SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows.

The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin; thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal; n = 30) and Aged (Primiparous; n = 20) dairy cows (Bos taurus) of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV; p value = 3.302e-07) and 0.95 (plasma; p value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (*p = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (***p = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle.

Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition.

SCOPE: Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. METHODS AND RESULTS: This study develops a pipeline using advanced proteomics and transcriptomics to enable cross-species comparison of milk and IF EVs. The number of nanoparticles per mL of IF is significantly reduced compared to unprocessed CM. 130 proteins and 514 miRNAs are differentially abundant between HM and CM EVs. While 90% of CM EV miRNAs are also identified in IF EVs, only 20% of CM EV proteins are identified in IF EVs. CONCLUSIONS: This workflow identifies key species-specific differences that can be used to optimize IF recipes and enhance infant nutrition. Improved preservation of EV functional molecular cargo in IF products is of critical importance to retaining molecular drivers of good health and should be the focus of future investigations.

Dynamics of inflammatory cytokine expression in bovine endometrial cells exposed to cow blood plasma small extracellular vesicles (sEV) may reflect high fertility.

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30-200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein-Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.

A Comparison of Blood Plasma Small Extracellular Vesicle Enrichment Strategies for Proteomic Analysis.

Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A 'gold-standard' method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

Characterisation of ReNcells CX and VM stimulated with interleukin-1ß and lipopolysaccharide.

Abnormal and dysregulated neuroinflammation has been linked to many neurological disorders and neurodegenerative diseases. Understanding the mechanisms of neuroinflammation, their impact on neurodevelopment and how neuroinflammation might be modulated, are currently considered to be critical to improving neurological treatment. ReNcell CX (originating from the cortical region) and VM (originating from the ventral mesencephalon) are human immortalised neural stem cell lines, that have the potential to be used as experimental models for investigating neuroinflammation in vitro. However, the information on the inflammation response of these cells is limited. This is especially more so for undifferentiated ReNcells. In this report we demonstrate using ELISA that cultured, undifferentiated ReNcell CX and VM produce significant amounts of IL-6 in response to IL-1ß treatment, but not to LPS treatment. Additionally, conventional RT-PCR showed that ReNcell CX cells expressed TNFR1 and NF-κB, whereas ReNcell VM expressed only NF-κB. Our results encourage further investigation into the relationship between 1L-1ß and IL-6 in both ReNcell CX and VM. Moreover, TNF-α treatment might potentially affect neuroinflammation in ReNcell CX, while activation of the NF-κB pathway could also play a critical part in neuroinflammation.

Proteomic profiling of plasma-derived small extracellular vesicles: a novel tool for understanding the systemic effects of tick burden in cattle.

Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.

Cattle ticks are a significant burden to cattle industries globally. Current methods to treat cattle ticks are costly and inefficient in the long term. It has been noted that while some cattle may exhibit a natural resistance to ticks, others carry a heavy tick burden. The study of small extracellular vesicles, or exosomes (EXs), isolated from cattle blood plasma provides a noninvasive way of analyzing changes at the cellular level and may be of use in understanding the systemic effects of tick burden or factors leading to natural resistance. The aim of this study was to assess high (HTR) and low tick resistance (LTR) cattle identified using a tick burden scoring system by analyzing the protein content of circulating EXs via qualitative proteomics analysis. We found that a class of proteins related to defense/immunity comprised 50% of proteins unique to HTR cattle, while this protein class was not detected in proteins unique to LTR cattle. Additionally, epidermal growth factor­calcium-binding protein domains were 2-fold increased in LTR cattle compared with HTR cattle, indicating a possible mechanism for widespread metabolic change. This is the first study to employ proteomic analysis of exosomal cargo as an approach to understanding the systemic effects of tick burden in cattle.

A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry.

