Poster # 001
Mithramycin A (MTM) is a potential auerolicacid-type polyketide natural product targeting EWS-FLI1, an ETS transcription factor that is dominant in Ewing Sarcoma. Despite the high potency of MTM, there are several limitations that hinder its success for clinical use. These limitations include a narrow therapeutic index, low selectivity resulting in hepatotoxicity, and poor pharmacokinetic properties. We previously introduced MTM 2’-oxime conjugation as a new MTM derivatization strategy and revealed this method to render new corresponding analogues with dramatically improved selectivity and pharmacokinetic properties but decreased potency compared to MTM. Herein we described the synthesis and SAR of 84 second generation analogues. Importantly, this study led to members that were equipotent to MTM in vitro but demonstrated up to nearly 10-fold improvements of in vitro selectivity and PK improvements of 3 orders of magnitude compared to the parent MTM. In vivo efficacy studies are currently underway with one of the best members of this series.
Poster # 002
Toxoplasma gondii is a single-cell obligate intracellular parasite that infects most warm-blooded animals including humans. In the human host, parasite exists in two stages fast-growing tachyzoites and slow-growing bradyzoites. One of the distinguishing morphological features of the bradyzoites is the accumulation of amylopectin granules (AGs), an insoluble starch-like glucose polymer that represents stored energy/biosynthetic potential for diverse physiological functions including replication, stage-conversion, and reactivation. The level and distribution of AGs in encysted bradyzoites is highly heterogeneous suggesting a dynamic process, impacted by both synthesis and turnover of AGs. Although AGs is believed to drive many processes central to the disease pathogenesis, very little is known about the basis of its heterogeneity, its physiological impact, and the regulatory enzymes. In order to understand that, we developed a novel image-based software “Amyloquant” to analyze the stored AG. In our study, we have also included two key enzymes of AG metabolism pathways, the glucan phosphatase TgLaforin (TGME49_205290) and its partner glucan dikinase TgGWD (TGME49_214260). We used biochemical, molecular biology, and imaging-based approaches and employed “Amyloquant” to understand the AG heterogeneity and its effect on the progression of the chronic infection. The current study and the techniques used will provide a platform to investigate the role of AG/AGs pathway enzymes in parasite stage transition, replication, reactivation, and whether it can be a potential drug target.
Poster # 003
Introduction: Myocardial infarction, commonly referred to as a "heart attack," is the primary cause of heart failure, affecting around 1 million individuals in the US annually. The severe and poorly manageable inflammation in damaged cardiac tissue leads to cardiac remodeling and potential heart failure. Our lab's pre-clinical research revealed that liposomal azithromycin (L-AZM) is more effective in reducing cardiac inflammation and minimizing cardiotoxicity compared to free azithromycin treatment. Given these promising results and the lack of efficient treatments, we aimed to optimize the L-AZM formulation utilizing microfluidics to advance it to the clinic.
Methods: We utilized a microfluidic system, which is known for reproducible and scalable manufacturing of liposomes. To optimize the L-AZM formulation, we considered microfluidic parameters, e.g., Total flow rate (TFR) and flow rate ratio (FRR), and formulation parameters like lipid composition and lipid-to-drug ratios.
Results and Discussion: Varying TFR from 5-20ml/min and FRR (organic:aqeous) from 1:2 to 1:5, we found that higher TFR and FRR led to smaller particle size and lower polydispersity index (PDI), with the best results at TFR 10ml/min and FRR 1:4, achieving a size of ~150 nm and PDI of 0.1. By comparing different lipid compositions and AZM concentrations, we determined that a DSPC:DSPG:Chol: AZM mole ratio of 1:1:1:0.5 is optimal for L-AZM.
Conclusion: We optimized the L-AZM formulation using a clinically relevant microfluidic system known for reproducibility and scalability. Further steps, including evaluating the formulation in larger animal models, are essential for advancing toward clinical use.
Poster # 004
Introduction: Light at night is all around us. It has been shown to affect cardiometabolic health in people. We tested the effect that mice housed in dim light at night (dLAN) had on the blood pressure (BP), heart rate (HR), core body temperature (Tb), and activity and if restricted feeding can prevent the dLAN effects.
Methods: Mice (n=6/sex) were implanted with telemetry devices to continuously record the BP, HR, Tb, and activity. Mice were initially housed in a 12 h light: 12 h dark cycle (LD, 200 lux: 0 lux) with ad libitum access to food (LD-ALF) for 7 days. Mice were then subjected to 12 h light: 12 h dim light cycle (dLAN-ALF; 200 lux: 5 lux) for two weeks. After 2 weeks mice dLAN mice were restricted to nighttime restricted-feeding (dLAN-RF).
Results: dLAN mice ate more food during the light cycle ‘wrong time of the day’ compared to mice housed in LD mice. Mice housed in dLAN had a smaller amplitude in the day/night rhythm of BP, HR, and Tb. Specifically, compared to mice housed in LD, mice housed in dLAN showed a 25-40% reduction in amplitude without affecting the overall mean. dLAN abolished the day/night differences between BP, HR, and Tb. Restricted feeding to the nighttime (active hour for mice) ameliorated the dLAN-induced effects on the BP, HR, and Tb and restored day/night difference in BP, HR, and Tb.
Conclusions: dLAN-induced disruptions in day/night rhythms in HR, BP, and Tb are prevented by restricted feeding.
Poster # 005
Introduction: Typically, injured tissues in mammals form scars during healing. However, spiny mice (Acomys cahirinus) are unique among rodents as they regenerate adult tissues and organs. For instance, they completely regenerate removed tissue in ear pinna holes, a useful model for studying musculoskeletal regeneration. Conversely, regular mice develop open holes that heal with fibrotic tissue. Unraveling the drivers of regenerative healing in spiny mice could guide strategies to shift human tissue healing from scarring to true regeneration. Inflammation, a hallmark of tissue injury, is orchestrated by two main macrophage populations: tissue-resident macrophages (TRMs) and infiltrating bone marrow-derived macrophages (InfMs). However, their distinct roles in tissue regeneration remain poorly understood.
Methods: We utilized clodronate drug packaged with liposomes (Clo-Lipo) to deplete macrophages in the ear pinna tissue. Regeneration effects were studied through measurements of ear closure, immuno-histochemistry, and biochemical assays.
Results: Notably, the permanent inhibition of musculoskeletal regeneration following an injury in spiny mice occurs specifically when TRMs, not InfMs, are ablated. The lack of TRMs brings about several changes, such as elevated and prolonged inflammation, delayed re-epithelization, decreased proliferation, and heightened collagen deposition, ultimately leading to fibrosis.
Discussion: The findings of this study definitively demonstrated that following an injury, TRMs promptly engage in safeguarding the injury site, facilitating debris removal, managing inflammation, and initiating diverse regeneration repair pathways. In aggregate, the data suggests that TRMs function as the tissue's guardians, playing a pivotal role as essential regulators of musculoskeletal regeneration in spiny mice.
