poster # 21

Introduction: PFOS is a widespread environmental pollutant present in 45% of USA drinking water. Due to its resistance to natural degradation, PFOS can accumulate in intestinal tissues and disrupt normal function. PFOS has been shown to contribute to carcinogenesis, but its effect in gastrointestinal tract (GI) is not well understood. It’s essential to elucidate PFOS's impact on GI to develop PFOS mitigation strategies. Methods: Wild-type mice were exposed to PFOS in drinking water for 7 weeks. RNAseq analysis was performed on intestinal tissues to assess gene expression changes. To investigate PFOS effects and perform qRT-PCR, western blot, proliferation assay, and immunofluorescence, we utilized mouse and human organoids, and a normal colon cell. Results: RNA-seq analysis revealed that PFOS exposure upregulates pathways related to cancer, lipid metabolism, and immune system in normal intestinal tissues. PFOS decreased expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential for production of β-hydroxybutyrate (βHB), a metabolite with anti-tumor properties. Moreover, PFOS increased the levels of proteins associated with colorectal cancer (CRC) initiation. HMGCS2 knockdown in normal colon cells increased cell proliferation, lipid accumulation, and expression of CRC-related proteins. Discussion: Our data suggests that PFOS may induce pathological changes in GI by elevating cancer-related proteins, potentially increasing CRC risk. PFOS downregulates HMGCS2, which may reduce βHB level and its protective effects on tissue homeostasis. Additional studies are needed to determine if βHB supplementation can prevent PFOS-induced changes. Dietary interventions, such as βHB supplementation, might improve the quality of life for individuals in PFOS-contaminated areas.

Light at night disrupted day-night rhythms in the heart rate and core body temperature in female diabetic mice

Abhilash Prabhat | Medicine | Physiology

Poster # 22

Introduction: Environmental factors affect the 24-hour regulation of the heart rate (HR) and core body temperature (Tb). Db/db mice have been shown to have irregular thermoregulation. We explored the effect of housing temperature and light conditions on the HR and Tb in db/db mice. 

Methods: Four to six-month-old db/db and control female mice were implanted with telemetry devices to continuously record the electrocardiogram (ECG), core body temperature (Tb), and activity. Mice were housed at room temperature (25 °C) followed by thermoneutrality (30 °C) in 12 h light: 12 h dark cycles (LD, 200 lux: 0 lux) with ad libitum access to food. Mice were then subjected to 12 h light: 12 h dim light cycles (dLAN-ALF; 200 lux: 5 lux) for two weeks. 

Results: db/db mice had blunted day/night rhythm in HR and Tb compared to control mice, as 50% of db/db mice did not show 24-hour variation in the HR. Thermoneutrality increased the day/night variation and 24-hour rhythmicity in the HR in diabetic mice. Specifically, thermoneutrality decreased mean HR in both the genotypes and increased amplitude in the HR of db/db mice. Thermoneutrality also increased core body temperature in db/db mice up to the control levels. dLAN disrupted the day/night differences between HR, Tb, and activity in db/db and the control mice. 

Discussion: Light at night has more effects on diabetic mice underscoring the critical role of environmental conditions such as light and housing temperature in 24-hour heart rate and core body temperature rhythm regulation in mice with metabolic disease.

Numerical Investigation of High Temperature Graphite Oxidation and Nitridation

Ahilan Appar | Engineering | Mechanical and Aerospace Engineering 

Poster # 24

Introduction: Understanding the oxidation and nitridation mechanisms of carbon is essential for designing reliable carbon-based heat-shield materials for hypersonic flight. The interactions between solid carbon and reactive gases, such as oxygen and nitrogen, directly influence the stability of graphite, a material widely used in aerospace applications. This study investigates these gas-surface interactions under conditions that mimic the hypersonic boundary layer.

Methodology: In this work, we aim to develop numerical models of the temperature-dependent reactivity of graphite in molecular oxygen at pressures of 2 kPa, expressed as reaction probabilities. To achieve this, we simulated the flow reactor experiments conducted by Anderson et al. using a particle-based stochastic method known as Direct Simulation Monte Carlo (DSMC). The results obtained from the DSMC simulations were compared and validated against experimental data. The validated model was then applied to study the behavior of atomic oxygen, accounting for diffusion limitations and measuring the concentration of atomic oxygen.

