Physicochemical Properties and Whey Proteomes of Camel Milk Powders Produced by Different Concentration and Dehydration Processes.

Camel milk powder production is an alternative to preserve the perishable milk for later-date consumption. However, the impacts of dehydration processes on bioactive compounds in camel milk are largely unknown. Hence, the present study attempted to compare the physicochemical properties and protein profiles of camel milk powders produced by different concentration and dehydration processes. Six camel milk powders were produced by freeze- and spray-drying methods in conjunction with two liquid concentration techniques, namely spray dewatering and reverse osmosis. The results of proteomic analysis showed that direct freeze-dried camel milk powder had the least changes in protein profile, followed by direct spray-dried powder. The camel milk powders that underwent concentration processes had more profound changes in their protein profiles. Among the bioactive proteins identified, lactotransferrin and oxidase/peroxidase had the most significant decreases in concentration following processing. On the contrary, glycosylation-dependent cell adhesion molecule 1, peptidoglycan recognition protein 1, and osteopontin increased in concentration. The results revealed that direct freeze drying was the most ideal method for preserving the bioactive proteins during camel milk powder production. However, the freeze-drying technique has cost and scalability constraints, and the current spray-drying technique needs improvement to better retain the bioactivity of camel milk during powder processing.

Comprehensive biochemical and proteomic characterization of seasonal Australian camel milk.

Although camel milk is increasingly becoming a popular alternative to bovine milk around the world including Australia, studies of Australian camel milk are still lacking. A comprehensive and systematic analysis of major nutritional components, physical properties, antimicrobial enzymes and whey proteomes of Australian camel milk obtained over four seasons was conducted, for the first time in present study. The composition and physical properties of Australian camel milk varied with season, milking frequency and yield. The highest lactoperoxidase and polyamine oxidase activity was observed in summer and winter, respectively. A total of 97 proteins were quantified, on a relative basis, across all the seasonal bulk milk samples. Summer camel milk contained higher amounts of functional whey proteins, such as lactotransferrin, peptidoglycan recognition protein 1, osteopontin and lactoperoxidase. These results contribute to a better understanding of the Australian camel milk and provide insights into processing of dairy products from this milk.

Digestibility of proteins in camel milk in comparison to bovine and human milk using an in vitro infant gastrointestinal digestion system.

The absence of ß-lactoglobulin, high ß-/αs-casein ratio and protective proteins make camel milk a promising alternative protein base for making human infant formulae. In this study, protein digestibility of camel milk was compared with that of bovine and human milk using an in vitro infant gastrointestinal digestion system. A low degree of gastric proteolysis was observed in all three kinds of milk, and a single clot was formed in camel milk. The soluble milk proteins remaining in the gastric digesta were digested rapidly and extensively in the intestinal phase, while the proteins in the camel milk clot were hydrolysed gradually. Despite several similarities, bioactive peptides unique to individual milk were identified in the three intestinal milk digesta. The results suggest that camel milk proteins are equally digestible as bovine and human milk proteins under infant gastrointestinal digestion conditions, and it may be a prospective substitute for infant formula base.

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.

Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

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.

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.