Poster # 006
Modern machine learning techniques are revolutionizing signal analysis. One of the most promising emerging tools is the implicit neural representation (INR); that is, a neural network approximation of a true physical signal. These continuous representations have several advantages over alternatives, such as discretized grids. For one, INRs can be more memory efficient than a discrete representation. Another advantage is that INRs can be continuously differentiable, meaning that its derivatives can be computed analytically using automatic differentiation. Physics-Informed Neural Networks (PINNs) take full advantage of automatic differentiation to include the laws of physics, generally expressed as differential equations, directly in the loss terms used to optimize the network. As such, PINNs offer a new tool for modeling physical systems, respecting both the data and the underlying physics.
Poster # 007
Introduction: Equid gammaherpesvirus 2 (EHV-2) is a benign and ubiquitous respiratory tract pathogen in horses. It resembles the shedding pattern of Equid alphaherpesvirus 1 (EHV-1), however, associated with severe complications of a myeloencephalopathy or pregnancy loss1,2. Surveillance early detection have focused on nasal swab analysis via PCR assays. This study aimed to assess alternative non-invasive sampling techniques as a method of early detection.
Methods: EHV-2 shedding horses were individually housed in a barn for 10hr on 2 consecutive days. Nasal swabs were collected at the beginning of each day and served as the gold standard using qPCR. Alternative samples included environmental surface samples (sample A; direct contact option); droplet catching devices which stayed for 6 hours outside the stall bars (sample B: airborne transmission), and air samples collected via 2 strategies: sample C- going from stall to stall (4 minutes/horse), and sample D- ‘continuously’ at a central point for 6 hours using a Micro Air Sampler at maximum capacity (50 L/minute).
Results: Nine horses on day 1 and 11 horses on day 2 were EHV-2 qPCR positive on the nasal swabs. 68% of samples A, and 59% of samples B were positive. The agreement between nasal swabs and sample A or B, varied insignificantly (P>0.05). Samples C and D were positive on both days.
Discussion: Air sampling has reliably detected EHV-2 genome copies using 2 different sampling protocols. However, to detect ‘one’ or the ‘first’ shedding horse sampling protocol adjustments and refinements are likely required 4.
Poster # 008
Introduction: Existing scholarship finds that in the past fifty years, affirmative action law has transformed into diversity law as race-based considerations in admissions have remained controversial. No longer is the primary way to incorporate more Black students into higher education race-conscious affirmative action aimed at remedying discrimination. In its place is diversity whose main aim is to provide all students with the “benefits of diversity.” The focus away from justice for historic and present discrimination and a move toward a “diversity must benefit all” narrative creates a disparity in the experiences of those students assumed to be Black or who identify as Black, as well as creating an undue burden on them to educate their white peers on issues of race and racism. This poster will examine how Black undergraduate students in two different diversity scholarship programs at a large Midwestern university are living through and understanding this legal shift and how macro processes of Supreme Court decisions make their way to the micro processes and daily interactions of Black and White students.
Methods, Results and Discussion: Using four years of ethnographic participant observation and 23 in-depth interviews this paper makes conclusions that Black students, by and large, have a legal consciousness of the burdens of diversity (which I term diversity-work), diversity’s focus on whites and the expectations placed on them to enact the “diversity benefits all” narrative, thereby showing a sophisticated understanding of the myth of rights as they pertain to diversity.
Poster # 009
Problem: The assessment of resilience among people with mental illnesses (MI) is an important research area, as it can inform the development of interventions to promote resilience and ultimately improve mental health outcomes. This study aims to assess the level of resilience among individuals with mental illnesses and compare resilience by demographic factors, childhood adversity, and social support.
Theoretical framework: The socio-ecological model emphasizes the importance of multiple levels of influence (i.e., individual, interpersonal, community, and societal factors) on resilience.
Methods and Design: Convenient sampling using an online survey was used to obtain data from 169 participants. We used the Connor-Davidson Resilience Scale (CD-RS) to measure resilience. Descriptive analyses, t-test and ANOVAs were performed to assess participants characteristics in relation to resilience. A linear regression analysis was used to determine factors that contribute to resilience.
Results: Participants reported moderate resilience scores. To establish social determinants of resilience among a community sample of people living with MI, a multiple linear regression model was conducted after checking all assumptions, with CD-RISC total score as a dependent variable, and age, employment status, yearly income, perceived social support, and the history of childhood trauma. The overall multiple linear regression model was significant (DF= 7.492, p<0.001). The combination of the six independent predictors can explain approximately 19% of the variance in the resilience scores among people living with MI.
Implications for psychiatric-mental health nursing: Understanding these associations can help mental health professionals provide more personalized and effective treatment and support for their patients. By assessing an individual's unique demographic, social, and childhood experiences, mental health professionals can develop treatment plans that address specific areas of need and promote greater resilience.
Implications for future research: Studying these associations can help identify the specific factors that promote resilience in people living with MI. Researchers can use this knowledge to develop and test interventions that target these factors and promote greater resilience in individuals with MI.
Poster # 010
Introduction: Wound healing is a multi-step process. Though platelet-rich plasma has been used to accelerate wound repair, functional understanding of how platelets affect wound healing is limited. We investigated the roles of platelet endo- and exocytosis in a full-thickness wound model.
Methods: Two dorsal 4-mm full-thickness circular excisions were made on mice defective in platelet α-granule biogenesis (Nbeal2-/- and Serglycin-/-), exocytosis (Munc13-4Jinx and VAMP8-/-), endocytosis (Arf6-/- and VAMP2/3∆), and wildtype mice (C57BL/6J). Wounds were measured daily. On days 3 and 7, wound sites were harvested for histology. Levels of bioactive molecules were analysed from extracts prepared from the wound tissue.
Results: Nbeal2-/- mice (defective granule biogenesis and cargo packaging) showed impaired wound healing with distinctive morphology. Platelet-specific Arf6-/- and VAMP2/3∆ mice (improper endocytic trafficking and fibrinogen uptake/storage) also showed slower wound healing. Munc13-4Jinx mice (no dense and delayed α-granule release) had faster healing. In histological sections, Arf6-/-, Munc13-4Jinx, and Serglycin-/- mice resembled wound healing and morphology of C57BL/6J mice. In Nbeal2-/-, VAMP2/3∆, and VAMP8-/- mice, healing progression was considerably behind, reflected by increased scab area and decreased re-epithelialisation in Nbeal2-/- mice. Over the course of healing, levels of various bioactive molecules decreased relative to wound resolution, except for FGF2 and MMP-9, reflecting wound remodelling. In Nbeal2-/- mice wound FGF2 and MMP-9 levels decreased as the wounds healed.
Conclusions: Platelets have specific roles in the progression of wound healing and may impact the presence and levels of bioactive molecules in the wounded tissue.
Supported by AHA (1020159), NIH/NHLBI (HL150818), and VA.
Poster # 011
Introduction: Somatic cell reprograming is understood to underlay the conversion of Schwann cells (SCs) into repair cells after peripheral nerve injury. However, most available information on repair mechanisms involving SCs rely on rodent models rather than in human models of nerve injury.
Methods: To address this question, donor-matched human nerves before and two weeks after transection were compared to examine injury-induced changes at the cellular and molecular levels. We combined single cell transcriptomics analysis (snRNAseq) and immunohistochemistry of fixed sural nerve from subjects undergoing experimental axotomy as part of a nerve transplantation trial (NCT02369003).
