FAF1 blocks ferroptosis by inhibiting peroxidation of polyunsaturated fatty acids.

Iron-dependent peroxidation of polyunsaturated fatty acids (PUFAs) leads to ferroptosis. While detoxification reactions removing lipid peroxides in phospholipids such as that catalyzed by glutathione peroxidase 4 (GPX4) protect cells from ferroptosis, the mechanism through which cells prevent PUFA peroxidation was not completely understood. We previously identified Fas-associated factor 1 (FAF1) as a protein directly interacting with free PUFAs through its UAS domain. Here we report that this interaction is crucial to protect cells from ferroptosis. In the absence of FAF1, cultured cells became sensitive to ferroptosis upon exposure to physiological levels of PUFAs, and mice developed hepatic injury upon consuming a diet enriched in PUFA. Mechanistically, we demonstrate that FAF1 assembles a globular structure that sequesters free PUFAs into a hydrophobic core, a reaction that prevents PUFA peroxidation by limiting its access to iron. Our study suggests that peroxidation of free PUFAs contributes to ferroptosis, and FAF1 acts upstream of GPX4 to prevents initiation of ferroptosis by limiting peroxidation of free PUFAs.

Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication.

Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.

Diverse roles of SIRT1 in cancer biology and lipid metabolism.

SIRT1, an NAD(+)-dependent deacetylase, has been described in the literature as a major player in the regulation of cellular stress responses. Its expression has been shown to be altered in cancer cells, and it targets both histone and non-histone proteins for deacetylation and thereby alters metabolic programs in response to diverse physiological stress. Interestingly, many of the metabolic pathways that are influenced by SIRT1 are also altered in tumor development. Not only does SIRT1 have the potential to regulate oncogenic factors, it also orchestrates many aspects of metabolism and lipid regulation and recent reports are beginning to connect these areas. SIRT1 influences pathways that provide an alternative means of deriving energy (such as fatty acid oxidation and gluconeogenesis) when a cell encounters nutritive stress, and can therefore lead to altered lipid metabolism in various pathophysiological contexts. This review helps to show the various connections between SIRT1 and major pathways in cellular metabolism and the consequence of SIRT1 deregulation on carcinogenesis and lipid metabolism.

Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release.

Background: Cancer cell evasion of the immune response is critical to cancer development and metastases. Clinicians' ability to kickstart the immune system to target these rogue cells is an ever-growing area of research and medicine. This study delved into the relationship between lipid metabolism, High Mobility Group Box 1 protein (HMGB1)-a pro-inflammatory damage-associated molecular pattern protein-and immune regulation within non-small cell lung adenocarcinoma (NSCLC). Method: To address this question, we used a combination of proteomics, molecular biology, and bioinformatic techniques to investigate the relationship between fatty acids and immune signals within NSCLC. Results: We found that the expression of stearoyl CoA desaturase 1 (SCD1) was decreased in NSCLC tumors compared to normal tissues. This emphasized the critical role of lipid metabolism in tumor progression. Interestingly, monounsaturated fatty acid (MUFA) availability affected the expression of programmed death ligand-1 (PD-L1), a pivotal immune checkpoint target in lung cancer cells and immune cells, as well as HMGB1, suggesting a novel approach to modulating the immune response. This study uncovered a complex interplay between SCD1, PD-L1, and HMGB1, influencing the immunological sensitivity of tumors. Conclusion: Our work underscores the critical importance of understanding the intricate relationships between lipid metabolism and immune modulation to develop more effective NSCLC treatments and personalized therapies. As we continue to explore these connections, we hope to contribute significantly to the ever-evolving field of cancer research, improving patient outcomes and advancing precision medicine in NSCLC.

Unsaturated fatty acid alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 trafficking.

Cancer cell evasion of the immune response is critical to cancer development and metastases. The ability of clinicians to kickstart the immune system to target these rogue cells is an ever-growing area of research and medicine. In this study, we delved into the relationship between lipid metabolism, High Mobility Group Box 1 protein (HMGB1), and immune regulation within non-small cell lung adenocarcinoma (NSCLC), shedding light on novel therapeutic avenues and potential personalized approaches for patients. We found that the expression of stearoyl CoA desaturase 1 (SCD1) was decreased in NSCLC tumors compared to normal tissues. This emphasized the critical role of lipid metabolism in tumor progression. Interestingly, monounsaturated fatty acid (MUFA) availability impacted the expression of programmed death receptor ligand -1 (PD-L1), a pivotal immune checkpoint target in lung cancer cells and immune cells, suggesting a novel approach to modulating the immune response. This study uncovered a complex interplay between HMGB1, SCD1, and PD-L1, influencing the immunological sensitivity of tumors. Our work underscores the importance of understanding the intricate relationships between lipid metabolism and immune modulation to develop more effective NSCLC treatments and personalized therapies. As we continue to explore these connections, we hope to contribute to the ever-evolving field of cancer research, improving patient outcomes and advancing precision medicine in NSCLC.

