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ARS Home » Southeast Area » Fayetteville, Arkansas » Poultry Production and Product Safety Research » Research » Publications at this Location » Publication #373499

Research Project: Antibiotic Alternatives for Controlling Foodborne Pathogens and Disease in Poultry

Location: Poultry Production and Product Safety Research

Title: Changes in thymosin Beta-4 during enteroid generation demonstrated by direct MALDI-TOF-MS

item ACHARYA, MOHAN - University Of Arkansas
item LIYANAGE, ROHANA - University Of Arkansas
item Rath, Narayan
item LAY, JACKSON - University Of Arkansas
item Donoghue, Ann - Annie

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/5/2020
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Introduction Thymosin beta4 (Tbeta4) is a widely occurring and evolutionarily conserved regulatory peptide which has been implicated in many biological functions involving wound healing, chemotaxis, tissue modeling, anti-inflammatory, and antimicrobial activities. Because of its interaction with monomeric G-actin that prevents its polymerization to F-actin it can affect cytoskeletal dynamics altering cellular function. In the course of generating chicken enteroids from intestinal villi, we noticed significant elevation in Tbeta4 peak in the enteric spheroids after 18- 24 h of culture using MALDI-MS. Hence, the objective was to find the Tbeta4 dynamic of chicken enteric organoids. Methods The crypt-villus enteroids were generated using mucosal tissues from chicken intestines reconstituted in DMEM-F12 medium then filtered through 70µm cell strainers to isolate villi. Parts of it cultured in medium containing serum and growth factors to make enteroids culturing for 24 h. The tissues were filtered to strain enteroids. The villi and the enteroids were washed with serum free medium twice, centrifuged, and the tissue pellets suspended with 70% ethanol. One ul of cell suspension from each prep was spotted, smeared on a MALDI target as a thin film, and overlaid with 1 uL saturated HCCA matrix solution and subjected to MALDI-TOF-MS in the 2-20 kDa range using Bruker-Ultraflex-MALDI-TOF-MS. The TBeta4 results were confirmed with immunohistochemistry (IHC) using anti-thymosin beta4. Preliminary Data In MALDI-TOF-MS spectra, majority of peaks (nominally) were common to both villi and the enteroids. However, there were some peaks with clearly different relative intensities in two samples. A conspicuous but consistent difference was noticed in a peak corresponding to m/z 4963 which in several of our earlier studies with chicken macrophages was identified as Tbeta4. The TBeta4 level was low in fresh villi but clearly present in organoids. This result was confirmed using IHC staining where the organoids showed to have higher densities of Tbeta4 compared with villi. Our results show that Tbeta4 expression in the intestinal villi may vary depending upon the intactness of the mucosal villi. We propose that the depletion of TBeta4 in wounded mucosa is related to its release from the cells freeing G-actin to undergo its polymerization which is an essential aspect of tissue repair. This interesting dynamics Tbeta4 during villi transformation to organoids was captured by MALDI and confirmed by immunohistochemistry. Novel Aspect Transitional depletion of TBeta4 levels may be important for wound healing.