Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

Chemoproteomic capture of RNA binding activity in living cells.

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

Macrophage acetyl-CoA carboxylase regulates acute inflammation through control of glucose and lipid metabolism.

Acetyl-CoA carboxylase (ACC) regulates lipid synthesis; however, its role in inflammatory regulation in macrophages remains unclear. We generated mice that are deficient in both ACC isoforms in myeloid cells. ACC deficiency altered the lipidomic, transcriptomic, and bioenergetic profile of bone marrow-derived macrophages, resulting in a blunted response to proinflammatory stimulation. In response to lipopolysaccharide (LPS), ACC is required for the early metabolic switch to glycolysis and remodeling of the macrophage lipidome. ACC deficiency also resulted in impaired macrophage innate immune functions, including bacterial clearance. Myeloid-specific deletion or pharmacological inhibition of ACC in mice attenuated LPS-induced expression of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß, while pharmacological inhibition of ACC increased susceptibility to bacterial peritonitis in wild-type mice. Together, we identify a critical role for ACC in metabolic regulation of the innate immune response in macrophages, and thus a clinically relevant, unexpected consequence of pharmacological ACC inhibition.

Feeding desensitizes A1 adenosine receptors in adipose through FOXO1-mediated transcriptional regulation.

OBJECTIVE: Adipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism. METHODS: We used Adora1 floxed mice with an inducible, adiponectin-Cre to generate FAdora1-/- mice, where F designates a fat-specific deletion of A1R. We used these FAdora1-/- mice along with specific agonists and antagonists of A1R to investigate changes in adenosine signaling within adipocytes between the fasted and fed state. RESULTS: We found that the adipose tissue response to adenosine is not static, but changes dynamically according to nutrient conditions through the insulin-Akt-FOXO1 axis. We show that under fasted conditions, FAdora1-/- mice had impairments in the suppression of lipolysis by insulin on normal chow and impaired glucose tolerance on high-fat diet. FAdora1-/- mice also exhibited a higher lipolytic response to isoproterenol than WT controls when fasted, however this difference was lost after a 4-hour refeeding period. We demonstrate that FOXO1 binds to the A1R promoter, and refeeding leads to a rapid downregulation of A1R transcript and desensitization of adipocytes to A1R agonism. Obesity also desensitizes adipocyte A1R, and this is accompanied by a disruption of cyclical changes in A1R transcription between fasting and refeeding. CONCLUSIONS: We propose that FOXO1 drives high A1R expression under fasted conditions to limit excess lipolysis during stress and augment insulin action upon feeding. Subsequent downregulation of A1R under fed conditions leads to desensitization of these receptors in adipose tissue. This regulation of A1R may facilitate reentrance into the catabolic state upon fasting.

Identification of ritanserin analogs that display DGK isoform specificity.

The diacylglycerol kinase (DGK) family of lipid enzymes catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Both DAG and PA are lipid signaling molecules that are of notable importance in regulating cell processes such as proliferation, apoptosis, and migration. There are ten mammalian DGK enzymes that appear to have distinct biological functions. DGKα has emerged as a promising therapeutic target in numerous cancers including glioblastoma (GBM) and melanoma as treatment with small molecule DGKα inhibitors results in reduced tumor sizes and prolonged survival. Importantly, DGKα has also been identified as an immune checkpoint due to its promotion of T cell anergy, and its inhibition has been shown to improve T cell activation. There are few small molecule DGKα inhibitors currently available, and the application of existing compounds to clinical settings is hindered by species-dependent variability in potency, as well as concerns regarding isotype specificity particularly amongst other type I DGKs. In order to resolve these issues, we have screened a library of compounds structurally analogous to the DGKα inhibitor, ritanserin, in an effort to identify more potent and specific alternatives. We identified two compounds that more potently and selectively inhibit DGKα, one of which (JNJ-3790339) demonstrates similar cytotoxicity in GBM and melanoma cells as ritanserin. Consistent with its inhibitor profile towards DGKα, JNJ-3790339 also demonstrated improved activation of T cells compared with ritanserin. Together our data support efforts to identify DGK isoform-selective inhibitors as a mechanism to produce pharmacologically relevant cancer therapies.

Work, physical activity, and metabolic health: Understanding insulin sensitivity of long-haul truck drivers.

BACKGROUND: Long-haul truck drivers are disproportionately exposed to metabolic risk; however, little is known about their metabolic health and the role of physical activity and other risk factors in metabolic outcomes. OBJECTIVE: This study compares truck drivers' insulin sensitivity, and associations between metabolic risk factors and insulin sensitivity, with those of the general population. METHODS: Survey, anthropometric, and biometric data were collected from 115 long-haul truckers, which were then compared to the general population data using the National Health and Nutrition Examination Survey (NHANES) dataset. The quantitative insulin sensitivity check index (QUICKI) was used to estimate insulin sensitivity. RESULTS: Truck drivers had lower QUICKI scores than the general population cohort. Sagittal abdominal diameter and exercise were predictive for QUICKI among combined cohorts. Waist circumference and perceived health were more predictive for QUICKI among truck drivers, and sagittal abdominal diameter and income were more predictive for QUICKI among the general population. CONCLUSIONS: Long-haul truckers appear to represent a subset of the general population regarding the impact of physical activity and other metabolic risk factors on QUICKI. Accordingly, comprehensive efforts which target these factors are needed to improve truckers' physical activity levels and other metabolic risks.

