Assessment of lipids and adrenal hormones in the Florida manatee (Trichechus manatus latirostris) from different habitats.

Florida manatees (Trichechus manatus latirostris), a federally protected species, are classified as threatened due to anthropogenic stressors. Manatees inhabit sites that are impacted by human activities that can negatively affect stress physiology and metabolism. Samples collected from healthy manatees (pregnant females, non-pregnant females, and males) at Crystal River and Indian River Lagoon in Florida, were assessed for adrenal hormones, proteins, glucose, and lipid content in plasma. The objective was to determine if healthy manatees sampled between 2010-2014 from the Indian River Lagoon exhibited evidence of stress compared to healthy manatees sampled between 2012-2019 from Crystal River. Plasma cortisol concentrations were not different in male and non-pregnant female manatees between sites but were elevated in pregnant manatees. Plasma aldosterone concentrations were elevated in Indian River Lagoon manatees relative to those at Crystal River, possibly due to differences in salinity and available freshwater between the two environments. Site differences were noted for plasma protein and glucose concentrations in manatees; additionally, differences between the sexes were also observed in glucose concentrations. Fifteen lipid subclasses, including oxidized lysophosphatidylcholines, oxidized phosphatidylcholines, oxidized triacylglycerols, were elevated in manatees from the Indian River Lagoon relative to manatees from Crystal River. Evidence of a stress response in healthy Indian River Lagoon manatees was lacking compared to Crystal River manatees. Differences in metabolites related to energy (glucose, protein, and lipids) may be related to site-specific variables, such as salinity and food availability/quality. This study generates novel data on plasma lipid profiles and provides cortisol, aldosterone, glucose, and protein values from healthy Florida manatees in two disparate sites that can be referenced in future studies. These data contribute to an improved understanding of manatee physiology to better inform population management.

Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis.

Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.

C Type Natriuretic Peptide Receptor Activation Inhibits Sodium Channel Activity in Human Aortic Endothelial Cells by Activating the Diacylglycerol-Protein Kinase C Pathway.

The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA's corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) was augmented after treating hAoEC with cANF compared to vehicle, suggesting EnNaC activity is down-regulated upon NPRC ligand binding in a DAG-PKC dependent manner. The reciprocal cross-talk between NPRC activation and EnNaC inhibition represents a feedback mechanism that presumably is involved in the regulation of endothelial function and aortic stiffness.

Lipid Profiles of Urinary Extracellular Vesicles Released during the Inactive and Active Phases of Aged Male Mice with Spontaneous Hypertension.

Hypertension remains a major problem, especially in the elderly, as it increases the risk for cardiovascular, coronary artery, cerebrovascular, and kidney diseases. Extracellular vesicles (EVs) play a role in the aging process and contribute to pathophysiology. Our goal was to examine differences in lipid profiles of urinary EVs (uEVs) collected during the inactive and active phases of aged mice and investigate whether these EVs regulate the density of lipid rafts in mouse cortical collecting duct (mpkCCD) principal cells. Here, we demonstrate the epithelial sodium channel (ENaC) inhibitor benzyl amiloride reduced systolic blood pressure in aged male mice during the inactive and active phases. Lipidomics data demonstrate differential enrichment of lipids between the two groups. For example, there are more phosphatidylethanolamine plasmalogens, particularly in the form of alkyl phosphatidylethanolamines, that are enriched in active phase uEVs compared to inactive phase uEVs from the same mice. Amiloride-sensitive transepithelial current increased more in mpkCCD cells challenged with uEVs from the active phase group. Moreover, more ENaC alpha protein was distributed to lipid raft fractions of mpkCCD cells challenged with active phase uEVs. Taken together, the identification of bioactive lipids associated with lipid rafts that are enriched in EVs released during the active phase of aged mice may offer clues to help understand lipid raft organization in recipient principal cells after EV uptake and increased renal ENaC activity, leading to a time-of-day dependent regulation of blood pressure in an aging model.

Increased endothelial sodium channel activity by extracellular vesicles in human aortic endothelial cells: putative role of MLP1 and bioactive lipids.

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17ß-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17ß-estradiol were highly correlated with R2 of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17ß-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants.

Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants,especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C3 or C4), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress.

Analysis of flooding-responsive proteins localized in the nucleus of soybean root tips.

