Progress 08/01/16 to 05/20/22

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral fellows, graduate students and undergraduate students, and a visiting scientist. One of the current doctoral students, Alice Ngu is a first-generation college student. Approximately 70% of the trainees are women, and the project has supported one Hispanic and two African American students. All program graduates have secured positions in industry or academe. How have the results been disseminated to communities of interest?Findings have been disseminated through presentations at invited seminars, international, national and local conferences, social media (Twitter, blogs), publications in peer-reviewed journals (see section Products), and through collaborations with partners in industry (PureTech Health, Inc., see Patent; and Land O'Lakes, Inc.) and foundations (Gates, SynGAP Research Fund). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1, Direct markers of milk microRNA nutrition: 1) We have devised a PCR protocol, RNase H2 PCR that allowed us to detect bovine microRNAs in human plasma after, but not before a milk meal. 2) We have identified potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage). 3) We have developed a transgenic mouse model (Exosome and Cargo tracking (ECT mouse), which allowed us to demonstrate that milk exosomes are transferred from dams to pups through the natural route of suckling and accumulate in liver and brain. 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We have shown that the microbial mRNAs are bioavailable in mice and humans. 5) We have developed milk exosome and microRNA labeling protocols which enabled us to assess the bioavailability and distribution of milk exosomes and their microRNA cargos in mice and pigs. Exosomes accumulated primarily in liver and brain, whereas distinct microRNAs showed unique distribution patterns (brain vs. liver/spleen vs. intestinal mucosa vs. spleen). 6) We have developed a protocol for sequencing microRNAs in exosomes purified from small volumes (0.5 mL) of human milk. The protocol enables us to explore the roles of exosomes and their microRNA cargos in preterm infants. 7) We have developed a protocol to deplete bovine milk of exosomes and microRNAs by using ultrasonication. This technical advance enables us to study phenotypes of milk exosome depletion.. Objective 2, Indirect markers of milk microRNA nutrition: 1) We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk. 2) We assessed effects of exosome- and RNA-defined diets on amino acid metabolism in mice. Effects were negligible and this line of research was abandoned. The studies were useful because they allowed us to demonstrate that our experimental diets, which we also used in our studies, do not alter food and water consumption and physical activity and did not affect liver and kidney health. 3) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We are in the process of assessing the bioavailability of these mRNAs in a human milk feeding study. Bovine mRNAs are excellent markers for milk intake because their sequence is specific for cows, i.e., they cannot be synthesized by humans. 4) We have identified polymorphisms and mutations in the gut microbiome, selected by milk exosomes. Objective 3, Functional markers of milk microRNA nutrition: 1) We have shown that microbial mRNAs in bovine milk exosomes bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. TLR activation by milk exosomes is a promising functional marker for milk exosome intake (10-fold activation compared to controls in TLR reporter cells). These observations are consistent with observations that mice fed an exosome and RNA-sufficient diet survived a challenge with influenza A, whereas mice fed an exosome and RNA-depleted diet died within 11 days of influenza A challenge. 2) We have identified phenotypes of dietary depletion of milk exosomes and microRNA cargos. The phenotypes include loss of fecundity, increased severity of symptoms of inflammatory bowel disease, increases susceptibility to challenges with influenza A virus and impaired cognitive performance in mice. Knockout of maternal exosome and microRNA biogenesis caused slow growth and leakiness of the gastrointestinal mucosa in wild-type pups fostered to the knockout dams. 3) We have developed genetically engineered bovine mammary alveolar MAC-T cells, which enable us to interrogate milk exosome-dependent pathways.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Sukreet S, Pereira Braga C, An TT, Adamec J, Cui J, Trible B, Zempleni J. Isolation of extracellular vesicles from byproducts of cheese making by tangential flow filtration yields heterogeneous fractions of nanoparticles. J Dairy Sci 104:9478-9493, 2021
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ogunnaike M, Wang, H, Zempleni J. Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery. Int J Pharm 610:121263, 2021
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang H, Wu D, Sukreet S, Delaney A, Belfort MB, Zempleni J. Quantitation of exosomes and their microRNA cargos in frozen human milk. JPGN Reports 3:e172, 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Mutai E, Ngu AKH, Zempleni J. Preliminary evidence that lectins in infant soy formula apparently bind bovine milk exosomes and prevent their absorption in healthy adults. BMC Nutr 8:7, 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Sukreet S, Pereira Braga C, An, TT, Adamec J, Cui J, Zempleni J. Ultrasonication of milk decreases the content of exosomes and microRNAs in an exosome-defined rodent diet. J Nutr 152:961-970, 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Sadri M, Wang H, Kuroishi T, Li Y, Zempleni J. Holocarboxylase synthetase knockout is embryonic lethal in mice. PLoS One 17:e0265539, 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhou F, Ebea P, Mutai E, Wang H, Sukreet S, Navazesh S, Dogan H, Li W, Cui J, Ji P, Ramirez DMO, Zempleni J. Small extracellular vesicles in milk cross the blood-brain barrier in murine cerebral cortex endothelial cells and promote dendritic complexity in the hippocampus and brain function in C57BL/6J mice. Front Nutr 9:838543, 2022
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (submitted)
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Khanam A. Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. Am J Physiol Cell Physiol 321:C606-C614, 2021
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: Ngu A, Zempleni J. Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs). Nutrition 2022 Conference (virtual), American Society for Nutrition; 6/14  6/16, 2022
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ong SL, Blenkiron C, Haines S, Acevedo-Fani A, Leite JAS, Zempleni J, Anderson RA, McCann MJ. Ruminant milk-derived extracellular vesicles: a nutritional and therapeutic opportunity? Nutrients 13:2505, 2021
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ngu A, Wang S, Wang H, Khanam A, Zempleni J. Milk exosomes in nutrition and drug delivery. Am J Physiol Cell Physiol 322:C865-C874, 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Khanam A, Yu J, Zempleni J. Class A scavenger receptor-1/2-mediated phagocytosis of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. NPOD 7th Annual Research Symposium, September 13th, 2021, Lincoln, NE [video poster, delivered by Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Wang S, Auchtung J, Zempleni J. Milk exosomes select genetic variants that decrease the toxicity of Clostridioides difficile. NPOD 7th Annual Research Symposium, September 13th, 2021, Lincoln, NE [video poster, delivered by Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ngu A, Zempleni J. Modification of MAC-T exosomes with expression of CD24 and CD47 and its reduction to be eliminated by bone marrow-derived macrophages. NPOD 7th Annual Research Symposium, September 13th, 2021, Lincoln, NE [video poster, delivered by Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zempleni J. The use of milk exosomes to increase the expression of Syngap1 in Syngap1 mice. SynGAP Research Fund, Palo Alto, CA. March 3, 2022 [talk, delivered by Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zempleni J. Novel bioactive compounds in milk: exosomes. Harold Hamm Diabetes Center at the University of Oklahoma, Oklahoma City, OK. March 21, 2022 [talk, delivered by Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zempleni J. Milk exosomes and the gut brain axis. Delivered in the symposium titled Extracellular Vesicles in GI Physiology and Beyond, by the American Physiological Society (Chairs: Sarah Andres, Lauren Davey), Philadelphia, PA. April 4, 2022 [talk]
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: Zempleni J, Ngu A. Bovine mammary alveolar MAC-T cells afford a tool for designing milk exosomes optimized for drug delivery. Gordon Research Conference Nanoscale Science and Engineering for Agriculture and Food Systems, Southern New Hampshire University, Hooksett, NH, June 19-24, 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Khanam A, Adamec J, Zempleni J. Time courses of milk-derived extracellular vesicles in murine plasma. Nebraska Research Days, University of Nebraska-Lincoln, Lincoln, NE, April 12, 2022 [poster]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Ngu A., Zempleni J. Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs). Spring 2022 Student Research Days, University of Nebraska-Lincoln; April 12, 2022 [poster]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mumtaz PT, Zempleni J. Extracellular vesicles from Bifidobacterium infantis are bioavailable in C57BL/6J mice and human intestinal Caco-2 cells. NPOD Annual Spring Research Retreat, April 19, 2022, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Ngu A, Zempleni J. Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs). NPOD Annual Spring Research Retreat, April 19, 2022, Lincoln, NE
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: Zempleni J. Genetically altered milk exosomes facilitate nutrition research and drug delivery. International Milk Genomics Cosnrtium Symposium 2022, October 18-20, 2022, University of California-Davis; Davis, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Wang S, Auchtung J, Zempleni J. Milk exosomes select mutations that decrease the toxicity of Clostridioides difficile. NPOD Annual Spring Research Retreat, April 19, 2022, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Chen T, Wang HC, Wang S, Zempleni J. Perilimilary of exploration of bovine milk exosome ncRNAs and its distribution in mouse. NPOD Annual Spring Research Retreat, April 19, 2022, Lincoln, NE