BACKGROUND: The present study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition. METHODS: The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated using Information-Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison is made between their proteomic profiles. RESULTS: IDA analyses revealed similarities and differences in protein content. About 229 and 239 proteins were identified in the human and bovine milk exosome proteome, respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common in both groups. These included proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophilin, perilipin-2, and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes. CONCLUSION: The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Cannabidiol and Neurodevelopmental Disorders in Children.

Neurodevelopmental and neuropsychiatric disorders (such as autism spectrum disorder) have broad health implications for children, with no definitive cure for the vast majority of them. However, recently medicinal cannabis has been successfully trialled as a treatment to manage many of the patients' symptoms and improve quality of life. The cannabinoid cannabidiol, in particular, has been reported to be safe and well-tolerated with a plethora of anticonvulsant, anxiolytic and anti-inflammatory properties. Lately, the current consensus is that the endocannabinoid system is a crucial factor in neural development and health; research has found evidence that there are a multitude of signalling pathways involving neurotransmitters and the endocannabinoid system by which cannabinoids could potentially exert their therapeutic effects. A better understanding of the cannabinoids' mechanisms of action should lead to improved treatments for neurodevelopmental disorders.

Exosomal Cargo May Hold the Key to Improving Reproductive Outcomes in Dairy Cows.

The reproductive status of dairy cows remains a challenge for dairy farmers worldwide, with impaired fertility linked to a significant reduction in herd profitability, due in part to impaired immunity, increased metabolic pressure, and longer postpartum anestrous interval (PPAI). Exosomes are nanovesicles released from a variety of cell types and end up in circulation, and carry proteins, bioactive peptides, lipids, and nucleic acids specific to the place of origin. As such, their role in health and disease has been investigated in humans and animals. This review discusses research into exosomes in the context of reproduction in dairy herds and introduces recent advances in mass-spectrometry (MS) based proteomics that have a potential to advance quantitative profiling of exosomal protein cargo in a search for early biomarkers of cattle fertility.

Preterm labor is characterized by a high abundance of amniotic fluid prostaglandins in patients with intra-amniotic infection or sterile intra-amniotic inflammation.

OBJECTIVE: To distinguish between prostaglandin and prostamide concentrations in the amniotic fluid of women who had an episode of preterm labor with intact membranes through the utilisation of liquid chromatography-tandem mass spectrometry. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry analysis of amniotic fluid of women with preterm labor and (1) subsequent delivery at term (2) preterm delivery without intra-amniotic inflammation; (3) preterm delivery with sterile intra-amniotic inflammation (interleukin (IL)-6>2.6 ng/mL without detectable microorganisms); and (4) preterm delivery with intra-amniotic infection [IL-6>2.6 ng/mL with detectable microorganisms]. RESULTS: (1) amniotic fluid concentrations of PGE2, PGF2α, and PGFM were higher in patients with intra-amniotic infection than in those without intra-amniotic inflammation; (2) PGE2 and PGF2α concentrations were also greater in patients with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (3) patients with sterile intra-amniotic inflammation had higher amniotic fluid concentrations of PGE2 and PGFM than those without intra-amniotic inflammation who delivered at term; (4) PGFM concentrations were also greater in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation who delivered preterm; (5) amniotic fluid concentrations of prostamides (PGE2-EA and PGF2α-EA) were not different among patients with preterm labor; (6) amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in cases with intra-amniotic inflammation; and (7) the PGE2:PGE2-EA and PGF2α:PGF2α-EA ratios were higher in patients with intra-amniotic infection compared to those without inflammation. CONCLUSIONS: Mass spectrometric analysis of amniotic fluid indicated that amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in women with preterm labor and intra-amniotic infection than in other patients with an episode of preterm labor. Yet, women with intra-amniotic infection had greater amniotic fluid concentrations of PGE2 and PGF2α than those with sterile intra-amniotic inflammation, suggesting that these two clinical conditions may be differentiated by using mass spectrometric analysis of amniotic fluid.

Differentially Expressed Extracellular Vesicle, Exosome and Non-Exosome miRNA Profile in High and Low Tick-Resistant Beef Cattle.

Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance. A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.

The Role of Exosomal Epigenetic Modifiers in Cell Communication and Fertility of Dairy Cows.

Abnormal uterine function affects conception rate and embryo development, thereby leading to poor fertility and reproduction failure. Exosomes are a nanosized subclass of extracellular vesicles (EV) that have important functions as intercellular communicators. They contain and carry transferable bioactive substances including micro RNA (miRNA) for target cells. Elements of the cargo can provide epigenetic modifications of the recipient cells and may have crucial roles in mechanisms of reproduction. The dairy industry accounts for a substantial portion of the economy of many agricultural countries. Exosomes can enhance the expression of inflammatory mediators in the endometrium, which contribute to various inflammatory diseases in transition dairy cows. This results in reduced fertility which leads to reduced milk production and increased cow maintenance costs. Thus, gaining a clear knowledge of exosomal epigenetic modifiers is critical to improving the breeding success and profitability of dairy farms. This review provides a brief overview of how exosomal miRNA contributes to inflammatory diseases and hence to poor fertility, particularly in dairy cows.

Prostaglandin and prostamide concentrations in amniotic fluid of women with spontaneous labor at term with and without clinical chorioamnionitis.

OBJECTIVE: Prostaglandins (PGs) are considered universal mediators for the process of physiological parturition. This is based on observations that amniotic fluid concentrations of PGs are elevated prior to and during the onset of labor (mostly utilizing immunoassays). Distinguishing PGs from similarly structured molecules (i.e. prostamides; PG-EA) is difficult given the cross-reactivity of available antibodies and the chemical similarity between these compounds. Herein, this limitation was overcome by utilizing mass spectrometry to determine PG and PG-EA concentrations in amniotic fluid of women with spontaneous labor at term and in those with clinical chorioamnionitis (CHAM), the most common infection-related diagnosis made in labor and delivery units worldwide. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry (LC MS/MS) was used to determine the PG and PG-EA content in amniotic fluid samples of women with spontaneous labor at term with (n = 14) or without (n = 28) CHAM. Controls included women who delivered at term without labor (n = 10). RESULTS: PGE2, PGF2α, and 13,14-dihydro-15-keto-PGF2α (PGFM) were higher in amniotic fluid of women with spontaneous labor at term than in those without labor. PGE2, PGF2α, and PGFM were also higher in amniotic fluid of women with CHAM than in those without labor. However, PGE2-EA and PGF2α-EA were lower in amniotic fluid of women with CHAM than in those without CHAM. The ratios of PGE2 to PGE2-EA and PGF2α to PGF2α-EA were higher in amniotic fluid of women with spontaneous labor at term with or without CHAM than in those without labor; yet, the ratio of PGF2α to PGF2α-EA was greater in women with CHAM than in those without this clinical condition. CONCLUSIONS: Spontaneous labor at term with or without CHAM is characterized by elevated amniotic fluid concentrations of prostaglandins (PGE2, PGF2α, and PGFM) but not prostamides. Quantification of these products by LC MS/MSlc==may potentially be of utility in identifying their physiological functions relevant to parturition. SUMMARY: Prostaglandins (PGs) are critical for the onset and progression of labor. Structural similarities of PGs and prostamides (PG-EA) prevents their specific identification by immunoassay. We utilized LC MS/MS to determine PG and PG-EA content in amniotic fluid (AF) of women with spontaneous labor at term with or without CHAM and women who delivered at term without labor. Higher aamniotic ffluid PG levels were observed in women with spontaneous labor with and without CHAM compared to women delivering without labor. PG-EA levels in amniotic fluid of women with spontaneous labor and CHAM were lower than in women with spontaneous labor without CHAM but not those without labor. Ratios of PGs to PG-EAs were higher in AF of women with labor and CHAM compared to those without labor. Delineation of these products by LC MS/MS may potentially be of utility in identifying their physiological functions relevant to parturition.

