Progress 02/01/24 to 01/31/25
Outputs
Target Audience:Graduate students and the PD of the project have presented scientific findings from the research project in national and international symposiums and conferences where the target audience was scientists and animal health professionals including cattle producers, veterinarians and ruminant nutritionists. Changes/Problems:We had several technical issues while troubleshooting thein vitroexperiments which needed the baseline prior to the animal experiment. Additionally, we spent more time than we initially anticipated developing a standardized protocol for extracellular vesicles (EVs)isolation and characterization from bovine colostrum and milk. Thus, some of our experiments have been delayed. Additionally, the PD of the project was on maternity leave in spring 2024 which prevented the PD to be on site for guidance of the graduate student assigned to the project. Therefore, our animal experiment needed to be postponed. What opportunities for training and professional development has the project provided?All the in vitro experiments performed to accomplishaim 1 including EVs isolation via ultracentrifugation protocols and microscopy studies were performed by training graduate students on the appropriate techniques. Additionally,one DVM student through the 2024Veterinary Research Scholar Program at the School of Veterinary Medicine at Texas Tech University was trained on the techniques used to achieve objective 1. Furthermore, the partial findings from the in vitro studies are being part of one of the PhD student's dissertation. How have the results been disseminated to communities of interest?Our research findings have been presented at local, national, and international conferences as follows: 2024 American Association of Extracellular Vesicles Association Annual Meeting - Houston, TX 2024 Tech4EVITa workshop - Italian Society for Extracellular Vesicles - Trieste, Italy 2024 High Plains Dairy Conference - Amarillo, TX 2024 CRWAD - The conference of research workers in animal diseases - Chicago IL What do you plan to do during the next reporting period to accomplish the goals?We plan to delineate the mechanisms underlying the functional role of EVs and their cargo including microRNAsin the immune system of dairy calves (objective 2) within vivo experiments (calf trial) that are yet to be performed. After sample colletion from the animal trial, we will perform laboratory analysis cinluding transcriptomic essays and data analysis to achieve aim 2.
Impacts
What was accomplished under these goals?
Within objective 1, we aimed to identify, quantify, and analyze extracellular vesicles (EVs) derived from bovine colostrum transporting microRNAs that are involved in the immune gene regulation and intestinal cell proliferation of neonatal calves. We were able to develop a standardized protocol to isolate EVS from defatted colostrum samples, as well as to characterize colostral-EVs using bovine specific antibodies. To accomplish this, we performed the following techniques: milk EVs were isolated by ultracentrifugation at 340,000 × g for 60 min with a fixed angle rotor. After EVs extraction, dynamic light scattering measurements were performed to check size distribution (Zetasizer Nano ZS90, Malvern). Further, Transmission Electron Microscopy with negative staining (1% uranyl acetate) confirmed the colostral EVs to fall within the desirable range of 40-150 nm. Cryo-Electron Microscopy showed nanoparticles with clear lipid bilayer boundaries, and the classical donut shape of EVs. Extracellular vesicles from colostrum of 4 different animals were also captured on Luni chips loaded with bovine specific anti-CD9 and anti-CD63 for single particle interferometric reflectance imaging on a Leprechaun instrument (Unchained Labs, Pleasanton, CA, US). Mean diameter of ~ 45 nm and particle concentration within a 2-fold range across the 4 samples were detected. We successfully detected CD63 and CD9-bound EVs, in both defatted colostrum samples, and previously isolated EVs by ultracentrifugation. These findings suggest that although the colostrum heterogeneity presents a challenge for EVs isolation, applications of these methods potentially contribute to the standardization of EV extraction from colostrum and allow us to better understand milk extracellular vesicles transfer from mother to offspring. We plan to delineate the mechanisms underlying the functional role of EVs and their cargo including microRNAsin the immune system of dairy calves (objective 2) within vivo experiments that are yet to be performed.
