Single-nucleus transcriptomics of epicardial adipose tissue from female pigs reveals effects of exercise training on resident innate and adaptive immune cells.

BACKGROUND: Coronary artery disease (CAD) is a leading cause of death in women. Epicardial adipose tissue (EAT) secretes cytokines to modulate coronary artery function, and the release of fatty acids from EAT serves as a readily available energy source for cardiomyocytes. However, despite having beneficial functions, excessive amounts of EAT can cause the secretion of proinflammatory molecules that increase the instability of atherosclerotic plaques and contribute to CAD progression. Although exercise mitigates CAD, the mechanisms by which exercise impacts EAT are unknown. The Yucatan pig is an excellent translational model for the effects of exercise on cardiac function. Therefore, we sought to determine if chronic aerobic exercise promotes an anti-inflammatory microenvironment in EAT from female Yucatan pigs. METHODS: Sexually mature, female Yucatan pigs (n = 7 total) were assigned to sedentary (Sed, n = 3) or exercise (Ex, n = 4) treatments, and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk (n = 7 total) and single nucleus transcriptomic sequencing (n = 2 total, 1 per exercise treatment). RESULTS: Based on the bulk transcriptomic analysis, exercise upregulated S100 family, G-protein coupled receptor, and CREB signaling in neurons canonical pathways in EAT. The top networks in EAT affected by exercise as measured by bulk RNA sequencing were SRC kinase family, fibroblast growth factor receptor, Jak-Stat, and vascular endothelial growth factor. Single nucleus transcriptomic analysis revealed that exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT, with fibroblast and macrophage populations predominant in O-Ex EAT and T cell populations predominant in N-Ex EAT. Unlike the findings for exercise alone as a treatment, there were not increased interactions between endothelial and mesenchymal cells in O-Ex EAT. Coronary artery occlusion impacted the most genes in T cells and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. CONCLUSIONS: According to bulk transcriptomics, exercise upregulated pathways and networks related to growth factors and immune cell communication. Based on single nucleus transcriptomics, aerobic exercise increased cell-to-cell interaction amongst immune, mesenchymal, and endothelial cells in female EAT. Yet, exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.

Single-nucleus transcriptomics of epicardial adipose tissue from females reveals exercise control of innate and adaptive immune cells.

Coronary artery disease (CAD) is a leading cause of death in women. Although exercise mitigates CAD, the mechanisms by which exercise impacts epicardial adipose tissue (EAT) are unknown. We hypothesized that exercise promotes an anti-inflammatory microenvironment in EAT from female pigs. Yucatan pigs (n=7) were assigned to sedentary (Sed) or exercise (Ex) treatments and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk and single nucleus transcriptomic sequencing (snRNA-seq). Exercise upregulated G-protein coupled receptor, S100 family, and FAK pathways and downregulated the coagulation pathway. Exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT with fibroblast and macrophage populations predominant in O-Ex EAT and T cell population predominant in N-Ex EAT. Coronary occlusion impacted the largest number of genes in T and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. In conclusion, aerobic exercise increased interaction amongst immune and mesenchymal and endothelial cells in female EAT. Exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.

Core metabolism plasticity in phytoplankton: Response of Dunaliella tertiolecta to oil exposure.

Human alterations to the marine environment such as an oil spill can induce oxidative stress in phytoplankton. Exposure to oil has been shown to be lethal to most phytoplankton species, but some are able to survive and grow at unaffected or reduced growth rates, which appears to be independent of the class and phylum of the phytoplankton and their ability to consume components of oil heterotrophically. The goal of this article is to test the role of core metabolism plasticity in the oil-resisting ability of phytoplankton. Experiments were performed on the oil- resistant chlorophyte, Dunaliella tertiolecta, in control and water accommodated fractions of oil, with and without metabolic inhibitors targeting the core metabolic pathways. We observed that inhibiting pathways such as photosynthetic electron transport (PET) and pentose-phosphate pathway were lethal; however, inhibition of pathways such as mitochondrial electron transport and cyclic electron transport caused growth to be arrested. Pathways such as photorespiration and Kreb's cycle appear to play a critical role in the oil-tolerating ability of D. tertiolecta. Analysis of photo-physiology revealed reduced PET under inhibition of photorespiration but not Kreb's cycle. Further studies showed enhanced flux through Kreb's cycle suggesting increased energy production and photorespiration counteract oxidative stress. Lastly, reduced extracellular carbohydrate secretion under oil exposure indicated carbon and energy conservation, which together with enhanced flux through Kreb's cycle played a major role in the survival of D. tertiolecta under oil exposure by meeting the additional energy demands. Overall, we present data that suggest the role of phenotypic plasticity of multiple core metabolic pathways in accounting for the oxidative stress tolerating ability of certain phytoplankton species.

PFASs pollution in Galveston Bay surface waters and biota (shellfish and fish) following AFFFs use during the ITC fire at Deer Park (March 17th-20th 2019), Houston, TX.