Results & Discussion: The experiments demonstrated that oxidation and nitridation processes do not significantly interfere with one another, allowing for independent analysis of their effects on graphite. However, limitations in generating atomic oxygen in flow reactors were identified, potentially affecting the accuracy of the results. To address these limitations, the DSMC method was proposed as a numerical approach to replicate and validate the experimental findings. Additionally, DSMC can be utilized to study the interactions of other gases with carbon, offering a more comprehensive understanding of gas-surface interactions critical for hypersonic applications. The initial study using DSMC confirmed the interdependence of species in a high-pressure environment on reaction probabilities.

Stable Isotope Resolved Metabolomics of cancer cells by Cation-Exchange Ion Chromatography Coupled to Ultra-High Resolution Mass Spectrometry

Mohamed Kaddah | Medicine | Center for Environmental and Systems Biochemistry 

Poster # 25

Introduction: Stable isotope-resolved metabolomics (SIRM) has emerged as a crucial tool in uncovering the altered metabolic pathways characteristic of pathological conditions such as cancer. Despite the advancements in the field, the chromatographic separation and precise determination of highly polar and ionic metabolites remain challenging. Although anionic IC-UHR-MS has been established, the study of cationic metabolites utilizing IC-UHR-MS for SIRM has not been published yet. Here, we present the first successful implementation of cation-exchange ion chromatography coupled to ultra-high resolution mass spectrometry (IC[+]-UHR-MS) for SIRM.

Method: For IC[+]-UHR-MS, an Orbitrap Fusion Tribrid mass spectrometer was coupled with an ion chromatography system to separate and analyze polar cationic and zwitterionic metabolites from liver cell extracts of mice administered with 13C6-glucose. The IC[+] electrochemically generates a methanesulfonic acid (MSA) gradient for cation chromatography, and a suppressor selectively removes MSA before entering the MS; analytes elute from the IC[+] in water with minimal electrospray ion suppression. 

Results: This method successfully quantified 26 polar metabolites, including acetylcholine, choline, trimethyllysine, nicotinamide riboside, thiamine, betaine, and several polyamines and short-chain acylcarnitines. The sensitivity of the method achieved a lower limit of quantification approaching 1 ng/mL (≈ 50 fmol on column).

Discussion: This technique marks a significant advancement in metabolomics by filling the gap left by anionic IC-UHR-MS, providing new insights into cancer cell metabolism. The IC[+]-UHR-MS approach offers a robust platform for the comprehensive analysis of cationic metabolites in biological samples, paving the way for future applications in biomedical research.

Circular RNAs encode proteins that contribute to Alzheimer’s disease

Giorgi Margvelani | Medicine | Biochemistry

Poster # 27

Introduction: Circular RNAs (circRNAs) are covalently closed RNAs present in all eukaryotes. generated through backsplicing that depends on intronic elements such as primate-specific Alu elements. Over 800,000 circRNA isoforms exist in humans. Recent studies showed that circRNAs are translated into proteins after undergoing epigenetic base modifications, which most often occurs under pathophysiological conditions. Thus, circRNAs generate a novel proteome that has not been studied and is associated with human disease, such as Alzheimer’s disease (AD). 

Methods: Using reporter genes, we found most circRNA are translated after A>I editing, but several circRNAs respond to m6A modification and eIF4A expression. These in vitro data were supported by immunohistochemistry using human tissue and circRNA-encoded protein specific antisera, as well as RNAseq.

Results: We analyze circRNAs generated form genes encoding microtubule associated protein tau, amyloid precursor protein, and presenilin 1 and found that in addition to the known methylation of adenosines (m6A), deaminating inosines to adenosines (A>I editing) strongly promotes circRNA translation during AD progression. Several circRNAs responded to eIF4A expression too, suggesting that circRNAs use different, possibly overlapping translational initiation mechanisms [PUBMED: 36533443, 37266374].

Discussion: Generally, we see an increase in circRNA encoded protein expression and aggregation in neurons during AD.  Circtau encoded proteins promote tau aggregation, and in some cases colocalize with tau tangles, hallmarks of AD. circAPP proteins encode an intracellular Cupper domain causing oxidative stress, and also encode beta amyloid containing intracellular proteins. We propose to circRNA encoded proteins, generated in response to inflammation, contribute to AD.