Progress 08/01/20 to 07/31/21

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral fellows, graduate students and undergraduate students, and a visiting scientist. One of the current doctoral students, Alice Ngu is a first-generation college student. Approximately 70% of the trainees are women, and the project has supported one Hispanic and two African-American students. All program graduates have secured positions in industry or research. How have the results been disseminated to communities of interest?Findings have been disseminated through presentations at invited seminars, international, national and local conferences, publications in peer-reviewed journals (see section Products), and collaborations with partners in industry (PureTech Health, Inc., see Patent; and Land O'Lakes, Inc.). What do you plan to do during the next reporting period to accomplish the goals?Objective 2 has been completed. We expect to complete the Objectives 1 and 3 (See "Preface" in "Accomplishments") by July 31, 2022. We have devised the following tentative timetable: Subject enrollment (ongoing): expected completion by July 31, 2021 Microbial cultures: expected completion by November, 15, 2021 Analysis of bacterial communities, transcriptome and metabolome: expected completion by May 31, 2022 Publication of research: expected completion by July 31, 2022 The manuscript regarding bacterial communities, their genetic variations and transcriptomics and metabolomics has been reviewed by Nature Communications and a revised manuscript will be considered for publication after the data generated during the no-cost extension have been added: Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (under revision)

Impacts
What was accomplished under these goals? Preface: In years 3 and 4 of funding, we made the important discovery that milk exosomes and their microRNA cargos alter bacterial communities in the mouse gut and select for genetic variants in gut bacteria in mice. This discovery has implications for objectives 1 (Assess direct markers of milk microRNA intake and status in humans) and 3 (Assess functional markers of milk microRNA intake and status in humans) in our project. The changes in the gut microbiome were associated with impaired cognitive performance in juvenile and adult mice. We have secured IRB approval to assess whether milk exosomes and their microRNA cargos also alter bacterial communities and select for genetic variants in gut bacteria in in infants and adults. These studies are important for completing objectives 1 and 3 and we expect to complete the studies during a one-year no cost extension that we were granted. Objective 1, Direct markers of milk microRNA nutrition: 1) We have devised a PCR protocol, RNase H2 PCR that allowed us to detect bovine microRNAs in human plasma after, but not before a milk meal. 2) We have completed a project, in which we assessed the effects of potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage). 3) We have developed a transgenic mouse model (Exosome and Cargo tracking (ECT mouse), which allowed us to demonstrate that milk exosomes are transferred from dams to pups through the natural route of suckling and accumulate in liver and brain. 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We have shown that the microbial mRNAs are bioavailable in mice and humans. 5) We have developed milk exosome and microRNA labeling protocols which enabled us to assess the bioavailability and distribution of milk exosomes and their microRNA cargos in mice and pigs. Exosomes accumulated primarily in liver and brain, whereas distinct microRNAs showed unique distribution patterns (brain vs. liver/spleen vs. intestinal mucosa vs. spleen). 6) We developed a protocol for sequencing microRNAs in exosomes purified from small volumes (0.5 mL) of human milk. The protocol enables us to explore the roles of exosomes and their microRNA cargos in preterm infants. 7) We developed a protocol to deplete bovine milk of exosomes and microRNAs by using microfluidization. This technical advance enables us to study the benefits of exosome-containing milk drinks in developing countries. 8) See above, Preface. Objective 2, Indirect markers of milk microRNA nutrition: 1) We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk. 2) We assessed effects of exosome- and RNA-defined diets on amino acid metabolism in mice. Effects were negligible and this line of research was abandoned. The studies were useful because they allowed us to demonstrate that our experimental diets, which we also used in our studies, do not alter food and water consumption and physical activity and did not affect liver and kidney health. 3) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We are in the process of assessing the bioavailability of these mRNAs in a human milk feeding study. Bovine mRNAs are excellent markers for milk intake because their sequence is specific for cows, i.e., they cannot be synthesized by humans. Objective 3, Functional markers of milk microRNA nutrition: 1) We have shown that microbial mRNAs in bovine milk exosomes bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. TLR activation by milk exosomes is a promising functional marker for milk exosome intake (10-fold activation compared to controls in TLR reporter cells). These observations are consistent with observations that mice fed an exosome and RNA-sufficient diet survived a challenge with influenza A, whereas mice fed an exosome and RNA-depleted diet died within 11 days of influenza A challenge. 2) We have identified phenotypes of dietary depletion of milk exosomes and microRNA cargos. The phenotypes include loss of fecundity, increased severity of symptoms of inflammatory bowel disease, increases susceptibility to challenges with influenza A virus and impaired cognitive performance in mice. Knockout of maternal exosome and microRNA biogenesis caused slow growth and leakiness of the gastrointestinal mucosa in wild-type pups fostered to the knockout dams. 3) See above, Preface.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J, Zhou F, Dogan H, Cui J. Divergence of gut bacteria through the selection of genetic variations by extracellular vesicles in milk. International Society for Extracellular Vesicles. Annual (virtual) conference, 5/18  5/21, 2021 [poster video presentation]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Khanam A, Yu J, Zempleni J. Loss of maternal microRNA biogenesis impairs gut health in wild-type pups fostered to Dicer knockout dams. Nutrition 2021 Conference (virtual), American Society for Nutrition; 6/7  6/10, 2021 [oral presentation]
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Wang S, Auchtung J, Zempleni J. Milk exosomes protect human microbiota associated-mice against Clostridioides difficile infection. Nutrition 2021 Conference (virtual), American Society for Nutrition; 6/7  6/10, 2021 [poster presentation]
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Ogunnaike M, Zempleni J. Bovine mammary alveolar Mac-T cells secrete exosomes with properties similar to bovine milk exosomes (BMEs). Nutrition 2021 Conference (virtual), American Society for Nutrition; 6/7  6/10, 2021 [poster presentation]
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. Keystone Virtual Symposia Optimizing Nutrition for Maternal, Newborn and Child Health, October 22, 2020 [ePoster]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. Keystone Virtual Symposia Optimizing Nutrition for Maternal, Newborn and Child Health, October 22, 2020 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Exosomes and microRNAs in maternal milk are important for growth and gut health during weaning in murine pups. Chapman University, CA. November 11, 2020 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. The role of milk exosomes and their RNA cargos in neonatal health. Life Span Diseases Mini Summit in the Child Health Research Institute, University of Nebraska Medical Center, November 13, 2020 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Resources in the Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules. Life Span Diseases Mini Summit in the Child Health Research Institute, University of Nebraska Medical Center, November 13, 2020 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health in neonate mice. American Society for Cell Biology Virtual, December 14-16, 2020 [ePoster]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J, Zhou F, Dogan H, Cui J. Divergence of gut bacteria through the selection of genetic variations by milk exosomes. Keystone Virtual Symposia The Microbiome: From Mother to Child, January 17-21, 2021 [video poster, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Invited presentation titled W-4002 progress report Zempleni lab: milk exosomes as part of the remote annual W-4002 Multistate group meeting; Ohio State University, Columbus, OH, January 27th, 2021 [talk]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Milk exosomes and their microRNA cargos: infants, gut and brain. Department of Nutritional Sciences, University of Michigan, MI. February 10, 2021 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Biological Activities of Natural Nanoparticles (Exosomes) in Milk. Department of Ophthalmology, Penn State Hershey Medical Center, Hershey, PA. February 17, 2021 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Novel bioactive compounds in milk: exosomes. Department of Animal Science, University of Nebraska-Lincoln, NE. April 6, 2021 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. NPODs transition from Phase I to Phase II. Rural Drug Addiction Research Center, University of Nebraska-Lincoln, NE. April 8, 2021 [talk, delivered by using Zoom]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Khanam A, Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake and clearance of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. NPOD 12th Annual Spring Research Retreat, April 14th, 2021, Lincoln, NE [video poster]
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Milk exosome-driven evolution of antibiotic-resistant gut pathogens. NIFA Program Directors Meeting, Kansas City, KS. May 4, 2021 [talk, delivered by using Zoom]
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Penberthy WT, Sadri M, Zempleni J. Chapter 23: Biotin. In: Present Knowledge in Nutrition. Marriott BP, Birt DF, Stallings VA, Yates AA (eds.), 11th edition. Elsevier & International Life Sciences Institute, London, UK, 2020, 289-303
• Type: Book Chapters Status: Other Year Published: 2020 Citation: Zempleni J. MicroRNAs and exosomes in human milk. In: Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules. McGuire S. (ed), Elsevier, Amsterdam, The Netherlands, 2020, 339-346
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. Milk exosomes and microRNAs cross the placenta and promote embryo survival in mice. Reproduction 160:501-509, 2020
• Type: Other Status: Published Year Published: 2021 Citation: Zempleni J. Letter to the editor (regarding Perspective: The Role of Human Breast-Milk Extracellular Vesicles in Child Health and Disease by D. OReilly et al., published in AN on 24 August 2020). Adv Nutr 12:280, 2021
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Sadri M, Wang H, Kuroishi T, Li Y, Zempleni J. Engineering of a conditional holocarboxylase synthetase knockout mouse (submitted)
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Sukreet S, Pereira Braga C, An TT, Adamec J, Cui J, Trible B, Zempleni J. Isolation of extracellular vesicles from byproducts of cheese making by tangential flow filtration yields heterogeneous fractions of nanoparticles (submitted)
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (submitted)
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Khanam A. Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake and clearance of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice (submitted)
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Zhao W, Zempleni J. Adipocyte-derived exosomes increase the growth of breast tumors in obese mice. Nutrition 2020 Conference, American Society for Nutrition; Seattle, WA, 5/30  6/2, 2020
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Mother-to-infant communication through milk exosomes: loss of maternal exosome and microRNA biogenesis impairs gut health in pups. EMBL Heidelberg. Inter-Organ Communication in Physiology and Disease. Heidelberg, Germany. March 15-18, 2020 [abstract accepted, meeting canceled due to the Corona virus pandemic]
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Mother-to-infant communication through milk exosomes: loss of maternal exosome and microRNA biogenesis impairs gut health in pups. International Society for Extracellular Vesicles, International Society for Extracellular Vesicles, Philadelphia, PA, May 20-24, 2020 [abstract accepted, meeting canceled due to the Corona virus pandemic]
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhou F, Zempleni J. Loss of maternal microRNA biogenesis impairs gut health in wild-type pups fostered to Dicer knockout dams. Nutrition 2020 Conference, American Society for Nutrition; Seattle, WA, 5/30  6/2, 2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. American Society for Exosomes and Microvesicles, November 17, 2020 (virtual meeting) [short talk, delivered by using Zoom]. Extracellular Vesicle and Circulating Nucleic Acid (EVCNA) Journal. 1:33-34, 2020