The use of aqueous film forming foams (AFFFs) as fire retardants is an critical point-source for per- and polyfluoroalkyl substances (PFASs) pollution into the aquatic environment. This study investigated PFASs pollution in the surface waters and biota (shellfish and fish) of Galveston Bay, following AFFFs use to extinguish a petrochemical fire (March 17th to 20th, 2019) of oil storage tanks at the International Terminals Company (ITC) in Deer Park (Houston, TX). The levels of up to twelve EPA priority PFASs were measured in surface waters and biota from March-November 2019. PFASs levels in surface waters showed mean total levels in March and April 2019 to be from 4× to ~300× higher than those measured in the following months. PFOS (perfluorooctanesulfonic acid) was the most abundant homolog measured at ≥66% of total PFASs. Maximal PFOS levels exceeded the State of Texas' water regulatory limit of 0.6 µg L-1 in 3% of the samples analyzed in March and April 2019. PFOS was also the most prominent homolog (≥66% of total PFASs) measured in eastern oysters (Crassostrea virginica), red drum (Sciaenops ocellatus), gafftopsail catfish (Bagre marinus), and spotted seatrout (Cynoscion nebulosus). A statistically significant elevation of PFOS body-burdens was measured in oysters and spotted seatrout in April and May 2019, respectively. A Hazard Ratio calculation for seafood safety suggests an advisory of 1-2 meals per week for gafftopsail catfish and red drum, and 2 meals per week for spotted seatrout to be protective for human exposure to PFOS. The levels in oysters indicated no immediate concerns for the dietary exposure of humans. Our results highlight a need for continual monitoring to assess the long-term fate and seafood advisories for PFASs.

Correlations between environmental salinity levels, blood biochemistry parameters, and steroid hormones in wild juvenile American alligators (Alligator mississippiensis).

American alligators (Alligator mississippiensis) inhabit freshwater wetlands that are vulnerable to salinization caused by anthropogenic alterations to freshwater flow, in addition to storm surges, sea level rise, and droughts. Salinization of coastal freshwater habitats is a growing concern in a changing climate due to increased frequency and intensity of storm surges and drought conditions. This study opportunistically sampled juvenile male and female wild alligators in various salinities each month excluding November, December, and January for one year at Rockefeller Wildlife Refuge in coastal Louisiana. Blood plasma biochemistry parameters including electrolyte levels were subsequently measured. In addition, levels of various renin-angiotensin-aldosterone system hormones, glucocorticoids, androgens, estrogens, and progestogens were analyzed using liquid chromatography and tandem mass spectrometry. Only males were sampled in hyperosmotic environments (> 10‰) during dry conditions in late summer 2018. In juvenile males, plasma Na+, Cl-, and the progestogen 17α,20ß-dihydroxypregnenone were significantly and positively correlated with environmental salinity. However, variation in glucocorticoids, androgens, and estrogens were not associated with hypersaline water while sex steroids showed significant seasonal variation. This study demonstrated significant correlation of environmental salinity with electrolyte levels and a sex steroid in wild juvenile alligators, and to our knowledge represents the first measurement of 17α,20ß-dihydroxypregnenone in alligators.

An integrated in vivo and in silico analysis of the metabolism disrupting effects of CPI-613 on embryo-larval zebrafish (Danio rerio).

CPI-613 is a mitochondrial metabolism disrupter that inhibits tricarboxylic acid (TCA) cycle activity. The consequences of TCA cycle disruption on various metabolic pathways and overall organismal physiology are not fully known. The present study integrates in vivo experimental data with an in silico stoichiometric metabolism model of zebrafish to study the metabolic pathways perturbed under CPI-613 exposure. Embryo-larval life stages of zebrafish (Danio rerio) were exposed to 1 µM CPI-613 for 20 days. Whole-organism respirometry measurements showed an initial suppression of O2 consumption at Day 5 of exposure, followed by recovery comparable to the solvent control (0.01% DMSO) by Day 20. Comparison of whole-transcriptome RNA-sequencing at Day 5 vs. 20 of exposure showed functional categories related to O2 binding and transport, antioxidant activity, FAD binding, and hemoglobin complexes, to be commonly represented. Metabolic enzyme gene expression changes and O2 consumption rate was used to parametrize two in silico stoichiometric metabolic models representative of Day 5 or 20 of exposure. Computational simulations predicted impaired ATP synthesis, α-ketoglutarate dehydrogenase (KGDH) activity, and fatty acid ß-oxidation at Day 5 vs. 20 of exposure. These results show that the targeted disruption of KGDH may also impact oxidative phosphorylation (ATP synthesis) and fatty acid metabolism (ß-oxidation), in turn influencing cellular bioenergetics and the observed reduction in whole-organism O2 consumption rate. The results of this study provide an integrated in vivo and in silico framework to study the impacts of metabolic disruption on organismal physiology.

The pharmaceutical prednisone affects sheepshead minnow (Cyprinodon variegatus) metabolism and swimming performance.