Results: We found SC activation associated with an increased expression of NGFR protein, a marker of repair SCs. However, myelin phagocytosis, downregulation of myelinating SC-associated genes, increased cell numbers and transcriptional reprogramming were not particularly evident two weeks after injury in SCs. Immunostaining analysis evidenced distinct populations of injury-responsive NGFR positive, S100B negative cells, which reacted to the injury by upregulating NGFR. Accordingly, snRNAseq revealed NGFR-expressing cells within 3 major groups of non-glial cells which reprogrammed their transcriptome in response to injury. These groups were identified for their content of mesenchymal stem cell genes, and injury-induced changes were consistent with epithelial-to-mesenchymal transition (EMT) and extracellular matrix remodeling mechanisms.
Discussion: These studies indicate an overall complex cellular response in injured human nerves involving both glial and non-glial cellular components. Future studies will address the identification of the reprogrammable non-glial cells, as they may enhance the therapeutic value of sural nerve and possibly other PNS-derived SC grafts.
Poster # 012
Background: Cellular neddylation regulate many oncogenic pathways and its pharmacological inhibition has potential as anticancer therapy. The main target of the neddylation cascade are the cullin ring ligases. Cullins besides their other functions also regulate the endosome maturation pathway. Our lab has developed a neddylation inhibitor which binds DCN1 protein. DCN1 is a component of the neddylation cascade which transfers nedd8 tag to cullins. NAcM-OPT kills cancer cells having amplified DCN1 protein and produces a characteristic vesicular phenotype in cells. The purpose of this study was to investigate this vesicular phenotype.
Methods: Immunocytochemistry, immunoprecipitation, western blot.
Results: NAcM -OPT inhibits the fusion of EGFR +ve vesicles with Lamp1+ve vesicles. This fusion is essential for EGFR regeneration and blocking this inhibits cell growth. EGF processing is delayed in cells and is accumulated. NAcM-OPT treatment increases KLHK22 protein in cells. KLHL22 is an adaptor protein in the neddylation cascade. Fusion of KLHL22 +ve vesicles with lamp1+ve vesicles was also inhibited. NAcM-OPT disrupts the autophagy flux. Fusion is inhibition between the autophagosome with the lysosome. All these results are more pronounced under starvation.
Discussion: Our results link the vesicular phenotype with growth inhibition. The accumulated vesicles contain EGFR/EGF causing delay in EGFR regeneration, cell growth. Autophagy is inhibited, explaining higher activity of NAcM-OPT under starvation.
Poster # 013
Introduction: Cerebrovascular dysfunction is frequently comorbid with Alzheimer’s disease (AD), yet the mechanistic consequences of this mixed pathology remain unclear. Recent work suggests that p38 alpha (p38α) MAPK, a regulator of neuroinflammation, may represent an effective target for AD therapies. For example, MW150, a small molecule p38α inhibitor, was shown to improve cognition and decrease cytokines in amyloidogenic mice. However, p38α inhibition in the context of mixed vascular and AD pathologies has yet to be thoroughly characterized. We therefore tested if MW150 could reduce neuroinflammation, synaptic dysfunction, and cognitive impairment in a model of mixed amyloid and cerebral small vessel disease (hyperhomocysteinemia [HHcy]).
Methods: Briefly, 5xFAD mice were given a B-vitamin-deficient diet for 8-weeks to induce HHcy, during which they also received intraperitoneal injections of saline vehicle or MW150. Endpoints included cytokine quantification, immunohistochemistry, neuroimaging, electrophysiology, and behavioral testing.
Results: HHcy diet altered cerebrovascular function, increased cytokines and glial cell activation, altered synaptic transmission, and worsened behavioral performance. Although no effect of MW150 was detected on inflammation or vascular dysfunction, the compound did increase synapse numbers, enhance LTP maintenance, and rescue population spike thresholds. Interestingly, these neuronal measures were also associated with improved performance on the Morris water maze and novel spatial recognition tests.
Discussion: These findings suggest that p38α can influence processes outside of neuroinflammation and also support further investigations of p38α inhibitors in the clinic. Future work will use similar techniques in other AD models to characterize the translatability of this approach across pathologies (i.e. HHcy and tau).
Poster # 014
Introduction: Job insecurity, food insecurity, and their connection are important social determinants of health, but unemployment-linked Medicaid enrollment attenuates the strength of this relationship, especially during the COVID-19 pandemic.
Methods: In states that expanded Medicaid under the Affordable Care Act against states that did not expand Medicaid, we compare the association between pandemic-linked job loss and food security indicators: 1) whether households got enough food, 2) whether households couldn’t afford to buy more food (i.e., food unaffordability), 3) receipt of free groceries or free meals, and 4) the amount of income spent on food. Our analytical sample are 1.2 million working-aged (27-64) adults in the Census Bureau’s 2020-2021 Household Pulse Survey.
Results: The negative association between pandemic-related job loss and food security was smaller in states that expanded Medicaid. In expansion states, the association between job loss and food sufficiency was 2 percentage points (PPT) lower (P<0.05), and the association was 2.6 PPT (P<0.05) lower for food unaffordability. However, there were no differences in the association between job loss and free food receipt or food spending in expansion relative to non-expansion states. Findings suggesting expanded access to Medicaid insulated households from food insecurity created by pandemic-linked job loss were more prevalent in older (aged 50-64) adults and non-metropolitan area residents.
Discussion: Beyond ensuring access to health care, Medicaid has important financial protections that can insulate against economic hardships such as food insecurity created by joblessness.
Poster # 015
Introduction: PFOS is one of the most detected per-and polyfluoroalkyl substances in water in Kentucky. PFOS can accumulate in the intestine and modulate intestinal homeostasis. Although some studies have highlighted the potential mechanisms of PFOS in cancer initiation and progression, there is not much known about the effect of PFOS in colorectal cancer (CRC) development. Therefore, the goal of this study is to investigate the PFOS effects on normal and pre-malignant intestinal epithelium. Methods: The effect of PFOS on the gene expression profile was assessed by RNA-Seq analysis in intestinal tissue of C57BL/6 mice. Tumor organoids were established from Apc/VillinCre adenoma mouse model. CRC cell lines, and human CRC tissue were used to assess the mechanisms involved in PFOS-induced intestinal alterations. Results: RNA-Seq analysis showed that PFOS exposure is associated with significant enrichment of genes involved in lipid metabolism, immune system regulation and pro-carcinogenesis signaling. We found that HMGCS2, a rate-limiting ketogenic enzyme, is significantly downregulated in intestinal tissues of PFOS-exposed mice and in human CRC tissues as compared to normal mucosa. We also show that PFOS upregulates FASN, a key gene of de novo lipogenesis, and PDL-1, a protein involved in immune regulation, in Apc/VillinCre organoids. Conclusion: Our data suggest that PFOS may contribute to CRC development through alterations in ketogenesis and upregulation of pro-carcinogenic pathways, such as de novo lipid synthesis and immune suppression. Delineating the effects of PFOS in CRC initiation and progression may contribute to development of interventional strategies to eliminate harmful effects of this pollutant.
Poster # 016
Introduction: Blood platelets, with their 5-8 mitochondria, are one of the most metabolically active cells in blood. Upon activation, they transition to a high energy-requiring state to mediate hemostasis, yet it is unclear how mitochondria contribute to that process and its sequelae.