Progress 08/01/19 to 07/31/20

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral associates, graduate students and undergraduate students, two technologists and a visiting scientist. To date, students and postdoctoral associates supported by this project were recognized with 26 travel and research awards, including five national research awards sponsored by the American Society for Nutrition. How have the results been disseminated to communities of interest?Findings have been disseminated through presentations at invited seminars, international, national and local conferences, publications in peer-reviewed journals, and collaborations with partners in industry (PureTech Health, Inc., and Land O'Lakes, Inc.).Research discoveries were also shared with the general public through regional media events and national news outlets. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 We will complete our studies of microbial messenger RNAs as markers for milk nutrition. Aim 2 We will complete our studies of bovine messenger RNAs as markers for milk nutrition. We will assess the mechanisms by which purine metabolism is altered and whether altered purine metabolism affects cognitive performance. (Purines play crucial roles in spatial learning and memory.) Aim 3 We will complete our studies of the binding of microbial and bovine RNAs to TLRs, and their beneficial effects on enhancing resistance to viral challenge.

Impacts
What was accomplished under these goals? Accomplishments Aim 1, Direct markers of milk microRNA nutrition 1) We have devised a PCR protocol, RNase H2 PCR that allowed us to detect bovine microRNAs in human plasma after, but not before a milk meal. 2) We have completed a project, in which we assessed the effects of potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage). 3) We have developed a transgenic mouse model (Exosome and Cargo tracking (ECT mouse), which allowed us to demonstrate that milk exosomes are transferred from dams to pups through the natural route of suckling and accumulate in liver and brain. 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We have shown that the microbial mRNAs are bioavailable in mice and humans. 5) We have developed milk exosome and microRNA labeling protocols which enabled us to assess the bioavailability and distribution of milk exosomes and their microRNA cargos in mice and pigs. Exosomes accumulated primarily in liver and brain, whereas distinct microRNAs showed unique distribution patterns (brain vs. liver/spleen vs. intestinal mucosa vs. spleen). 6) We developed a protocol for sequencing microRNAs in exosomes purified from small volumes (0.5 mL) of human milk. The protocol enables us to explore the roles of exosomes and their microRNA cargos in preterm infants. 7) We developed a protocol to deplete bovine milk of exosomes and microRNAs by using microfluidization. This technical advance enables us to study the benefits of exosome-containing milk drinks in developing countries. Aim 2, Indirect markers of milk microRNA nutrition 1) We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk. 2) We assessed effects of exosome- and RNA-defined diets on amino acid metabolism in mice. Effects were negligible and this line of research was abandoned. The studies were useful, because they allowed us to demonstrate that our experimental diets, which we also used in our studies, do not alter food and water consumption and physical activity and did not affect liver and kidney health. 3) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We are in the process of assessing the bioavailability of these mRNAs in a human milk feeding study. Bovine mRNAs are excellent markers for milk intake because their sequence is specific for cows, i.e., they cannot be synthesized by humans. Aim 3, Functional markers of milk microRNA nutrition 1) We have shown that microbial mRNAs in bovine milk exosomes bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. TLR activation by milk exosomes is a promising functional marker for milk exosome intake (10-fold activation compared to controls in TLR reporter cells). These observations are consistent with observations that mice fed an exosome and RNA-sufficient diet survived a challenge with influenza A, whereas mice fed an exosome and RNA-depleted diet died within 11 days of influenza A challenge. 2) We have identified phenotypes of dietary depletion of milk exosomes and microRNA cargos. The phenotypes include loss of fecundity, increased severity of symptoms of inflammatory bowel disease, increases susceptibility to challenges wit influenza A virus and impaired cognitive performance in mice. Knockout of maternal exosome and microRNA biogenesis caused slow growth and leakiness of the gastrointestinal mucosa in wild-type pups fostered to the knockout dams. 3) We have shown that milk exosomes select for polymorphisms and mutations in the gut microbiome. We are studying whether the genetic changes have a consequence for the host metabolism.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mutai E, Ramer-Tait AE, Zempleni J. MicroRNAs in bovine milk exosomes are bioavailable in humans but do not elicit a robust pro-inflammatory cytokine response. BMC exRNA 2:2, 2020
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Liu, B, Lu Y, Chen X, Muthuraj, PG, L X, Pattabiraman M, Zempleni J, Kachman SD, Natarajan SK, Yu J. Protective role of shiitake mushroom-derived exosome-like nanoparticles in D-galactosamine and lipopolysaccharide-induced liver injury in mice. Nutrients 12:477, 2020
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Dogan H, Shu J, Xu Z, Cui J. Elucidation of Molecular Links between Obesity and Cancer through MicroRNA Regulation
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Shao D, Wang Y, Cui J,. Human Body Fluid Proteome: Quantitative Profiling and Computational Prediction. Briefings in Bioinformatics
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cao M, Li HQ, Zhao J, Cui J, Hu GH. Identification of age- and gender-associated long noncoding RNAs in the human brain with Alzheimer's disease. Neurobiology of Aging. 2019, 81:116-126
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Li HQ, Dogan H, Cui J#. A New Approach to Batch Effect Removal Based on Distribution Matching in Latent Space. IEEE International Conference on Bioinformatics and Biomedicine (BIBM) 2019. San Diego, CA, November. 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dogan H, Hakguder Z, Scott S, Cui J#. Unraveling MicroRNA-Gene Regulatory Networks in Breast Cancer with Integrative Directed and Undirected Graphical Models. BIBM 2019 ERLBD workshop
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ryan A. Grove, Deepak Madhavan, Cory H.T. Boone, Camila Pereira Braga, Zuzana Papackova, Hannah Kyllo, Kaeli Samson, Kristina Simeone, Timothy Simeone, Tomas Helikar, Corrine K. Hanson, Jiri Adamec: Aberrant energy metabolism and redox balance in seizure onset zones of epileptic patients, Journal of Proteomics, 2020, 103812, ISSN 1874-3919
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Leiferman A, Shu J, Upadhyaya B, Cui J, Zempleni J. Storage of extracellular vesicles in human milk, and microRNA profiles in human milk exosomes and infant formulas. J Pediatr Gastroenterol Nutr 69:235-238, 2019
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhou F, Paz HA, Shu J, Sadri M, Cui J, Fernando SC, Zempleni J. Dietary bovine milk exosomes and their RNA cargos elicit changes in bacterial communities in C57BL/6 mice. Am J. Physiol Gastrointest Liver Physiol 317:G618-G624, 2019
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wu D, Kittana H, Shu J, Kachman SD, Cui J, Ramer-Tait AE, Zempleni J. Dietary Depletion of milk exosomes and their microRNA cargos elicits a depletion of miR-200a-3p and elevated intestinal inflammation and CXCL9 expression in Mdr1a-/- mice. Curr Develop Nutr 3:122, 2019
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zeynep Hakguder, Chunxiao Liao, Jiang Shu, Kaiyue Pan, Cui J. Genome-Scale MicroRNA Target Prediction through Clustering with Dirichlet Process Mixture Model. BMC Genomics. 2018
