Despite the significant advancements in breast cancer (BCa) research and potential therapeutic targets, mortality rates for African American (AA) women remain high compared to other ethnic groups. In the United States, BCa is the second leading cause of cancer-related deaths and is commonly classified by the expression of molecular markers. Treatment and prognosis are determined by the presence and absence of the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2), and, more recently, the androgen receptor (AR). AA women are more likely to be diagnosed with triple-negative breast cancer (TNBC) and experience a higher risk of BCa mortality than European American women (EA), creating a survival disparity. Biological and non-biological factors are implicated in the difference between EA and AA women contributing to the racial disparities in BCa mortality. Potential implications of this disparity in AA women have been linked to treatment, health care access, socioeconomic status (SES), stress, genetics, and comorbidities. Due to the fraction of available chemotherapeutics for TNBC and the survival disparity in AA women, identifying mechanisms contributing to this aggressive, hard-to-treat BCa is warranted. Studies examining psychological stress and its connection has been shown to contribute to the etiology and aggressiveness of BCa. A direct link has been made in animal models between induced psychological stress and tumor development.Specifically, psychological stress has resulted in higher catecholamine levels and is associated with cancer, resulting in higher incidence, aggression, and mortality. Norepinephrine and epinephrine signal through β-Adrenergic receptors (β-ARs), and these interactions have been shown to decrease apoptosis in prostate cancer cells, increasing malignancy in BCa through the p38 MAPK signaling pathway, and promote BCa stem-like cells. Inhibitors of β-ARs or beta-blockers have been shown to improve the efficacy of chemotherapeutics and decrease the risk of heart failure in cancer patients receiving anthracycline therapy. In this study, we use four TNBC cells (MDA-MB-231, HCC-1143, MDA-MB-468, HCC-1806) that differ in the percentage of West African Ancestry (WAA); we identify differential expression of the ADRB2 protein across all cell lines utilizing flow cytometry. We also show that the non-selective inhibitor of ADRB1 and ADRB2, propranolol, increases the efficacy of Doxorubicin in some cells, which is determined by IC50. We find that the agonist norepinephrine (NE) has no significant impact on cellular growth but impacts the cell's response to Doxorubicin. Here we show that beta-blockers may provide a clinical benefit in combination with Doxorubicin which is dependent upon cell line, and that the β-AR is a promising target for treating TNBC. Citation Format: Alanna Burwell, Portia Andrews, Dayami Lopez, K. Sean Kimbro. The role of beta-adrenergic receptor agonists and inhibitors on the efficacy of Doxorubicin in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1592.

