Item No. 1 of 1

INVESTIGATOR: Farnell, Y. Z.

PERFORMING INSTITUTION:
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

THE ULTRACENTRIFUGE SYSTEM: A CRITICAL TOOL TO ENHANCE THE PERFORMANCE OF AGRICULTURAL RELATED RESEARCH

NON-TECHNICAL SUMMARY: Researchers in agriculture-related fields often must work not only with plant and animal cells, but also with the biological structures and chemical components within those cells. Ultracentrifugation is a fundamental and widely used tool to separate these tiny structures both from the cells themselves and from each other according a property called differential density. This way, as the centrifuge spins a liquid, it will force different structures with different density to either stay in the liquid or sediment at the bottom of the tube into what's known as a pellet. Ultracentrifugation provides a simple but versatile method to isolate structures of interest, like extracellular vesicles (exosomes), viruses, subcellular organelles, and large pieces of DNA, making it an essential platform to isolate specific molecules as well as quantify them. This proposal seeks funds to help support replacement of an aged and mechanically unsound ultracentrifuge used by a diverse group of researchers. Projects that will benefit from the replacement of the ultracentrifuge range from studies of human metabolic disorders to cattle and poultry diseases to the creation of drought-resistant crops. Obtaining a state-of-the-art ultracentrifuge system (in this case, the proposed Beckman Optima XPN ultracentrifuge) will have an immediate and profound impact on current and planned research into a variety of important agricultural problems. The device will improve routine analyses of small RNAs and proteins associated with exosomes, molecular markers reflective of plant and animal health, and pathogens of livestock and crops. Replacement of the current ultracentrifuge, which is increasingly unreliable and unserviceable, will also reduce a significant bottleneck that limits the rate of progress on several important projects. Lastly, it will allow these research teams to explore new approaches and projects that require a high-quality method of ultracentrifugation.

OBJECTIVES: Project #1. Identification of the exosomal biomarkers for cardiovascular disease in a porcine model with the metabolic syndrome.1) to purify exosomes from conditioned media of primary cardiac fibroblasts from both male MetS/diabetic pigs and nondiabetic control littermates; 2) to examine their characteristics by transmission electron microscope and Western Blot analysis; and 3) to compare the differences of exosomal protein profiles from cardiac fibroblasts of diabetic and nondiabetic pigs by mass spectrometric analysis.Project #2. Development of Molecular Markers to Determine the Sex of a Chick Embryo. 1) To see if there are exosomes present in amniotic fluid of a chick embryo; 2) Identify exosomse by their phenotypes and biochemical composions.

APPROACH: General Methods: We are going to isolate and purify exosomes by differential centrifugtion method. for isolation of exosomes. The ultracentriguge supported by this grant (Beckman ultracentrifuge Optima XPN 90 - IVD with SW-32TI and SW-55TI rotors) will be extensively utilized. The phenotype of purified exosomes will be confirmed by transmission electron microscope. Western blot analysis will be performed by standard exosomal protein markers such as CD63, Tsg101 and HSP 70. Hopefully, the preliminary data supported by this grant will be used to compete future federally-funded proposal, such as US Poultry & Egg Association, USDA Agriculture Food Safety Research Initiative Exploratory grant and Mississippi Poultry Association.Efforts: I will be invited as a guest speaker at Molecular Genetics (BCH 8643) from Department of Biochemistry and Molecular Biolgy to talk about the extracellular vesicles (exosomes). Most of students are not familier with this knowledge. Undergradaute students will be recruited as direct individual study in exosome reserach.Evaluation-plan/steps for swine research: Data generated with the help of this equipement grant will aid in competing for the upcoming Grant-in-aid from (Greater Southeast Affiliate) American Heart Association, Institute Academic Research Enhancement Awards form National Heart, Lung, and Blood (NIH NHLBI R15), and Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Animal Species (USDA and NIH).Evaluation-plan/steps for chicken research: This will be the first report of exosome from amniotic fluid of chick embyro. I am using my start-up funding to get the preliminary data. With the help of USDA AFRI equipment grant, the data collected from Method section above will be published in peer review journal such as Poultry Science. Then I will apply USDA AFRI Seed grant in Spring 2016.