Flooding stress has negative impact on soybean cultivation as it severely impairs plant growth and development. To examine whether nuclear function is affected in soybean under flooding stress, abundance of nuclear proteins and their mRNA expression were analyzed. Two-day-old soybean seedlings were treated with flooding for 2 days, and nuclear proteins were purified from root tips. Gel-free proteomics analysis identified a total of 39 flooding-responsive proteins, of which abundance of 8 and 31 was increased and decreased, respectively, in soybean root tips. Among these differentially regulated proteins, the mRNA expression levels of five nuclear-localized proteins were further analyzed. The mRNA levels of four proteins, which are splicing factor PWI domain-containing protein, epsilon2-COP, beta-catenin, and clathrin heavy chain decreased under flooding stress, were also down-regulated. In addition, mRNA level of a receptor for activated protein kinase C1(RACK1) was down-regulated, though its protein was accumulated in the soybean nucleus in response to flooding stress. These results suggest that several nuclear-related proteins are decreased at both the protein and mRNA level in the root tips of soybean under flooding stress. Furthermore, RACK1 might have an important role with accumulation in the soybean nucleus under flooding-stress conditions.

Lipidomics and plasma hormone analysis differentiate reproductive and pregnancy statuses in Florida manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are protected as a threatened species, and data are lacking regarding their reproductive physiology. This study aimed to (1) quantify plasma steroid hormones in Florida manatees from two field sites, Crystal River and Indian River Lagoon, at different gestational stages and to (2) identify individual lipids associated with pregnancy status. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis was used to measure plasma steroid hormones and lipids. Pregnant female manatees were morphometrically distinct from male and non-pregnant female manatees, characterized by larger body weight and maximal girth. Progesterone concentrations in manatees were also elevated during early gestation versus late gestation. Cholesterol, an important metabolic lipid, and precursor for reproductive steroids, was not different between groups. Mass spectrometry quantified 949 lipids. Plasma concentrations of glycerophospholipids, glycerolipids, sphingolipids, acylcarnitines, and cholesteryl esters were associated with pregnancy status in the Florida manatee. Most of the lipid species associated with pregnancy were triacylglycerides, phosphatidylethanolamines, and ether-linked phosphatidylethanolamines, which may serve as energy sources for fetal development. This research contributes to improving knowledge of manatee reproductive physiology by providing data on plasma steroid hormones relative to reproductive status and by identifying plasma lipids that may be important for pregnancy. Elucidation of lipid species directly associated with pregnancy has the potential to serve as a diagnostic approach to identify pregnant individuals in fresh and archived samples. These biochemical and morphometric indicators of reproductive status advance the understanding of manatee physiology.

Effects of the organochlorine pesticide metabolite p,p'-DDE on the gastrointestinal lipidome in fish: A novel toxicity pathway for a legacy pollutant.

Though phased out from use in the United States, environmental contamination by organochlorine pesticides (OCPs) remains a widespread issue, especially around intensive agricultural regions. OCPs, such as dichlorodiphenyltrichloroethane (DDT) and its primary metabolite, dichlorodiphenyldichloroethylene (DDE), have been detected in soils, sediments, surface waters, and biota decades after their discontinued use. As OCPs are persistent and can bioaccumulate in fats, these compounds can transfer and magnify across food webs. Freshwater predatory fish and birds can accumulate high OCP concentrations, leading to a myriad of deleterious impacts on organismal health. Studies have found evidence of reproductive disruption in predatory fish, such as the largemouth bass (LMB; Micropterus salmoides), associated with DDT and DDE exposure. DDE can act through estrogenic pathways and induce the expression of estrogenic signals in male animals; however, the molecular mechanism of disruption is unclear. Recently, metabolomics research has revealed corollary relationships between lipid signals and organic pollutant toxicity. Here, a two-month feeding experiment on LMB was conducted to assess the interactions of DDE (as p,p'-DDE) in food with gut and liver lipid signaling. Targeted lipidomic analysis revealed global alterations in the abundance of tissue lipids, especially cholesteryl esters and phospholipids, in LMB exposed to low levels of p,p'-DDE. Results from these studies indicate that p,p'-DDE may act through disruption of normal lipid homeostasis to cause toxicity in freshwater fish.

Trace elements and lipidomic datasets of stranding samples in the blubber of Turpsios truncatus from the Yucatan coast: Association with biological features.