Methods: To dissect the roles of mitochondria in vivo, we generated two platelet-specific knockout mouse lines deleting TFAM and QPC. TFAM is a DNA binding protein important to maintain the integrity of the mitchondrial genome. QPC is a core protein subunit of Complex III of Oxidative Phosphorylation that generates ATP.
Results: Platelets from these animals showed the predicted defects in bioenergetics and also had significant bleeding phenotypes in three different injury models. Our studies definitely show how mitochondrial activity is important for clot formation and yield insights into the mechanisms underlying the increased thrombotic risks seen in various metabolic syndromes.
Poster # 017
Introduction: Global navigation satellite system (GNSS) receivers on unmanned aircraft systems (UAS) are typically equipped with standard or differential fix configurations, offering horizontal and vertical accuracies of approximately ±5 meters and ±15 meters, respectively. While spatial error is not as critical for temperature and relative humidity gradients, low positioning accuracy is problematic for meteorological applications involving barometric pressure.
Methods: This study aimed to improve our understanding of GNSS position accuracy in UAS-based atmospheric observation. The primary objective encompassed three specific goals: (1) developing a GNSS accuracy testing system, (2) deploying it on a multirotor UAS, and (3) evaluating static and dynamic accuracy of L1 and L1/L2 GNSS receivers in both Real-Time Kinematic (RTK) and non-RTK modes.
Results: Results indicated significant differences across receiver configurations and deployment strategies. RTK receivers displayed minimal mean error and consistent standard deviations, while non-RTK receivers showed greater average error and variability, especially in elevation.
Discussion: Though the study did not conclusively confirm a consistent reduction in accuracy due to UAS deployment, findings suggest that RTK receivers substantially enhance accuracy and mitigate measurement variability attributable to timing or treatment.
Future work entails conducting tests over a full day to monitor satellite movements and accuracy fluctuations resulting from changes in time of day, weather patterns, and environmental conditions, as well as incorporating vector control techniques to consider UAS orientation.
In conclusion, this research contributed insights into GNSS accuracy for UAS-based atmospheric observations and underscored the importance of considering receiver configurations and deployment strategies for reliable atmospheric observations.
Poster # 018
Background: Dietary lipid absorption is essential for systemic lipid homeostasis. We previously demonstrated a role for Dennd5b in intestinal lipid absorption. Female Dennd5b-/- mice have reduced appearance of dietary triglycerides (TG) in plasma and are less susceptible to diet-induced obesity. In humans, DENND5B gene variants are correlated with BMI in females, but not males.
Objective: Determine if the Dennd5b-/- mouse model recapitulates the sex disparity observed in humans with DENND5B polymorphisms and determine the fate of unsecreted TG in intestinal tissue.
Methods and Results: A non-absorbable fatty acid (FA) tracer was used to quantify the impact of Dennd5b-deficiency on lipid absorption in male and female mice. We observed a relatively modest reduction in lipid absorption efficiency in Dennd5b-/- mice in both sexes, despite a complete absence of plasma TG after oil gavage. We hypothesized that this was due to metabolic utilization of TG by enterocytes. Metabolic cage studies showed that both wildtype and Dennd5b-/- mice shift toward utilization of FAs when fed high-fat diet. Electron microscopy (EM), revealed large electron dense structures that resemble autophagosomes in Dennd5b-/- enterocytes. Western blots revealed an increase in Lc3-II:Lc3-I ratio in Dennd5b-/- intestine, indicating increased autophagy. Levels of the FA sensing transcription factor, Hnf4g, and its target gene involved in beta oxidation, Cpt1a, were increased in Dennd5b-/-. EM also revealed altered mitochondrial morphology in Dennd5b-/- enterocytes.
Conclusions: We conclude that unsecreted TG in the Dennd5b-/- enterocytes are degraded by autophagy, liberating free FAs which are utilized in mitochondrial oxidation. The sexually dimorphic impact of Dennd5b may not be as prominent in mice as it is in humans. Overall, our findings demonstrate that Dennd5b plays a critical role in regulating lipid metabolism in the intestinal tissue that can impact systemic metabolic health.
Poster # 019
Introduction: In cells, DNA acts as the instruction manual for all cellular functions. Through base-pair interactions, DNA can replicate with fidelity. These same base-pair interactions can be used to artificially design, or program, strands of DNA to perform intricate functions like mathematics. In this project, a DNA-based Chemical Reaction Network (D-CRN) was constructed to perform fractional multiplication as a proof-of-concept.
Methods: DNA sequences were designed using Piperine, a program that converts CRNs into nucleotide sequences. The CRN utilizes strand-displacement to compute the desired mathematic function. The DNA duplexes in the CRN are synthesized with a fluorophore and quencher. As specific concentrations of input strands are added, DNA strands from the duplexes are displaced, the fluorescence increases, and the mathematical equation can be solved using the detected concentration of the displaced products.
Results: Using a spectrum of input values between 0 and 1, we demonstrate that the D-CRN we have designed accurately performs fractional multiplication.
Discussion: Programmable DNA lays the foundation for a variety of industrial and medical applications. In this project, we demonstrate that D-CRN can be used to perform fractional multiplication. Future D-CRN can be easily adapted to perform other mathematic functions, and as we continue to refine these networks, they can be developed for applications requiring precise spatial and temporal control at the molecular level.
Poster # 020
Introduction: In large volume muscle injuries, therapeutic myogenic progenitor cells (MPCs) cannot reach the injury site and initiate repair. Many tissue construct designs for these injuries start as two-dimensional layers of aligned myotubes prior to being scaled in three dimensions. Through the binding affinity of biotin and streptavidin molecules, we pre-position MPCs on an ECM substrate in straight-line patterns separated with small gaps, mimicking small-scale muscle injuries and initiating the MPC migratory nature to align and form myotubes across the gaps.
Methods: The binding affinity of biotin and streptavidin molecules allows for intentional positioning of MPCs in patterned structures. Streptavidin has four binding sites, which can link biotin conjugated to the ECM substrate and cell surface. After MPCs are seeded on the patterned substrate, centrifugation washing maximizes cell removal outside of the patterned regions (weak adhesion) while minimizing cell removal within the patterned regions (strong adhesion). Cell patterns are placed in differentiation media for 6 days, allowing MPCs to migrate between patterns and form aligned myotubes.
Results: During differentiation, patterned MPCs migrated unidirectionally perpendicular (90°) to the original biotin-streptavidin surface patterns. Conversely, MPCs seeded without biotin-streptavidin patterning did not exhibit any established directionality. Cell staining demonstrated that the cell biotinylation, seeding, and centrifugation processes did not impact viability or long-term functionality.
Discussion: The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary cell patterns. By creating repetitions of patterned cells and spacings, we have demonstrated rapid MPC migration and alignment, which differentiate into a long-range two-dimensional layer of aligned myotubes.
Poster # 021
Introduction: Thrombin, the enzyme which generates blood clot, is produced by the prothrombinase complex, composed of coagulation factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor-α (TFPIα) and activated protein C (APC), and their common cofactor protein S (PS). Here, we compared the mechanistic action of the TFPIα/APC/PS anticoagulant system on physiologic membrane surfaces (phospholipids/PLs, platelets, and endothelial cells/ECs).