PROGRESS: 2016/03 TO 2017/02
Target Audience:The information and research result produced with this grant will be beneficial for private and public sectors. Changes/Problems:I have been relocated to Department of Poultry Science at Texas A&M University sinceAugust 1, 2016, and do not know if I will have the access to Reeport System under my MSU account (yf70@msstate.edu), which may not exist in Feb. 2017. Please contact me through yfarnell@tamu.edu and (my office phone) 979-847-7346 if needed. What opportunities for training and professional development has the project provided?Two undergradute students, Kellie Mitchell and Tina Trans from Biochemistry Departement,were beneficial from these project. They have graduated and are in medical and pharmaceutical school, respectively. Dan Zhao has been trained and was benificial from this funding support. She graduated from MS in Spring 2016, and right now is pursuing her Ph. D at Texas A&M University. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2016/03 TO 2017/02
What was accomplished under these goals? USDA Equipment Grant has supported us with the purchase of ultracentrifugation system that can be used to isolatenano-size extracellular vesicles, exosome. With the help of this support, we have made several progress in severalareas related to project 1 and 2, as outline below. PI: Dr. Yuhua Farnell Aug, 2013- July 2016, Mississippi State University Aug. 2016 - present, Texas A&M University Project 1: Identification of the exosomal biomarkers for cardiovascular disease in a porcine model with the metabolicsyndrome Impact: The knowledge gained from our experiments will improve our comprehension of intercellular communication indiabetic hearts, and assist in biomarker discovery. Such a test could help save potentially hundreds of thousands or evenmillions of lives, particularly in the most at risk populations, by allowing treatment of heart disease before lives are at risk - orworse, lost. Results and Outcomes (due to the length of the animal treatment, we are still processing samples until the end of this year): 1. Animal Care and Sample Collection. This project is in collaboration with Dr. Michael Sturek from Dept. of Cellular &Integrative Physiol., Indiana University School of Medicine. A total of 10 Ossabaw pigs (6-month-old) were assigned to twodiet groups for 6 or 8 months. Lean control swine will receive a daily diet (standard chow) with a total caloric content of 2200kcal. The MetS/diabetic group will receive a high-energy diet of 6000 kcal/day as described previously. Body weight wasmonitored weekly. The blood pressure, blood glucose, glucose tolerance, and insulin sensitivity testing were performed.Serum and fresh hearts were collected and shipped via Fedx. 2. Isolation, Culture and Conditioned Media of Cardiac Fibroblast. Primary fibroblasts were collected from control leanand MetS pig hearts. Briefly, after collection, hearts were rinsed in saline solution, minced and digested with collagenase.Conditioned media from primary cells will then be collected, centrifuged at 2000 x g for 20 min, and filtered with 0.22 µmvacuum-driven filtration system. Samples were stored at -80ºC 3. Exosome Isolation. Exosome from conditioned media (b) and serum were purified by differential ultracentrifugation.The resulting exosome pellet was re-suspended in cold PBS. 4. Transmission Electron Microscopy (TEM) and The NS 300 nanoparticle characterization system. The phenotypeof exosomes was identified through TEM. Their sizes were in the range from 50-10 nm. The concentration of exosome inserum from MetS were three times higher than those from controls. 5. Western Blot Analysis. Western blot analysis confirmed the presence of CD63 protein and heat shock protein 70(HSP70), two well-characterized exosomal protein markers Project 2: Development of Molecular Markers to Determine the Sex of a Chick Embryo Impact: Current methods of determining gender are slow, and are somewhat inaccurate and labor intensive. Incubating eggsand hatching chicks that will have to eventually be euthanized is a significant cost to the integrator. Therefore, developing ameans to determine the sex of a chicken embryo in ovo could significantly reduce industry costs. Results and Outcomes: 1. We have found the presence of exosomes in amniotic fluid of a chick embryo on Day 17. 2. We are in the process to characterize the exosomes from male and female chicks.

