ARC25 Agenda – Sat, Feb 1

Landon Teer1, Neha Anil1, Dominic Armagno1, Bradley J. Mahaffey1, Sihan Sun2, Charles Froman-Glover3, Akshitkumar Mistry4, Kavitha Yaddanapudi5, and Joseph Chen1

1Department of Bioengineering, University of Louisville, Louisville, KY, 2Department of Biology, University of Louisville, Louisville, KY, 3Department of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, KY, 4Department of Neurosurgery, University of Louisville, Louisville, KY, 5Division of Immunotherapy, Department of Surgery, University of Louisville, Louisville, KY

Introduction/Background. Glioblastoma (GBM) is an incurable and highly aggressive cancer with a dismal prognosis. GBM progression is coupled with proneural-to-mesenchymal transitions (PMT) which worsen survival in part through heightened cell invasiveness. Notably, mesenchymal GBM rapidly invades the surrounding tissue despite dense physical barriers in the brain parenchyma. Recent work has suggested that efficient confined migration is critical to bypassing these physical constraints; however, the underlying mechanisms driving robust confined migration remain unclear. Here, we utilize a biophysical lens to investigate the relationship between mechanical alterations associated with GBM mesenchymal transformation and functional confined migration.

Hypothesis/Goal of Study. We hypothesize that PMT in GBM coincides with reduced nuclear stiffness to promote increased cell invasion through confined microenvironments.  

Methods and Results. GBM cells (U251s and U87s) were treated with 10 ng/ml TGF-β1 for 72 hours and assessed for changes in protein expression, cytoskeletal organization, mechanical rigidity, and confined migration. Biophysical properties were assessed through AFM mapping, and confined migration was analyzed via microchannel devices at varying channel widths to simulate increasing levels of confinement. Nuclear mechanics were modulated with RO-3306. TGF-β1 treatment successfully induced mesenchymal transitions in both cell lines, leading to decreased expression and reorganization of Lamin A/C and F-actin/G-actin. AFM revealed reductions in cell stiffness in both U251 and U87, especially in the nucleus. Mesenchymal GBM cells migrated more rapidly through confined environments; however, nuclear stiffening via RO-3306 dramatically reduced confined migration speed. 

Discussion/Conclusions. In this study, we demonstrate that TGF-β1 induced mesenchymal transitions and accompanying reductions in structural proteins, resulting in softer cells. Increased nuclear compliance facilitated expedited navigation of constricted environments, but subsequent pharmacological stiffening of the nucleus restored baseline speeds. Future studies dissecting this relationship may aid in the development of diagnostic tools and novel, biophysical targets for pharmacological intervention.

Citation/Acknowledgements. The authors gratefully acknowledge the following funding sources: P20GM135004 and 1R15CA274344.

Nicholas Mellen

Pediatrics, University of Louisville, Louisville, KY

Introduction/Background. Biological systems are complicated across spatial scales. Ca2+ imaging is a recording modality that offers access to network dynamics with cellular resolution, and thus is ideally suited to providing students with insights about biological organization across scales. There are three main barriers to entry to deploying optical recording methods in a teaching laboratory: the cost of synthetic indicators; the cost of cameras; the absence of software to extract signals from optical recordings. In this presentation a platform and work-flow will be presented that overcomes all three barriers.

Hypothesis/Goal of Study. The germline-GCaMP6F mouse expresses the genetically encoded Ca2+ indicator GCaMP6F in every cell of its body. These mice have normal behavior, lifespan and breeding patterns, thus if a mating pair can be maintained at even a rudimentary vivarium facility, imaging experiments can be carried out on heart, kidneys, brain, or any other organ system from offspring mice for as long as the colony is maintained. In addition, genetically encoded Ca2+ indicators are resilient to photobleaching and phototoxicity, enabling long-term optical recordings.

Methods and Results. Economies of scale have drastically lowered camera prices, with monochrome cameras suitable for optical recordings at up to 50 Hz starting at below $200.00. Similarly, LED light-sources have largely displaced Xenon lamps, starting at below $100. Finally, commercially available microscopy workstations for fluorescence microscopy can be purchased for as little as $800. These methods are being disseminated by others: 3D printing schematics and detailed instructions for an open-source epifluorescence microscope for Ca2+ imaging incorporating micromanipulators and LED light-source with a total cost of $1200 has recently been published (Ryan et al., 2020).

