Melt electrowriting scaffolds with fibre-guiding features for periodontal attachment.

Periodontal regeneration requires the re-attachment of oblique and perpendicular periodontal ligament (PDL) fibres to newly formed cementum and alveolar bone, which has proven elusive with existing approaches. In this study, multiple fibre-guiding biphasic tissue engineered constructs were fabricated by melt electrowriting. The biphasic scaffolds were 95 % porous and consisted of a pore size gradient bone compartment and periodontal compartment made of fibre-guiding channels with micro-architectural features ranging from 100 to 60 µm aimed to direct PDL fibre alignment and attachment. In vitro evaluations over 3 and 7 days demonstrated a marked improvement in collagen fibre orientation (over 60 % fully aligned) for scaffolds with micro-architecture ≤100 µm. The biphasic scaffolds were placed on a dentine slice and implanted ectopically, and this demonstrated that all micro-channels groups facilitated oblique and perpendicular alignment and attachment on the dentine with a mean nuclei angle and mean collagen fibre angle of approximately 60° resembling the native periodontal ligament attachment. A further in vivo testing using a surgically created rodent periodontal model highlighted the 80 µm micro-channel group's effectiveness, showing a significant increase in oblique PDL fibre attachment (72 %) and periodontal regeneration (56 %) when compared to all other groups onto the tooth root compared to control groups. Further to this, immunohistochemistry demonstrated the presence of periostin in the newly formed ligament indicating that functional regeneration occurred These findings suggest that scaffold micro-architectures of 100 µm or below can play a crucial role in directing periodontal tissue regeneration, potentially addressing a critical gap in periodontal therapy. STATEMENT OF SIGNIFICANCE: Periodontal regeneration remains a significant clinical challenge. Essential to restoring dental health and function is the proper attachment of the periodontal ligament, which is functionally oriented, to regenerated bone and cementum. Our research presents an innovative biphasic scaffold, utilizing Melt Electrowriting to systematically guide tissue growth. Distinct from existing methods, our scaffold is highly porous, adaptable, and precisely guides periodontal ligament fibre attachment to the opposing tooth root and alveolar bone interfaces, a critical step for achieving periodontal functional regeneration. Our findings not only bridge a significant gap in biomaterial driven tissue guidance but also promise more predictable outcomes for patients, marking a transformative advancement in the field.

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.

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.

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.

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.

Circulating exosomes may identify biomarkers for cows at risk for metabolic dysfunction.

Disease susceptibility of dairy cows is greatest during the transition from pregnancy to lactation. Circulating exosomes may provide biomarkers to detect at-risk cows to enhance health and productivity. From 490 cows, animals at high- (n = 20) or low-risk (n = 20) of transition-related diseases were identified using plasma non-esterified fatty acid and ß-hydroxybutyrate concentrations and liver triacylglyceride concentrations during the two weeks post-calving. We isolated circulating exosomes from plasma of dairy cows at low-risk (LR-EXO) and high-risk (HR-EXO), and analyzed their proteome profiles to determine markers for metabolic dysfunction. We evaluated the effects of these exosomes on eicosanoid pathway expression by bovine endometrial stromal (bCSC) and epithelial (bEEL) cells. HR-EXO had significantly lower yield of circulating exosomes compared with LR-EXO, and unique proteins were identified in HR-EXO and LR-EXO. Exposure to LR-EXO or HR-EXO differentially regulated eicosanoid gene expression and production in bCSC and bEEL cells. In bCSC, LR-EXO exposure increased PGE2 and PGD2 production, whereas HR-EXO exposure increased PTGS2 gene expression. In bEEL, HR-EXO exposure caused a decrease in PGE2, PGF2α, PGD2, PGFM and TXB2 production. The unique presence of serpin A3-7, coiled-coil domain containing 88A and inhibin/activin ß A chain in HR-EXO, indicates potential biomarkers for cows at-risk for metabolic diseases. Our results are in line with the health status of the cow indicating a potential diagnostic role for exosomes in enhancing cows' health and fertility.

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha.

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1ß and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1 µg/mL LPS, 10 ng/mL IL-1ß and 50 ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1ß and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1ß and PTGES2 when treated with IL-1ß. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE2, PGF2α, PGE2-EA and PGF2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1ß, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.

Regulation of inflammatory mediator expression in bovine endometrial cells: effects of lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha.

An abnormal uterine environment can influence maternal-fetal communication, conception rate and disrupt normal embryo development, thereby affecting fertility and the reproductive performance of dairy cows. Animal variability means that development of endometrial cell lines with appropriate characteristic are required. We evaluated the effect of an infectious agent (i.e., bacterial lipopolysaccharide; LPS) and proinflammatory mediators (i.e., Interleukin 1 beta; IL-1ß, and tumor necrosis factor alpha; TNFα) on inflammatory mediator gene expression and production by bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. Expression of CXCL8/IL8, IL1A, IL1B, and IL6 cytokine genes was significantly upregulated in both epithelial and stromal cells when treated with LPS and IL-1ß. LPS treatment of epithelial cells (compared with treatment by IL-1ß and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Whereas, in stromal cells, IL-1ß treatment (compared with LPS and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Interestingly, bEEL and bCSC cells treated with IL-1ß increased IL1B gene expression, suggesting that IL-1ß may act unusually in an autocrine-positive feedback loop. Cytokine production was stimulated by these agents in both cell types. We suggest that the characteristics of these two cell lines make them excellent tools for the study of intrauterine environment.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Proteomic content of circulating exosomes in dairy cows with or without uterine infection.