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cui J, Shu, J. Circulating microRNA trafficking and regulation: computational principles and practice. Briefings in Bioinformatics. 2019 Aug 28
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Dogan H, Shu J, Xu Z, Cui J#. Elucidation of Molecular Links between Obesity and Cancer through MicroRNA Regulation
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Li HQ, Dogan H, Cui J#. A New Approach to Batch Effect Removal Based on Distribution Matching in Latent Space. IEEE International Conference on Bioinformatics and Biomedicine (BIBM) 2019. San Diego, CA, November. 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 10. Dogan H, Hakguder Z, Scott S, Cui J#. Unraveling MicroRNA-Gene Regulatory Networks in Breast Cancer with Integrative Directed and Undirected Graphical Models. BIBM 2019 ERLBD workshop
• Type: Other Status: Other Year Published: 2018 Citation: Zempleni J. Milk exosomes. Program Officials of the Gates Foundation. August 7, 2018 [delivered remotely by Zoom]
• Type: Other Status: Other Year Published: 2018 Citation: Zempleni J. Bovine milk exosomes: bioavailability, distribution and interactions with the gut microbiome. PureTech Health, Inc., Boston, MA, August 23, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J, Upadhyaya B, Xia M, Moriyama H, Ohtsuka M. Milk exosomes accumulate in the intestinal mucosa and peripheral tissues in wild-type pups nursed by exosome and cargo tracking dams. American Society for Exosomes and Microvesicles, Baltimore Marriott Waterfront Conference Center, Baltimore, MD, October 20-24, 2018
• Type: Other Status: Other Year Published: 2019 Citation: Zempleni J. UNL & PureTech Health, Inc: milk exosomes in drug delivery. Presentation to the NUtech Ventures Board of Directors, March 1, 2019, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Biological activities of milk exosomes and their RNA cargos. In the symposia titled Nutrition, Extracellular Vesicles and RNA Cargos, Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 9, 2019
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Sukreet S, Adamec J, Cui J, Zempleni J. Galactose and sialo-galactose modifications in glycoproteins on the surface of bovine milk exosome are essential for exosome uptake in non bovine species. UNL Spring Research Fair, Lincoln, NE, April 15, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhou F, Sadri M, Zempleni J. Impaired maternal milk exosomes and microRNAs biogenesis decrease gut health and food intake in mouse pups. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Shu J, Braga CP, Cui J, Adamec J, Zempleni J. Bovine mRNAs in cow's milk exosomes are bioavailable and translated into peptides in non-bovine species. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cui J. Unraveling exosome-enabled cancer signaling: An integrated genomic approach, Abstract in Proceedings of the American Association of Cancer Research (AACR), Atlanta, GA, March 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Shu J, Gao T, Cui J. Identification of Exosomal microRNA Motifs using Graph Algorithms. In: Nebraska Center for the Prevention of Obesity Diseases; 2017 Mar, Lincoln, NE
• Type: Book Chapters Status: Awaiting Publication Year Published: 2020 Citation: Penberthy WT, Sadri M, Zempleni J. Chapter 23: Biotin. In: Present Knowledge in Nutrition. Birt D (ed.), 11th edition. International Life Sciences Institute, Washington, DC
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Cui J. Bioinformatics Databases and Tools on Dietary microRNA. In The Handbook of Nutrition, Diet and Epigenetics. Edited by Vinood B. Patel and Victor R. Preedy. Springer, 2019
• Type: Book Chapters Status: Submitted Year Published: 2020 Citation: Voshall A, Cui, J. Functional discovery of non-coding RNA through computational inference. Epigenetics & Noncoding RNA section of Major Reference Work on Comprehensive Foodomics, Elsevier
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zempleni J, Sukreet S, Zhou F, Wu D, Mutai E. Milk-derived exosomes and metabolic regulation. Ann Rev Anim Biosci 7:245-262, 2019
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Casavale KO, Ahuja J, Wu X, Li Y, Quam J, Olson R, Pehrsson P, Allen L, Balentine D, Hanspal M, Hayward D, Hines EP, McClung JP, Perrine C, Brown Belfort M, Dallas D, German B, Kim J, McGuire M, McGuire M, Morrow A, Neville M, Nommsen-Rivers L, Rasmussen KM, Zempleni J, Lynch CJ. NIH workshop on human milk composition: summary and visions. Am J Clin Nutr 110:769-779, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Exosomes in milk. Grand Challenges Meeting, sponsored by the Gates Foundation, Berlin, Germany, October 15-18, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Biotin metabolism. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 2, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Pursuing a graduate education in US. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 2, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Obesity research in the United States. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 3, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Dietary exosomes and their RNA cargos as novel bioactive food compounds. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 5, 2018
• Type: Other Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Effects of milk exosomes and their RNA cargos on anti-viral response and postnatal growth as part of the annual W-4002 Multistate group meeting; UC-Davis, Davis, CA. February 7th, 2019
• Type: Other Status: Published Year Published: 2019 Citation: Zempleni J. Invited lecture titled Dietary exosomes in the graduate class Exosomes: Molecular Mechanisms and Biomedical Applications (ME 340.714) at Johns Hopkins University. Baltimore, MD, March 27th, 2019
• Type: Other Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Biological activities of milk exosomes and their RNA cargos across and within species boundaries in the Department of Biological Chemistry at Johns Hopkins University. Baltimore, MD, March 27th, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Zhang Y. Zempleni J, McCarthy JJ, Young KC, Roberts MD, Kavazis AN. Bovine Milk Exosome Depletion Affects Skeletal Muscle and Liver in Young Growing Rats. American College of Sports Medicine, Orlando, FL, May 28  June 1, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Sadri M, Zhou F. Knockout of maternal Tsg101 and Dicer impair gut health in suckling wild-type pups. American Society for Exosomes and Microvesicles, Asilomar, CA, October 6-10, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Milk exosomes. Session Exosomes & microRNA at the 7th International Conference on Food Factors, Kobe, Japan, December 4th, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Concluding Remarks. Session Exosomes & microRNA at the 7th International Conference on Food Factors, Kobe, Japan, December 4th, 2019
• Type: Other Status: Published Year Published: 2019 Citation: Cui J. A Next Step for Bioinformatics: Integrated Machine Learning and Stochastic Modeling for Understanding Disease-related Cell Signaling and Regulation. University of Florence, Italy, 10/2019
• Type: Other Status: Published Year Published: 2018 Citation: Cui J. Multi-omics integration towards understanding non-coding RNA regulation in human disease. The 3rd Midwest Bioinformatics conference, Columbia, MO, 04/11/2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Shu J, Cui J. A large-scale metagenomic analysis using OSG, Open Science Grid All Hands Meeting 2017, San Diego, CA, March 7, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Shu J, Cui J. MiRDR-OSG: MicroRNA dynamic regulation analysis utilizing open science grid. Workshop on High Throughput Computing in Bioinformatics and Biomedicine using Open Science Grid, BIBM 2017, Kansas City, MO, November 13, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Hakguder Z, Cui J. A New Statistical Model for Genome-Scale MicroRNA Target Prediction. IEEE International Conference on Bioinformatics and Biomedicine (BIBM) 2017, Kansas City, MO, November 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aditya Immaneni, Juan Cui, Glucose-Insulin Stand-Alone Simulation Tool Design and Implementation in OSG. BIBM BIOSG 2017 Workshop