Effect of Paclitaxel Loaded Poly(caprolactone) Nanoparticles on Endometrial Cancer Cell Migration

Lauren Mehanna | Engineering | Chemical Engineering 

Poster # 28

Introduction: Endometrial cancer (EC) is the most common female reproductive cancer worldwide. Biodegradable poly(caprolactone) (PCL) nanoparticles are promising for localized drug delivery to EC tissue. PCL particles can be loaded with Paclitaxel (PTX), a common chemotherapy, or Rhodamine B (RHO), a fluorescent dye to understand particle drug loading and release. The epithelial-to-mesenchymal transition states that cancer cells become more migratory as they become more metastatic. We hypothesize that PTX-PCL nanoparticles will suppress cancer cell migratory behavior or create chemotherapeutic resistance in EC cell lines.  

Methods: PTX-PCL and RHO-PCL particles were synthesized using a single emulsion solvent evaporation method. Particle formation and size characteristics were visualized using SEM imaging. Release kinetics were determined by quantifying the concentration of RHO or PTX released in aqueous media over time using a plate reader. Migration of KLE (Stage III EC) and HEC-1A (Stage I EC) cells were tracked in vitro using a scratch assay. 

Results: SEM imaging confirms sub-micron spheres formed for both RHO-PCL and PTX-PCL formulations. Release studies revealed effective drug/dye loading during synthesis and release, with a burst release within the first 12 h, followed by sustained release thereafter. Scratch assay negative controls (media only) showed that KLE (more metastatic) migrated faster to close the gap compared to HEC-1A (less metastatic). 

Discussion: RHO and PTX were successfully encapsulated in PCL particles and effectively released, with burst and sustained release profiles. More metastatic EC cell lines had increased cell migratory behavior in vitro, following EMT. These results demonstrate potential in using PTX-PCL as a localized therapeutic treatment to slow EC cell migration.

Germination of Cannabis sativa L. in Controlled Environment Versus a Field Environment 

Andrea Webb | Agriculture, Food and Environment | Integrated Plant and Soil Sciences

Poster # 29

Introduction: Cannabis sativa L. (industrial hemp), is a historically important crop, with uses for fiber, grain, and/or oil. With the US Congressional ban on Cannabis sativa L.  in the 1970’s, no research was conducted on industrial hemp until the 2018 Farm Bill. Since 2018, research has shown the possibility of industrial hemp becoming a successful crop in Kentucky. There is still a large amount of research to be conducted, specifically investigating the germination rates of commercial seed lots. Industrial hemp seed can be highly variable, resulting in poor establishment of the crop.  Some of these seeds can decrease by approximately 50% after being stored for one year. 

Material and Methods: Eight cultivars of industrial hemp seed were selected and evaluated in a controlled environment setting versus a field setting for percent germination. Seed were germinated at ¬¬30°C for 16 hours with no supplemental light and at 20°C for eight hours with supplemental light for seven days within a growth chamber. At seven days total percent germination was calculated. The same eight cultivars were then direct seeded into a standard field setting, germination counts were taken 10 days following seeding and total percent germination was calculated. 

Results: Data analysis shows that germination percentage within a controlled environment ranged from 96-81% for the eight cultivars. In a field setting germination percentages ranged from 17-45%. 

Discussion: With differences in germination in a controlled environment setting to a field setting altering by as much as 76% leads to the investigation of Cannabis sativa L. seed physiology. 

Amylin vasculopathy impairs cerebral Aβ efflux through altering cerebral vasodilation

Ravichandra S Davargaon | Medicine | Pharmacology and Nutritional Sciences

Poster # 30

Background: Impaired interstitial fluid drainage in the brain is indicated by the presence of perivascular β-amyloid (Aβ) deposits and is attributed to alterations in contractility and relaxation of vascular smooth muscle cells (SMCs). The brain microvasculature in Alzheimer disease (AD) accumulates amyloid-forming amylin secreted from the pancreas. Here, we tested the hypothesis that cerebrovascular amylin deposits perturbs cerebral Aβ efflux by impairing cerebral vasodilation.