Discussion/Conclusions. In my own laboratory, I have developed machine-vision algorithms for the near real time extraction of bounded regions of pixels whose luminance values fluctuate synchronously, consistent with physiological Ca2+ signaling(Mellen and Tuong, 2009). This software can be compiled as an executable running on a Windows workstation, for the price of a run-time engine (<$600). Other open-source algorithms solutions can be installed free of charge (Pnevmatikakis, 2019).

Mahdi Yazdanpour1 and Yangyang Tao2

1College of Arts and Sciences, Northern Kentucky University, Highland Heights, KY, 2College of Informatics, Northern Kentucky University, Highland Heights, KY

Introduction/Background. Limb loss significantly impacts the quality of life for over 2.3 million Americans and 57.7 million individuals worldwide, leading to reduced mobility, loss of independence, and psychological challenges. While traditional prosthetics provide functional support, they are often limited by imprecise control mechanisms, the lack of sensory feedback, and high costs.

To address these limitations, we propose the development of a non-invasive, AI-driven brain-controlled prosthetic arm equipped with a hybrid Brain-Computer Interface (H-BCI) and a real-time sensory feedback system.

The proposed system leverages advanced EEG-based signal decoding using a hybrid deep learning model that combines Convolutional Neural Networks (CNNs) and Recurrent Neural Networks (RNNs). To improve accuracy and address inter-user variability, Transfer Learning will be employed to enhance model generalization across subjects. Generative Adversarial Networks (GANs) will further augment training datasets by generating synthetic EEG data. Sensory feedback will be restored through tactile and thermal sensors integrated with haptic feedback mechanisms, allowing users to interact naturally with their environment. Additionally, the prosthetic arm will feature a cost-effective, modular design produced using 3D printing technology, ensuring adaptability to varying amputation levels and accessibility for underserved populations.

Hypothesis/Goal of Study. This project aims to significantly enhance the quality of life for upper-limb amputees by delivering a transformative and accessible solution that integrates advanced AI and non-invasive brain-computer interface technologies. Beyond its practical applications, this research will provide unique educational opportunities for undergraduate students, equipping the next generation of researchers and innovators in neuroengineering and assistive technologies.

Methods and Results. Our previous work, funded by the KYNETIC grant, focused on designing an early prototype of the prosthetic arm with additional joints and printing it using PLA material without sensory feedback. This initial version employed a machine learning-based BCI model, though we did not conduct human trials at this stage.

With the support of the KY INBRE grant, we aim to advance this project by developing a new, more detailed design using lightweight Nylon 12 for improved durability and precision. This updated design will integrate sensory feedback capabilities to enhance user experience. Additionally, we will leverage advanced deep learning techniques, combining two neural network models with Generative Adversarial Networks (GANs) for synthetic data generation to enhance training datasets, enabling more robust and accurate EEG signal interpretation. This approach will address the challenge of small, imbalanced datasets in BCI applications, improving the robustness and accuracy of EEG signal interpretation for more intuitive prosthetic control. This phase will include human testing to move the project closer to commercialization.

Discussion/Conclusions. Our neuroprosthetic arm is uniquely positioned in the market as the first commercially viable, non-invasive EEG-based prosthetic with integrated sensory feedback. Unlike EMG-based devices, which rely on muscle activity and require frequent calibration, our device directly interprets brainwave patterns through a Hybrid Brain-Computer Interface (H-BCI). This approach provides a more stable and intuitive control experience by bypassing the physical limitations of muscle-based systems, such as fatigue and sensor misalignment. Additionally, the sensory feedback system enables users to perceive tactile sensations, including pressure, temperature, and texture, allowing for more natural and precise interactions with objects. Our innovation offers substantial potential for intellectual property protection, with several patentable components.

Citation/Acknowledgements. KYNETIC (Kentucky Network for Innovation & Commercialization) Cycle 5

Michael Martin1 and Belinda Petri2

1Micro/Nano Technology Center, University of Louisville, Louisville, KY, 2Neuroscience Training, University of Louisville, Louisville, KY

Introduction/Background. The cost of nucleotide sequencing has dropped from $100 million in 2003 for a whole human genome to less than ~$1000, surpassing the pace of Moore’s Law in the semiconductor industry.  One important advance in the field has been driven by the development of nanopore technologies.  At its essence, strands of nucleotides are fed through a single pore in a dielectric membrane while applying a small voltage, the resulting current from the flow of electrolyte ions is then modulated  in a manner unique to the nucleotides occupying the aperture.  Although simple in principle, in practice the technology took roughly 25 years of development to bring to market.  