Progress 08/01/18 to 07/31/19

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral fellows, graduate students and undergraduate students, and a visiting scientist. One of the doctoral students, Mahrou Sadri was selected a finalist at the annual meeting of the American Society for Nutrition's (ASN) Graduate Student Competition and the overall winner of the Emerging Leaders Competition, section Bioactive Food Compounds. Another student, Di Wu is a finalist in the Emerging Leaders Competition during ASN's Nutrition 2019 meeting in June of 2019. One of the undergraduate students, Alice Ngu is a first generation college student and graduated in December of 2018. She will begin her Ph.D. studies in August of 2019. How have the results been disseminated to communities of interest?Findings have been disseminated through invited presentations; at international, national and local conferences; publications in peer-reviewed journals (see section Products); and collaborations with partners in industry (PureTech Health, Inc., see Patent; and Land O'Lakes, Inc.). In addition, findings have been shared with the lay audience though publications in the New Zealand Herald and the Knowable Magazine (associated with Annual Reviews). What do you plan to do during the next reporting period to accomplish the goals?Aim 1 We intend to focus studies of RNAs on microbial and bovine messenger RNAs as markers for milk nutrition (see Aim 1, #4) and complete an ongoing milk feeding study in humans and mice. For example, we intend to assess the time courses of mRNAs in human plasma and murine tissues. We have leveraged our ECT mouse (see Aim 1, #3) to initiate collaborations with scientists from Harvard University, Purdue University, University of Pennsylvania, University of California-San Francisco, University of Nebraska-Lincoln and the Ottawa Heart Institute (Canada). Aim 2 Milk exosomes and their cargos triggered a robust change in purine metabolism in human and animal models (see Aim 2, #1). Purines play crucial roles in spatial learning and memory. We will assess the mechanisms by which purine metabolism is altered and whether altered purine metabolism affects cognitive performance. (Purines play crucial roles in spatial learning and memory.) We will identify the regions in the brain in which milk exosomes and their microRNA cargos accumulate. In the context of polymorphisms and mutations selected for in gut microbiota, we will assess the biological relevance of these genomic changes for the function of gut microbiota and the appearance of amino acid metabolites in host plasma and tissues. Aim 3 We will continue our studies of the binding of microbial and bovine RNAs to TLRs. These studies will include an assessment of the antiviral activities of microbial RNAs in mice challenged with influenza A. In addition, we will assess the binding of microbial and bovine RNAs to RNA-binding proteins, which might reveal new biological functions of milk exosomes and their RNA cargos.

Impacts
What was accomplished under these goals? Aim 1, Direct markers of milk microRNA nutrition 1) We have devised a PCR protocol, RNase H2 PCR that allowed us to detect bovine microRNAs in human plasma after, but not before a milk meal (Wang et al., published). 2) We have completed a project, in which we assessed the effects of potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage) (Wang et al., published). 3) We have developed a transgenic mouse model (Exosome and Cargo tracking (ECT mouse), which allowed us to demonstrate that milk exosomes are transferred from dams to pups through the natural route of suckling and accumulate in liver and brain (Manca et al., published; Upadhyaya et al., manuscript in preparation; 2 conference presentations by Upadhyaya et al.). 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We have shown that the microbial mRNAs are bioavailable in mice and are currently assessing the bioavailability in humans by using RNA-sequencing (Wu et al., 1 conference presentation; Wu et al., manuscript in preparation). 5) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We used mRNA-sequencing and RT-qPCR technologies to demonstrate that humans absorb microbial mRNA encapsulated in milk exosomes. We used fluorophore-labeled microbial mRNA to demonstrate that microbial mRNA accumulates primarily in the intestinal mucosa and liver in mice. Bovine mRNA might be an excellent marker for milk intake, because many of them cannot be synthesized by humans (Wu et al., 1 conference presentation; Wu et al., manuscript in preparation). 6) We have developed milk exosome and microRNA labeling protocols which enabled us to assess the bioavailability and distribution of milk exosomes and their microRNA cargos in mice and pigs. Exosomes accumulated primarily in liver and brain, whereas distinct microRNAs showed unique distribution patterns (brain vs. liver/spleen vs. intestinal mucosa vs. spleen) (Manca et al., published). Aim 2, Indirect markers of milk microRNA nutrition 1) We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk (Aguilar-Lozano et al., published). 2) We assessed effects of exosome- and RNA-defined diets on amino acid metabolism in mice and also had only moderate effects in rats (Leiferman et al., published; Parry HA et al., published). Effects were negligible and this line of research was abandoned. The studies were useful, because they allowed us to demonstrate that our experimental diets, which we also used in our other studies, do not alter food and water consumption and physical activity and did not affect liver and kidney health (Leiferman et al., published). 3) We conducted a metagenomics analysis of murine gut microbes cultured in the presence or absence of milk exosomes ex vivo. Both presence and absence of milk exosomes caused a selection of polymorphisms and mutations compared with reference genomes in gut bacteria. The pathways affected by these polymorphisms and mutations clustered in amino acid metabolism, suggesting that microbiome-derived amino acid metabolites might be indirect markers for milk exosomes intake (see Next Reporting Period). Aim 3, Functional markers of milk microRNA nutrition We have shown that microbial mRNAs in bovine milk exosomes bind to Toll-like receptors (TLRs) and also activate the anti-viral NF-kB pathway. TLR and NF-kB activation by milk exosomes show promise as functional marker for milk exosome intake (10-fold activation compared to controls in TLR reporter cells). These observations are consistent with observations that mice fed an exosome and RNA-sufficient diet survived a challenge with influenza A, whereas mice fed an exosome and RNA-depleted diet died within 11 days of influenza A challenge. It remains to be determined whether activation of TLR and NF-kB have any value as functional markers of milk microRNA nutrition (see below).

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Haun CT, Zhang Y, Roberson PA, Zempleni J, Ferrando AA, Vecchetti IJ Jr., McCarthy JJ, Young KC, Roberts MD, Kavazis AN. Bovine milk extracellular vesicles (EVs) modification elicits skeletal muscle growth in rats. Front Physiol 10:436, 2019
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Wu D, Kittana H, Shu J, Kachman SD, Cui J, Ramer-Tait AE, Zempleni J. Dietary Depletion of milk exosomes and their microRNA cargos elicits a depletion of miR-200a-3p and elevated intestinal inflammation and CXCL9 expression in Mdr1a-/- mice. (under revision for transfer from Journal of Nutrition to Current Developments in Nutrition)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Upadhyaya B, Xia M, Moriyama H, Ohtsuka M. Milk exosomes accumulate in the intestinal mucosa and peripheral tissues in wild-type pups nursed by exosome and cargo tracking dams. International Society for Extracellular Vesicles, International Society for Extracellular Vesicles, Miyakomesse, Kyoto, Japan, April 24-28, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Development of an exosome and cargo tracking mouse. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ebea P, Sukreet S, Zempleni J. Uptake of RNA cargos in bovine milk exosomes by murine brain endothelial cells. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by bone marrow derived macrophages. NPOD 4th Annual Symposium 2018, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sukreet S, Adamec J, Cui J, Zempleni J. Identification of Glycoproteins on the Surface of Cows Milk Exosomes that Mediate the Uptake of Exosomes into Human Cells. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhou F, Shu J, Fernando S, Cui J, Zempleni J. Bovine milk exosomes select polymorphisms in murine intestinal microbiome in vitro. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d and miR-21-5p cargos cross the placenta and contribute toward embryonic development and survival in C57BL/6 mice. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhao W, Zempleni J. The role of adipose derived-exosomes in breast tumorigenesis in obese mice. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Exosomes in milk. Grand Challenges Meeting, sponsored by the Gates Foundation, Berlin, Germany, October 15-18, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Biotin metabolism. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 2, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Pursuing a graduate education in US. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 2, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Obesity research in the United States. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 3, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Dietary exosomes and their RNA cargos as novel bioactive food compounds. Visiting Professor, University of Yogyakarta, Yogyakarta, Indonesia, November 5, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Effects of milk exosomes and their RNA cargos on anti-viral response and postnatal growth as part of the annual W-4002 Multistate group meeting; UC-Davis, Davis, CA. February 7th, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited lecture titled Dietary exosomes in the graduate class Exosomes: Molecular Mechanisms and Biomedical Applications (ME 340.714) at Johns Hopkins University. Baltimore, MD, March 27th, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Biological activities of milk exosomes and their RNA cargos across and within species boundaries in the Department of Biological Chemistry at Johns Hopkins University. Baltimore, MD, March 27th, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. Nebraska Spring Research Fair; Lincoln, NE, April 15, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow derived macrophages. Nebraska Spring Research Fair; Lincoln, NE, April 15, 2019
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ebea P, Sukreet S, Zempleni J, Bovine milk exosomes and miR-34a cargo cross the blood brain barrier and elimination by microglial accumulation is minimal. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Development of an exosome and cargo tracking mouse model. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Swanson B, Zempleni J, Nordgren T, Romberger D, Heires A. Bovine milk-derived exosomes enhance inflammation and promote M1 polarization following agricultural dust exposure in mice. J Nutr Biochem 3:110-120, 2018