Methods: Using transgenic rats expressing amyloid-forming human amylin in the pancreas (HIP rats) (aged 16-months) and wild-type (WT) littermates that express non-amyloidogenic rat amylin, we conducted comparative analyses of cerebral blood flow (CBF), pressure myography in isolated pial arteries and vascular SMC oxidative stress experiments.

Results: Longitudinal brain MRI measurements revealed consistent structural alterations that progressed more rapidly with aging in HIP vs. WT rats, leading to 14.9% reduction in CBF in HIP rats. Plasma nitrite and nitrate, stable nitric oxide (NO) end products, were increased in HIP vs. WT by 84.7% and 24.87%, respectively. Pressure myography experiments using pial arteries showed that both WT and HIP arteries developed arterial tone (e.g. pressure-induced constriction); however, arteries from HIP rats show significant elevations (56.9-142.3%) in arterial tone compared to WT rats at physiologically-relevant intravascular pressures (e.g. 60-100 mmHg). Consistent with these results, vascular SMCs from HIP rats showed elevated (12.6% increase) lipid peroxidation, which was replicated in SMCs incubated with exogenous human amylin (29.6%).  Increased lipid peroxidation contributes to oxidative stress in the vascular wall and reduces NO bioavailability, altering vasodilatory function. Both arginase activity and expression (of Arginase 1 and 2) were increased in brain microvascular lysates from HIP rats compared to those from WT by 17.6%, 63.9%, and 57.8%, respectively, suggesting arginase-NO dysregulation. A possible impact of increased blood amylin concentration on cerebrovascular arginase-NO regulation was further tested in brain microvascular lysates from rats intravenously injected with amyloid-forming human amylin (55.0% reduction in arginase activity).

Conclusion: Our results indicate perivascular Aβ deposits in the setting of AD are potentially linked to amylin vasculopathy and altered spontaneous contraction/relaxation of cerebrovascular SMCs. Future experiments will focus on delineating molecular markers of amylin-induced alterations of SMC contractile phenotype. 

Exploring the Age Sensitivity of Young Children's Social-Emotional Development Through Psychological Network Analysis

Yuyan Xia | Health Sciences | Physical Therapy

Poster # 31

The period between ages 3 and 5 is pivotal, characterized by rapid advancements in children's social-emotional development. To utilize the NSCH 3–5-year-old young children sample, this study employed innovative Psychological Network Analysis to explore how age influences the network of social-emotional competencies in young children. By comparing the networks across three age groups and examining their centrality indices, the results reveal disparities in the components of social-emotional development among 3-, 4-, and 5-year-olds, especially noting a significant difference between the 3- and 5-year-old groups and the 4- and 5-year-old groups. Notably, compared to ages 3 and 4, the interconnectedness and focus of social and emotional competencies evolve distinctly by age 5. 

C–H Amination Chemistry Mediated by Trinuclear Cu(I) Sites Supported by Tetramethylguanidinyl Ligand Scaffold Featuring Pnictogen Elements (Sb, Bi), and an Arene Platform

Meenakshi Sharma | Pharmacy | Pharmaceutical Sciences

Poster # 32

Introduction: Transition-metal catalyst frameworks supported by tripodal [TMG3trphen] ligands mediate nitrene transfer from nitrogen sources such as PhI=NR (PhI=NTs or PhINTces) to a diverse group of aliphatic and aromatic hydrocarbons and olefins. These reactions are categorized as amination and aziridination reactions. 

Methods: Novel tripodal ligands and their complexes with late first- and second-row transition metals (Cu, Ag, Au) with different axial atoms1 such as Sb and Bi and benzene platform2 have been designed to impart weaker axial ligand field, which, in turn, enhances the electrophilicity of nitrene, potentially affording more reactive and site-selective aminated products.

Results and Discussion: The trinuclear copper catalysts [TMG3trphenSbCu3(μ2-Cl)3] and [TMG3trphenBiCu3(μ2-Cl)3] have shown promising results towards aziridination of substituted styrenes with excellent yields while the reactivity of the silver catalyst [TMG3trphenSb] Ag3Cl3 is comparatively low. The copper complexes are also reactive for the selective amination of various hydrocarbons at benzylic and tertiary C–H sites. The congener of [TMG3trphenSbCu3(μ2-Cl)3], [TMG3trphenSbCu3(μ2-Br)3] [TMG3trphenSbCu3(μ2-I)3] have also been synthesized to obtain comparative data for the catalytic reactions mediated by nitrene generated in situ.

