Bluefield State College

Abstract Purpose of the study: The dietary compound Capsaicin is the hot and pungent ingredient of chili peppers. Preliminary data in our laboratory have found that capsaicin displays robust growth-inhibitory activity in a diverse array of human cancers. However, the clinical applications of capsaicin as a viable anti-cancer drug are hindered by its adverse side effects, such as gastric irritation, nausea, stomach cramps and a burning sensation in the gut. Clinical trials which explored the pain-relieving activity of capsaicin found that patients who orally ingested capsaicin discontinued taking the drug due to its unpleasant side effects. The primary aim of our studies was to rationally design non-pungent capsaicin analogs which would retain the anti-cancer activity of the parent molecule. Experimental procedures. (SAR) studies have shown that the pharmacophore of capsaicin may be divided into three regions: REGION A, B, AND C. The addition of long-chain unsaturated fatty acyl groups in REGION C of capsaicin generated non-pungent compounds which displayed improved pain-relieving properties, relative to capsaicin. We synthesized a panel of compounds which contained a long chain unsaturated fatty acyl side chain in Region C of the capsaicin molecule. Subsequently, we determined the growth-inhibitory activity of these compounds in human ovarian cancer cells (in cell culture and chicken chorioallantoic membrane (CAM) models. Results: The non-pungent Region C capsaicin analog DOHEVANIL displayed greater pro-apoptotic activity than capsaicin in human ovarian carcinoma cells. Most interestingly, DOHEVANIL did not impact the growth of normal human liver and kidney cells. Conclusions: The non-pungent Region C capsaicin analog DOHEVANIL may be a promising non-pungent capsaicin analog which displays robust growth-suppressive activity in human ovarian cancer cells. Support or Funding Information Funding for our study was supported by the NIH R15-AREA Grant (2R15CA161491-02 and 2R15CA161491-03), the Women's Health T3: 3P20GM103434-23W1 (PI: Dr. G Rankin) to PD and MAV and the NIAID-AI151970 grant to TEL. Furthermore, this study was supported in part by the West Virginia IDeA Network of Biomedical Research Excellence (WV-INBRE) grant (NIH grant P20GM103434; PI: Dr. G. Rankin), the National Institute of General Medical Sciences of the National Institutes of Health under the award number P30GM122733. Citation Format: Kushal J. Modi, Rama S. Gadepalli, John R. Rimoldi, Timothy E. Long, Yi Charlie Chen, Kathleen C. Brown, Stephen D. Richbart, Justin C. Meritt, Sarah L. Miles, Piyali Dasgupta. Structure activity relationship (SAR) studies identify novel non-pungent capsaicin analogs which display robust growth-inhibitory activity in human ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6930.

Background: Escherichia coli (E. coli) infections are becoming difficult to treat due to the bacterium's biofilm-forming capabilities and rising resistance to multiple antibiotics, posing a growing clinical challenge. This study assessed the antimicrobial resistance and biofilm formation by Escherichia coli isolates from patients at a hospital in Tigray, Northern Ethiopia.Method: From patients exhibiting signs of bacterial infection, while excluding recent antibiotic users or those with incomplete data, 417 clinical samples comprised of 84 blood, 108 pus, and 225 urine samples were obtained in a cross-sectional study. The combination disc method was used to test extended-spectrum beta-lactamase (ESBL) production, and Ampicillin C (AmpC) enzyme presence was confirmed with cefoxitin and cefotaxime discs. Data analysis was conducted with SPSS version 22 software, applying ANOVA and logistic regression, with significance set at p<0.05.Result: Among the 417 samples, 109 (26.1%) tested positive for Escherichia coli. These isolates showed high resistance to ampicillin (84.4%) but lower resistance to meropenem (9.17%). ESBL was detected in 46.8% of isolates and AmpC in 54%, with 48 (44%) isolates positive for both. Strong biofilm formation occurred in 76% of isolates, while only 2.75% were weak producers. Biofilm strength correlated significantly with prior antibiotic use (p=0.028), ward type (p=0.001), and use of indwelling devices (p=0.000).Conclusion: In northern Ethiopia, Escherichia coli isolates showed resistance to major antibiotic classes like beta-lactams, fluoroquinolones, and aminoglycosides. This high resistance and biofilm development highlight the critical need for interventions to curb resistance spread, with a focus on antibiofilm research and enhanced infection prevention measures.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