Progress 08/01/17 to 07/31/18

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral fellows, graduate students and undergraduate students, and a visiting scientist. One of the doctoral students, Mahrou Sadri was selected a finalist at the annual meeting of the American Society for Nutrition's Graduate Student Competition and the overall winner of the Emerging Leaders Competition, section Bioactive Food Compounds. One of the undergraduate students, Alice Ngu is a first generation college student and will graduate in December of 2018. She has verbally committed to pursuing a doctoral degree, starting in May of 2019. How have the results been disseminated to communities of interest?Findings have been disseminated through presentations at invited seminars, international, national and local conferences, publications in peer-reviewed journals (see section Products), and collaborations with partners in industry (PureTech Health, Inc., see Patent; and Land O'Lakes, Inc. (pending)). What do you plan to do during the next reporting period to accomplish the goals?Aim 1 We intend to focus studies of RNAs on microbial and bovine messenger RNAs as markers for milk nutrition (see Aim 1, #4) and complete an ongoing milk feeding study in humans. The human feeding study assesses the bioavailability effects of immune-related microRNAs and their effects on cytokine production by human immune cells. We have leveraged our ECT mouse (see Aim 1, #3) to initiate collaborations with scientists from Harvard University, Purdue University, University of Pennsylvania, University of California-San Francisco, University of Nebraska-Lincoln and the Ottawa Heart Institute (Canada). Aim 2 Milk exosomes and their cargos triggered a robust change in purine metabolism in human and animal models (see Aim 2, #1). Purines play crucial roles in spatial learning and memory. We will assess the mechanisms by which purine metabolism is altered and whether altered purine metabolism affects cognitive performance. (Purines play crucial roles in spatial learning and memory.) Aim 3 We will continue our studies of the binding of microbial and bovine RNAs to TLRs. In addition, we will assess the binding of microbial and bovine RNAs to RNA-binding proteins, which might reveal new biological functions of milk exosomes and their RNA cargos.