Targeting lipid metabolism in the treatment of ovarian cancer.

Cancer cells undergo alterations in lipid metabolism to support their high energy needs, tumorigenesis and evade an anti-tumor immune response. Alterations in fatty acid production are controlled by multiple enzymes, chiefly Acetyl CoA Carboxylase, ATP-Citrate Lyase, Fatty Acid Synthase, and Stearoyl CoA Desaturase 1. Ovarian cancer (OC) is a common gynecological malignancy with a high rate of aggressive carcinoma progression and drug resistance. The accumulation of unsaturated fatty acids in ovarian cancer supports cell growth, increased cancer cell migration, and worse patient outcomes. Ovarian cancer cells also expand their lipid stores via increased uptake of lipids using fatty acid translocases, fatty acid-binding proteins, and low-density lipoprotein receptors. Furthermore, increased lipogenesis and lipid uptake promote chemotherapy resistance and dampen the adaptive immune response needed to eliminate tumors. In this review, we discuss the role of lipid synthesis and metabolism in driving tumorigenesis and drug resistance in ovarian cancer conferring poor prognosis and outcomes in patients. We also cover some aspects of how lipids fuel ovarian cancer stem cells, and how these metabolic alterations in intracellular lipid content could potentially serve as biomarkers of ovarian cancer.

Exploring the Scope and Dimensions of Vaccine Hesitancy and Resistance to Enhance COVID-19 Vaccination in Black Communities.

BACKGROUND: The long history of distrust that characterizes the relationship between the Black/African-American population and the US Medical community makes COVID-19 vaccine hesitancy of great concern. A needs assessment of the Black/African-American community assessed willingness and explored the perceptions of community members regarding COVID-19 vaccination. METHODS: The study used a mixed-methods approach. Respondents (n = 183) were surveyed with a web-based questionnaire. They were asked whether there would get vaccinated for COVID-19 barring any access or cost-related challenges. Perceptions of community members regarding vaccination were explored through one-on-one interviews (n = 30) and eight focus groups (n = 49), with participants drawn from across various demographic characteristics. Survey responses were summarized using frequencies and proportions. A thematic analysis was conducted on the qualitative data. RESULTS: Thirty-four percent of respondents indicated "Yes" (willing to get vaccinated); 26.8% indicated "No", while 37.1% expressed hesitancy ("Maybe" or "I don't know"). Themes emerging from the qualitative data are grouped into three broad categories: vaccine accessibility (transportation, information, navigating healthcare system); vaccine hesitancy (with sub-categories of compliance, complacency and confidence); and vaccine "resistance" (conspiracy theories, conflicting beliefs, distrust of Government, trustworthiness of Health care). CONCLUSION: Findings demonstrate a nuanced expansion of "vaccine hesitancy" to delineate groups with varying issues and perspectives. Interventions to enhance vaccination rates in Black/African-American communities should incorporate components that assure accessibility at the minimum, but also address non-access-related issues. Priority should be given to enhancing vaccine literacy, information-sharing as efficacy and safety data emerge, and addressing specific concerns identified through community-engaged outreach efforts.

Shifting post production patterns: exploring changes in New Zealand's seafood processing industry.

This paper examines the changing nature of New Zealand's seafood companies' production practices. The past 15 years has seen the offshore outsourcing of post-harvest fish gain unprecedented momentum. The growth in offshore processing is a further stage in an increasingly globalised fisheries value chain. Fish is head and gutted, frozen and then transported to processing sites in China where it is thawed, value-added processed and refrozen for export to the original sourcing country or third country markets. Reasons advanced by the industry for this shift in production practices include quota reductions, increasing production costs and the sale of trawlers.

Predictive power of SARS-CoV-2 wastewater surveillance for diverse populations across a large geographical range.