Founded in 2005, Oxford Nanopore Leads the field with an extensive portfolio of intellectual property and multiple sequencing products.  The MinION, their flagship product, features a single flow cell with up to 2048 individual nanopores reading up to 400 bases per second. While much has been written about the methods of operation and the molecular biology behind its products, the company has understandably remained secretive concerning the details of the devices’ implementation.

Hypothesis/Goal of Study. Here we present a curiosity driven exploration of MinION RNA sequencing chip’s design using metrology tools available in the Micro/Nano Technology Center.   

Methods and Results. The flow cell was carefully dissected beneath an optical microscope to reveal the nanopore chip.  The chip was then imaged in higher resolution using both optical and electron microscopy.  Further, compositional analysis of the various device layers was performed using energy dispersive x-ray spectroscopy.

We discovered a 100-micrometer thick top polymer layer that was micropatterned with a high-aspect ratio array of microwells (100 micrometers in diameter) in two distinct layers. The wells featured a series of gear-like teeth facing inward.  Beneath the micropatterned polymer layer we found a silicon chip with silver electrodes (one per well) designed to sense the pico-ampere sized signals.

Discussion/Conclusions. The nanopore chip represents an impressive fusion of molecular biology and microfabricated structures.  We speculate that the top polymer layer is composed of SU-8, a high aspect ratio photoresist, and that the gear-like structures stabilize the biomimetic membrane into which the protein nanopores are embedded.  The underlying sensor chip likely combines a custom-engineered preamplifier and patch clamp system used in electrophysiology experiments. Devices such as the MinION nanopore chip are just the first generation of technologies that will continue to accelerate base reading rates and potentially extend to proteomics and molecular manufacturing.

Citation/Acknowledgements. Micro/Nano Technology Center

Leah Bishop, Mackenzie Feltner, Asia Good, Taylor Easybuck, and Christine Perdan Curran
Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY

Introduction/Background. Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants produced by burning fossil fuels, cooking foods at high temperature, and during wildfires. Prospective studies children exposed to PAHs during early brain development found persistent adverse effects on behavior. Benzo[a]pyrene is one of the most well-characterized PAHs, and its metabolism is well characterized. We use a mouse model with genetic differences in BaP metabolism to identify those at highest risk. Our prior studies found greater susceptibility in mice lacking the enzyme CYP1A2. The follow-up studies reported here were designed to see if offspring exercise could mitigate some of the adverse neurological effects.

Hypothesis/Goal of Study. We hypothesized that BaP-exposed mice receiving regular exercise would resemble control mice compared with BaP-exposed mice that did not exercise.

Methods and Results. Pregnant dams were treated with 10mg/kg/day BaP in corn oil-soaked food from mid-gestation to weaning at postnatal day 25. The exercise group had free access to running wheels for 1h/day from P30-P60 when behavioral testing began. We assessed open field locomotor activity and rearing using a square plexiglass chamber (41 cm × 41 cm) for 60 min and a Photobeam Activity System (San Diego Instruments). There was a significant treatment x sex x exercise interaction (P = 0.035) with BaP-exposed females that exercised having higher activity levels and more time spent in the open central region compared with BaP-exposed females that did not exercise. The opposite trend was seen for BaP-exposed males with non-exercised males showing hyperactivity compared with all other groups. The opposite trend was also seen in corn oil-treated females. The exercised females were significantly less active than the non-exercised controls.

Discussion/Conclusions. Our data indicate that females exposed to BaP benefitted the most from regular exercise, because their activity patterns most closely matched the control behavior.

Citation/Acknowledgements. This project was supported by grants P20GM103436-24 (KY INBRE) from the National Institute of General Medical Sciences, and R15ES020053, R15ES030541 and R03ES035480 from the National Institute of Environmental Health Sciences (Curran).

Carly McPherson1, Lily Faulkenberg1, Jaime Heckler1, Isaiah Hisler1, Camille Ratcliff1, Carmelo Venero2, Gordon Baylis1, Alex Morgan3, and Patrick Ledwidge1

1Department of Psychological Sciences, Western Kentucky University, Bowling Green, KY, 2College of Medicine, University of Kentucky, Bowling Green, KY, 3Department of Allied Health, Sport and Wellness, Baldwin Wallace University, Berea, OH 

Background. This study examined the effects of anticipation, stimulus congruency, and limb dominance on vertical tibial accelerations during the plant phase of a sidestep run-to-cut maneuver. Unplanned sidesteps produce higher vertical tibial accelerations than planned sidesteps. In traditional Flanker tasks, incongruent flanker stimuli elicit higher conflict monitoring and attentional resource allocation than congruent stimuli.