Background: Serum lipids, including total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c), increase during pregnancy. Serum Proprotein Convertase Subtilisin Kexin 9 (PCSK9) is a vital regulator in lipoprotein metabolism. Circulating PCSK9 downregulates the LDL receptor on the surface of liver cells inhibiting clearance of LDL-c. Objective: To determine the influence of weeks of pregnancy and obesity on circulating levels of essential lipid lipoproteins and PCSK9 in women with normal, uncomplicated pregnancies and deliveries. Methods: We performed a comprehensive lipid and lipoprotein profile during each trimester of pregnancy in 70 mostly Caucasian women with uncomplicated normal pregnancies and deliveries. Based on their first trimester BMI, we placed them into one of three categories: (<25 kg/m(2) n=23, 25-30 kg/m(2) n=25, or >30 n=22) kg/m(2). Cholesterol, triglycerides, LDL cholesterol (LDL-c), non-HDL particles, and lipoprotein(a) were measured by spectrophotometry, ion mobility, and immunoturbidimetric assays. Elisa assay determined PCSK9 (active and total). Homeostatic Model Assessment (HOMA-IR) assessed insulin resistance in the second and third trimesters of pregnancy. Results: Total and active PCSK9, LDL-c, and nonHDL particle concentrations were higher than reported for non-pregnant normal values, increased after the first trimester of pregnancy, and were highest from mid-gestation to the last trimester of pregnancy in the overweight and the obese. Conclusion: PCSK9 levels rise as normal pregnancy progresses. Levels are higher in persons who are obese, even after adjustment for insulin resistance. Defining normal PCSK9 levels during pregnancy must adjust for gestational age and BMI. (C) 2022 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Abstract Background Type 2 Diabetes (T2D) has been considered an inflammatory disease that correlates with elevated responses cytokines. The increasing prevalence of T2D worldwide, is accompanied by the higher incidence of T2D globally and nationally. African Americans (AA) compared to other ethnic groups, have a higher prevalence of obesity (49.6%) and 1.4-2.3-fold higher in T2D. IL-7 is resident to the liver and been previously reported to drive inflammation in several chronic (rheumatic) inflammatory autoimmune responses. Obese patients with high levels of HbA1c are at higher risk of inflammatory diseases compared with non- obese individuals. This suggests that high blood sugar levels and subsequent inflammation may impact the prevalence of circulating cholesterol levels of LDL and or HDL. PCSK9 regulates the LDL-receptor and could potentially be a link between inflammation and active PCSK9. Preliminary data in our lab show elevated levels of IL-7 (>8 pg/mL) significantly correlate with various serum biomarkers of lipid metabolism, inflammation, and liver damage Methods A total of 239 serum samples were collected from AA: 172 women and 67 men. All participants had an BMI > 30. This group consists of 68 normal HbA1c and 171 high HbA1c participants. Clinical metabolic parameters were determined using standard commercial kits. Cytokines were measured using Luminex X-MAP® technology. Cytokine levels were compared using unpaired t-test, followed by Pearson's correlation coefficient (r) to determine the correlation between cytokine expression and clinical metabolic parameters. Results We found that men with high HbA1c significantly correlate with BMI (P= 0.0052) and increased levels of IL-7, which was not seen in women. Women with both normal and high HbA1c levels significantly correlate with IL-7 and liver damage markers AST; normal HbA1c (P= 0.0036), high HbA1c (P= 0.0194) and ALT; high HbA1c (P=0.0174). These correlations are not seen in men supporting the possible influence of blood sugar levels on liver function in women. When normal HbA1c and high HbA1c participants are combined, all women IL-7 levels significantly correlate with active PCSK9 (P= 0.02). This suggests high circulating LDL contribute to active PCSK9, which is not present in men. Conclusion Our findings suggest increased circulating levels of IL-7 based on individuals HbA1c significantly correlate with markers of inflammation, lipid metabolism and liver damage of obese individuals with T2D, while identifying a definitive difference between men and women. These findings establish IL-7 as playing a role in inflammatory responses in obese AA with T2D and potentially being a mediator of obesity-associated inflammation. IL-7 is an indicator of sex being a variable that influences innate and adaptive immune responses, resulting in sex-specific outcomes from multiple diseases including T2D. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Purpose: Obesity, a complex chronic disorder characterized by the enlargement of adipose tissue, has a multifactorial etiology. Adipose tissue is now recognized as an active tissue in the regulation of inflammation. Sex chromosome genes and hormones influences immune responses between males and females. Inflammation is rampant in obesity due to the expansion of visceral adipose tissue leading to insulin resistance resulting in type-2 diabetes (T2D). Differences in sex may lead to varied immune responses to T2D. Methods: A total of 116 serum samples were collected from African Americans: 68 women and 48 men. All participants had a BMI > 30. This group consists of 49 normal HbA1c and 71 high HbA1c participants. This study was designed to determine the impact of current circulating glucose on current serum IL-3 and IL-7 levels. Results: Serum cytokine levels are influenced by circulating high glucose and it varies based on sex. We found in women, IL-3 and IL-7 levels were upregulated 1.7-fold in the presence of high circulating glucose. In men, IL-3 levels were downregulated 1.5-fold and IL-7 levels downregulated 1.3-fold in the presence of high circulating glucose. IL-3 and IL-7 serum levels are also correlated with several lipid parameters. Conclusion: IL-3 and IL-7 are members of a complex network of cytokines that play a role in chronic inflammation. Inflammatory signaling impact several diseases including obesity, T2D, atherosclerosis and dyslipidemia. A better understanding of the pathological signaling of cytokines will help facilitate our understanding of inflammation in these diseases.