Impacts
What was accomplished under these goals? Aim 1, Direct markers of milk microRNA nutrition 1) We have devised a PCR protocol, RNase H2 PCR that allowed us to detect bovine microRNAs in human plasma after, but not before a milk meal (Wang et al., published). 2) We have completed a project, in which we assessed the effects of potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage) (Wang et al., published). 3) We have developed a transgenic mouse model (Exosome and Cargo tracking (ECT mouse), which allowed us to demonstrate that milk exosomes are transferred from dams to pups through the natural route of suckling and accumulate in liver and brain (Manca et al., under revision; Upadhyaya et al., manuscript in preparation; 2 conference presentations by Upadhyaya et al.). 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We have shown that the microbial mRNAs are bioavailable in mice and are currently assessing the bioavailability in humans by using RNA-sequencing (Wu et al., 1 conference presentation). 5) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We are in the process of assessing the bioavailability of these mRNAs in a human milk feeding study. Bovine mRNA might be an excellent marker for milk intake, because many of them cannot be synthesized by humans (Wu et al., 1 conference presentation). 6) We have developed milk exosome and microRNA labeling protocols which enabled us to assess the bioavailability and distribution of milk exosomes and their microRNA cargos in mice and pigs. Exosomes accumulated primarily in liver and brain, whereas distinct microRNAs showed unique distribution patterns (brain vs. liver/spleen vs. intestinal mucosa vs. spleen) (Manca et al., under revision). Aim 2, Indirect markers of milk microRNA nutrition 1) We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk (Aguilar-Lozano et al., revision submitted). 2) We assessed effects of exosome- and RNA-defined diets on amino acid metabolism in mice. Effects were negligible and this line of research was abandoned. The studies were useful, because they allowed us to demonstrate that our experimental diets, which we also used in our studies, do not alter food and water consumption and physical activity and did not affect liver and kidney health (Leiferman et al., in press). Aim 3, Functional markers of milk microRNA nutrition We have shown that microbial mRNAs in bovine milk exosomes bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. TLR activation by milk exosomes is a promising functional marker for milk exosome intake (10-fold activation compared to controls in TLR reporter cells). These observations are consistent with observations that mice fed an exosome and RNA-sufficient diet survived a challenge with influenza A, whereas mice fed an exosome and RNA-depleted diet died within 11 days of influenza A challenge. We are assessing whether regular consumption of dairy by healthy adults alters the response of human peripheral blood mononuclear cells (PBMC) ex vivo compared to dairy avoiders. It would be premature to draw preliminary conclusions as to whether this assay will be useful to assess milk exosome and RNA intake.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang L, Sadri M, Giraud D, Zempleni J. RNase H2-dependent PCR and elimination of confounders in sample collection, storage and analysis strengthen evidence that microRNAs in bovine milk are bioavailable in humans. J Nutr 148:153-159, 2018
• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins. J Nutr Biochem
• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. J Nutr Biochem
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Fratantonio D, Shu J, Howard K, Baier SR< Giraud D, Cui J, Zempleni.MicroRNAs from chicken eggs are bioavailable and alter gene expression in peripheral blood mononuclear cells in humans
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Zhou F, Paz HA, Shu J, Sadri M, Cui J, Fernando SC, Zempleni J. Dietary bovine milk exosomes and their RNA cargos elicit changes in the composition of the intestinal microbiome and the hepatic transcriptome in C57BL/6 mice
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Aguilar-Lozano A, Baier SR, Grove R, Shu J, Giraud D, Mercer KE, Cui J, Badger TM, Adamec J, Andres A, Zempleni J. Concentrations of purine metabolites are elevated in human fluids from adults and infants and in livers from mice fed diets depleted of bovine milk exosomes and their RNA cargos
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Manca S, Upadhyaya B, Mutai E, Desaulniers AT, Cederberg RA, White BR, Zempleni J. Milk exosomes are bioavailable and distinct microRNA cargos have unique distribution patterns
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Cui J. Zempleni J. RNase H2-dependent PCR Detects Bovine MicroRNAs in Human Plasma. [reply: letter to the editor]
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J,* Zhou F, Wu D, Shu J, Paz H, Cui J, Fernando S. The communication of animal and bacterial kingdoms through exosomes and their RNA cargos in bovine milk. 22. International Conference. Functional Foods Center, September 22-23, 2017, Harvard Medical Center, Boston, MA
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fratanotonio D,* Shu J, Cui J, Zempleni J. MicroRNAs in chicken egg exosomes: content and bioavailability in healthy humans. 22. International Conference. Functional Foods Center, September 22-23, 2017, Harvard Medical Center, Boston, MA. *Presenter
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Bioavailability, distribution and phenotypes of bovine milk exosomes in non-bovine species. The American Dairy Science Association, Annual Meeting, Knoxville, TN, June 24-27, 2018 (presentation date 6/25/2018)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zempleni J, Aguilar-Lozano A, Sadri M, Manca S, Wu D, Zhou F, Mutai E, Sukreet S. Biological activities of extracellular vesicles and their cargos from bovine and human milk in humans and implications for infants. J Nutr 147:3-10, 2017
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zempleni J. Milk exosomes: beyond dietary microRNAs. Nutr Genes 12:12, 2017
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Zempleni J, Sukreet S, Zhou F, Wu D, Mutai E. Milk-derived exosomes and metabolic regulation. Ann Rev Anim Biosci
• Type: Book Chapters Status: Submitted Year Published: 2018 Citation: Zempleni J. MicroRNAs and exosomes in human milk. In: Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules. McGuire S. (ed), Elsevier
• Type: Other Status: Published Year Published: 2017 Citation: Zempleni J. Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules. Agricultural Research Division at the University of Nebraska-Lincoln, August 11, 2017
• Type: Other Status: Published Year Published: 2017 Citation: Zempleni J. Invited seminar titled Biological activities of bovine milk exosomes and their RNA cargos in non-bovine species. University of Nebraska Medical Center, Department of Cellular and Integrative Physiology, Omaha, NE, September 1, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu D, Upadhyaya B, Shu J, Paz H, Fernando S, Cui J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. Opening ceremony speaker. American Society for Exosomes and Microvesicles, Asilomar Conference Center, Pacific Beach, CA, October 8-12, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fratantonio D, Zempleni J. MicroRNAs in chicken egg exosomes are bioavailable in humans and contribute toward spatial learning and memory in mice. American Society for Exosomes and Microvesicles, Asilomar Conference Center, Pacific Beach, CA, October 8-12, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hailey A. Parry1, C. Brooks Mobley1, Petey W. Mumford1, Matthew A. Romero, Yufeng Zhang, Janos Zempleni, Kaelin C. Young, Michael D. Roberts, and Andreas N. Kavazis. Dairy Exosome Effects on Mitochondria Function and Antioxidant Enzymes in Growing Male and Female Rats. Experimental Biology 2018, April 21-25, 2018, San Diego, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Nordgren TM, Zempleni J, Heires AJ, Romberger DJ. Dietary exosomes amplify the inflammatory effects of agricultural dust exposure in mice. 2018 American Thoracic Society International conference, May, 18-23, 2018, San Diego, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargo cross the placenta and contribute toward embryo development in C57BL/6 mice. Nutrition 2018 Conference, American Society for Nutrition; Boston, MA, June 9-12, 2018
• Type: Other Status: Published Year Published: 2017 Citation: Zempleni J, Sadri M, Mutai E, Manca S. The bioavailability and distribution of exosomes and their RNA cargos from bovine and porcine milk in mice. 9th exRNA Communications Consortium Investigators Meeting. Rockville, MD. November 6/7, 2017
• Type: Other Status: Other Year Published: 2017 Citation: Fratantonio D, Zempleni J. Chicken egg exosomes and their cargos are bioavailable and dietary depletion affects spatial learning and memory and gene expression in the hippocampus in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Sadri M, Mutai E, Zempleni J. Analysis of plasma by RNase H2 PCR provides evidence that microRNAs in bovine milk are bioavailable in humans. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Manca S, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Sukreet S, Silva-Resende B, Wu D, Shu J, Adamac J, Cui J, Zempleni J. Sonication and short-term incubation causes a specific loss of RNA cargos in bovine milk exosome. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos affects gene expression but not amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Mutai E, Ramer-Tait A, Zempleni J. Immunomodulatory microRNAs in bovine milk are bioavailable and synergize with concanavalin A in the stimulation of cytokine secretion by peripheral blood mononuclear cells ex vivo in humans. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
• Type: Other Status: Published Year Published: 2017 Citation: Wu D, Shu J, Upadhyaya B, Cui J, Zempleni J. Bioavailability of microbial RNAs in bovine milk exosomes in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017
• Type: Other Status: Published Year Published: 2017 Citation: Opening ceremony speaker at the meeting of the American Society for Exosomes and Microvesicles. Zempleni J, Zhou F, Wu D, Upadhyaya B, Shu J, Paz H, Fernando S, Cui J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. Asilomar Conference Center, Pacific Beach, CA, October 8-12, 2017
• Type: Other Status: Published Year Published: 2017 Citation: Invited presentation Exosomes and RNA cargos in human milk at the NIH-sponsored workshop titled Workshop on Human Milk Composition-Biological, Environmental, Nutritional, and Methodological Considerations Meeting. Bethesda, MD, November 16-17, 2017
• Type: Other Status: Published Year Published: 2017 Citation: Wu D, Shu J, Upadhyaya B, Cui J, Zempleni J. Bioavailability of microbial RNAs in bovine milk exosomes in mice. University of Nebraska, Fall 2017 Research Fair, November 8, 2017
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. W-3002 Multistate group meeting. February 8/9, 2018,Tucson, AZ
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Cross-kingdom communication: bovine milk exosomes talk to the gut microbiome talk to the host. Invited seminar in the Interdepartmental Nutrition seminar series. Speaker chosen by the Nutrition Science Graduate Student Organization as Spring Seminar 2018 Speaker. Purdue University, Lafayette, IN, March 9, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait AE, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
• Type: Other Status: Published Year Published: 2018 Citation: Sukreet S, Silva B, Wu Di, Shu J, Adamec J, Cui J, Zempleni J. Sonication and short term incubation causes specific cargos loss in bovine milk exosomes. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
• Type: Other Status: Published Year Published: 2018 Citation: Zhou F, Paz H, Fernando S, Sadri M, Zempleni J. A diet defined by its content of bovine milk exosomes alters the composition of the intestinal microbiome in C57BL/6 mice. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
• Type: Other Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargos cross the placenta contribute toward embryonic development and survival in C57BL/6 mice. UNL Research Fair, April 10, 2018, Lincoln, NE
• Type: Other Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait AE, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Fratantonio D, Shu J, Cui J and Zempleni J. Chicken egg exosomes and their cargo are bioavailable and dietary depletion affects the hippocampus gene expression in mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by bone marrow derived macrophages. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Sukreet S, Silva B, Wu Di, Shu J, Adamec J, Cui J, Zempleni J. Sonication and incubation of milk causes a moderate loss of exosomes and substantial changes in exosomal RNA, lipid and protein cargos. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Mutai E, Ramer-Tait A, Zempleni J. Bioavailability of immunomodulatory microRNAs in bovine milk and cytokine secretion by peripheral blood mononuclear cells ex vivo in humans. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J, Zhou F, Shu J, Wu D, Upadhyaya B, Cui J, Paz H, Fernando S. Milk exosomes interface with the microbiome. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
• Type: Other Status: Published Year Published: 2018 Citation: Zhou F, Giraud D, Wu D, Brown DM, Zempleni J. Microbial mRNAs in bovine milk exosomes activate interferon-beta in mice. Nebraska Center for the Prevention of Obesity Diseases, April 11th, 2018, Lincoln, NE. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Generation of transgenic exosome and cargo tracking mice. NPOD 10th Annual Retreat, April 11, 2018, Lincoln, NE
• Type: Other Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargos cross the placenta contribute toward embryonic development and survival in C57BL/6 mice. NPOD 10th Annual Retreat, April 11, 2018, Lincoln, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait A, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Exosome and cargo tracking mice. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
• Type: Other Status: Published Year Published: 2018 Citation: Ebea P, Sukreet S, Zempleni J. Uptake of RNA cargos in bovine milk exosomes by murine brain endothelial cells. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
• Type: Other Status: Published Year Published: 2018 Citation: Zempleni J, Wu D, Ramer-Tait A. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. NIFA Program Directors meeting; Boston, MA, June 9th, 2018
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Zhang Y. Zempleni J, Young KC, Roberts MD, Kavazis AN. Dietary Exosomes Affect Growth and Skeletal Muscle Physiology in Young Male and Female Rats. American College of Sports Medicine, San Diego, CA, September 5-8. 2018

Progress 08/01/16 to 07/31/17

Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in biomedical science and dairy production and processing, policy makers, pharmaceutical industry, journalists and lay public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of postdoctoral fellows, graduate students and undergraduate students, and a visiting scientist. How have the results been disseminated to communities of interest?Findings have been disseminated through presentations at invited seminars, international, national and local conferences, publications in peer-reviewed journals, and collaborations with partners in industry. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 We intend to focus studies of RNAs on microbial and bovine messenger RNAs as markers for milk nutrition. We have started a new collaboration with Dr. Robert Raffai (UC San Francisco) and will explore whether lipoproteins are carriers of RNAs in human plasma/ Aim 2 We intend to explore whether metabolites other than purines are robust markers for milk intake. Studies of amino acid flux are currently in progress. We have obtained two plasmids that will allow us to track the trafficking of milk exosomes in mice, and will assess the usefulness of this approach to this project's overarching theme. Aim 3 We will continue our studies of the binding of microbial and bovine RNAs to TLRs. In addition, we will assess the binding of microbial and bovine RNAs to RNA-binding proteins, which might reveal new biological functions of milk exosomes and their RNA cargos.