Altered productions of prostaglandins and prostamides by human amnion in response to infectious and inflammatory stimuli identified by mutliplex mass spectrometry.

INTRODUCTION: Prostaglandins are critical for the onset and progression of labor in mammals, and are formed by the metabolism of arachidonic acid. The products of arachidonic acid, 2-arachidonoylglycerol (2-AG), and anandamide (AEA) have a similar lipid back bone but differing polar head groups, meaning that identification of these products by immunoassay can be difficult. MATERIALS AND METHODS: In the current study, we present the use of mass spectrometry as multiplex method of identifying the specific end products of arachidonic and anandamide metabolism by human derived amnion explants treated with either an infectious agent (LPS) or inflammatory mediator (IL-1ß or TNF-α). RESULTS: Human amnion tissue explants treated with LPS, IL-1ß, or TNF-α increased production of prostaglandin E2 (PGE2; p < 0.05) but decreased PGFM. Overall, PGE2 production was greater compared to the other prostaglandins and prostamides irrespective of treatment. CONCLUSIONS: The findings of the current study are in keeping with the literature which describes amnion tissues as predominantly producing PGE2. The use of mass spectrometry for the differential identification of prostaglandins, prostamides, and other eicosanoids may help better elucidate mechanisms of preterm labor, and lead to new targets for the prediction of risk for preterm labor and/or birth.

The Role(s) of Eicosanoids and Exosomes in Human Parturition.

The roles that eicosanoids play during pregnancy and parturition are crucial to a successful outcome. A better understanding of the regulation of eicosanoid production and the roles played by the various end products during pregnancy and parturition has led to our view that accurate measurements of a panel of those end products has exciting potential as diagnostics and prognostics of preterm labor and delivery. Exosomes and their contents represent an exciting new area for research of movement of key biological factors circulating between tissues and organs akin to a parallel endocrine system but involving key intracellular mediators. Eicosanoids and enzymes regulating their biosynthesis and metabolism as well as regulatory microRNAs have been identified within exosomes. In this review, the regulation of eicosanoid production, abundance and actions during pregnancy will be explored. Additionally, the functional significance of placental exosomes will be discussed.

The physiology of intrapartum fetal compromise at term.

Uterine contractions in labor result in a 60% reduction in uteroplacental perfusion, causing transient fetal and placental hypoxia. A healthy term fetus with a normally developed placenta is able to accommodate this transient hypoxia by activation of the peripheral chemoreflex, resulting in a reduction in oxygen consumption and a centralization of oxygenated blood to critical organs, namely the heart, brain, and adrenals. Providing there is adequate time for placental and fetal reperfusion between contractions, these fetuses will be able to withstand prolonged periods of intermittent hypoxia and avoid severe hypoxic injury. However, there exists a cohort of fetuses in whom abnormal placental development in the first half of pregnancy results in failure of endovascular invasion of the spiral arteries by the cytotrophoblastic cells and inadequate placental angiogenesis. This produces a high-resistance, low-flow circulation predisposing to hypoperfusion, hypoxia, reperfusion injury, and oxidative stress within the placenta. Furthermore, this renders the placenta susceptible to fluctuations and reduction in uteroplacental perfusion in response to external compression and stimuli (as occurs in labor), further reducing fetal capillary perfusion, placing the fetus at risk of inadequate gas/nutrient exchange. This placental dysfunction predisposes the fetus to intrapartum fetal compromise. In the absence of a rare catastrophic event, intrapartum fetal compromise occurs as a gradual process when there is an inability of the fetal heart to respond to the peripheral chemoreflex to maintain cardiac output. This may arise as a consequence of placental dysfunction reducing pre-labor myocardial glycogen stores necessary for anaerobic metabolism or due to an inadequate placental perfusion between contractions to restore fetal oxygen and nutrient exchange. If the hypoxic insult is severe enough and long enough, profound multiorgan injury and even death may occur. This review provides a detailed synopsis of the events that can result in placental dysfunction, how this may predispose to intrapartum fetal hypoxia, and what protective mechanisms are in place to avoid hypoxic injury.