PUBLICATIONS (not previously reported): 2016/03 TO 2017/02
1. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A provisional abstract book of ISEV 2016. the 5th Annual Meeting of International Society of Extracellular Vesicles; 2016May 06; Rotterdam, The Netherlands. http://dx.doi.org/10.3402/jev.v5.31552: Journal of Extracellular Vesicles 2016, 5:31552; c2016. Comparative analyses of differential miRNA expression profiles between extracellular and intracellular components ofSCN2.2 cells. Farnell YZ, Zhao D, Hsu C, Arick T. Suprachasmatic nuclei cells (SCN2.2) are crucial in mammalian clock pacemaker, and secret soluble factors that drive oscillation of peripheral tissues or cells. The mechanisms of the synchronization are not fully understand, however, extracellular vesicle-mediated communication has been implicated in the intercellular transferring of miRNAs in circadian clock. Thus, this study explored the miRNA expression profile between extracellular matrix, including SCN2.2cell-derived exosome and exosome-dep
2. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: The 80th Mississippi Academy of Sciences Annual Meeting, February 18-19, 2016, Hattiesburg, MS 39402. (*Ms. Mitchell, undergraduate student, has won the 1st place for undergraduate research section) Characterization of exosomes from cardiac fibroblasts in Ossabaw pigs with metabolic syndrome. Mitchell, K*., Stewart, J., Zhao, D., Koniaris, L., Sturek, M., Alloosh, M., and Farnell, Y. Cardiovascular disease (CVD) is the leading cause of death worldwide, and patients with metabolic syndrome (MetS) are at higher risk to develop CVD. However, there is no current method to detect CVD and many MetS patients are unaware of their illnesses until advanced complications like heart attacks and strokes occur. A biomarker for CVD in diabetic patients could aid in early diagnostics and treatment of CVD and prevent the complications. In this study we searched for such a biomarker on vesicles (exosomes) secreted by card
3. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: The 80th Mississippi Academy of Sciences Annual Meeting, February 18-19, 2016, Hattiesburg, MS 39402.Proteomic analysis of exosomes derived from mammalian circadian clock cells. Zhao, D., Li, J., Pechan, T., Pechanova, O., Earnest, D., Farnell, M., and Farnell, Y. Suprachiasmatic nuclei (SCN) is the master circadian pacemaker in mammals that generates coordinated rhythms and drives oscillations in other peripheral tissues. We have identified that the conditioned media (CM) of SCN2.2 cells confer molecular rhythmicity to co-cultured fibroblasts via some diffusible factors. However, the mechanism of diffusible factors transfer from the SCN2.2 cells to other cells is currently unknown. One potential mechanism is through exosomes. Exosomes, secreted by various cells, are nanometer-sized vesicles that contain distinct subsets of RNAs and proteins.They play important roles in cell signaling and intercellular commu

PROGRESS: 2016/03/01 TO 2017/02/28
Target Audience:The information and research result produced with this grant will be beneficial for private and public sectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate student and one graduate student have been trained. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?This is equipment grant that helped us purchase the Beckman Ultracentrifugation System in May 2016 to get more preliminary data. We will try to get additional funding to analyze the biochemical contents, such as proteins and microRNAs, inside of exosomes for both projects.

IMPACT: 2016/03/01 TO 2017/02/28
What was accomplished under these goals? USDA Equipment Grant has supported us with the purchase of ultracentrifugation system that can be used to isolate nano-size extracellular vesicles, exosome. With the help of this support, we have made several progress in several areas related to project 1 and 2, as outline below. Dr. Yuhua Farnell, Aug, 2013- July 2016, Mississippi State University Aug. 2016 - present, Texas A&M University Project 1: Identification of the exosomal biomarkers for cardiovascular disease in a porcine model with the metabolic syndrome Impact: The knowledge gained from our experiments will improve our comprehension of intercellular communication in diabetic hearts, and assist in biomarker discovery. Such a test could help save potentially hundreds of thousands or even millions of lives, particularly in the most at risk populations, by allowing treatment of heart disease before lives are at risk - or worse, lost. Results and Outcomes (due to the length of the animal treatment, we are still processing samples until the end of this year): Animal Care and Sample Collection. This project is in collaboration with Dr. Michael Sturek from Dept. of Cellular & Integrative Physiol., Indiana University School of Medicine. A total of 10 Ossabaw pigs (6-month-old) were assigned to two diet groups for 6 or 8 months. Lean control swine will receive a daily diet (standard chow) with a total caloric content of 2200 kcal. The MetS/diabetic group will receive a high-energy diet of 6000 kcal/day as described previously. Body weight was monitored weekly. The blood pressure, blood glucose, glucose tolerance, and insulin sensitivity testing were performed. Serum and fresh hearts were collected and shipped via Fedx. Isolation, Culture and Conditioned Media of Cardiac Fibroblast. Primary fibroblasts were collected from control lean and MetS pig hearts. Briefly, after collection, hearts were rinsed in saline solution, minced and digested with collagenase. Conditioned media from primary cells will then be collected, centrifuged at 2000 x g for 20 min, and filtered with 0.22 µm vacuum-driven filtration system. Samples were stored at -80ºC Exosome Isolation. Exosome from conditioned media (b) and serum were purified by differential ultracentrifugation. The resulting exosome pellet was re-suspended in cold PBS. Transmission Electron Microscopy (TEM) and The NS 300 nanoparticle characterization system. The phenotype of exosomes was identified through TEM. Their sizes were in the range from 50-10 nm. The concentration of exosome in serum from MetS were three times higher than those from controls. Western Blot Analysis. Western blot analysis confirmed the presence of CD63 protein and heat shock protein 70 (HSP70), two well-characterized exosomal protein markers Project 2: Development of Molecular Markers to Determine the Sex of a Chick Embryo Impact: Current methods of determining gender are slow, and are somewhat inaccurate and labor intensive. Incubating eggs and hatching chicks that will have to eventually be euthanized is a significant cost to the integrator. Therefore, developing a means to determine the sex of a chicken embryo in ovo could significantly reduce industry costs. Results and Outcomes: We have found the presence of exosomes in amniotic fluid of a chick embryo on Day 17. We are in the process to characterize the exosomes from male and female chicks.