Publications
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Progress 02/01/23 to 01/31/24
Outputs
Target Audience:Dairy ranchers and producers; dairy nutritionists; researchers/scientists in neonatal nutrition and animal health;researchers/scientistsin calf health. Changes/Problems:Due to challenges regarding the EV-microRNAs isolation from bovine colostrum including optmization of ultracentrifugation protocols for EV pellets isolation from bovine colostrum our in vitro studies were delayed. The findings presented at the conferences in 2023 were partial findings from the in vitro studies. Further in vitro studies will be performed in year 2 to accomplish all the objectives described in this research proposal. Additionally, primary intestinal cells from bovine will be used for the in vitro experiments in year 2. What opportunities for training and professional development has the project provided?During this grant year 1, all the in vitro experiments performed to accomplished aim 1 including cell culture studies, EV isolation via ultracentrifugation protocols and microscopy studies were performed by graduate students and by one DVM student through the 2023 Veterinary Research Scholar Program at the School of Veterinary Medicine at Texas Tech University. Therefore, the studies performed to accomplish the aim 1 of this research project was used to trained different graduate students in a diverse pool of laboratory techniques. Additionally, the partial findings from the in vitro studies are being part of one of the PhD student's dissertation. The in vitro work performed under study aim 1 has been presented atnational and international conferences during 2023. Thus, this research project has also contributed to the professional growth of the students that had oral and poster presentations. How have the results been disseminated to communities of interest?Thein vitrowork performed under study aim 1 has been presented atlocal, national, and international conferences during 2023 including: 2023 ASAS Southern Section organized by the American Society of Animal Sciences in North Carolina. 2023 Immunology conference organized by the American Association of Immunologists. May 11-15 2023 in Washington DC. 2023 ADSA Annual Meeting organized by the American Dairy Science Association held in Canada June 25-28 2023. 2023Veterinary Scholars Symposium inSan Juan Puerto Rico. August 3rd--5th 2023. 2023 Gradute students presentations at the Texas Tech University 3MT Thesis Competition in Lubbock, TX. 2023 Phi Zeta research Day at School of Veterinary Medicine Texas Tech University in Amarillo, TX. 2023 Kick-Off Event by Texas Center for Comparative Cancer Research (TC3R) held in February 2023 in Amarillo, TX. What do you plan to do during the next reporting period to accomplish the goals?For year 2 of this funded research grant we plan to performed the calf in vivo trial as stated in the grant proposal. Additionally, we plan to evaluate the microRNAs isolated from bovine colostrum in the intestinal cell trafcking by performing additional in vitro studies to evaluate target responses by such microRNAs in bovine intestinal cells.
Impacts
What was accomplished under these goals?
Within objective 1 of this study, we aimed to identify specific miRNAs present in bovine milk that modulate the immune system's response to infection. To address this, RAW-Blue™ Cells, a mouse macrophage reporter cell line was challenged with E. coli lipopolysaccharide (LPS) and incubated with a pool of miRNAs isolated from bovine milk through a 24-hour period. After incubation, QUANTI-Blue™ assay was used to detect the activation of the transcription factor NF-κB by the different treatments. Raw-Blue™ cells treated with miRNAs induced to some extend the signaling pathways leading to the activation of NF-κB relative to control cells (P<0.01). Bovine-milk derived miRNAs suppressed LPS-induced NF-κB activity in a dose-dependent manner (P=0.01). To identify the top expressed miRNAs that are driving such effects in vitro, real-time qPCR assays were performed in the milk samples. The miRNA-profiling revealed the presence of miRNAs involved in tumor suppression and cell apoptosis. Overall, our preliminary findings indicated that miRNAs derived from bovine milk have the potential to modulate the immune response to LPS in vitro. Additionally, we were able to quantify and image the EVs isolated from bovine colostrum using ultracentrifugation protocol from 20mL of bovine colostrum per cow (n = 10 Holstein cows) and subsequently subjected to staining for electron microscopy (EM).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Fernanda Rosa, Rafaela Santos, Tomas Lugo, Audrey Brown, Ulrich Bickel; Extracellular vesicles derived miRNAs from bovine milk modulate the inflammatory response to lipopolysaccharide in vitro. J Immunol 1 May 2023; 210 (1_Supplement): 167.38. https://doi.org/10.4049/jimmunol.210.Supp.167.38
