Acidification effects on isolation of extracellular vesicles from bovine milk
Autoři:
Md. Matiur Rahman aff001; Kaori Shimizu aff002; Marika Yamauchi aff002; Hiroshi Takase aff004; Shinya Ugawa aff005; Ayaka Okada aff002; Yasuo Inoshima aff001
Působiště autorů:
The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu, Japan
aff001; Laboratory of Food and Environmental Hygiene, Cooperative Department of Veterinary Medicine, Gifu University, Gifu, Gifu, Japan
aff002; Department of Medicine, Sylhet Agricultural University, Sylhet, Bangladesh
aff003; Core Laboratory, Graduate School of Medical Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan
aff004; Department of Anatomy and Neuroscience, Graduate School of Medical Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan
aff005; Education and Research Center for Food Animal Health, Gifu University (GeFAH), Gifu, Gifu, Japan
aff006; Joint Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu, Japan
aff007
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0222613
Souhrn
Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.
Klíčová slova:
Biology and life sciences – Nutrition – Diet – Beverages – Milk – Breast milk – Anatomy – Body fluids – Physiology – Biochemistry – Proteins – Phosphoproteins – Casein – Nucleic acids – RNA – Non-coding RNA – Natural antisense transcripts – MicroRNAs – Biomarkers – Genetics – Gene expression – Gene regulation – Cell biology – Cellular structures and organelles – Vesicles – Medicine and health sciences – Research and analysis methods – Microscopy – Electron microscopy – Transmission electron microscopy – Engineering and technology – Mechanical engineering – Rotors
Zdroje
1. Kalra H, Drummen GPC, Mathivanan S. Focus on extracellular vesicles: Introducing the next small big thing. Int J Mol Sci. 2016; 17: 170. doi: 10.3390/ijms17020170 26861301.
2. Lässer C, Alikhani VS, Ekström K, Eldh M, Paredes PT, Bossios A, et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med. 2011, 9: 9. doi: 10.1186/1479-5876-9-9 21235781.
3. Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, et al. Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. Sci Rep. 2018, 8: 3945. doi: 10.1038/s41598-018-22142-x 29500443.
4. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007, 9: 654–659. doi: 10.1038/ncb1596 17486113.
5. Pap E, Ṕallinger É, Ṕasztói M, Falus A. Highlights of a new type of intercellular communication: microvesicle-based information transfer. Inflamm Res. 2009, 58: 1–8. doi: 10.1007/s00011-008-8210-7 19132498
6. Escudier B, Dorval T, Chaput N, André F, Caby MP, Novault S, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J Transl Med. 2005, 3: 10. doi: 10.1186/1479-5876-3-10 15740633.
7. Dai S, Wei D, Wu Z, Zhou X, Wei X, Huang H, et al. Phase I clinical trials of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer. Mol Ther. 2008, 16: 782–790. doi: 10.1038/mt.2008.1 18362931.
8. Ohno SI, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, et al. Systemically injected exosomes targeted to EGFR deliver antitumor MicroRNA to breast cancer cells. Mol Ther. 2013, 21: 185–191. doi: 10.1038/mt.2012.180 23032975.
9. Wang JG, Williams JC, Davis BK, Jacobson K, Doerschuk CM, Ting JPY, et al. Monocytic microparticles activate endothelial cells in an IL-1beta-dependent manner. Blood. 2011, 118: 2366–2374. doi: 10.1182/blood-2011-01-330878 21700772.
10. Oehmcke S, Mörgelin M, Malmström J, Linder A, Chew M, Thorlacius H, et al. Stimulation of blood mononuclear cells with bacterial virulence factors leads to the release of pro-coagulant and pro-inflammatory microparticles. Cell Microbiol. 2012, 14: 107–119. doi: 10.1111/j.1462-5822.2011.01705.x 21951918.
11. Skog J, Würdinger T, Rijn SV, Meijer DH, Gainche L, Esteves MS, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008, Biol 10: 1470–1476. doi: 10.1038/ncb1800 19011622.
12. Wang H, Hou L, Li A, Duan Y, Gao H, Song X. Expression of serum exosomal micro RNA-21 in human hepatocellular carcinoma. Biomed Res Int. 2014. https://doi.org/10.1155/2014/864894 24963487.
13. Reinhardt TA, Lippolis JD, Nonnecke BJ, Sacco RE. Bovine milk exosome proteome. J Proteom. 2012, 75: 1486–1492. doi: 10.1016/j.jprot.2011.11.017 22129587.
14. Hata T, Murakami K, Nakatani H, Yamamoto Y, Matsuda T, Aoki N. Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs. Biochem Biophys Res Commun. 2010, 396: 528–533. doi: 10.1016/j.bbrc.2010.04.135 20434431.
15. Izumi H, Kosaka N, Shimizu T, Sekine K, Ochiya T, Takase M. Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. J Dairy Sci. 2012, 95: 4831–4841. doi: 10.3168/jds.2012-5489 22916887.
16. Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, et al. Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. Sci Rep. 2017, 7: doi: 10.1038/s41598-017-06288-8
17. Melnik BC, John SM, Schmitz G. Milk is not just food but most likely a genetic transfection system activating mTORC1 signaling for postnatal growth. Nutr J. 2013, 12: 103. doi: 10.1186/1475-2891-12-103 23883112.
18. Crookenden MA, Walker CG, Peiris H, Koh Y, Heiser A, Loor JJ, et al. Proteins from circulating exosomes represent metabolic state in transition dairy cows. J Dairy Sci. 2016, 99: 7661–7668. doi: 10.3168/jds.2015-10786 27320663.
19. Cai M, He H, Jia X, Chen S, Wang J, Shi Y, et al. Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus. Cell Stress Chaperon. 2018, 23: 663–672. doi: 10.1007/s12192-018-0876-3 29383581.
20. Yamada T, Shigemura H, Ishiguro N, Inoshima Y. Cell infectivity in relation to bovine leukemia virus gp51 and p24 in bovine milk exosomes. PLoS One. 2013, 8. e77359. doi: 10.1371/journal.pone.0077359 24146982.
21. Mather IH, Keenan TW. Origin and secretion of milk lipids. J Mammary Gland Biol Neoplasia. 1998, 3: 259–273. 10819513.
22. McMahon DJ, Oommen BS. Supramolecular structure of the casein micelle. J Dairy Sci. 2008, 91: 1709–1721. doi: 10.3168/jds.2007-0819 18420601.
23. Lönnerdal B. Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr. 2003, 77: 1537S–1543S. doi: 10.1093/ajcn/77.6.1537S 12812151.
24. Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles. 2013, 2: 1–6. doi: 10.3402/jev.v2i0.19861 24009896.
25. Yamada T, Inoshima Y, Matsuda T, Ishiguro N. Comparison of methods for isolating exosomes from bovine milk. J Vet Med Sci. 2012, 74: 1523–1525. doi: 10.1292/jvms.12-0032 22785357.
26. Van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014, 12: 1182–1192. doi: 10.1111/jth.12602 24818656.
27. Ban JJ, Lee M, Im W, Kim M. Low pH increases the yield of exosome isolation. Biochem Biophys Res Commun. 2015, 461: 76–79. doi: 10.1016/j.bbrc.2015.03.172 25849885.
28. Somiya M, Yoshioka Y, Ochiya T. Biocompatibility of highly purified bovine milk derived extracellular vesicles. J Extracell Vesicles. 2018, 7: 1440132. doi: 10.1080/20013078.2018.1440132 29511463.
29. Yamauchi M, Shimizu K, Rahman M, Ishikawa H, Takase H, Ugawa S, et al. Efficient method for isolation of exosomes from raw bovine milk. Drug Dev Ind Pharm. 2018, 45: 359–364. doi: 10.1080/03639045.2018.1539743 30366501.
30. Schacterle GR, Pollack RL. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973, 51: 654–655. doi: 10.1016/0003-2697(73)90523-x 4735559.
31. Vaswani K, Koh YQ, Almughlliq FB, Peiris HN. A method for the isolation and enrichment of purified bovine milk exosomes. Reprod Biol. 2017, 17: 341–348. doi: 10.1016/j.repbio.2017.09.007 29030127.
32. Benmoussa A, Lee CHC, Laffont B, Savard P, Laugier J, Boilard E, et al. Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. J Nutr. 2016, 146: 2206–2215. doi: 10.3945/jn.116.237651 27708120.
33. Shandilya S, Rani P, Kumar S, Singh D. Small Interfering RNA in Milk exosomes is resistant to digestion and crosses the intestinal barrier in vitro. J Agric Food Chem. 2017, 65: https://doi.org/10.1021/acs.jafc.7b031239506−9513.
Článek vyšel v časopise
PLOS One
2019 Číslo 9
- S diagnostikou Parkinsonovy nemoci může nově pomoci AI nástroj pro hodnocení mrkacího reflexu
- Je libo čepici místo mozkového implantátu?
- Pomůže v budoucnu s triáží na pohotovostech umělá inteligence?
- AI může chirurgům poskytnout cenná data i zpětnou vazbu v reálném čase
- Nová metoda odlišení nádorové tkáně může zpřesnit resekci glioblastomů
Nejčtenější v tomto čísle
- Graviola (Annona muricata) attenuates behavioural alterations and testicular oxidative stress induced by streptozotocin in diabetic rats
- CH(II), a cerebroprotein hydrolysate, exhibits potential neuro-protective effect on Alzheimer’s disease
- Comparison between Aptima Assays (Hologic) and the Allplex STI Essential Assay (Seegene) for the diagnosis of Sexually transmitted infections
- Assessment of glucose-6-phosphate dehydrogenase activity using CareStart G6PD rapid diagnostic test and associated genetic variants in Plasmodium vivax malaria endemic setting in Mauritania
Zvyšte si kvalifikaci online z pohodlí domova
Všechny kurzy