The COVID-19 pandemic has exacerbated the disparities in healthcare delivery in the US. Many communities had, and continue to have, limited access to COVID-19 testing, making it difficult to track the spread and impact of COVID-19 in early days of the outbreak. To address this issue we monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA at the population-level using municipal wastewater influent from 19 cities across the state of Minnesota during the COVID-19 outbreak in Summer 2020. Viral RNA was detected in wastewater continually for 20-weeks for cities ranging in populations from 500 to >1, 000, 000. Using a novel indexing method, we were able to compare the relative levels of SARS-CoV-2 RNA for each city during this sampling period. Our data showed that viral RNA trends appeared to precede clinically confirmed cases across the state by several days. Lag analysis of statewide trends confirmed that wastewater SARS-CoV-2 RNA levels preceded new clinical cases by 15-17 days. At the regional level, new clinical cases lagged behind wastewater viral RNA anywhere from 4-20 days. Our data illustrates the advantages of monitoring at the population-level to detect outbreaks. Additionally, by tracking infections with this unbiased approach, resources can be directed to the most impacted communities before the need outpaces the capacity of local healthcare systems.

A novel wastewater-based epidemiology indexing method predicts SARS-CoV-2 disease prevalence across treatment facilities in metropolitan and regional populations.

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin's Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin's Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF's) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102-1.2 × 109 genome copies/L). Lag analysis of trends in Melvin's Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15-17 days at the statewide level and by up to 25 days at the regional/county level. Melvin's Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.

Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study.

Bartonella bacilliformis (B. bacilliformis), Bartonella henselae (B. henselae), and Bartonella quintana (B. quintana) are bacteria known to cause verruga peruana or bacillary angiomatosis, vascular endothelial growth factor (VEGF)-dependent cutaneous lesions in humans. Given the bacteria's association with the dermal niche and clinical suspicion of occult infection by a dermatologist, we determined if patients with melanoma had evidence of Bartonella spp. infection. Within a one-month period, eight patients previously diagnosed with melanoma volunteered to be tested for evidence of Bartonella spp. exposure/infection. Subsequently, confocal immunohistochemistry and PCR for Bartonella spp. were used to study melanoma tissues from two patients. Blood from seven of the eight patients was either seroreactive, PCR positive, or positive by both modalities for Bartonella spp. exposure. Subsequently, Bartonella organisms that co-localized with VEGFC immunoreactivity were visualized using multi-immunostaining confocal microscopy of thick skin sections from two patients. Using a co-culture model, B. henselae was observed to enter melanoma cell cytoplasm and resulted in increased vascular endothelial growth factor C (VEGFC) and interleukin 8 (IL-8) production. Findings from this small number of patients support the need for future investigations to determine the extent to which Bartonella spp. are a component of the melanoma pathobiome.

Pedagogy of the Black academic.

I am just starting my career as a cancer biologist, but I have always been a Black man in America. This means that I have always inhabited a world that generally disregarded my existence in some form or another. It is June 17th, 2020 and protests have been happening for weeks since the killing of George Floyd in Minneapolis. The current state of America may be uneasy for some, but for many Americans, the looming threat of exclusion and violence has been an unwelcome companion since birth. This letter is not about a single person, but the Black academic's experience of race inside and outside of the academy during a time of social upheaval. I have trained in a variety of institutions, big and small, and all the while acutely aware of the impact of my Blackness on my science. The intent of the following is to provoke the reader to reflect on how we as a nation can move toward radically positive change and not incremental adjustments to the status quo. The views expressed are my own and are the result of years of personal experience observing the anti-Black standard in America.

Potential Links between YB-1 and Fatty Acid Synthesis in Clear Cell Renal Carcinoma.

