Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues.

MicroRNAs (miRs, miRNAs) play central roles in gene regulation. Previously, we reported that miRNAs from somatic cell content, and handling by consumers on the degradation of miRNAs in milk; we also quantified miRNAs in dairy products. Pasteurization and homogenization caused a 63% loss of miR-200c, whereas a 67% loss observed for miR-29b was statistically significant only in skim milk. Effects of cold storage and somatic cell content were quantitatively minor (<2% loss). Heating in the microwave caused a 40% loss of miR-29b but no loss of miR-200c. The milk fat content had no effect on miRNA stability during storage and microwave heating. The concentrations of miRNAs in dairy products were considerably lower than in store-bought milk. We conclude that processing of milk by dairies and handling by consumers causes a significant loss of miRNAs.

Workers exposed to organic dusts from concentrated animal feeding operations (CAFOs) are at risk for developing airway inflammatory diseases. Available preventative and therapeutic measures for alleviating dust-induced lung disease are inadequate. Because omega-3 fatty acids can mitigate inflammatory processes, we aimed to determine whether nutritional supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) could reduce the airway inflammatory consequences of exposures to organic dust. Aqueous extracts of organic dusts from swine CAFOs (ODE) were utilized. In DHA-pretreated human bronchial epithelial cells, lung fibroblasts, monocyte cell cultures, and precision-cut murine lung slices, we found that DHA pretreatment dose-dependently decreased ODE-induced inflammatory cytokine production. To determine the in vivo significance of DHA, C57BL/6 mice were orally administered DHA for seven days prior to treatment with intranasal ODE or saline inhalations. Animals treated with 2 mg DHA demonstrated significant reductions in ODE-induced bronchial alveolar lavage neutrophil influx and pro-inflammatory cytokine/chemokine production compared to mice exposed to ODE alone. Collectively, these data demonstrate that DHA affects several lung cells to reduce the airway inflammatory response to organic dust exposures. Dietary supplementation with DHA may be an effective therapeutic strategy to reduce the airway inflammatory consequences in individuals exposed to agriculture dust environments.

Purpose Piezo1 mechanosensitive ion channels play an important role in mechanotransduction in all vertebrates and have been identified in a variety of tissues and cell types including neurons. Previously, we have identified Piezo1 mechanosensitive ion channels in zebrafish and mammalian retinal ganglion cells (RGCs). The principle aim of this study is to test whether activation of Piezo1 channels can produce neurodegeneration of zebrafish and mammalian RGCs. Methods For zebrafish Ca2+ imaging was performed using a genetically‐encoded Ca2+ indicator, GCaMP6f, and was targeted predominantly to RGCs using the Tg(elavl3: GCaMP6f) zebrafish line, and confirmed by backfilling the optic nerve with Texas Red hydrazide. In order to activate Piezo1 channels in RGCs, cytosolic [Ca2+]i changes were measured in response to application of the Piezo1 selective agonist, Yoda1. Immunoblotting and immunohistochemical localization for Piezo1 were used to determine the expression profile in rodent and zebrafish retina using retinal wholemounts and vertical sections. To examine apoptosis, a short‐term organotypic eyecup culture preparation was used. Eyecups were incubated in oxygenated Ringer’s solution (for 1–8 hrs) with various concentrations of Yoda1 (0.1–100μM). Following treatment with Yoda1, eyecups were fixed, sectioned, and prepared for immunohistochemistry. TUNEL assays was used to label apoptotic cells in fixed vertical retinal sections, and TUNEL‐positive cells were quantified using ImageJ software. All imaging was acquired on a Nikon C2+ confocal microscope. Results Calcium imaging experiments revealed that Yoda1 evokes a dose‐dependent activation of Piezo1 ion channels in GCAMP6f expressing RGCs, which was reduced in the presence of GsMTx‐4, a mechanosensitive channel antagonist. Immunohistochemical findings show strong Piezo1 immunoreactivity in RGCs and their axons in rodent and zebrafish retina. Moreover, Yoda1 evoked a concentration‐dependent increase in TUNEL‐positive labeling of RGCs in both zebrafish and rodent retina, implicating a common Ca2+‐mediated pathway for Piezo1 channel induced RGC death. Conclusions This study provides the first evidence for Piezo1 channel‐mediated RGC death and suggests that there is a potential link between Piezo1 channel activation on RGCs, increased [Ca2+]i influx and apoptosis. Therefore, changes in cell stiffness and pressure acting through Piezo1 mechanosensitive channels could contribute to the neurodegenerative effects observed in optic neuropathies, such as ocular injury and glaucoma. Support or Funding Information HMC CURE Zebrafish Pilot Grant, Penn State University‐Hershey College of Medicine; Pennsylvania Department of Health, Tobacco CURE Funds

Female clinical populations are generally protected from obesity-related blood pressure and metabolic complications such as hypertension and type II diabetes, despite increased adiposity compared to males. Similar cardiovascular protection has been observed in obese female mice produced by high fat diet (HFD) feeding, a well-established animal model of obesity. The impact of HFD on glucose homeostasis and insulin action in relation to gender differences, however, remains unclear. In this study, we tested the hypothesis that female mice would be protected from HFD-induced glucose intolerance and insulin resistance. To test this, five-week old male and female C57BL/6J mice were placed on control chow diet or 60% HFD for 12 weeks (n=11 chow male; n=6 chow female; n=15 HFD male; n=5 HFD female). Body composition was measured and glucose tolerance testing (GTT; 2g/kg of 50% dextrose) and insulin tolerance testing (ITT; 0.75 units/kg) were performed during the last week of diet. Females had lower body mass compared with males over the 12-week study period. Both genders increased body mass to a similar extent in response to chow diet (24.6±1.7 male vs. 26.5±2.3% female; p=0.517), and HFD (85.0±2.1 males vs. 79.8±6.7% females; p=0.332). HFD females had similar adiposity compared with males (22.9±0.7 males vs. 22.5±1.6% females; p=0.780). HFD reduced insulin sensitivity in male mice [area under the curve (AUC) for blood glucose levels during ITT: -5903±662 chow vs. -1748±1024 HFD; p=0.005] and female mice (-6599±966 chow vs. -2904±1507 HFD; p=0.041), with no differences between genders (p=0.567). HFD also resulted in glucose intolerance in male (AUC for blood glucose levels during GTT: 40262±1769 chow vs. 52800±2502 HFD; p=0.001) and female (36951±2270 chow vs. 55507±3582 HFD; p=0.001) mice, with no gender differences (p=0.582). These data suggest that in contrast to our hypothesis, HFD female mice develop obesity-related insulin resistance and glucose intolerance to a similar extent as males. These findings have important implications for use of this mouse model to study impact of gender on metabolic function in obesity.