1 Organometallics 2024, 43, 634–652. 

2 Dalton Transactions 2024, submitted.

Functional Elastic Fibers Were Newly Generated in Chronic Aortic Dissection

Sohei Ito | Medicine | Physiology

Poster # 33

Introduction: Aortic dissection (AD) is often mortal in the acute phase, whereas its chronic phase may be also clinically problematic due to vascular complications, such as aortic rupture and aneurysm formation. Despite considerable attention to the acute phase in previous studies, substantial knowledge gaps persist regarding the pathophysiology of chronic AD. This study aims to determine the pathophysiology of chronic AD focusing on biomechanical and histological alterations using a mouse model.

Methods and Results: To induce AD, β-aminopropionitrile (BAPN) was administered to 4-week-old male C57BL/6J mice. After 4 weeks of BAPN administration, intramural hematoma was evident in the descending aorta, indicating acute AD. After 12 weeks, significant aneurysmal changes occurred, characterized by wall thickening, discoloration, and organized thrombus, indicating the presence of chronic AD. Subsequently, biomechanical functions of the AD wall were evaluated by ultrasound at 4 and 12 weeks. The aortic wall exhibited increased elasticity and decreased distensibility in acute AD, which became more pronounced in the chronic phase. Notably, histological analysis displayed that significant de novo elastic fibers formed surrounding the false lumen. These new fibers were thinner and denser compared to native elastic fibers in normal aortas. Confocal 3D imaging revealed a spider-web structure of the new fibers. Furthermore, electron microscopy demonstrated de novo fibers were composed of uneven granules. Elastin mRNA was co-localized with αSMA and MYH11, suggesting that smooth muscle cells synthesize these fibers.

Conclusion: De novo elastic fibers with biomechanical functions were newly synthesized in the false lumen of chronic AD in mice.

Exploring the Role of ABL1/2 Kinases in Enhancing MAPK Inhibitor Resistance via ZEB1/NCAD Stabilization

Bhuvanesh Kalal | Medicine | Pharmacology and Nutritional Sciences

Poster # 34

Melanoma is the fifth most common cancer among adults globally, and is projected to increase by 50% by 2040, with a 68% rise in associated deaths. Constitutive activation of the MAPK pathway which occurs primarily via hyperactive mutations in BRAF (50% of cases) or NRAS (15-25% of cases), drives the development of metastatic melanoma. Targeted therapy using BRAF/MEK inhibitors increases median overall survival for BRAF-mutant metastatic melanoma patients (from 9 to 14 months); however, resistance inevitably develops. For patients with NRAS-mutant melanomas, no effective targeted therapy exists as BRAFi paradoxically increase melanoma growth, and MEKi provide little survival benefit  due to intrinsic and acquired resistance. 

Melanomas dynamically shift between different transcriptional states. Downregulation of epithelial-mesenchymal transition (EMT) transcription factors (TF), SNAI2 and ZEB2, and upregulation of  TWIST1 and ZEB1, drives an aggressive invasive, metastatic phenotype. Our lab previously showed that ABL kinases, (ABL1/2) play critical roles in tumor progression, drug resistance, and metastasis, and their kinase activities and/or expression are increased in resistant melanoma cell lines and patient samples. Here, we show that increased expression of ZEB1 and N-cadherin (NCAD) and reduced expression of SNAI2, ZEB2, MITF, and in some cases E-cadherin, correlate with resistance to MAPK inhibitors (MAPKi). Moreover, silencing or inhibiting ABL1/2 with nilotinib or the highly specific allosteric inhibitor (GNF-5) reduces ZEB1 and NCAD expression in resistant cells and, in some cases, coordinately induces SNAI2 and E-cadherin. Conversely, expression of constitutively active forms of ABL1/2 into parental melanoma cells increases ZEB1 and NCAD expression and promotes MAPKi resistance. RT-qPCR assays demonstrate that these effects are not mediated by changes in ZEB1/NCAD mRNAs. In contrast, cycloheximide assays show that ABL1/2 promote ZEB1 and NCAD protein stability, and MG-132 (proteasome inhibitor) treatment rescues nilotinib-mediated degradation of ZEB1 and NCAD. ABL1/2 regulate protein stability via a number of different mechanisms in other cell contexts. We found that ZEB1 and ABL1/2 are in the same complex, but ABL1/2 do not stabilize ZEB1 by impacting previously identified ZEB1 E3 ligases, such as SIAH1. We currently are focused on identifying the mechanism by which ABL1/2 stabilize ZEB1/NCAD, and are using siRNA screens to identify ZEB1/NCAD E3 ligase(s) regulated by ABL1/2.