High usage of the synthetic glucocorticoids (GCs) has led to significant presence of this pharmaceutical group in surface waters where it can affect non-target organisms such as fish. Assessment of a fish's metabolism and swimming performance provide reliable sub-lethal measures of effects of GCs on oxygen-requiring processes and ability to swim. In this study, we determined time-dependent (7, 14 and 21 days) effects of the synthetic GC prednisone (1 µg L-1) on sheepshead minnow (SHM) (Cyprinodon variegatus). Standard (SMR), routine (RMR) and maximum (MMR) metabolic rate, metabolic scope (MS), excess post-exercise oxygen consumption (EPOC), cost of transport (COT) and critical swimming speed (Ucrit) were determined. Twenty-one days exposure to prednisone resulted in significantly higher SMR, RMR, MMR, MS, EPOC and COT compared with 7d and 14d prednisone fish. However, Ucrit was not significantly different between prednisone and solvent control exposed fish (within 7d, 14d, 21d groups). SMR, RMR and MMR were lower in the 7d and 14d prednisone exposed fish compared with their solvent control groups. In contrast, SMR, RMR and MMR were all significantly higher in the 21d prednisone exposed fish compared with solvent control. EPOC was significantly higher in 14d prednisone exposed fish and trending higher in 21d and 7d prednisone exposed fish compared with their solvent controls. EPOC was significantly higher in 21d compared with 7d prednisone exposed fish. A significantly higher COT was seen in the 21d compared with 7d and 14d prednisone fish. Collectively, this study showed time-dependent effects of prednisone on SHM metabolism and swimming performance.

Short-term exposure to 12‰ brackish water has significant effects on the endocrine physiology of juvenile American alligator (Alligator mississippiensis).

American alligators (Alligator mississippiensis) mainly inhabit freshwater habitats but can be exposed to a wide range of salinities during storm surges, droughts or from alterations in freshwater flows. Although some salinization events last weeks, others only last a few days. This study assessed changes in the endocrine function of the renin-angiotensin-aldosterone system (RAAS) and steroid hormone production (steroidogenesis) in juvenile alligators exposed to brackish water (12‰) for 7 days. We quantified plasma levels of angiotensin II and the corticosteroids (aldosterone, corticosterone and 11-deoxycortisol). Various progestogens, androgens, and estrogens were further assessed. The protein expression for the RAAS enzymes, renin and angiotensin converting enzyme (ACE), was quantified immunohistochemically in kidney and lung tissue, respectively, and histology was performed on kidney, lung and gonad tissues. Finally, blood biochemistry parameters such as electrolyte levels and diagnostic indicators for dehydration, renal, and hepatic function were measured. Corticosterone, 11-deoxycortisol, Na+, Cl-, total protein, albumin, uric acid, and cholesterol levels were all significantly elevated in alligators exposed to brackish water compared with alligators in freshwater. The levels of 17ß-estradiol and estrone were significantly lowered while histology showed alterations in gonad tissue in the brackish water exposed group. In contrast, while there were no effects of exposure on aldosterone levels, angiotensin II was significantly reduced in brackish water exposed alligators. These results correlated with significantly decreased expressions for both renin and ACE in kidney and lung tissue. Overall, this study showed that short-term exposure of alligators to 12‰ brackish water has significant endocrine effects on juvenile alligators.

Child-feeding strategies are associated with maternal concern about children becoming overweight, but not children's weight status.

BACKGROUND: Research suggests that parents use specific child-feeding strategies to influence their child's weight based on perceptions and concerns about their child's overweight risk, but limited data are available on ethnically diverse low-income populations. OBJECTIVE: This cross-sectional study examined associations between mothers' perception and concern about children's weight, child-feeding strategies, and child overweight in an ethnically diverse population. SUBJECTS: Mothers of preschool children (n=967) who participated in a federally funded nutrition program were asked how they fed their child, how they perceived child's weight, and whether or not they were concerned about their child becoming overweight. STATISTICAL ANALYSES PERFORMED: Logistic regression to calculate odds of maternal perception/concern given child weight, feeding strategy given maternal perception/concern, and child overweight given feeding strategy. RESULTS: Only 21% (n=23/108) of overweight preschoolers were perceived as overweight. Maternal perception of overweight was not associated with feeding strategies. About 53% (n=76/144) of Hispanic, 42% (n=23/55) of African-American, and 29% (n=223/768) of white mothers reported concern about their child becoming overweight. Mothers reporting concern were more likely to restrict child's intake of select foods (odds ratio 5.94; 95% confidence interval 1.74 to 20.28) and less likely to pressure child to eat (odds ratio 0.39; 95% confidence interval 0.15 to 0.99); however, these strategies did not predict child overweight. CONCLUSIONS: Mothers concerned about their child becoming overweight were more likely to restrict children's intake of specific foods and less likely to pressure their child to eat; however, this study did not detect an association between feeding strategies and child overweight.