The Yucatan coastal zone is an area that contributes to many anthropogenic activities resulting in substantial contamination (metals, pesticides) in aquatic organisms. The dolphin is an excellent sentinel animal used in studying contamination in this area. Some substances found in dolphins have been identified as toxic causing alterations in the properties of membranes and produce lipid peroxidation especially heavy metals. The dataset presented here is associated with the research article paper entitled "Trace element and lipidomic analysis of bottlenose dolphin blubber from the Yucatan coast: Lipid composition relationships". In this article, we presented the trace element concentrations found in blubber and their comparison with other studies performed in mammal marine organisms. Lipidomic characterization of bottlenose dolphin blubber and their association with trace elements and the differences related to biological characteristics were presented. This data provides a correlation analysis between trace element concentrations, lipid species and body length and the lipid differences related to biological characteristics such as growth stage, stranding code, and the presence of stomach contents. We used Spearman correlation analysis to identify the association with body length, trace elements and lipids. Wilcoxon rank-sum test was used to determine differences in lipids related to stranding code (3: moderate decomposition, 4: advanced decomposition), growth stage (juveniles and adults) and whether they showed presence of stomach contents or not. The data indicates that Cr, Cd and Zn concentrations were higher compared to concentrations found in blubbler of T. truncatus from other studies (See Table 3). Cr, Co, As and Cd were found in higher concentration in larger organisms compared to smaller ones. The results of correlation between lipids and body length showed a decrease in some ceramides (CER, DCER, HCER), sterols (CE), glycerolipids (TAG, DAG) and phosphatidylethanolamines (LPE, PE) in larger dolphins (Table 4). Dolphins with advanced decomposition (code 4) showed lower concentrations of phosphatidylethanolamines (PE) compared with organisms with moderate decomposition (code 3). Organisms with empty stomachs showed higher concentrations of phosphoethanolamines suggesting a preferential metabolism of energy-rich lipids over structural lipids. The information in these datasets may contribute to understanding the potential associations of trace elements, lipids and their associations with biological characteristics.

Plant cell organelle proteomics in response to abiotic stress.

Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.

Proteome analysis of gut and salivary gland proteins of fifth-instar nymph and adults of the sunn pest, Eurygaster integriceps.

In the digestive system of the sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), the salivary gland has a key role in extra oral digestion and the gut is the main site for digestion of food. In this study, proteomics was used to study the role of proteins involved in digestion. The amount of feeding on wheat grain by adult insects increased by comparison to fifth-instar nymphs. Proteins of the gut and salivary gland in adults and fifth-instar nymphs were analyzed 1 day after feeding. The proteins related to digestion, metabolism, and defense against toxins were accumulated in the gut of adult insects. Three plant proteins including serpin, dehydroascorbate reductase, and ß-amylase were accumulated in guts of adults. In the salivary gland, phospholipase A2 and arginine kinase were increased in adults. Heat shock protein 70 increased in the gut of fifth-instar nymphs. Proteomic analysis revealed that most of changed proteins in digestive system of sunn pest were increased in adults. This study provided more targets derived from gut and salivary gland for pest management.

Trace element and lipidomic analysis of bottlenose dolphin blubber from the Yucatan coast: Lipid composition relationships.

Bottlenose dolphins (Tursiops truncatus) are found in coastal and estuarine ecosystems where they are in continuous contact with multiple abiotic and biotic stressors in the environment. Due to their role as predators, they can bioaccumulate contaminants and are considered sentinel organisms for monitoring the health of coastal marine ecosystems. The northern zonal coast of the Yucatan peninsula of Mexico has a high incidence of anthropogenic activities. The principal objectives of this study were two-fold: 1) to determine the presence of trace metals and their correlation with lipids in bottlenose dolphin blubber, and 2) to use a lipidomics approach to characterize their biological responses. Levels of trace elements (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, Pb) were analyzed using ICP-MS and lipids were measured using a targeted lipidomics approach with LC-MS/MS. Spearman correlation analysis was used to identify associations between lipids and trace elements. The influences of gender, stranding codes, presence of stomach content, growth stages and body length were also analyzed. Blubber lipid composition was dominated by triacylglycerols (TAG). Our results demonstrated the presence of heavy-metal elements such as Cd and As, which were correlated with different lipid species, mainly the ceramides and glycerophospholipids, respectively. Organisms with Cd showed lower concentrations of ceramides (CER, HCER and DCER), TAG and cholesteryl esters (CE). Trace elements Cr, Co, As and Cd increased proportionately with body length. This study provides a novel insight of lipidomic characterization and correlations with trace elements in the bottlenose dolphin which might contribute to having a better understanding of the physiological functions and the risks that anthropogenic activities can bring to sentinel organisms from coastal regions.

Endocrine, immune and renal toxicity in male largemouth bass after chronic exposure to glyphosate and Rodeo®.