Bovine milk is composed of a variety of biologically active compounds including mi-croRNAs (miRNA), which are found within extracellular vesicles (EVs). Extracellular vesicle-derived miRNAs may play a crucial role in the regulation of the immune response. Thus, this study aimed to identify specific miRNAs present in bovine milk that modulate the immune systems response to infection. To address this, RAW-Blue" Cells, a mouse macrophage reporter cell line was challenged with E. coli lipopolysaccharide (LPS) and incubated with a pool of miRNAs isolated from bovine milk through a 24-hour period. After incubation, QUANTI-Blue" assay was used to detect the activation of the transcription factor NF-?B by the different treatments. Raw-Blue" cells treated with miRNAs induced to some extend the signaling pathways leading to the activation of NF-?B relative to control cells (P<0.01). Bovine-milk derived miRNAs suppressed LPS-induced NF-?B activity in a dose-dependent manner (P=0.01). To identify the top expressed miRNAs that are driving such effects in vitro, real-time qPCR assays were performed in the milk samples. The miRNA-profiling revealed the presence of miRNAs involved in tumor suppression and cell apoptosis. Overall, our preliminary findings indicated that miRNAs derived from bovine milk have the potential to modulate the immune response to LPS in vitro.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Audrey Brown, Rafaela Santos, Yeseul Ahn, Ulrich Bickel, Fernanda Rosa, 68 Characterization of Bovine Extracellular Vesicles Isolated from Colostrum, Journal of Animal Science, Volume 101, Issue Supplement_1, May 2023, Pages 5556, https://doi.org/10.1093/jas/skad068.066
Bovine colostrum is composed of a variety of biologically active compounds including microRNAs (miRNA), which are found within extracellular vesicles (EVs). Newborn calves are agammaglobulinemic. Hence, the ability of the bovine neonate to fight off pathogens is therefore almost entirely dependent upon colostrum ingestion. Extracellular vesicle-derived miRNAs may have a crucial role in the regulation of the immune response. Thus, we aimed to characterize the EVs morphology from bovine colostrum using stimulated emission depletion (STED) super resolution microscopy. Bovine colostrum samples (approx. 3-4 h after parturition) were collected from primiparous Holstein dairy cows (n = 10) at a commercial dairy farm. Colostrum samples (15 mL) were subjected to a series of centrifugation steps to achieve defatted supernatant and to label EV pellets with EV specific markers. The EV pellets were resuspended in 100�L of 1 x PBS for EV staining methods or resuspended in 1 mL of trizol reagent for further miRNA isolation. The EVs suspensions were incubated with a primary polyclonal antibody anti-CD63 and with a secondary antibody (Alexa Fluor 594) for approximately 90 minutes each. Labeled EV-antibody complex were analyzed using confocal and STED microscopy. Based on the confocal and super resolution STED imaging capability obtained from the EVs-derived from bovine colostrum (Figure 1), labeled EVs with anti-CD63 and the enhanced resolution of STED over conventional confocal imaging, allowed to image the EVs at their actual size (30-200 nm). Further in-depth view on EV morphology, marker proteins expressed on the membrane, and size will contribute to our understanding of EVs-derived from colostrum and their functions. This will be crucial in studies to evaluate EVs uptake by intestinal cells for the correct identification of compartments involved in subcellular trafficking.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Conference Proceedings: 2023 Veterinary Scholars Symposium at San Juan Puerto Rico. https://www.aavmc.org/wp-content/uploads/2023/07/Abstracts_Rev_465.pdf