Abstract Heart disease is the principal cause of death and disability for both men and women in the US, accounting for 40% of all annual deaths. African American populations are disproportionately burdened with metabolic diseases, due in part to cholesterol metabolism deficiencies. Elevated low density lipoprotein (LDL) cholesterol levels and inflammation promote atherogenic conditions which lead to heart disease. Proprotein convertase subtilisin/kexin-9 (PCSK9) is a biomarker which enhances athrogenic progression by controlling the number of LDL receptor molecules expressed at the plasma membrane. PCSK9 indirectly regulates LDL-cholesterol levels. Previous reports show some patients do not respond well to general anti-cholesterolemic treatments. We believe this is due to altered PCSK9 activity, which is currently not being evaluated. We have developed a novel assay to detect active PCSK9. A1AT is a SERPIN family member whose primary objective is inhibition of proteases. Specific levels of A1AT are required to maintain metabolic homeostasis. Based on this, we hypothesized that a specific ratio between A1AT serum levels and PCSK9 activity levels would eliminate statin intolerance/resistance, regulating LDL-cholesterol metabolism congruently. Using this novel active PCSK9 detection assay, we provide evidence that A1AT interacts with PCSK9 in the medium of C3A hepatic-like cells, preventing the formation of PCSK9/LDL receptor complexes in vitro. There was an approximate 20% inhibition in PCSK9-LDL receptor complex formation when liver cells were treated with recombinant A1AT (rA1AT). A dose dependent response analysis proved 200ng/ml of rA1AT had an 46% reduction in PCSK9 activity. We determined PCSK9 activty and A1AT levels correlate with key diabetic factors in humans, suggesting that A1AT could effect diabetes progression.

High levels of cholesterol, especially as low-density lipoprotein (LDL), are a well-known risk factor for atherosclerotic-related diseases.The key atherogenic property of LDL is its ability to form atherosclerotic plaque.Proprotein convertase subtilisin/kexin-9 (PCSK9) is an indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane, especially in the liver.Herein, we performed a combination of affinity chromatography, mass spectrometry analysis and identification, and gene expression studies to identify proteins that interact with PCSK9.Through these studies, we identified three proteins, alpha-1-antitrypsin (A1AT), alpha-1-microglobulin/bikunin precursor (AMBP), and apolipoprotein H (APOH) expressed by C3A cells that interact with PCSK9.The expression levels of A1AT and APOH increased in cells treated with MITO+ medium, a condition previously shown to affect the function of PCSK9, as compared to treating with Regular (control) medium.However, AMBP expression did not change in response to the treatments.Additional studies are required to determine which of these proteins can modulate the expression/function of PCSK9.The identification of endogenous modulators of PCSK9's function could lead to the development of novel diagnostic tests or treatment options for patients suffering hypercholesterolemia in combination with other chronic metabolic diseases.

Proprotein convertase subtilisin-kexin 9 (PCSK9) appears to be involved in multiple processes. A ProtoArray Human Protein Microarray was used to identify proteins interacting with biotinylated PCSK9. Fifteen novel proteins interacting with PCSK9 were identified using this technique. Only two of these proteins, sterol carrier protein 2 and hepatoma-derived growth factor, related protein 3, have known functions. The identification of proteins that could affect the expression/function of PCSK9 is of great interest due to potential implications in personalized medicine for hypercholesterolemic patients.

The majority of the low-density lipoprotein (LDL) receptors present in the body are expressed in the liver. Therefore, plasma LDL levels significantly correlate with changes in the activity of the hepatic LDL receptor. Based on this, there is a need to understand the regulatory mechanisms that control the hepatic expression of the low-density lipoprotein (LDL) receptor. Herein, we have prepared a functional rat LDL receptor minigene construct that can produce mRNA after splicing. Sequence analysis suggests that this construct has the potential to code for a truncated version of LDL receptor protein. This minigene could be used as a research tool to identify small molecules, natural products, and regulators of the LDL receptor gene that could be developed into LDL receptor-specific activators for therapeutic use.

Hypercholesterolemia is a cardio-metabolic disease caused by a disruption in low density lipoprotein (LDL) cholesterol metabolism. Proprotein convertase subtilisin-kexin 9 (PCSK9) is an indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane. Serum levels of PCSK9, along with associated mutations, dictate the severity of the hypercholesterolemic condition. Although several treatment options are available to treat hypercholesterolemia, a cost-effective and quick test to best qualify patients for appropriate treatment is currently lacking. We have developed and validated a diagnostic tool to be used in a clinical setting which measures PCSK9 levels in a currently unreported manner. The results from this tool will give predictive instructions to physicians as to which hypercholesterolemic treatment will best suite the patient, preventing the development of alternative diseases (such as Type 1 and Type 2 diabetes) currently being observed by the trial-and-error methods of treatment. We have used disease (diabetic) and non-disease (non-diabetic) state human samples to validate the use of our diagnostic test for clinical use. The human samples were used in clinical trials to measure the effectiveness of the diagnostic tools in personalized medicine. The results showed the diagnostic tool has the ability to determine which patients have genetic mutations in PCSK9 that will result in the progression of their diabetic condition under generalized Statin recommendations.