Resistance to targeted therapy is often accompanied by the EMT-TF switch, which drives a more invasive and metastatic phenotype. ABL1/2 promotes invasion and metastasis of resistant cells; however, unexpectedly, ABL1/2 don't promote invasion via ZEB1 as silencing ZEB1 paradoxically potentiates the ability of constitutive active ABL1/2 to increase invasion. Thus, current experiments are focused on identifying the biological role of ZEB1 and NCAD upregulation in resistant cells. Importantly, the pathway we identified is clinically relevant as single-sample Gene Set Enrichment Analysis demonstrates a robust positive correlation between ABL1/2 kinase activity and ZEB1 transcriptional activity (assessed using downstream target gene sets sourced from the IPA Ingenuity Knowledge Base) in samples from melanoma patients. In summary, our work identifies a new role for ABL1/2 in regulating the stability of transcription factors with critical roles not only in invasion and metastasis but also resistance to therapy.

Dissecting the expression and function of Angiopoietin-1 in brain tumors

Jaldeep Langhnoja | University of Cincinnati | Pharmacy | Pharmaceutical Sciences

Poster # 35

Angiopoietin-1 (Angpt1) is a secreted protein that can promote angiogenesis and vascular stability in development and certain disease states through activation of the Tie2 receptor. However, little is known about its expression or function across brain tumor types. Surveying its expression across pediatric and adult brain tumors we show Angpt1 is upregulated in glial brain tumors, including high-grade gliomas and ependymomas, and that expression patterns correlate with cell-type expression patters we recently described in the normal developing and adult brain. To examine the cell type and location of Angpt1 expression within tumors we have generated electroporation based high-grade glioma and ependymoma mouse models in Angpt1-GFP reporter knock-in mice. Angpt1-GFP expression is found mainly in tumor cells within both tumor models, with varying expression in high-grade glioma models based on driver mutations and molecular subtype. Single-cell transcriptomic studies in these mouse models are underway to further delineate expression across tumor and non-tumor cell types. Moreover, we are leveraging this flexible electroporation-based mouse brain tumor modeling platform to examine Angpt1 function within brain tumors. Preliminary data shows that including a DNA plasmid encoding Cre-recombinase induces recombination and loss of Angpt1 in our electroporation-based brain tumor models, and ongoing studies aim to delineate its function in tumor pathogenesis, including tumor angiogenesis and blood-brain barrier function.

Soluble IL-2R Impairs Mitochondrial Respiration in Muscle Cells of Post-COVID Fatigue Patients

Laura Brown | Medicine | Physiology

Poster #36

Background: Post-acute sequelae of COVID (PASC) continue to affect many patients for weeks and even months after recovering from the initial SARS-CoV-2 infection. Recent studies indicate that pathological changes in skeletal muscle may play a significant role in ongoing pain and fatigue. This study aimed to explore the underlying mechanisms of PASC-related fatigue by assessing skeletal muscle function and circulating factors in affected individuals.

Methods: We conducted a cross-sectional case-control study involving patients with fatigue-associated PASC who had experienced mild to moderate COVID-19 without hospitalization. High-resolution respirometry, immune marker analysis, and western blotting were used to assess human tissues, and in vitro studies were used to validate our findings.

Results: Skeletal muscle biopsies from PASC participants showed lower mitochondrial respiration and content compared to healthy controls. This reduced respiratory capacity was associated with significantly higher levels of the T cell-specific receptor, soluble IL-2 receptor alpha subunit (sIL2R), in the bloodstream. In vitro experiments confirmed that sIL2R directly impairs mitochondrial oxygen consumption and lowers the protein levels of mitochondrial complex III subunits in cultured muscle cells. These findings suggest a link between systemic immune dysregulation and muscle-specific mitochondrial dysfunction in PASC.