Exosomes from dairy cows of divergent fertility; Action on endometrial cells.

Abnormalities in endometrial function contribute to poor fertility and reproductive failure. Exosomes are small lipid vesicles that contain transferable bioactive substances; they participate in intercellular signaling and may have critical roles in reproductive mechanisms, including endometrial remodeling in preparation for pregnancy. In this study, we evaluated the effects of exosomes from heifers with high and low genetic merit for fertility on inflammatory mediator expression by bovine endometrial epithelial and stromal cell lines. Co-incubation of exosomes from low, compared with high, fertility heifers upregulated the gene expression of pro-inflammatory IL1A and IL8 (CXCL8) but downregulated IL4 gene expression in epithelial cells. In contrast, stromal cells co-incubated with exosomes from low, compared with high, fertility heifers downregulated the gene expression of CXCL9, CXCL10, and CX3CL1. Our findings demonstrated that circulating exosomes from high fertility heifers did not alter endometrial inflammatory mediator gene expression. In contrast, circulating exosomes from low fertility heifers enhanced endometrial expression of inflammatory mediators, which may contribute to aberrant inflammation, leading to a reduced fertility in low fertility heifers. However, an in-depth investigation is required to elucidate the role of exosomes in regulating endometrial remodeling events required for enhanced reproductive performance and fertility in dairy cows.

A Method for the Isolation of Exosomes from Human and Bovine Milk.

SCOPE: Milk provides a natural means of nutrient supply to infants. Exosomes are an important component of milk that are not only being studied for their promise in translational medicine but also in infant nutrition. They also play important roles in intercellular communication and immune function in mammary glands and are able to transfer their materials to the recipient. Therefore, the isolation of high-quality exosomes is an important aspect of exosome research. METHODS AND RESULTS: This study is a technical study, which provides a detailed methodology for the isolation and enrichment of exosomes from milk. In this study, we evaluate the suitability of using the exosome enrichment method that we have recently published for bovine milk, on human milk. We initially isolated extracellular vesicles from human and bovine milk on a fresh set of samples, using ultracentrifugation, and then exosomes were subsequently enriched via size exclusion chromatography (SEC). Following isolation and enrichment, exosomes from both species were characterized by particle concentration (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), and the presence of exosomal markers (immunoblotting and mass spectrometry using information dependant acquisition (IDA)). The key exosomal characteristics of spherical/donut-shaped morphology, the presence of exosomal markers, e.g., FLOT-1 and the tetraspanins, CD9 and CD81), and particle concentration were confirmed in both human and bovine milk exosomes. CONCLUSION: We conclude that our robust exosome enrichment method, previously published for bovine milk, is suitable for use on human milk.

The Interplay between the Endocannabinoid System, Epilepsy and Cannabinoids.

Epilepsy is a neurological disorder that affects approximately 50 million people worldwide. There is currently no definitive epilepsy cure. However, in recent years, medicinal cannabis has been successfully trialed as an effective treatment for managing epileptic symptoms, but whose mechanisms of action are largely unknown. Lately, there has been a focus on neuroinflammation as an important factor in the pathology of many epileptic disorders. In this literature review, we consider the links that have been identified between epilepsy, neuroinflammation, the endocannabinoid system (ECS), and how cannabinoids may be potent alternatives to more conventional pharmacological therapies. We review the research that demonstrates how the ECS can contribute to neuroinflammation, and could therefore be modulated by cannabinoids to potentially reduce the incidence and severity of seizures. In particular, the cannabinoid cannabidiol has been reported to have anti-convulsant and anti-inflammatory properties, and it shows promise for epilepsy treatment. There are a multitude of signaling pathways that involve endocannabinoids, eicosanoids, and associated receptors by which cannabinoids could potentially exert their therapeutic effects. Further research is needed to better characterize these pathways, and consequently improve the application and regulation of medicinal cannabis.