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows.

The use of SWATH to analyse the dynamic changes of bacterial proteome of carbapanemase-producing Escherichia coli under antibiotic pressure.

Antibiotic resistance associated with the clinically significant carbapenemases KPC, NDM and OXA-48 in Enterobacteriaceae is emerging as worldwide. In Australia, IMP-producing Enterobacteriaceae are the most prevalent carbapenemase-producing Enterobacteriaceae (CPE). Genomic characteristics of such CPE are well described, but the corresponding proteome is poorly characterised. We have thus developed a method to analyse dynamic changes in the proteome of CPE under antibiotic pressure. Specifically, we have investigated the effect of meropenem at sub-lethal concentrations to develop a better understanding of how antibiotic pressure leads to resistance. Escherichia coli strains producing either NDM-, IMP- or KPC-type carbapenemases were included in this study, and their proteomes were analysed in growth conditions with or without meropenem. The most significant difference in the bacterial proteomes upon the addition of meropenem was triggered amongst NDM-producers and to a lower extent amongst KPC-producers. In particular, HU DNA-binding proteins, the GroEL/GroES chaperonin complex and GrpE proteins were overexpressed. These proteins may thus contribute to the better adaptability of NDM- and KPC-producers to meropenem. A significant meropenem-induced increase in the expression of the outer membrane protein A was only observed in IMP-producers, thus demonstrating that carbapenemase-mediated resistance relies on far more complex mechanisms than simple inactivation of the antibiotic.

Mid-to-Late Gestational Changes in Inflammatory Gene Expression in the Rat Placenta.

BACKGROUND: The placenta plays an important role during pregnancy providing maternal blood supply from the uterus to the developing fetus. The structure and function of the placenta changes with gestation, as the fetus develops and its demands change. This study aims to elucidate changes in cytokine and chemokine gene expression throughout mid-to-late gestation in rat placenta. METHODS: Sprague Dawley rats were time-mated, and placentae were obtained from 6 pregnant dams at 4 different gestational periods: E14.25, E15.25, E17.25, and E20. Changes in placental gene expression were measured by microarray analysis. Differentially expressed inflammatory genes were functionally categorized by pathway analysis. To validate the microarray results, a subset of genes was analyzed by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort of 22 rats. RESULTS: Changes in messenger RNA (mRNA) expression of various cytokines, chemokines, and genes of the tumor growth factor ß and tumor necrosis factor family were analyzed in rat placentae at E14.25, E15.25, E17.25, and E20. Forty-six genes were differentially expressed, and of these 21 genes had increased expression in late gestation (E20). The gestational age pattern of gene expression was confirmed by qPCR in the validation cohort. CONCLUSION: The observed acute, prelabor changes in the expression of these genes during gestation warrant further investigation to elucidate their role in pregnancy and parturition.

Exosome enrichment by ultracentrifugation and size exclusion chromatography.

Exosomes are a subset of extracellular vesicles (EVs) that have important roles in intercellular communication. They contain and carry bioactive molecules within their membranes which are delivered to target cells. Reproducible isolation and enrichment of these exosomes will aid in evaluation of cellular communication. We present an approach that involved the pre-processing of plasma, combined with ultracentrifugation (UC) and size exclusion chromatography (SEC) to isolate EVs and subsequently enrich exosomes. Four variations of this approach (denoted methods I to IV) were compared. Coupling an ultracentrifugation method with size exclusion chromatography (Method II) provided the best yield by nanoparticle tracking analyses (NTA), the presence of the exosomal markers CD63, Flotillin-1 and TSG-101 (immunoblotting) and showed exosome morphology using transmission electron microscopy (TEM). This method provides an efficient way to enrich the exosomes from blood (plasma), which could be potentially employed for clinical diagnostic assessment and therapeutic intervention.

A method for the isolation and enrichment of purified bovine milk exosomes.

Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC. This method was compared to buoyant density gradient centrifugation, a widely used method of enrichment. Exosomes were characterised by particle concentration and size (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), presence of exosomal markers (immunoblotting) and protein concentration (bicinchoninic acid assay, BCA). Proteomic profiles of exosomal fractions were analyzed by mass spectrometry using Information Dependant Acquisition. Milk exosomal fractions were shown to contain exosomal markers flotillin-1 (FLOT-1) and tumor susceptibility gene-101 (TSG-101). The new method produced a higher yield of exosomes compared to buoyant density gradient centrifugation. Pooled exosomal fractions exhibited intact morphology by TEM. The use of SEC confirmed the fractionation of exosomes based on size while minimizing the interference with proteins. Tetraspanins CD9 and CD81 were observed via mass spectrometry in exosomal fractions. This new and efficient method confirmed the signatures for exosomes derived from unpasteurized bovine milk. Purification of exosomes is a foundational technique in the study of biomarkers for pathological conditions and effective drug delivery systems.