Discussion: This study offers new insights into the pathophysiology of PASC, highlighting sIL2R as a potential therapeutic target for mitigating mitochondrial deficits associated with PASC-related fatigue. These findings pave the way for developing targeted interventions.

O-GlcNAcylation underlies the activation of sodium-glucose cotransporter 1 in diabetic hearts

Vivek Pandey | Medicine | Pharmacology and Nutritional Sciences

Poster #37

Rationale: Type-2 diabetes (T2D) greatly increases the risk of developing heart failure. Recent evidence indicates that the cardiac expression and function of the sodium-glucose cotransporter 1 (SGLT1) is elevated in disease states, including T2D, leading to structural and functional remodeling of the heart. The mechanisms underlying SGLT1 activation in the diabetic heart are currently unknown.

Objective: To investigate if SGLT1 undergoes the O-linked attachment of β-N-acetylgluco¬samine (O-GlcNAcylation), a post-translational modification that is exacerbated in T2D, and its effect on SGLT1 activity in diabetic hearts.

Methods/Results: SGLT1 co-immunoprecipitates with O-GlcNAc in heart homogenates and HL-1 cardiomyocyte lysates. Enhancing global protein O-GlcNAcylation in HL-1 cells via si-RNA-mediated silencing of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, resulted in increased amounts of O-GlcNAc that precipitate with SGLT1. SGLT1 function, measured as the SGLT1-mediated Na+ influx, was significantly increased in HL-1 cells with silenced OGA. These data suggest that SGLT1 undergoes O-GlcNAcylation and this modification results in SGLT1 activation. Using rats that express human amylin in the pancreatic β-cells (HIP rats) as a model of late-onset T2D and their wild-type littermates as controls, we found that a larger fraction of SGLT1 co-immunoprecipitates with O-GlcNAc in hearts from T2D rats compared to controls. The SGLT1-mediated Na+ influx was larger in myocytes from T2D vs. control rats. Increasing total O-GlcNAcylation with Thiamet-G in control myocytes resulted in a larger Na+ influx. In reverse experiments, inhibition of O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine crystalline reduced SGLT1-mediated Na+ uptake in myocytes from T2D rats. Inhibition of O-GlcNAcylation in T2D myocytes reduced Ca2+ spark frequency and this effect was significantly less pronounced with SGLT1 blocked. These results suggest that O-GlcNAcylation affects myocyte Na+ regulation by activating SGLT1, contributing to myocyte Na+ overload and its downstream effects on sarcoplasmic reticulum Ca2+ leak in T2D.  

Conclusion: Exacerbated O-GlcNAcylation underlies SGLT1 activation in diabetic hearts.  Preventing O-GlcNAcylation may limit myocyte Na+ overload and the frequency of pro-arrhythmogenic Ca2+ sparks in myocytes from T2D rats.

Serum Biomarkers in Surgically Treated Degenerative Cervical Myelopathy Patients

Cassandra Wolsh | Medicine | Spinal Cord and Brain Injury Research Center

Poster #38

Introduction: Degenerative cervical myelopathy (DCM) is the largest cause of spinal cord injury in adults. Decompression surgery is the standard treatment but surgery does not always result in a patient improvement. The goal is to identify serum based biomarkers that associate with the quality of functional recovery following surgery to appropriately recommend future patients as candidates for surgery treatment.

Methods: Here, we have screened for markers of spinal damage in serum from DCM patients before receiving decompression surgery and 6 months after surgery. These markers include Glial fibrillary acidic protein (GFAP), Apolipoprotein E (ApoE), Neuron-Specific Enolase (NSE), Neurofilament Heavy Chain (NF-H), Amyloid Beta Peptide (AB40, AB42) and others. Functional recovery is measured here by modified Japanese Orthopaedic Association scale (mJOA) score. We hypothesize that an increase in markers for neuronal damage before surgery will correlate to lower recovery rate and correlate to increased severity before surgery, as measured by mJOA. 