Hypothesis. Vertical accelerations will be highest in the condition that requires the greatest level of conflict monitoring: the unplanned-incongruent condition. The effect of limb dominance on vertical accelerations was explored, but no hypothesis was formed because of inconsistencies in the literature.

Methods and Results. Methods: Right-leg dominant athletes (n = 19) were fitted with accelerometers on bilateral tibial tuberosities. Participants completed 192 trials where they jogged (4.5 mph) and performed a sidestep change of direction (“cut”) at the end of a 12-foot runway. A flanker stimulus (e.g., < < > < <) appeared during the run phase; the middle arrow dictated cut direction. Trials randomly varied based on cut direction’s anticipation (unanticipated v. anticipated via pre-cue), Flanker congruency (congruent v. incongruent), and Flanker direction (left v. right). Peak vertical tibial accelerations were recorded during the plant phase of the sidestep. 

Results: There were no interactions between anticipation, congruence, and direction (p’s > .10). Vertical accelerations were higher when cut direction was anticipated than unanticipated, F(1,18) = 3.41, p = .081. Accelerations were higher when planting on non-dominant limb (right cuts) than dominant limb (left cuts), F(1,14) = 4.90, p= .044.

Conclusion. Vertical accelerations were greater for non-dominant plants, likely caused by the unnatural tendency to plant on the non-dominant limb. Although failing to reach statistical significance, anticipated trials produced greater accelerations, contrary to hypotheses. This may be due to participants’ likelihood to focus on the physical plant and increased confidence in the anticipated condition. 

Citation/Acknowledgements. KY INBRE (NIGMS grant # 8P20GM103436); WKU Office of Research and Creative Activity.

LeaAnn King, Christina Gogzheyan, Leah Bishop, Deaysha Fox, Taylor Easybuck, Alexandria Easton, Kalyani Abbaraju, Joseph Ashley, Duong Pham, Mackenzie Feltner and Christine Perdan Curran

Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY

Introduction/Background. Benzo[a]pyrene (BaP) is a carcinogen and neurotoxicant found in grilled foods, cigarette smoke, vehicle exhaust, and other sources of air pollution. Evidence is accumulating that exposure to BaP and related chemicals have adverse effects on children exposed during pregnancy and early life, so we designed experiments to determine if exercise could mitigate those adverse effects. We previously showed that both maternal and offspring exercise increased levels of brain-derived neurotrophic factor (BDNF) in the offspring. BDNF is essential to normal neuronal development and function.

Hypothesis/Goal of Study. We hypothesize that exercise will reduce cognitive impairments in mice exposed to BaP during early brain development.

Methods and Results. We examined the effect of both maternal and offspring exercise using 1h of daily, voluntary running on exercise wheels. Dams exercised two weeks prior to mating and from gestational day 0-10 (G0-G10). Offspring exercised from postnatal day 30 (P30-P60). Dams were treated with 10mg/kg/day of BaP in corn oil-soaked cereal or the corn oil vehicle as controls. We used novel object recognition and Morris water maze to test hippocampal dependent learning and memory. In our preliminary analysis, we found BaP-exposed offspring that exercised performed better in the Morris water maze. Differences were only significant on one day of testing for each of the three hidden platform phases; however, the trends were consistent across the entire test period. There were no significant differences in novel object recognition.

Discussion/Conclusions. Our preliminary results indicate that offspring exercise has positive benefits on hippocampal dependent learning and memory.

Citation/Acknowledgements. This project was supported by grants P20GM103436-24 (KY INBRE) from the National Institute of General Medical Sciences, and R15ES020053, R15ES030541 and R03ES035480 from the National Institute of Environmental Health Sciences (Curran).