The Impact of Work Organization, Job Stress, and Sleep on the Health Behaviors and Outcomes of U.S. Long-Haul Truck Drivers.

Background. Compared with other occupations, long-haul truck drivers (LHTD) engage in excessively unhealthy behaviors and experience disproportionately poor health outcomes. Health promotion efforts targeting LHTDs focus on improving individual-level behaviors; however, this occupation is replete with adverse work organization characteristics, high job stress, and compromised sleep health, which are hypothesized to cause poor health behaviors and outcomes among LHTDs. Therefore, the purpose of this study was to explore the connections between work characteristics, job stress, and sleep outcomes, and health behaviors and physical and mental health outcomes among LHTDs. Method. This was a cross-sectional study, using interviewer-administered surveys with LHTDs (n = 260). Bivariate correlation analysis was used to explore the associations among work organization, job stress, sleep health, and health behaviors and outcomes. Logistic regression analyses were used to determine whether these work organization, job stress, and sleep factors predicted health behaviors and outcomes. Results. Long work hours of more than 11 hours daily (odds ratio [OR] = 2.34) resulted in increased odds of high caffeine consumption. High job stress (OR = 0.48) and poor sleep quality (OR = 0.42) led to decreased odds for spending at least 1 hour daily for cooking/eating. Low sleep duration, less than 7 hours daily (OR = 2.55), led to increased odds of a physical health diagnosis. Both high job stress (OR = 3.58) and poor sleep quality (OR = 2.22) resulted in increased odds of a mental health diagnosis. Conclusion. Health promotion efforts targeting LHTDs need to be coupled with upstream policy, environmental, and systems-level change, especially at the governmental and trucking industry levels.

Occupational health disparities among U.S. long-haul truck drivers: the influence of work organization and sleep on cardiovascular and metabolic disease risk.

OBJECTIVE: The organization of work has undergone vast transformations over the past four decades in the United States and has had profound impacts on worker health and wellbeing. The profession of commercial truck driving is one of the best examples. Particularly for long-haul truck drivers, changes in work organization have led to disproportionately poor physiological, psychological, and sleep health outcomes. METHODS: The present study examined disparities in cardiometabolic disease risk among long-haul truck drivers and the general population, and the influence of work organization and sleep in generating these outcomes. Researchers collected survey data from 260 drivers, and blood assay samples from 115 of those drivers, at a large highway truck stop in North Carolina. Comparisons were made for cardiovascular and metabolic risk against the 2011-2012 National Health and Nutrition Examination Survey (NHANES). In addition, logistic regression was used to explore predictive relationships between work organization and sleep and risk for cardiovascular and metabolic disease. RESULTS: There were statistically significant mean differences between the long-haul truck driver sample and the NHANES sample for both cardiovascular (3.71 vs. 3.10; p <0.001) and metabolic (4.31 vs. 3.09; p <0.001) disease risk. The truck driver sample was less physically active and had lower HDL cholesterol along with greater levels of smoking, BMI, and metabolic syndrome diagnosis. More years of driving experience and poor sleep quality were statistically significant predictors for both cardiovascular and metabolic disease risk. CONCLUSIONS: Study findings implicate elements of the occupational milieu experienced by long-haul truck drivers that induce disproportionate cardiometabolic disease risk. Sleep quality, largely compromised by poor work conditions and workplace environments, plays a significant role in increased risks for cardiometabolic disease. There is an urgent need for longitudinal studies of this critical occupational sector as well as intervention research centered on policy and systems level change.

Risks endemic to long-haul trucking in North America: strategies to protect and promote driver well-being.

Long-haul truck drivers in North America function in a work context marked by excess physical and psychological workload, erratic schedules, disrupted sleep patterns, extreme time pressures, and these factors' far-reaching consequences. These work-induced stressors are connected with excess risk for cardiometabolic disease, certain cancers, and musculoskeletal and sleep disorders, as well as highway crashes, which in turn exert enormous financial burdens on trucking and warehousing companies, governments and healthcare systems, along with working people within the sector. This article: 1) delineates the unique work environment of long-haul truckers, describing their work characteristics and duties; (2) discusses the health hazards of long-haul trucking that impact drivers, the general population, and trucking enterprises, examining how this work context induces, sustains, and exacerbates these hazards; and (3) proposes comprehensive, multi-level strategies with potential to protect and promote the health, safety, and well-being of truckers, while reducing adverse consequences for companies and highway safety.