Glyphosate is the most used herbicide worldwide, with no historical comparison. It is used for genetically modified crops, and particularly in Florida, it is used as a sugar cane ripener. An aquatic formulation (Rodeo®) is used to treat aquatic weeds in waterbodies and drainage canals. Because of its extended use, glyphosate can run off or be sprayed directly into waterbodies, and chronically expose aquatic wildlife. Exposure in animal models has been associated with kidney and liver damage and glyphosate has been suggested as an endocrine disruptor. We exposed adult male largemouth bass for 21 days to two doses of glyphosate and Rodeo® (chemically equivalent concentration of glyphosate) at 0.5 mg L-1 and 10 mg L-1 and to a clean water control (n=4 fish/tank in quadruplicate). Concentrations during the experiment were corroborated with UHPLC-MS/MS. Total RNA was isolated from the trunk kidney and head kidney. RNA-seq was performed for the high doses compared to controls. Transcripts were analyzed with fish and mammalian pathway analyses software. Transcripts mapped to Zebrafish metabolic pathways using PaintOmics showed steroid hormone biosynthesis in the trunk kidney as the most significantly enriched pathway. Steroid hormones were measured in plasma by UHPLC-MS/MS. Total androgens were significantly reduced at 0.5 mg L-1 of glyphosate and at equivalent concentrations in Rodeo® compared to controls. 11-ketotestosterone and estrone concentrations were significantly reduced in all doses. A gene involved in the conversion of testosterone to 11-ketotestosterone was down-regulated by glyphosate. Using the mammalian pathway analysis algorithm, cellular processes associated with T-cell activation/development and intracellular pH were significantly enriched in the trunk kidney by glyphosate and Rodeo® exposure. Endocrine disruption was corroborated at the hormone and gene expression levels. Rodeo® and glyphosate share gene expression pathways, however, Rodeo® had more pronounced effects in largemouth bass.

Comprehensive analysis of mitochondria in roots and hypocotyls of soybean under flooding stress using proteomics and metabolomics techniques.

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

Lipidomics reveals multiple stressor effects (temperature × mitochondrial toxicant) in the zebrafish embryo toxicity test.

Aquatic organisms are exposed to multiple stressors in the environment, including contaminants and rising temperatures due to climate change. The objective of this study was to characterize the effect of increased temperature on chemical-induced toxicity and lipid profiles during embryonic development and hatch in fish. This is important because temperature and many environmental chemicals modulate cellular metabolism and lipids, both of which play integral roles for normal embryonic development. As such, we employed the zebrafish embryo toxicity test for multiple stressor exposures, using the mitochondrial toxicant 2,4-Dinitrophenol (DNP; 6-30 µM) in conjunction with different temperature treatments (28 °C and 33 °C). We found a positive relationship between temperature and lethality at lower DNP concentrations, suggesting temperature stress can increase toxicant sensitivity. Next, we used LC-MS/MS for lipidomics following exposure to sublethal stressor combinations. It was determined that temperature stress at 33 °C augmented DNP-induced effects on the lipidome, including the upregulation of bioactive lipids involved in apoptosis (e.g., ceramides). These data reveal potential implications for climate change and sensitivity to environmental pollution and demonstrate the utility of lipidomics to characterize metabolic pathways underlying toxicity. Data such as these are expected to advance adverse outcome pathways by establishing multiple stressor networks that include intermediate lipid responses.

Tempol Alters Urinary Extracellular Vesicle Lipid Content and Release While Reducing Blood Pressure during the Development of Salt-Sensitive Hypertension.

Salt-sensitive hypertension resulting from an increase in blood pressure after high dietary salt intake is associated with an increase in the production of reactive oxygen species (ROS). ROS are known to increase the activity of the epithelial sodium channel (ENaC), and therefore, they have an indirect effect on sodium retention and increasing blood pressure. Extracellular vesicles (EVs) carry various molecules including proteins, microRNAs, and lipids and play a role in intercellular communication and intracellular signaling in health and disease. We investigated changes in EV lipids, urinary electrolytes, osmolality, blood pressure, and expression of renal ENaC and its adaptor protein, MARCKS/MARCKS Like Protein 1 (MLP1) after administration of the antioxidant Tempol in salt-sensitive hypertensive 129Sv mice. Our results show Tempol infusion reduces systolic blood pressure and protein expression of the alpha subunit of ENaC and MARCKS in the kidney cortex of hypertensive 129Sv mice. Our lipidomic data show an enrichment of diacylglycerols and monoacylglycerols and reduction in ceramides, dihydroceramides, and triacylglycerols in urinary EVs from these mice after Tempol treatment. These data will provide insight into our understanding of mechanisms involving strategies aimed to inhibit ROS to alleviate salt-sensitive hypertension.

Chronic exposure to glyphosate in Florida manatee.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate's and AMPA's concentrations in Florida manatee plasma (n = 105) collected during 2009-2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees' plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees' immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel-based and LC MS/MS-based proteomics approaches.

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.