PUBLICATIONS: 2016/03/01 TO 2017/02/28
1. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A provisional abstract book of ISEV 2016. the 5th Annual Meeting of International Society of Extracellular Vesicles; 2016 May 06; Rotterdam, The Netherlands. http://dx.doi.org/10.3402/jev.v5.31552: Journal of Extracellular Vesicles 2016, 5: 31552; c2016. Comparative analyses of differential miRNA expression profiles between extracellular and intracellular components of SCN2.2 cells. Farnell YZ, Zhao D, Hsu C, Arick T. Suprachasmatic nuclei cells (SCN2.2) are crucial in mammalian clock pacemaker, and secret soluble factors that drive oscillation of peripheral tissues or cells. The mechanisms of the synchronization are not fully understand, however, extracellular vesicle-mediated communication has been implicated in the intercellular transferring of miRNAs in circadian clock. Thus, this study explored the miRNA expression profile between extracellular matrix, including SCN2.2 cell-derived exosome and exo
2. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: The 80th Mississippi Academy of Sciences Annual Meeting, February 18-19, 2016, Hattiesburg, MS 39402. (Ms. Mitchell, undergraduate student, has won the 1st place for undergraduate research section) Characterization of exosomes from cardiac fibroblasts in ossabaw pigs with metabolic syndrome. Mitchell, K*., Stewart, J., Zhao, D., Koniaris, L., Sturek, M., Alloosh, M., and Farnell, Y. Cardiovascular disease (CVD) is the leading cause of death worldwide, and patients with metabolic syndrome (MetS) are at higher risk to develop CVD.? However, there is no current method to detect CVD and many MetS patients are unaware of their illnesses until advanced complications like heart attacks and strokes occur.? A biomarker for CVD in diabetic patients could aid in early diagnostics and treatment of CVD and prevent the complications.? In this study we searched for such a biomarker on vesicles (exosomes) secreted by
3. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: The 80th Mississippi Academy of Sciences Annual Meeting, February 18-19, 2016, Hattiesburg, MS 39402. Proteomic analysis of exosomes derivd from mammalian circadian clock cells Zhao, D*., Li, J., Pechan, T., Pechanova, O., Earnest, D., Farnell, M., and Farnell, Y. Suprachiasmatic nuclei (SCN) is the master circadian pacemaker in mammals. , generates coordinated rhythms and drives oscillations in other peripheral tissues. We have identified that the conditioned media (CM) of SCN2.2 cells confer molecular rhythmicity to co-cultured fibroblasts via some diffusible factors. However, the mechanism of diffusible factors transfer from the SCN2.2 cells to other cells is currently unknown. One potential mechanism is through exosomes. Exosomes, secreted by various cells, are nanometer-sized vesicles that contain distinct subsets of RNAs and proteins. They play important roles in cell signaling and intercellular com