Heart disease and diabetes are both in the top ten for leading causes of death in the United States with both being linked to obesity. Studying human metabolism can prove difficult due to ethical concerns with human subjects but zebrafish larvae (Danio rerio) are optically transparent. Zebrafish larvae at seven days post fertilization also have fully formed digestive tracts and are actively seeking new food due to exhausting their supply of egg yolk. Free feeding environments are typically the standard when introducing new food to the larvae but lack versatility in their imaging. The Walters lab microfluidic chip can image larvae before, during, and after their introduction to the diet. To prove the two types of feeding environments were compared by using fluorescent digestive markers mixed into high fat diets. The free fed larvae were put into a well filled with diet and allowed to feed for three hours while sat on a orbital shaker inside of a 28 degrees Celsius incubator. After three hours, these larvae were rinsed out and imaged. The microfluidic chip larvae were mounted into the chip and fed the diet while inside and under the same Zeiss Discovery V8 microscope the freely fed larvae were imaged under. Future predictions for this data were that the larvae would consume slightly less diet inside of the microfluidic chip than the larvae in free feed. Results for this are still ongoing but seem to lead towards supporting the hypothesis.

&#x0D; &#x0D; &#x0D; More than a third of the adults in the Appalachia are affected by obesity, which is associated with diets that are high in fats and cholesterol as well as the development of many diseases such as diabetes, hypertension and cardiovascular diseases. These diets lead tochronic inflammation and cellular damage from production of free radicals such as Reactive Oxygen Species (ROS). We compared the respirometry outcomes in a 7 days post- fertilization zebrafish (Danio rerio) larvae after different diet conditions. Larvae were either fed nothing at all, a low fat diet (LFD), a high fat diet (HFD), or a HFD plus a drug that mitigates oxidative stress (Anethole Trithione and mito-TEMPO). We expected that the HFD would cause the mitochondria to be put under more stress due to oxidative phosphorylation of lipids and would decrease in the larvae treated with the drugs. Respirometry was obtained using the XFe24 Extracellular Flux Analyzer (Agilent Technologies) and a modified Cell Mito Stress Test. We found basal respiration is higher in the HFD and lower in the other two treatment diets, indicating that high fat diets do produce more oxidative stress in mitochondria, and the drugs tested are able to successfully mitigate it. The analysis of drug interaction readings is in progress. In the future, we will test other drugs that can affect mitochondrial metabolism (i.e. Curcumin, Pitastatin, Mdivi-1 and PPPA).&#x0D; Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.&#x0D; &#x0D; &#x0D;

Kaitlyn Cook*, Tesfaye Belay, Ph.D*., *Department of Applied Science and Mathematics, Bluefield State College, Bluefield WV 24701 Production of CD4+ T Cell Subsets in a Beta2-Adrenergic Receptor Knockout Stress Mouse Model during Chlamydia muridarum Genital Infection&#x0D; Chlamydia is the most widely spread sexually transmitted disease, with more than one hundred million new cases occurring every year worldwide. Studies show the effect of stress promoting infection, but little is known about Chlamydia and the impact of stress. Cold-induced stress was used to simulate stress in mice to understand the relationship between the stress hormone norepinephrine (NE) and the immune system. This study aims to determine the role of beta2-adrenergic receptor (β2-AR) under stressful conditions during infection. We hypothesize the β2-AR suppresses T helper cell 1 (Th1) and promotes the production of Th2 cytokines. Following stressing, infection, sacrificing, and dissection of mice, the spleen, lymph node, and genital tract cells were proliferated for 72 hours. ELISA data for IL-13 concentration shows stressed knockout (KO) mice had 273.6pg/mL compared to stressed wildtype (WT) mice with 120.6pg/mL. Various treatment of CD4+ T cells resulted in production of TNF- α, ranging from 595pg/mL to 398.7pg/mL. Viability of CD4+ T cells treated with β2-AR agonist Fenoterol, and antagonist ICI 118,551 were analyzed. The viability of CD4+ T cells treated with NE was 70.7% in KO, compared to 69.1% and 70.2% for the agonist and antagonist, respectively. NE, Fenoterol, and ICI 118,551 treatment resulted in 72.2%, 83.6% and 84.4% respectively for WT. No significant difference is found between the agonist and antagonist. Chlamydia muridarum isolation from the genital tract of treatment groups is underway. The ELISA data implies that β2-AR mice restored increased production of protective cytokines more than their WT counterparts.