Ragan Adkins, Darini Nagarajan, Lanayshia Edison, and Sham Kakar1,2

1James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, 2Department of Physiology, University of Louisville School of Medicine, Louisville, KY

Introduction/Background. Cachexia is a complex syndrome frequently observed in cancer patients, with its occurrence ranging from 42% to 80% depending on the cancer stage. It accounts for 30% of deaths in these patients. Cachexia can lead to poor appetite, weight loss, and a decline in muscle strength. Previous studies from our laboratory have shown that cancer induced cachexia can be alleviated by Withaferin A, resulting in restored skeletal muscle strength and organ function. It is believed that cancer-induced cachexia can lead to pathological changes within the kidneys. Angiotensin II serves as a model to induce kidney cachexia mimicking cancer induced cachexia. This study aims to identify if Withaferin A can serve as therapeutic treatment option for Angiotensin II induced kidney cachexia. This study aims to identify the cachexia signaling pathways and inflammatory markers activated in renal damage, as well as determine whether WFA can reverse this damage. We explored the role of angiotensin II (Ang II) in activating downstream signaling pathways related to inflammatory cytokines such as IL-1β and TNF-α. Additionally, we analyzed the expression of AT1A, AT1B, and the TLR4 receptors using QPCR. The fibrosis pathway was also investigated, focusing on genes such as collagen I and collagen IV. Our results demonstrated that treatment with Withaferin A restored kidney function by reducing inflammation and fibrosis. These findings suggest that WFA has the potential to improve kidney function impaired by elevated Ang II levels.

Hypothesis/Goal of Study. Cachexia, a multifactorial syndrome characterized by severe body weight, fat, and muscle loss, poses significant clinical challenges, particularly in chronic diseases such as cancer and heart failure. Recent studies have implicated Angiotensin II (Ang II) in the pathogenesis of cachexia, notably through its ability to induce oxidative stress and inflammatory responses. This study aims to elucidate the specific pathways involved in the upregulation of Angiotensin II-induced cachexia in a mouse model, with a particular focus on three critical pathways: the AT1A receptor pathway, the Toll-Like Receptor 4 (TLR4) pathway, and associated cytokine pathways. The following pathways were the focus of present research to understand the complexity of Angiotensin II-induced cachexia in the mouse model, and to evaluate the effects of Withaferin A.

AT1A and TLR4 Receptor Pathways
Angiotensin II is known to exert its effects through receptor-mediated mechanisms involving the AT1A receptor and the TLR4 receptor. The AT1A receptor, a primary receptor for Ang II, is integral in mediating vasoconstrictive and pro-fibrotic responses. Concurrently, TLR4, a pivotal receptor of the innate immune system, plays a significant role in recognizing pathogen-associated molecular patterns and initiating inflammatory responses. Studies have demonstrated that Ang II induces oxidative stress and an inflammatory response, which are key factors in renal damage and fibrosis. The TLR4 pathway, in particular, is instrumental in these processes, as it activates downstream signaling cascades that lead to inflammation and tissue damage. Moreover, TLR4 is implicated in apoptotic pathways, where uncontrolled apoptosis is an indicator of acute organ injury.

Cytokine Pathways and Hyperinflammatory Response
Given the involvement of TLR4 and Ang II receptor pathways in inflammatory and fibrotic processes, our research then focused on the cytokines associated with hyperinflammatory pathways and cytokine storms. Cytokine storms, characterized by the excessive release of pro-inflammatory cytokines, have been linked to severe tissue damage and organ failure, including kidney damage. Specifically, the cytokines IL-1 Beta, IL-18, and TNF-alpha were investigated due to their known roles in mediating inflammatory responses and contributing to the severity of cytokine storms. Elevated levels of these cytokines can exacerbate inflammation and promote a vicious cycle of tissue damage and fibrosis.

Fibrosis Pathways in Kidney Damage
Fibrosis, a common pathological consequence of chronic inflammation, is particularly detrimental in the context of kidney damage. The activation of cytokine pathways by Ang II and TLR4 not only triggers inflammatory responses but also promotes fibrotic pathways, leading to the deposition of extracellular matrix proteins and scarring of renal tissue. This process impairs kidney function and can result in chronic kidney disease. Understanding the molecular mechanisms behind fibrosis is crucial for developing therapeutic interventions to mitigate renal damage and preserve organ function.

Methods and Results.
Mouse Model
A pharmacological model of cachexia independent of tumors was used by continuously infusing Ang II. Eleven-week-old female C57BL/6J mice from Jackson Laboratory were randomly divided into two groups for baseline measurements of body weight, forelimb strength, and total grip strength. Osmotic minipumps (Alzet model 1004) were implanted in the suprascapular region to deliver either Ang II (1000 ng/kg/min) or sterile saline at 0.1 μL/hour for 4 weeks. After one week, mice in both groups received i.p. injections of WFA (4 mg/kg) or vehicle (10% DMSO, 90% Glycerol Trioctanoate) every three days for the last three weeks of the study.