According to the National Institutes of Health, clear cell renal cell carcinoma (ccRCC) is the most common type of Renal Cell Carcinoma (RCC), making up approximately 75% of total renal carcinoma cases. Clear cell Renal Cell Carcinoma is characterized by a significant accumulation of lipids in the cytoplasm, which allows light from microscopes to pass through giving them a "clear" phenotype. Many of these lipids are in the form of fatty acids, both free and incorporated into lipid droplets. RCC is typically associated with a poor prognosis due to the lack of specific symptoms. Some symptoms include blood in urine, fever, lump on the side, weight loss, fatigue, to name a few; all of which can be associated with non-specific, non-cancerous, health conditions that contribute to difficult diagnosis. Treatment of RCC has typically been centered around radical nephrectomy as the standard of care, but due to the potentially small size of lesions and the possibility of causing surgically induced chronic kidney disease, treatments have shifted to more cautious, less invasive approaches. These approaches include active surveillance, nephron-sparing surgery, and other minimally invasive techniques like cryotherapy and renal ablation. Although these techniques have had the desired effect of reducing the number of surgeries, there is still considerable potential for renal impairment and the chance that tumors can grow out of control without surgery. With the difficulty that surrounds the treatment of ccRCC and its considerably high mortality rate amongst urological cancers, it is important to look for novel approaches to improve patient outcomes. This review looks at available literature and our data that suggests the lipogenic enzyme stearoyl-CoA desaturase may be more beneficial to patient survival than once thought. As our understanding of the importance of lipids in cell metabolism and longevity matures, it is important to present new perspectives that present a new understanding of ccRCC and the role of lipids in survival mechanisms engaged by transformed cells during cancer progression. In this review, we provide evidence that pharmacological inhibition of lipid desaturation in renal cancer patients is not without risk, and that the presence of unsaturated fatty acids may be a beneficial factor in patient outcomes. Although more direct experimental evidence is needed to make definitive conclusions, it is clear that the work reviewed herein should challenge our current understanding of cancer biology and may inform novel approaches to the diagnosis and treatment of ccRCC.

First year allopathic medical student attitudes about vaccination and vaccine hesitancy.

First year medical students at an allopathic medical school regional campus were asked to complete a 10-question survey at both the beginning and the conclusion of a required course on immunology, hematology and oncology. The survey was designed to solicit student attitudes about vaccination and the students' level of comfort with and exposure to vaccine hesitant patients. Surveys were administered to five consecutive classes from 2013 to 2017. Total response rate for completion of both surveys was 58.0% (178/307). Pre- and post-course surveys were administered to assess whether curricular experiences altered the students' perceptions about vaccinations and their ability to counsel vaccine hesitant patients. Curricular elements were presented in several different formats aimed at increasing student knowledge about vaccinations and student capacity to work with vaccine hesitant patients and families (problem-based learning, didactic lecture and interactive panel discussion). In the pre-survey, the majority of students reported having already encountered people who decline vaccinations (78.8%). Additionally, in the pre-survey the majority of medical students expressed strong support for vaccination (99.6% agreed with the statement that Vaccinations are a great public health accomplishment) and strong disagreement with both of the assertions that childhood vaccines cause autism and vaccine preservatives cause adverse health effects. In response to questions about comfort level while talking with patients about vaccine choices, baseline responses demonstrated a moderately high level of comfort. Post-course surveys revealed a statistically significant increase in student-rated comfort level in talking with patients about vaccine choices (pre-survey 79.2% report comfort versus post 97.8%; p < 0.001). Though this study is limited to student self-reporting, survey responses suggest that targeted curricular elements can improve medical student confidence in counseling patients about vaccinations. Future curricular elements designed to directly observe student performance could provide verification of counseling skill acquisition.

Y-box binding protein 1 acts as a negative regulator of stearoyl CoA desaturase 1 in clear cell renal cell carcinoma.

Y-box binding protein 1 (YB-1) is a regulatory protein associated with oncogenesis and poor prognosis in patients with cancer. In the cell, YB-1 functions as a DNA and RNA binding protein that promotes or suppresses expression of target genes. The cancer-promoting activity of YB-1 is mediated through its activation of oncogenes and repression of tumor suppressor genes. Lipogenic enzyme stearoyl-CoA desaturase (SCD1) drives the production of endogenous monounsaturated fatty acids (MUFAs) in cells and protects against toxic buildup of saturated fatty acids. Clear cell renal cell carcinoma (ccRCC) is often characterized by aberrantly high SCD1 expression and cytosolic accumulation of unsaturated fatty acids. In the present study, a proteomics screen of cells treated with inhibitors of SCD1 supported a potential relationship between YB-1 and SCD1. It was revealed that the presence of MUFAs led to increased protein synthesis and increased expression of high molecular weight forms of YB-1 in ccRCC cells, but not in non-tumorigenic cells. Ectopic expression of YB-1 led to decreased expression levels of SCD1 protein and mRNA in ccRCC cell lines. Conversely, targeted knockdown of YB-1 increased SCD1 mRNA abundance. Analysis of ccRCC patient data from The Cancer Proteome Atlas database showed YB-1 expression was negatively associated with survival, whereas SCD1 was associated with improved survival. These data suggested an antagonistic relationship between YB-1 and SCD1 that may influence survival of patients with ccRCC.