Impacts
What was accomplished under these goals? Aim 1, Direct markers of milk microRNA nutrition 1) RNase H2 PCR: The vast majority of bovine and human microRNAs have an identical nucleotide sequence, or sequences differ by as little as one nucleotide. This makes it extremely difficult to distinguish microRNAs obtained through endogenous synthesis and dietary sources (bovine milk). We have devised a PCR-based protocol that allows us to distinguish microRNAs for which the nucleotide sequence differs by as little as on nucleotide, RNase H2 PCR. Using this protocol we detected bovine microRNAs in human plasma after, but not before, a milk meal. 2) We have completed a project, in which we assessed the effects of potential confounders of microRNA analysis in plasma (sample collection, hemolysis, circadian rhythm, storage). 3) We have succeeded developing an antibody specific for a protein (casein) in exosomes from bovine milk. We are currently testing whether the antibody will allow us to detect bovine exosomes in human plasma. We have devised the following back-up plan, in case the anti-casein strategy failed. We have identified hundreds of proteins on the surface of bovine milk exosomes by mass spectrometry, and will develop antibodies to the most abundant protein species. 4) Using RNA sequencing, we have discovered that about 20% of the messenger RNAs in bovine milk exosomes are of microbial origin. We are in the process of assessing the bioavailability of these microbial mRNAs in a human milk feeding study. Microbial mRNA might be an excellent marker for milk intake, particularly mRNAs from microbes abundant in milk (Pseudomonas fluorescens). 5) Using RNA sequencing, we have identified about 3600 bovine messenger RNAs in bovine milk exosomes. We are in the process of assessing the bioavailability of these mRNAs in a human milk feeding study. Bovine mRNA might be an excellent marker for milk intake, because many of them cannot be synthesized by humans. Aim 2, Indirect markers of milk microRNA nutrition We have identified a robust indirect marker of milk exosome and microRNA intake, purines and their metabolites. Studies in animals, human infants (breastfed vs. formula) and healthy adults (dairy consumers v. dairy avoiders) consistently showed elevated levels of purines in subjects with a low intake of milk. Aim 3, Functional markers of milk microRNA nutrition We have shown that microRNAs in bovine milk bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. We are currently testing the activation of TLRs by milk microRNAs in TLR reporter cell lines and TLR reporter mice. The latter is done in conjunction with an influenza A virus challenge of mice to determine whether pro-inflammatory effects of milk, mediated by binding to TLRs (unpublished), has beneficial effects regarding anti-viral response.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zempleni J, Wu D, Shu J, Zhou F, Sadri M, Jarecke J, Fernando S, Cui J, Adamec J. Non-canonical pathways of signaling and metabolic regulation by extracellular vesicles from cows milk. American Society for Exosomes and Microvesicles, Asilomar Conference Center, Pacific Beach, CA, October 20-24, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu D, Manca S, Sadri M, Fernando S, Paz H, Shu J, Cui J. Bovine milk exosomes and their cargos may regulate metabolism through non-canonical pathways in non-bovine species. 2017 ISEV (International Society for Extracellular Vesicles) Workshop on Diet, Environment and Extracellular Vesicles, January 27, 2017, La Trobe University, Melbourne, Australia
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Debate #1: Are dietary RNA/EVs detected in circulation? Do they have any functional role? Pros and cons. 2017 ISEV (International Society for Extracellular Vesicles) Workshop on Diet, Environment and Extracellular Vesicles, January 28, 2017, La Trobe University, Melbourne, Australia
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Leiferman AL, Aguilar A, Mutai E, Adamec J, Zempleni J. Dietary depletion of bovine milk exosomes elicits changes in amino acid metabolism in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhou F, Paz AH, Sadri M, Fernando CS, Zempleni J. A diet defined by its content of bovine milk exosomes alters the composition of the intestinal microbiome in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manca S, Giraud D, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mutai E, Zhou F, Zempleni J. Depletion of dietary bovine milk exosomes impairs sensorimotor gating and spatial learning in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sukreet S, Zhang H, Adamec J, Cui J, Zempleni J. Identification of glycoproteins on the surface of bovine milk exosomes and intestinal cells that facilitate exosome uptake in human colon carcinoma Caco-2 cells. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Belak EE, Nordgren TM, Heires AJ, Zempleni J, Romberger DJ. Serum exosomes modulate monocyte and macrophage inflammatory responses. American Thoracic Society, May 2017, Washington, DC
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu, D, Manca S, Sadri M, Fernando S, Paz H, Shu J, Cui J. Bovine milk exosomes and their cargos may regulate metabolism through non-canonical pathways in non-bovine species, San Diego, CA, March 25/26, 2017. Abstract published in: Functional Foods and bioactive compounds in health and disease. Martirosyan D (ed.), Volume 21, Proceedings of the 21st International Conference, Food Science Publisher, Dallas, TX, p. 144-145
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manca S, Zhou F, Sadri M, Shu J, Jarecke J, Aguilar-Lozano A, Leiferman A, Grove R, Paz H, Adamec J, Fernando S, Cui J, Zempleni J.* Biological activities of extracellular vesicles and their cargos from bovine milk in non-bovine species. International Society for Extracellular Vesicles, Toronto, Canada, May 18-21, 2017. *Presenter
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zempleni J, Aguilar-Lozano A, Sadri M, Manca S, Wu D, Zhou F, Mutai E, Sukreet S. Biological activities of extracellular vesicles and their cargos from bovine and human milk in humans and implications for infants. J Nutr 147:3-10, 2017
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Zempleni J. Milk exosomes: beyond dietary microRNAs. Nutr Genes (in press)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cui J, Zhou B, Ross S, Zempleni J. Nutrition, microRNAs and human health. Minireview based on the symposium Nutrition, microRNAs and human health held 5 April 2016 at the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2016 in San Diego, CA. The symposium was sponsored by the American Society for Nutrition (ASN), and the ASN Nutrition Science Council. Adv Nutr 8:105-112, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zempleni J. Delivery of functional RNA cargos by dietary exosomes from cows milk in C57BL/6 mice. 6th International Conference of Genomics and Pharmacogenomics, September 12-14, 2016, Berlin, Germany; organized by omics International
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Wu D, Shu J, Grove R, Boone C, Cui J, Adamec J, Zempleni J. mRNA cargos in cows milk exosomes are templates for translation. Nebraska Center for the Prevention of Obesity Diseases. Lincoln, NE, September 21th, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhou F, Mutai E, Sadri M, Fernando S, Zempleni J. Bovine milk exosome depletion alters the gut microbiome and behavior in mice. Nebraska Center for the Prevention of Obesity Diseases. Lincoln, NE, September 21th, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Aguilar-Lozano AG, Baier S, Adamec J, Sadri M, Giraud D, Zempleni J. Depletion of dietary microRNAs from cows milk causes an increase of purine metabolites in human body fluids and mouse livers. Nebraska Center for the Prevention of Obesity Diseases. Lincoln, NE, September 21th, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mutai E, Ramer-Tait A, Zempleni J. Effects of extracellular vesicles and their cargos from cow's milk on the release of cytokines by human peripheral blood mononuclear cells ex vitro. Nebraska Center for the Prevention of Obesity Diseases. Lincoln, NE, September 21th, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sukreet S, Zhang H, Adamec J, Cui J, Zempleni J. Identification of glycoproteins on the surface of cows milk exosomes that mediate the uptake of exosomes into human colon carcinoma caco-2 cells. Nebraska Center for the Prevention of Obesity Diseases. Lincoln, NE, September 21th, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Non-canonical pathways of signaling and metabolic regulation by extracellular vesicles from bovine milk. University of Nebraska Medical Center, Department of Internal Medicine; March 9, 2017, Omaha, NE
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Leiferman AL, Aguilar A, Grove R, Shu J, Cui J, Adamec J, Zempleni J. Dietary depletion of bovine milk exosomes elicits changes in amino acid metabolism in C57BL/6 mice. UNL Spring Research Fair; Lincoln, NE, April 5, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mutai E, Ramer-Tait A, Zempleni J. Effects of extracellular vesicles and their cargos from cow's milk on the release of cytokines by human peripheral blood mononuclear cells ex vitro. UNL Spring Research Fair; Lincoln, NE, April 5, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Non-canonical pathways of signaling and metabolic regulation by extracellular vesicles from bovine milk. Baylor University College of Medicine & Nutrition Research Center; April 6, 2017, Houston, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Are dietary RNAs bioavailable? RNA Group of Houston, Baylor University College of Medicine, UT Southwestern, University of Houston, and MD Anderson; April 6, 2017, Houston, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Invited presentation titled Biological activities of bovine milk exosomes and their cargos in non-bovine species as part of the annual W-3002 Multistate group meeting; Oregon State University, Portland, OR. May 23th, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Molecular signatures of exosomes and their RNA cargos from bovine milk in humans and mice. NIFA Program Directors meeting; Las Vegas, NV. June 24th, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Bioavailability of immune-relevant RNAs from bovine milk exosomes and its implications for pro-inflammatory responses in humans and mice. NIFA Program Directors meeting; Las Vegas, NV. June 24th, 2017