Fibrosis Analysis with Histological Samples and Trichrome Staining
Kidney samples from each group of animals were transversely sectioned before being mounted in OCT and then stored at −80 °C until use. Sections were cut to a thickness of 4 microns using an Epredia Microm HM525 cryostat at −20 °C and mounted on glass microscope slides (two sections on each slide). Slides were subjected to hematoxylin and eosin staining (H&E) for structural determination and Masson’s trichrome staining for collagen deposition as an index of fibrosis using a trichrome staining kit. First, equilibrate all materials and reagents to room temperature and gently agitate. The trichrome staining procedure went as follows. Preheat Bouin’s Fluid to 56-64°C in a fume hood or a well-ventilated area. Place the slide in the preheated Bouin’s Fluid for 60 minutes, followed by a 10-minute cooling period. Rinse the slide in tap water until clear, then rinse once in distilled water. Mix equal parts of Weigert’s (A) and (B) and stain the slide with this mixture for 5 minutes. Rinse the slide in running tap water for 2 minutes. Apply Biebrich Scarlet/Acid Fuchsin Solution to the slide for 15 minutes, then rinse in distilled water. Differentiate in Phosphomolybdic/Phosphotungstic Acid Solution for 25 minutes or until the collagen is no longer red. Without rinsing, apply Aniline Blue Solution for 15 minutes. Rinse the slide in distilled water, then apply Acetic Acid Solution (1%) for 3-5 minutes. Dehydrate the slide quickly in 2 changes of 95% alcohol, followed by 2 changes of absolute alcohol. Finally, mount in synthetic resin. Following trichrome staining, imaging of each section was performed using the 3D HISTECH Panoramic Slide Scanner following the manufacturer’s protocol for slide preparation and imaging. WGA imaging was performed using a Nikon Eclipse Ti confocal microscope.

Total RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA from kidney tissues was extracted and purified using the RNAeasy Fibrous Tissue Mini Kit from Qiagen (cat # 74704) (Germantown, MD, USA 20874) according to the manufacturer’s instructions. Briefly, approximately 25–30 mg of kidney tissue was homogenized using a polytron homogenizer using the tissue extraction buffer from the kit. The total RNA was purified using the column provided in the kit following the manufacturer’s instructions. The total RNA was quantitated using spectrophotometer. The first-strand cDNA was synthesized using a commercially available cDNA synthesis kit (iScript cDNA Synthesis Kit from BioRad Cat # 170-8891) (Hercules, CA, USA 94547) using 1 μg of total RNA for each sample. Quantitation of mRNA expression was analyzed using the SYBR Green dye and specific primers for each gene in the CFX- Connect Real-Time System (Bio-Rad, Hercules, CA, USA 94547). Beta-actin gene primers were used as an internal control to normalize the levels of expression of each gene as described previously.

Discussion/Conclusions. The findings of our study illustrate a significant increase in circulating Angiotensin II, indicative of cancer-induced cachexia and kidney damage. However, the administration of Withaferin A (WFA) successfully alleviated these elevated levels.

Despite initial anticipations from previous research suggesting WFA inhibits Ang-II induced fibrosis, real-time PCR data on gene expression levels of Collagen I, Collagen IIII, and TGF-Beta were inconclusive. Nonetheless, we assert that WFA plays a crucial role in mitigating kidney damage resulting from elevated Angiotensin II levels. This assertion is supported by initial data involving IL1-Beta, AT1A, and TLR4.

Our study underscores the complexity of pathways responsible for Angiotensin II-induced kidney damage and the potential for WFA to mediate these pathways. However, we faced significant sample-to- sample variation, impacting the statistical analysis. To address this, future research should incorporate more tissue samples to verify preliminary findings and reduce the impact of outliers.

While our findings are preliminary and not definitive, they highlight an urgent need for therapeutic targets to alleviate cancer/AngII-induced cachexia. This study serves as a foundation for future research aimed at identifying signaling pathways associated with renal damage due to cancer cachexia and the therapeutic potential of Withaferin A.

Citation/Acknowledgements. KY INBRE Grant Number: P20GM103436