Objectives: Alzheimer's disease (AD) is a neurodegenerative disorder that affects millions of individuals.Moreover, hypothyroidism has been identified as one of the risk factors that may contribute to the development of AD.Here, we investigated whether there was a correlation among expression levels of proteins involved in the formation of AD lesions, neurite outgrowth, and thyroid hormone levels.Methods: Cells were grown in media supplemented with different levels of 3,5,3'-triiodothyronine (T3) and then processed for neurite outgrowth and to prepare RNA samples.RNA samples were analysed using quantitative real-time PCR.Protein levels were measured using in cell-Western blotting analysis.Results: By using neurite outgrowth studies, it was demonstrated that T3 treatment enhanced neurite outgrowth in NS-1 cells in a time-and dosedependent manner.Quantitative real-time PCR studies further confirmed that NS-1 cells expressed substantial levels of TRα and significantly less TRβ, either of which could be responsible for the T3-dependent effects on neurite outgrowth.Although the overall tau protein expression was not affected in response to T3 treatment, the splicing of tau exon 10 was impacted in the direction of producing more tau molecules that excluded the exon (tau 3R). Conclusion:The results of this study are critical not only to understand the probable link between hypothyroidism and AD but also in providing the basis for future prevention and treatment of AD in hypothyroid patients.

Alzheimer's disease [AD] is not only the most common neurodegenerative disease but is also currently incurable. Proprotein convertase subtilisin/kexin-9 [PCSK9] is an indirect regulator of plasma low density lipoprotein [LDL] levels controlling LDL receptor expression at the plasma membrane. PCSK9 also appears to regulate the development of glucose intolerance, insulin resistance, abdominal obesity, inflammation, and hypertension, conditions that have been identified as risk factors for AD. PCSK9 levels also depend on age, sex, and ethnic background, factors associated with AD. Herein, we will review indirect evidence that suggests a link between PCSK9 levels and AD.

Since the human genome project in 2003, the view of personalized medicine to improve diagnosis and cure diseases at the molecular level became more real. Sequencing the human genome brought some benefits in medicine such as early detection of diseases with a genetic predisposition, treating patients with rare diseases, the design of gene therapy and the understanding of pharmacogenetics in the metabolism of drugs. This review explains the concepts of pharmacogenetics, polymorphisms, mutations, variations, and alleles, and how this information has helped us better understand the metabolism of drugs. Multiple resources are presented to promote reducing the gap between scientists, physicians, and patients in understanding the use and benefits of pharmacogenetics. Some of the most common clinical examples of genetic variants and how pharmacogenetics was used to determine treatment options for patients having these variants were discussed. Finally, we evaluated some of the challenges of implementing pharmacogenetics in a clinical setting and proposed actions to be taken to make pharmacogenetics a standard diagnostic tool in personalized medicine.

Heart disease ends the life of more people than any other disease in the United States. High levels of low density lipoprotein (LDL)-cholesterol cause heart diseases by increasing the formation of atherosclerotic plaques. Proprotein convertase subtilisin/kexin-9 (PCSK9) indirectly regulates plasma LDL levels by controlling the LDL receptor expression at the plasma membrane. PCSK9 also appears to modulate glucose intolerance, insulin resistance, abdominal obesity, inflammation, and hypertension. The magnitude of PCSK9's involvement in the onset of these metabolic abnormalities appears to be associated with age, sex, and ethnic background. Another regulator, the inducible degrader of the LDL receptor (IDOL), works by enhancing the ubiquitination of the LDL receptor. Herein, we will review the functions and regulatory mechanisms of PCSK9. The effects of PCSK9 on the LDL receptor, the relationship of this convertase with IDOL, and treatments currently available against hypercholesterolemia are also discussed.