Methods: Thrombin generation (TG) was measured in plasma and platelet-rich plasma (PRP) by calibrated automated thrombography, prothrombinase activity was assayed chromogenically, and FVa cleavage by immunoblotting.
Results: Inhibitory antibodies against TFPI and PC had an additive effect on plasma TG which was mimicked by inhibiting PS alone. Similar results were obtained in plasma supplemented with ECs or in PRP. However, in PRP, the antibodies reversed TG beyond what was seen with coagulation-initiation alone. In purified protein systems, on PLs or ECs, physiological TFPIα concentrations had no effect on TG, unless APC was present. The greatest inhibition was observed when TFPIα, APC, and PS were all present. However, prothrombinase on activated platelets was protected from inhibition in any conditions, as measured either by TG or FVa cleavage.
Discussion: On PL or EC membranes in the plasma environment, APC, TFPIα, and PS cooperate synergistically to inhibit prothrombinase activity, preventing excessive clot propagation. Conversely, platelets protect prothrombinase from inhibition, supporting a procoagulant environment within the clot.
Poster # 022
Introduction: Structure-based drug discovery depends on understanding the complex interplay between biomolecular structures and their functions. However, the complexity arising from the high dimensionality of biomolecular structures presents a challenge in developing meaningful mathematical models. A typical human biomolecule consists of thousands of atoms, occupying an 18,000-dimensional space, making direct physical calculations and integration into deep neural networks impractical due to increased machine learning (ML) dimensions. Biomolecule size variations further complicate the application of ML models.
Methods: We propose an approach that combines differential geometry, graph theory, and advanced machine learning techniques. This involves encoding chemical and biological insights into differentiable manifolds through molecular surfaces and leveraging the inherent graph-like nature of biomolecules. Our models predict the binding affinity between a ligand (drug candidate) and its target protein, a crucial aspect of drug discovery, and undergo comparison with established datasets and prevailing methods.
Results: Our models demonstrate enhanced predictive accuracy, surpassing existing methods. The synergy between differential geometry, graph theory, and advanced machine learning enables us to capture vital biomolecular features while addressing high-dimensional challenges. Our approach effectively handles diverse biomolecule sizes and intricate binding scenarios.
Discussion: The integration of differential geometry, graph theory, and advanced machine learning holds promise for advancing structure-based drug discovery. By reducing dimensionality and capturing key biomolecular insights, our models provide a pragmatic solution to the complexities posed by high-dimensional biomolecular structures. The demonstrated performance improvement underscores the significance of our approach in predicting protein-ligand binding affinity, highlighting its potential to expedite drug discovery efforts.
Poster # 024
Although wastewater disposal in evaporation ponds may be a cost-effective strategy for wastewater management, it overlooks water recycling and can possibly pose significant threats to the surrounding environment. The development of photothermal materials with abundant functional groups capable of adsorbing toxic contaminants offers a promising opportunity for detoxification and potentially solar-driven recycling of wastewater disposed in evaporation ponds. In this study, ultralight aerogels based on chitosan (Ch), a natural biodegradable polymer, functionalized with mussel-inspired polydopamine (PDA) and carbon nanotubes (CNT) were fabricated through a facile approach and examined for the removal of arsenic from water. Three aerogels, namely chitosan, CNT/Ch, and PDA@CNT/Ch were fabricated and characterized using SEM, FTIR, TGA, XPS, and BET surface area analysis. Adsorption isotherms, thermodynamics, and kinetics were systematically investigated, revealing the feasibility of the fabricated aerogels for arsenic removal from aqueous solutions. The UV-Vis and photothermal analysis indicated excellent light-absorption and light-to-heat conversion performance of the functionalized aerogels. Arsenic adsorption by aerogels occurred rapidly and reached equilibrium within 30 to 60 min and was well-fitted by a pseudo-second-order kinetics model. The Langmuir model well described the adsorption isotherm, and the maximum adsorption capacities were found to be 31.5, 36.6, and 38.7 mg/g at neutral pH for chitosan, CNT/Ch, and PDA@CNT/Ch, respectively. The aerogels were regenerated using 0.1 mol/L NaOH and the removal efficiency of the modified aerogels remained as high as >91% after five adsorption-desorption cycles. This study broadens the scope of utilizing aerogels for the detoxification and potential recycling of wastewater from diverse sources and effluents disposed of in evaporation ponds.
Poster # 025
Introduction: Low temperatures cause physiological challenges for ectotherms, and as a result winter conditions can have a strong impact on species distributions. Ectotherms can use evolutionary adaptations or adaptive phenotypic plasticity (i.e., thermal acclimation) to mitigate thermal stress. Under quantitative genetic theory, traits like thermal tolerance are under the influence of both genotype and environment, and the presence of a genotype-environment interaction indicates that phenotypic plasticity can evolve. This study was designed to determine the extent to which distinct types of thermal acclimation show genetic variation, determine whether distinct types of thermal acclimation independent traits, and identify molecular mechanisms that underlay variation in acclimation capacity. We assessed critical thermal minimum (CTmin) of various lines of the Drosophila Genetic Reference Panel (DGRP) after distinct acclimation pre-treatments.
Methods: To assess CTmin, we used a vertical jacketed column connected to a temperature-controlled fluid bath, and a type T thermocouple was used to measure the temperature inside of the column at the precise temperature when the fly lost neuromuscular coordination and fell through a collection funnel. For a subset of lines with variable acclimation responses, RNA-sequencing was used to reveal molecular mechanisms that associate with this variation.
Results: All types of acclimations were genetically variable, with short-term rapid hardening responses being the most variable. There was a slight correlation between adult and developmental acclimation capacity, and we observed considerable gene expression differences between lines with distinct acclimation capacity.
Discussion: These results are useful to understand the evolutionary genetics of insect thermal plasticity and response to environmental perturbation like climate change.
Poster # 026
INTRODUCTION: Osteosarcoma (OS) is the most common primary bone malignancy that affects children and young adults and approximately 3,970 new cases are being diagnosed with 2140 deaths in the USA alone in 2023. Recent studies have targeted the tumor microenvironment macrophages (M2) which help in tumor progression but M1 macrophages can show anti-cancer properties and show repolarization of M2 macrophages.
METHODS and RESULTS: Human PBMCs were used to prepare cisplatin-loaded M1 macrophage-based engineered vesicles (C-MEVs). Electron microscopy showed a round morphology with a size range of 150-200nm. TEM suggested their internalization into the tumor cell mitochondria. The OS cells showed sensitivity against C-MEVs with lower IC50 and an equal tendency to cause DNA damage as compared with free cisplatin. The orthotropic OS tumor model was developed in nude mice and the primary tumor was observed by 2 weeks post-injection. Upon C-MEV treatment, they showed localization in the primary tumor and the liver of the mouse post 72 hours of treatment. Histopathological analysis suggested distinguished M1 and M2 macrophages in the tumor and lungs of mice. Ki67 was used as a proliferation marker. These initial parameters suggested the use of engineered vesicles for targeted therapy against OS.