The Mediterranean diet is associated with higher levels of polyunsaturated fatty acids (PUFAs) as well as lower incidences of cardiac disease and colon cancer (1). Due the presence of multiple double bonds, PUFAs such as alpha-linoleic acid (ALA), are thought to generate fewer harmful metabolites, specifically the Reactive Oxidative Species (ROS). Conversely, monounsaturated fatty acids (MUFAs) may lead to relatively increased ROS. In this study we compare two high-fat diets: the MUFA diet containing oleic acid (1 mM C18:1 plus 1 mM cholesterol) and the PUFA diet containing ALA (1 mM C18:3 plus 1 mM cholesterol). We compared gene expression from the dissected intestines of six days post-fertilization zebrafish (Danio rerio) larvae after feeding the PUFA and MUFA diets for 3 hours. We measured ROS response indirectly via the increased expression of ROS response genes (GSTO1, GSR, and cyp1a). Our preliminary results show upregulation of the ROS response genes GSTO1, GSR, and cyp1a in the presence of MUFA and await further replicates to become significant. . These data support the hypothesis that dietary fatty acid saturation plays a role in oxidative stress within enterocytes. In the future we plan to measure ROS responsiveness directly via respirometry. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College and the McNair Scholars Program.

NPC1L1 transport protein is located in the lumen of the intestines and is critical for cholesterol absorption. The reason why we study NPC1L1 is because cholesterol absorption in the intestines affect obesity and diabetes which is still under investigation. Our goal is to investigate the mechanism by which the protein NPC1L1 absorbs cholesterol we are creating an npc1l1 -/- (knockout) line of zebrafish. We hypothesize that the npc1l1 -/- larval zebrafish will show reduced intestinal cholesterol absorption when feed a high fat diet compared to wild type zebrafish larvae. We are screening two zebrafish lines that carry mutations within the NPC1L1 sequence. These zebrafish lines were obtained from the Zebrafish International Resource Center. Parent fish carrying the mutation are identified by fin clipping, DNA extraction, and DNA sequencing of the potential mutant locus within NPC1L1.After identifying the mutation we will amplify the DNA fragments. The DNA fragments will then be sent off to Marshall Genomics Core for sequencing. The mutation sequence will be compared to a wild type sequence. Then the zebrafish that has been confirmed a carrier will be outcrossed with a wild type to eliminate background mutations and then intercrossed to make a homozygous carrier. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

Active Hexose Correlated Compound (AHCC) has been shown to have many immunostimulatory and anti-cancer activities in mice and in humans. As a natural product, AHCC has potential to create safer adjuvant therapies in cancer patients. Acute Myeloid Leukemia (AML) is the least curable and second-most common leukemia in adults. AML is especially terminal to those over 60 years old, where median survival is only 5 to 10 months, due to inability to receive intensive chemotherapy. Hence, the purpose of this study was to investigate the effects of AHCC on AML cells both in vitro and in vivo. Results showed that AHCC induced Caspase-3-dependent apoptosis in AML cell lines as well as in primary AML leukopheresis samples. Additionally, AHCC induced Caspase-8 cleavage as well as Fas and TRAIL upregulation, suggesting involvement of the extrinsic apoptotic pathway. In contrast, monocytes from healthy donors showed suppressed Caspase-3 cleavage and lower cell death. When tested in a murine engraftment model of AML, AHCC led to significantly increased survival time and decreased blast counts. These results uncover a mechanism by which AHCC leads to AML-cell specific death, and also lend support for the further investigation of AHCC as a potential adjuvant for the treatment of AML.