Statins are the first-line treatment for hypercholesterolemic patients. Herein, the effects of three statins on complex formation between proprotein convertase subtilisin-kexin 9 (PCSK9) and the low density lipoprotein receptor (LDLR), a critical step for the PCSK9-dependent degradation of LDLR in the lysosome, were examined. Human hepatocyte-like C3A cells grown in control (containing 10% fetal bovine serum) or MITO+ (supplemented with BD™ MITO + serum extender) medium were also treated with atorvastatin (Atorv), lovastatin (Lov), or pravastatin (Prav) for 24 h. RNA and protein expression studies and determinations of PCSK9/LDLR complex formation were performed. As expected, the statins increased the expression of PCSK9 and LDLR independently of the medium employed. Interestingly, Atov and Lov caused increases in PCSK9/LDLR complex formation, whereas Prav decreased complex formation when compared to cells treated without drugs. These results may explain why Prav works better for statin intolerant patients than other statins such as Atorv and Lov.

Niemann-Pick C1-Like 1 (NPC1L1) is expressed in human liver, but its mechanism of action and how it is regulated in this tissue is not well-understood. Herein, we decided to examine the effects of the inhibitor of NPC1L1, ezetimibe, in the presence and absence of a low cholesterol medium (MITO+ medium), on the expression of NPC1L1 in the human hepatocytelike C3A cell line. It was demonstrated that ezetimibe and MITO+ medium were able to independently decrease NPC1L1 protein expression while increased the mRNA expression of this transporter. Treating with ezetimibe and MITO+ medium together additively decreased NPC1L1 protein levels, but their effects on NPC1L1 mRNA expression were counteracting. Cycloheximide studies showed that these treatments enhanced the stability of the NPC1L1 protein in an additive manner, supporting the possibility that the translation efficiency of the NPC1L1 mRNA was decreased under these conditions or that the factors required to degrade the NPC1L1 transporter were inhibited by the presence of cycloheximide. In addition, the data confirmed that growing the cells in flasks coated with poly-D-lysine (should induce polarization) was required for maximum expression levels of NPC1L1 protein but not for localization of this transporter to the apical membrane. The information derived from this research is critical to our understanding of the role of hepatic NPC1L1 in removing cholesterol from the bile and in cholesterol homeostasis in general.

The hepatic LDL receptor is the major determinant of plasma LDL levels, and as a result, a greater understanding of the regulatory mechanisms that control LDL receptor expression and function is essential. Herein, we optimized a biotinylation assay that was able to differentiate between cell surface (plasma membrane) and intracellular LDL receptors. We also tested monensin, a chemical that prevents the recycling of the LDL receptors to the cell surface and enhances the number of receptors that can bind and internalize LDL. Herein, it was confirmed the effects of monensin on the ability of the LDL receptor to bind LDL, and demonstrated for the first time, using the biotinylation assay, that monensin did not affect the LDL receptor expression levels at the plasma membrane or inside the cell. This was confirmed using immunocy to chemistry and Western blotting analysis. Monensin treatment may enhance the distribution of the LDL receptor to clathrin-coated pits explaining the higher binding of LDL, but not of VLDL, to the cells. This effect of monensin did not require upregulation of the LDL receptor expression. This indicates that the biotinylation assay described herein, in combination with monensin treatment, could be used in future studies to determine the effects of different treatments/drugs on the plasma membrane distribution/function of the hepatic LDL receptors.

The aim of this study was to evaluate the effects of extender composition and osmolality on post-thaw motility of Brycon orbignyanus sperm. Eight extenders comprising combinations of two compositions (NaCl and glucose) and four osmolalities (285, 325, 365 and 405mOsmkg(-1)) were tested. Methyl glycol was used as cryoprotectant. Diluted sperm was loaded into 0.25ml straws, frozen in a nitrogen vapor vessel (dry shipper) and stored in a liquid nitrogen vessel. Straws were thawed in a water bath at 60 degrees C for 3s and sperm was immediately evaluated for motility rate and velocities (curvilinear=VCL; straight line=VSL; average path=VAP). Seminal plasma osmolality was also determined (249mOsmkg(-1)). Both extender composition and osmolality affected post-thaw sperm motility. In general, sperm cryopreserved in NaCl was of better motility than that in glucose, and at lower osmolalities better than at higher ones. High post-thaw quality, with motility above 60% and VCL above 140ms(-1), was observed only in samples frozen in NaCl at 285mOsmkg(-1). Different from most of the sperm from freshwater species that need a cell membrane protector (such as sugars), B.orbignyanus sperm should be frozen in an ionic solution for better protection during freezing and thawing processes. Furthermore, this solution should be prepared at an osmolality just above seminal plasma osmolality. Cryopreserved sperm can be used both for aquaculture purposes and for conservational programs, since B.orbignyanus is a threatened species.