DISCUSSION: Cisplatin is the most versatile drug used to treat cancer patients but is toxic at higher doses. Our results showed the better efficacy of C-MEVs over free cisplatin on OS cells in vitro and in vivo. This vesicle-based immunotherapy has shown a promising treatment strategy against OS with lower toxicity and targeted therapy.
Poster # 027
Introduction: Lampreys and hagfish, known as cyclostomes, are the only living jawless vertebrates and represent a lineage that diverged from our own ~500 million years ago. They provide a comparative perspective on the evolution of vertebrates but also undergo programmed DNA loss, a phenomenon where DNA is eliminated from somatic cells during embryogenesis while being retained in the germline (eggs and sperm). We aim to study germline-retained genomic material across different cyclostome species and see if there is a common part inherent to all cyclostomes.
Methods: The reconstruction of cyclostome genomes, especially in germline-retained regions, is complicated by various complex structures and repetitive elements. Hybrid genome assembly approaches with long reads and chromatin contact data from sperm DNA were used to build chromosome-scale assemblies for several cyclostomes. We also employed “k-mer”-based methods to separately assemble high-copy repeats. These assemblies were used to compare DNA from somatic and germline cells.
Results: We identified germline-specific sequences including hundreds of important genes across several lamprey and hagfish species. Some of these genes are shared across several species, and have evolved specialized functions in germline since duplicating from their somatically-retained homologs. Computational analyses and in situ validation reveal that germline-enriched repeats comprise a large fraction of germline-specific chromosomes.
Discussion: While cyclostomes rely on programmed elimination of DNA as a normal feature of their development, similar processes lead to chromosome instability in humans. Studies of DNA loss further our understanding of genetic functions that contribute to germ cell biology and the regulation of genomic rearrangements.
Poster # 028
Background: Circulating, pro-inflammatory lymphocytes invading the ischemic and perilesional tissue is a key neuroinflammatory response, exacerbating stroke outcome. However, few studies focus on neuroinflammation in remote brain regions.
Aim: We investigated whole-brain regional distribution of intravenously injected lymphocytes (CD19+ B-cells, CD8+ T cytotoxic (Tc)-cells) following transient middle cerebral artery occlusion (tMCAo) in young male mice.
Method: e450-labeled naïve B- or Tc-cells transferred i.v. at 7, 24, 48, and 72h post-tMCAo, with sacrifice on day 4. Serial two-photon tomography of cell localization in coronal sections was registered to Allen Atlas prior to unbiased supervised machine learning (pixel-wise random forest model via ilastik).
Results: Post-stroke B-cell localization was evident in regions closely related to neurogenesis (i.e., vascular organ of lamina terminalis, subfornical organ, area postrema; all p≤0.05) compared to vehicle controls. Tc-cells migrated through nuclei associated with central regulation of physiological functions, energy homeostasis, respiration, blood pressure, and learning and memory (i.e., arcuate nucleus of the hypothalamus, nucleus of the solitary tract, parabrachial nuclei, dorsal raphe nucleus; all p≤0.05, versus vehicle controls). Both B-cells and Tc-cells had a high affinity for the lateral ventricle, a neurogenic niche that, with the choroid plexus, supports the CNS entry of immune cells.
Conclusions: Long-term neuroinflammatory mechanisms could either stimulate or hinder stroke recovery depending on the time of activation, leukocyte subset, or the nuclei in which immune cells migrate. Our results provide detailed large-scale automated quantification of post-stroke region-specific neuroinflammation, confirming subacute recruitment of systemic lymphocytes into remote brain regions mediating stroke pathology and recovery.
Poster # 029
Hemp seeds are recognized as excellent sources of essential fatty acids, proteins, vitamins, and dietary fiber. However, limited research has been conducted on the dietary fiber composition of hempseeds, despite the established nutritional value of seed oil and proteins. Some recent studies have provided evidence of hempseed fiber profiling, but they have either focused on soluble fibers or provided incomplete profiling of polysaccharides. The objective of this study was to conduct a comprehensive chemical and structural analysis of the cell wall polysaccharides in ten hempseed cultivars, namely NWG 2730, Enecterol, Yuma, Lara, Fedora 17, Henola, Orion 33, Futura 75, Futura 83, and USO 31. The experiment involved drying, defatting, milling, and de-starching the hempseeds, followed by the isolation of water-soluble and insoluble polysaccharides. The monosaccharide composition of the isolated polysaccharides were determined using high-performance anion-exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD) quantification after Saeman hydrolysis and methanolysis. The analysis of the linkages among sugar units in the polysaccharide chains was performed by methylation followed by gas chromatography-mass spectrometry (GC-MS) analysis. Furthermore, lignin contents were determined as acetyl bromide soluble lignin (ABSL). The results revealed that all cultivars contained comparable amounts of moisture, fat, fibers (soluble and insoluble), and lignin contents. The hemp grain cell wall polysaccharides consisted of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid. Glucose and xylose, which represent cellulose, xylans, and xyloglucans, were found to be the most abundant in water-insoluble polysaccharides. The water-soluble polysaccharides contained abundant amounts of galactose, galacturonic acid, arabinose, rhamnose, and mannose, indicating the presence of galactomannan, arabinogalactans, and pectins (homogalacturonans and rhamnogalacturonans). The linkage analysis revealed the presence of both linear and branched arabinans, as well as xyloglucans with F-motifs. A key significance of this study is the potential utilization of hemp grain as a food source for the gut microbial population in humans, as they are explored as a rich source of hemicelluloses and pectins. Degradation of these polysaccharides leads to the production of short-chain fatty acids, which have wide-ranging health benefits for the host.
Poster # 030
Colonization by Helicobacter pylori strains carrying the cag type IV secretion system (cag-T4SS) is the strongest known risk factor for the development of severe gastric diseases in humans. In addition to delivering the bacterial oncoprotein CagA, LPS intermediates, and peptidoglycan to host cells, recent evidence demonstrates that cag T4SS activity stimulates human TLR9 raising the hypothesis that microbial DNA is translocated into host cells. We demonstrate that chromosomal fragments delivered to the host cell cytoplasm via cag T4SS-dependent trans-kingdom conjugation activate the cytoplasmic DNA sensor cGMP-AMP synthase to stimulate STING signaling. Using ChIP-seq workflows, we observed that microbial DNA co-purified with cGAS predominantly cluster around oriC, suggesting that DNA translocation is mechanistically coupled to chromosomal replication. Using a reverse genetics approach and robust TLR9 stimulation assays, we discovered several key ‘uncoupling mutations’ that selectively disarm DNA translocation without disrupting CagA or peptidoglycan injection into host epithelial cells. We demonstrate that multiple non-cag-T4SS components are directly involved in trans-kingdom conjugation, including DNA integrase/recombinases, periplasmic DNA processing proteins, FtsK/SpoIIIE-related DNA mobilization proteins, and Agrobacterium tumefaciens VirD2 endonuclease orthologs. Finally, we show that transfer DNA is processed and coupled to the cag-T4SS apparatus prior to host cell contact to establish a ‘ready-to-fire’ nanomachine and demonstrate that DNA loading is dependent upon FtsK. Collectively, our data suggest that the H. pylori cag-T4SS co-opts orphaned components of divergent DNA conjugation systems, DNA segregation and mobilization machinery, and phage resistance mechanisms to deliver immunostimulatory nucleic acid cargo to gastric cells.