Culturally Responsive Health Promotion to Address Health Disparities in African American Men: A Program Impact Evaluation.

African American (AA) men continue to experience worse health outcomes compared to men of other races/ethnicities. Community-based interventions are known to be effective in health promotion and disease prevention. The program objectives were to (a) increase knowledge and risk awareness of targeted conditions, (b) change health-care-seeking attitudes toward regular primary care among AA men, and (c) improve their lifestyle-related health behaviors by leveraging the influence of women in their lives. The community-engaged educational intervention targeted both men and women and included eight 90-min sessions per cohort. Topics included prostate cancer, cardiovascular disease, diabetes, mental health, health-care access, and healthy lifestyle. Sessions were both didactic and interactive. A pre-/post-intervention questionnaire assessed knowledge. Interviews were conducted with male participants and a focus group discussion (FGD) with women to assess program impact. Interview and FGD transcripts were analyzed for themes and recommendations. Major themes were-increased knowledge/awareness of risk associated with chronic conditions, change in health-care-seeking attitudes, increased self-efficacy to engage the health-care system, and lifestyle changes. Other impacts reported were building community/social support, a safe and enabling learning environment, and enhanced community health status overall. Recommendations included having extended, more in-depth sessions, targeting the younger generation, smaller cohort sizes, and more community-based health programming. Community-engaged health promotion using a cohort model as well as including women can be effective in increasing knowledge, enhancing self-efficacy, and providing the much-needed social support. These can influence health-related behaviors and thus contribute to improving health outcomes for AA men.

Dishevelled: A masterful conductor of complex Wnt signals.

The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity [1-3]. The Dsh gene (Dsh/Dvl, in Drosophila and vertebrates respectively) gained popularity when it was discovered that it plays a key role in segment polarity during early embryonic development in Drosophila [4]. Subsequently, the vertebrate homolog of Dishevelled genes were identified in Xenopus (Xdsh), mice (Dvl1, Dvl2, Dvl3), and in humans (DVL1, DVL2, DVL3) [5-10]. Dishevelled functions as a principal component of Wnt signaling pathway and governs several cellular processes including cell proliferation, survival, migration, differentiation, polarity and stem cell renewal. This review will revisit seminal discoveries and also summarize recent advances in characterizing the role of Dishevelled in both normal and pathophysiological settings.

A novel MeCP2 acetylation site regulates interaction with ATRX and HDAC1.

Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used to selectively decrease H3K27me3 repressive marks on a subset of repressed target gene promoters analyzed. Lastly, we show that RKO cells over-expressing MeCP2 mutant show reduced proliferation compared to those over-expressing MeCP2-wildtype. Our study demonstrates the importance of acetylated lysine residues and suggests their key role in regulating MeCP2 function and its ability to bind transcriptional regulators.

Frizzled 7 expression is positively regulated by SIRT1 and ß-catenin in breast cancer cells.

The Wnt signaling pathway is often chronically activated in diverse human tumors, and the Frizzled (FZD) family of receptors for Wnt ligands, are central to propagating oncogenic signals in a ß-catenin-dependent and independent manner. SIRT1 is a class III histone deacetylase (HDAC) that deacetylates histone and non-histone proteins to regulate gene transcription and protein function. We previously demonstrated that SIRT1 loss of function led to a significant decrease in the levels of Dishevelled (Dvl) proteins. To further explore this connection between the sirtuins and components of the Wnt pathway, we analyzed sirtuin-mediated regulation of FZD proteins. Here we explore the contribution of sirtuin deacetylases in promoting constitutive Wnt pathway activation in breast cancer cells. We demonstrate that the use of small molecule inhibitors of SIRT1 and SIRT2, and siRNA specific to SIRT1, all reduce the levels of FZD7 mRNA. We further demonstrate that pharmacologic inhibition of SIRT1/2 causes a marked reduction in FZD7 protein levels. Additionally, we show that ß-catenin and c-Jun occupy the 7 kb region upstream of the transcription start site of the FZD7 gene, and SIRT1 inhibition leads to a reduction in the occupancy of both ß-catenin and c-Jun at points along this region. This work uncovers a new mechanism for the regulation of FZD7 and provides a critical new link between the sirtuins and FZD7, one of the earliest nodal points from which oncogenic Wnt signaling emanates. This study shows that inhibition of specific sirtuins may provide a unique strategy for inhibiting the constitutively active Wnt pathway at the level of the receptor.