Abstract Title: Characterization of bovine extracellular vesicles and microRNA immunoregulation
Authors: Audrey Brown, Rafaela Santos, Ulrich Bickel, Fernanda Rosa
Affiliations: School of Veterinary Medicine, Texas Tech University (Brown, Santos, Rosa), Texas Tech University Health Sciences Center School of Pharmacy, Amarillo, TX (Bickel)
Abstract: Immunologically active compounds such as extracellular vesicles (EVs) containing microRNA (miRNA) can be found in bovine colostrum. Thus, this study aimed to characterize the morphology of EVs via super resolution stimulated emission depletion (STED) microscopy and to determine whether miRNA regulates the NF-K? signal transduction pathway. To test this, colostrum from fresh Holstein cows were collected and stored at -80�C until further analysis. Extracellular vesicles from colostrum samples were isolated using ExoQuick� ULTRA EV isolation kit and then incubated with a primary polyclonal antibody (anti-CD63) and with a secondary antibody (Alexa Fluor 594). To characterize EV morphology, STED microscopy was used to visualize labeled EV-antibody complexes. Subsequently, EVs pellets were subjected to microRNA isolation and purification using ExoQuick� RNA kit. To evaluate the immunological effect of miRNA over time, a colorimetric enzyme assay (QUANTI-Blue", Invivogen) was used to measure the activation of the NF-K? pathway upon incubation with RAW-Blue" Cells, a mouse macrophage reporter cell line known to express toll-like receptors (TLR) including TLR7 and TLR8. On day one, treatment group [RAW-Blue" Cells + miRNA] and control group [RAW-Blue" Cells alone] were incubated in a 96-well-plate overnight at 37�C with 5% CO2. On day two, all groups received QUANTI-Blue" solution, and absorbance readings were taken at 620 and 655 nm over 24-hour period. The NF-K? activation was detected in the cells treated with bovine miRNA which led us to conclude that miRNAs can regulate immunological pathways via endosomal TLR binding independently of onset infections.
Research Grant Funding: Agriculture and Food Research Initiative Competitive Grant no. 2023-67015-39081 from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Conference Proceedings: 2023 American Dairy Science Association� Annual Meeting
https://www.adsa.org/Portals/0/SiteContent/Docs/Meetings/2023ADSA/Abstracts_BOOK_2023.pdf
Abstract number: 1026M
J. Dairy Sci. Vol. 106, Suppl. 1; page 217
Abstract Title: Activation of immune signaling pathways by microRNAs extracted from bovine colostrum.
Authors: R. Santos; A. Brown; Y.Ahn; U. Bickel; F. Rosa
Affiliations: Texas Tech University, School of Veterinary Medicine, Amarillo, TX; Texas Tech University, Health Sciences Center, School of Pharmacy, Amarillo, TX.
Abstract: MicroRNAs (miRNAs) are non-coding RNA sequences that attach to
targeted mRNA resulting in cellular and protein regulation. MicroRNAs in milk can serve as biomarkers of inflammation and might help
neonatal immune system regulation. This study aimed to elucidate the
activation of immune signaling pathways by miRNAs extracted from
bovine colostrum. The miRNA was isolated from bovine colostrum (n =
10 samples) using commercial kits and quantified using real-time qPCR
assays. After exposing RAW-Blue Cells, a mouse macrophage cell line,
to E. coli lipopolysaccharide (LPS), cells were incubated with a pool
of miRNAs. The transcription factor NF-kB activity was measured for
24 h post-challenge using spectrophotometry absorbance. Statistical
analysis was performed using the PROC MIXED procedure of SAS
with treatment and time (repeated measurements) as the fixed effects and
well within treatment as the random effect. Significance was declared
at P < 0.05. Our results demonstrated that the miRNA dose (1?g and
0.1?g) triggered the activation of the transcription factor of inflammation NF-kB in a dose-dependent manner (P = 0.01). Several miRNAs
including let-7a-5p, miR-1013p, miR-29C-3p, miR-215p, miR-142,
and miR-3403p were identified in the bovine colostrum samples used
for this in vitro assay. Such miRNAs are involved in immune signaling
regulation and cell apoptosis. Thus, our findings indicate that miRNAs
extracted from bovine colostrum can potentially enhance the immune
system (i.e., regulate immune pathways) of neonatal calves if transferred
via colostrum.
Key Words: microRNAs, colostrum, bovine
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