Poster # 031
CD8+ T cells from HLA-A2.1-transgenic mice, but not wild-type mice, immunized with the amino-terminus region (aa 41-152) of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii secreted large amounts of perforin and granzyme B in response to GRA6Nt through antigen presentation by HLA-A2.1 in vitro. When their CD8+ T cells were transferred into chronically infected NSG mice expressing HLA-A2.1, cerebral cyst burden of the recipients of only HLAA2.1-transgenic T cells became significantly less than that of control mice with no cell transfer. Thus, GRA6Nt activates CD8+ T cells capable of removing T. gondii cysts through antigen presentation by human HLA-A2.1.
Poster # 032
Introduction: The purpose is to examine tobacco use, secondhand smoke (SHS) exposure and infant feeding practices including breastfeeding (BF) duration in mothers living in rural Kentucky communities. Women in rural communities have higher rates of tobacco use and SHS exposure as well as lower rates of BF initiation and duration compared to their urban counterparts. Research is lacking on the association of tobacco use, SHS exposure and infant feeding status in rural communities.
Methods: This study uses a cross-sectional retrospective design and purposive cluster sampling with stratification by strength of municipal smoke-free laws and tobacco use and/or exposure status. Women between 18-45 years of age currently residing in one of the six identified rural Kentucky counties: Knott, Owsley and Perry (strong smoke-free laws) and Bath, Menifee, Morgan (no smoke-free laws), who have given birth to a live infant within the past two years and speak English are eligible. Recruitment methods include ResearchMatch, local health departments and community organizations. Measures include demographics; infant feeding practices (prior BF history and infant feeding status); tobacco use (previous and current; cigarettes and e-cigarettes); SHS exposure (home, workplace and vehicle); lung cancer (prior screening, personal and family history and worry); depression; anxiety and alcohol and substance abuse (prenatal and current).
Results: As an ongoing study, we will present preliminary findings. We will present descriptive statistics, bivariate associations and multi-level modelling at study conclusion.
Discussion: We will present implications for policy change, public health campaigns, and environmental health practice.
Poster # 033
Global sweet potato yields and quality face a grave threat from Sweet Potato Virus Disease (SPVD). However, the intricate workings of pathogenesis and dynamics of SPVD remain shrouded. Here, we leverage single-cell transcriptomics, using virus-infected plants collected from the field and virus-free plants generated through meristem-derived callus regeneration, and successfully constructed maps of host and viral infection for SPVD. Our results revealed that the virus preferentially accumulates in palisade mesophyll cells (PMCs), where it suppresses defense signaling and facilitates synergy of co-infecting viruses. Furthermore, the viruses induce photooxidative stress and disrupt chloroplast ultrastructure by recruiting PMC-specific transcriptional regulators, resulting in reduced tuber starch biosynthesis and yield. Overexpressing a PMC-specific transcriptional repressor resulted in phenotypes that mirrored viral infected plants. We identified cell type specific transcription factor hubs that form gene regulatory networks. The single-cell resolution maps of SPVD illuminated molecular mechanisms that are unlikely to be uncovered by bulk transcriptomics.
Poster # 034
Introduction: Aortopathies, specifically life-threatening aortic aneurysms, and dissections, have a critical but understudied connection with platelets. While platelets are acknowledged for their involvement in clotting, recent research suggests they may also play a role in aortopathies.
Methods: Our study draws from novel techniques involving mouse models with defects in platelet exocytosis. We intend to understand platelet involvement in Angiotensin II- (AngII) and β-aminopropionitrile- (BAPN) triggered aortic conditions. Preliminary findings show increased platelet activity in mice subjected to AngII and BAPN treatments. In our approach, we'll explore the role of Vesicle Associated Membrane Protein 8 (VAMP8) and neurobeachin-like 2 (NBEAL2) in the AngII model. Aim 2 utilizes the antiplatelet drug, clopidogrel, to gauge its therapeutic potential against BAPN-induced conditions.
Results: Initial data reveals heightened platelet activation at aneurysm sites in AngII-treated mice and aortas impacted by BAPN. We anticipate that by inhibiting specific platelet functions or modifying their activity, the progression of aortic conditions can be controlled.
Discussion: Considering the severe risks associated with aortic aneurysms and dissections, understanding platelets' involvement may offer vital strategies for prevention and treatment. Our research aims to shed light on the role of platelets in aortopathies, suggesting potential pathways for effective medical interventions.
Poster # 035
Introduction: Aortic dissection (AD) is a life-threatening disease with disruption of the extracellular matrix. Elastic fibers are a major component of the extracellular matrix and are vital for aortic integrity. Elastic fibers have a long half-life and are minimally synthesized in adulthood. Despite the important role in structural stability, it remains unknown whether new elastic fibers could be formed following ADs in adults.
Methods and Results: To induce ADs, β-aminopropionitrile (BAPN) was administered to male C57BL/6J mice at 4 weeks of age. Aortas were harvested at 4, 8, and 12 weeks of BAPN administration. BAPN administration for 4 weeks led to relatively fresh hematoma with modest luminal dilatation in the descending aorta, indicating development of acute ADs. Mice with BAPN administration for 8 or 12 weeks displayed severe aortic dilatation with thrombus and aortic wall remodeling, suggesting chronic ADs. Using these tissues, we then performed Verhoeff’s iron hematoxylin staining to evaluate the presence of elastic fibers during AD progression. As expected, elastic fibers adjacent to the true lumen were disrupted in both acute and chronic ADs. Of note, in chronic ADs, elastic fibers were observed surrounding the false lumen. Compared to elastic fibers surrounding the true lumen, these new fibers were thinner, denser, and less organized. In situ hybridization revealed Eln and Fbn1 mRNA were distributed in proximity to new elastic fibers. In addition, immunostaining found the hyperplasia of αSMA-positive cells in the wall of the false lumen. Importantly, these cells were coincident with de novo elastic fibers. These results are consistent with smooth muscle cells being the primary cell type generating elastic fibers in chronic ADs. To investigate the molecular mechanism driving de novo elastic fiber formation, elastogenic genes were evaluated by RT-qPCR. Aortic Eln and Fbn1 mRNA were increased significantly in the chronic phase, but not in the acute phase. Genes related to elastic fiber assembly, such as Fbln5, Emilin1, and Lox, were also upregulated in chronic ADs.
Conclusion: Elastic fiber formation is reactivated, with enhanced expression of elastogenic genes and SMC hyperplasia, in response to AD progression.
Poster # 036
Let's imagine we are a scientist on a quest to develop a new medicine. Your goal is to create a powerful drug that can kill cancer cells without harming the heart and normal cells. Let's break down our journey step by step in simpler term. In our body, there's a special process called "neddylation." Think of it as attaching a magical tag called NEDD8 to certain proteins. These tagged proteins, like cullins, become part of a team, working together to control how our cells clean up and dispose of waste. The cullins, when tagged with NEDD8, turn into powerful warriors called cullin-RING ubiquitin E3 ligases (CRLs). Sometimes in cancer, certain proteins can go rogue and start causing trouble. We need a way to stop them. Here's where the neddylation pathway comes in. It's like the switch that makes these rogue proteins super-strong and causes them to create even more chaos. So, if we can turn off this switch, those troublesome proteins lose their power, and we can bring things back to order.