Results: Most, but not all, patients have a significantly improved mJOA score following 6 months of recovery from surgery. Our preliminary results indicate that ApoE and NSE have a significant correlation to severity of DCM symptoms before surgery. Furthermore, we find that ApoE and NF-H have significant correlations to change in mJOA score from pre-surgery to 6-months after surgery.

Discussion: Eventually, these biomarkers may be utilized as a diagnostic tool to identify early signs of spinal cord damage in DCM as well as other spinal cord injuries. Serum based diagnostics will be more accessible to patients, allow earlier identification of disease so patients can get treatment as soon as possible, and aid physicians in recommending the correct treatment following spinal cord injury. Our goal is to eventually provide a non-invasive, accessible and cost-effective diagnostic tool that will facilitate earlier and more accurate prognosis of disease progression and treatment outcomes.

Healing the Broken Heart: Development and Optimization of a Liposomal Azithromycin Formulation

Abdullah Masud | Medicine | Opthalmology and Vision Sciences

Poster #39

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide, largely due to poorly controlled inflammation in injured cardiomyocytes, which impairs the healing process. Azithromycin, a well-known antibiotic, has recently shown promise in reducing inflammatory responses in mouse models of MI. However, its therapeutic potential as an anti-inflammatory agent is limited by dose-related toxicity and off-target effects. To overcome these challenges, our group has developed a liposomal formulation of Azithromycin (L-AZM) that significantly enhances its anti-inflammatory efficacy while reducing toxicity. The optimized L-AZM formulation is designed to meet specific quality attributes and ensure reproducible, high-throughput manufacturing, which are crucial for clinical translation. Our initial formulation, composed of an equimolar mixture of DSPC, DSPG, and cholesterol, demonstrated moderate Azithromycin encapsulation efficiency (EE) of 30-50%, likely due to the drug's high molecular weight (MW 785), which causes it to leak from the liposomal bilayer. To improve drug retention within the bilayer membrane, we employed an ion-pairing strategy using cationic Azithromycin and anionic lipids in a microfluidic channel. By optimizing microfluidic parameters such as flow mixing rate and mixing ratio, we developed a lead formulation with an EE exceeding 90%, an acceptable particle size (100-200 nm), and a low polydispersity index (PDI < 0.1). This optimized L-AZM formulation, combined with positive pre-clinical data, positions it as a promising candidate for clinical application in post-MI therapy.

Development of Machine Learning Models Predicting Neurological Improvement and One-Year Mortality in Traumatic Cervical Spinal Cord Injury Patients

Harshit Arora | Medicine | Neurosurgery

Poster #40

INTRODUCTION: Traumatic cervical spinal cord injury (tSCI) is associated with high morbidity and mortality globally, frequently observed in motor vehicle collisions and falls in elderly population. The American Spinal Injury Association (ASIA) Impairment Scale is used to assess SCI severity, yet the impact of radiological factors on outcomes remains underexplored. This study aims to develop machine learning (ML) models to predict mortality and neurological improvement at one-year follow-up.

METHODOLOGY: 103 patients who sustained isolated tSCI were included in this retrospective study. Key variables included demographic, clinical, and radiological data, with outcomes focused on one-year mortality and ASIA grade conversion. Six ML models (Multinomial, Random Forest, k-NN, SVM, Decision Tree and Naïve Bayes) were developed and evaluated using leave one out cross-validation and five-fold-cross-validation techniques. Model performances were assessed using ROC-AUC and individual accuracies.

RESULTS: Average age of study participants was 51.9 years. Amongst the study cohort, 23 (22.3%) patients were deceased at one-year follow-up. Amongst the others, ASIA grade conversion was observed in 66.7% of the patients. The SVM and Naïve Bayes models reported higher accuracies when predicting one-year mortality (AUC = 0.980 and 0.971 respectively). Similarly, ASIA grade conversion prediction showed increased accuracy in the Decision Tree (AUC = 0.987) and Naïve Bayes (AUC = 0.984) models.

CONCLUSION: Our study highlights the effectiveness of ML models in predicting one-year mortality and neurological outcomes in tSCI patients. These models, with high accuracy, offer valuable tools for risk stratification and clinical management, paving the way for improved prognostication and patient care.