Using a high throughput screening and structural optimization strategies, we discovered a way to potently inhibit the neddylation process with a small molecule, our hero UK-1 (IC50= 60 nM). But here's the catch: our hero, UK-1, has a side effect. It can affect the heart and cause a dangerous heart rhythm problem. We needed to make UK-1 safer. So, we went back to the drawing board, started research and modified UK-1's structure to create a new version called superhero TK_006. This new hero doesn't mess with the heart's rhythm but kills cancer cells and suit with extra safety features. So, in our quest for a cancer-fighting medicine, we discovered a new and safer hero: TK_006.
Poster # 037
Introduction: Skeletal muscle remodeling involves intricate interactions among diverse mononuclear cell populations. Satellite cells hold particular significance, as they play a central role in coordinating cellular responses during muscle adaptation and growth. However, the mechanisms governing communication between satellite cells and neighboring cells remain poorly understood. Recent attention has focused on exosomes, recognized as pivotal mediators of intercellular communication, presenting a promising avenue to bridge this knowledge gap.
Methods: To investigate the in vivo dynamics of intercellular exosome transfer, we designed a novel transgenic mouse model expressing a satellite cell-specific exosome reporter: human-CD63-GFPf/f. We investigate the origin of the exosome and track the direction of the transfer to target cells by employing, advanced cellular sorting techniques, immunohistochemical techniques, and confocal microscopy. Additionally, the ExoView R100 platform was utilized to analyze the characteristic of human-CD63-GFP-tagged exosomes released by satellite cells.
Results: We established a vibrant GFP fluorescence in the majority of satellite cells in the human-CD63-GFPf/f mouse model, facilitating efficient exogenous human-CD63-GFP transfer to various non-satellite mononuclear cells. Exosome characterization indicated an approximate count of 4000 particles per spot, with sizes ranging from 50 to 100nm. Through immunohistochemical and confocal microscopy approaches, the distribution of CD63-GFP+ puncta within muscle cross sections and individual myofibers was visualized, both in the absence and presence of hypertrophic stimuli.
Discussion: Our findings highlight the model's potential in uncovering satellite cell communication within the muscle microenvironment. Future studies must identify recipient cells, comprehend essential exosome-carried signaling molecules, and unravel their impact on recipient cell programming.
Poster # 038
Introduction: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a common technique to separate macromolecules, such as proteins, by their molecular weights. In addition to molecular weight, SDS-PAGE gels can be modified, referred to as Phos-tag gels, to separate the proteins by their phosphorylation patterns. Separation by weight is often visualized by staining the gels. On the other hand, Western blotting is preferred for separation by phosphorylation status. The densitometry analysis of the bands is performed to determine the relative quantities of the proteins or phosphorylation patterns. The difficulty in quantification depends on how well the bands are resolved. As the bands start to overlap a better approach is required to determine relative quantities of proteins.
Methods: GelBox, a program written and developed in MATLAB App Designer (MathWorks, Natick, MA), to analyze gels with and without overlapping lanes. It provides an interactive analysis space for users and can handle up to three bands simultaneously. It uses optimization schemes to fit a function, in the form of a summation of Gaussian functions, to the resulting densitometry profile. The interface does not require future users to have prior experience in MATLAB language. Analysis, result export, and data visualization are interactively performed through the user interface.
Results: A titin gel and two Phos-tag gels for RLC and cTnI are presented as examples. While titin gel and RLC Phos-tag gel resulted in two bands, cTnI Phos-tag gel resulted in three bands. GelBox successfully quantified the titin isoform ratios and the phosphorylation patterns.
Poster # 039
CRISPR/Cas9 technology holds great potential for cancer treatment due to its versatility in editing disease-associated genes, including those promoting tumor growth, drug resistance, and immune suppression. However, uncontrolled gene editing in normal tissue can induce genotoxicity. The lack of technology controlling in vivo CRISPR/Cas9 activity hurdles its clinical translation. Here we develop a platform consisting of magnetic nanoparticles (MNP) and baculoviral vectors (BV) for site-specific activation of CRISPR/Cas9 gene editing for cancer immunotherapy. BV was conjugated to MNP through electrostatic interaction between the viral envelope and the TAT peptides on the surface of MNPs. Our study confirmed that when mixed with MNP-TAT, BV transduction was significantly enhanced in mouse colon cancer MC38 cells. Next, we selected the sgRNA targeting mouse PD-L1 through bioinformatics tools and in vitro screening. MC38 infected with BV packaging Cas9 nuclease and the sgRNA had a lower PD-L1 expression (45.5%) compared with untreated cells. Furthermore, the splenocytes cocultured of BV-infected MC38 cells showed higher cell cytotoxicity than those with untreated MC38 cells (37.3% vs. 28.7%). Intratumoral injection with MNP-BV promoted CD3+CD8+ T cell infiltration in the MC38 tumors and significantly inhibited tumor growth. Our study demonstrates MNP-BV as an efficient and controllable gene editing platform for cancer immunotherapy in solid tumors.
Poster # 040
In the ever-evolving landscape of education, equity remains a prime concern. The release of the third version of the Assessment, Evaluation, and Programming System for Infants and Children (AEPS-3) has raised significant considerations concerning the accuracy of child development measures, especially when transitioning from its predecessor, AEPS-2. The main objective of this study is to apply score equating under the Rasch framework. This is done to ensure continuity and comparability between the scores of AEPS-2 and AEPS-3. This study employed the method of the common item non-equivalent design, featuring fixed parameter calibration equating (known as "anchoring"), utilized within the Rasch framework. An operational score conversion table for the six developmental areas from AEPS-2 to AEPS-3 in 0-3 years old levels was provided. The process of score equating between AEPS-2 and AEPS-3 serves as a prime example of how educational measures can be adapted over time without losing their comparative value. This case not only underscores the importance of consistent and accurate measures in early childhood education but also offers a blueprint for future version transformations in this field.
Poster # 041
Introduction: Programmed genome rearrangement (PGR) is a phenomenon that has been found in an increasing amount of vertebrate taxa. Understanding how these changes manifest at the cellular level is therefore of interest in both developmental and cancer biology. The sea lamprey Petromyzon marinus provides a model for addressing these questions, as twenty percent of its genome is purged from somatic cells. The removed regions include genes known to be involved in germline differentiation, meaning that PGR in lamprey may serve to block cells from acquiring germline features. We were thus interested in determining how early we could detect the effects of PGR on somatic cells.
Methods: We hypothesized that primordial germ cells are differentiated before the onset of PGR and could thus be identified by a suite of transcriptional and histological characteristics. To accomplish these, we have utilized scRNA-seq, histochemical staining, and in situ hybridizations to better understand the development of primordial germ cells.
Results: Our scRNA-seq data have revealed clustering by the time that PGR commences (36 hpf), but differences in gene expression at this point are mostly quantitative. By 48 hpf, these differences are considerable and candidate genes can be identified, allowing us to map identities on the early embryo. On the histological level, lamprey primordial germ cells are almost indistinguishable from somatic cells at these stages.
Discussion: Our results here provide not only to our understanding of PGR and germline development in lamprey, but also to a framework to study early development in non-traditional model organisms.