Progress 02/01/14 to 01/31/17
Outputs
Target Audience:Data generated during the reporting period was shared with the scientific community at the annual USDA Project Directors meeting in Chicago. The results will also be communicated to the general public after the publication of the work in peer reviewed journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student received training in cell biology, biochemistry and molecular biology under this project, which will help his career in biomedical research. This student attended USDA annual project directors' meeting which provided an opportunity to interact with other scientists thereby promoting exchange of ideas. The presentation and written communication skills of this student showed significant improvement. This student is now in a position to work independently as well as train and help other students. The student is in the process of submitting dissertation for the award of PhD. How have the results been disseminated to communities of interest?A poster was presented in the annual USDA Project Directors meeting in Chicago. Information about the project was communicated to Michigan Technological University marketing department which was published in TechToday. We are currently preparing two manuscripts based on this project work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Anti-inflammatory effects of rice callus suspension culture (RCSC) on three inflamed normal colon epithelial cell lines, InEpC, NCM 356 and CCD 841 CoN was investigated. Cells were treated with a pro-inflammatory cytokine cocktail for 24 hours followed by RCSC treatment at varying dilutions for 24-96 hours and were subjected to fluorescent microscopy analysis. InEpC cells treated with 1:5 dilution of RCSC showed significant decrease in cytoplasmic reactive oxygen species (ROS), while mild increase was observed in nuclear generated ROS. NCM 356 treated with 1:40 dilution of RCSC showed increase in both cytoplasmic generated ROS and nuclear generated ROS. CCD 841 CoN treated with 1:40 dilution of RCSC revealed a similar pattern of nuclear generated ROS as observed in NCM 356. Flow cytometry analysis of the above RCSC treatments largely supported the fluorescent microscopy data. Flow cytometry analysis of propidium iodide staining was suggestive of decreased cell death in RCSC treated groups in NCM 356. Several changes in gene expression were observed in NCM 356 cell line with five genes upregulated two-fold or higher and ten genes downregulated following 48-hour treatment with RCSC versus inflammatory control. However, only two of the upregulated and four of the downregulated genes showed the same expression pattern in RCSC treated versus inflammatory plus plant medium control. Gene expression analysis on CCD 841 CoN cell line identified nine genes upregulated and fifteen genes downregulated in RCSC treated versus inflammatory control. However, only eight of the upregulated and six of the downregulated genes showed the same expression pattern in RCSC treated versus inflammatory plus plant medium control. Based on upregulated and downregulated genes, we hypothesize that RCSC acts as a novel anti-inflammatory agent for the gastrointestinal tract, which is mediated through nuclear factor kappa beta (NF-Κβ), interleukin 2 (IL-2), and AKT dependent pathways. Blueberry extract showed significant anti-oxidative effects on NCM 356 and CCD 841 CoN cells treated for 24 hours with the pro-inflammatory cytokine cocktail followed by 48 hours treatment with blueberry extract (0.5 mg/ml). Blueberry extract treatment resulted in reduction in both cytoplasmic and nuclear ROS to cells treated only with pro-inflammatory cocktail and pro-inflammatory cocktail plus plant medium, based on fluorescent microscopy and flow cytometry analysis. Blueberry is a better antioxidant than RCSC. It prevents cytokine induced cell death but not as efficiently as RCSC. RCSC was concentrated and resuspended in different solvents: methanol, acetone, and water. Samples were applied to Thin Layer Chromatography (TLC; silica gel plates) with methanol/ chloroform as the running solvent. Two or more major compounds with different Rf values were observed. RCSC resuspended in methanol was separated through a gravity column/silica and 25 fractions were collected. These fractions were analyzed using high performance liquid chromatography (HPLC) on a C18 reverse phase column. Fractions 10-18 with same peaks were combined and assayed for bioactivity in the cell line, SW620. Treated samples showed significantly higher fluorescence signal compared to control. Draft versions of two manuscripts are ready and will be submitted for publication in peer-reviewed journals.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Kyle M. Driscoll, Nafeesa Rahman, Ramakrishna Wusirika, Aparna Deshpande (2015) Bioactive Compounds in Rice Callus
Culture and Blueberry Extract as Anti-Inflammatory Agents of the Gastrointestinal Tract. USDA/NIFA AFRI/NRI Function and Efficacy of Nutrients/Bioactive Components for Optimal Health, and Improving Food Quality Program Directors Meeting. Oral Presentation. July 2015.
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2017
Citation:
Kyle Driscoll PhD dissertation.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:Data generated during the reporting period will be used for research publications for communicating the results to the scientific community. The results will also be communicated to the general public through University's TechToday and other marketing resources. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student received training in cell biology, biochemistry and molecular biology under this project, which will help his career in biomedical research. This student attended USDA annual project directors' meeting which provided an opportunity to interact with other scientists thereby promoting exchange of ideas. The presentation and written communication skills of this student showed significant improvement. This student is now in a position to train and help other students. How have the results been disseminated to communities of interest?A poster was presented in the annual USDA Project Directors meeting in Chicago. Information about the project was communicated to Michigan Technological University marketing department which was published in TechToday. We are currently preparing two manuscripts based on this project work. What do you plan to do during the next reporting period to accomplish the goals?Flow cytometry analysis will be completed for RCSC and blueberry extract treated samples. This will help in quantitative comparative analysis of the anti-inflammatory effects of RCSC and blueberry extract. High performance liquid chromatography (HPLC) fraction(s) with better anti-inflammatory activity, if any, will be identified followed by isolation and characterization of bioactive compounds. We will submit one to two manuscripts for publication in peer-reviewed journals.
Impacts
What was accomplished under these goals?
We investigated the anti-inflammatory potential of rice callus suspension culture (RCSC) on three inflamed normal colon epithelial cell lines, InEpC, NCM 356 and CCD 841 CoN. Cells were treated with a pro-inflammatory cytokine cocktail followed by RCSC treatment at varying dilutions for 24-96 hours. InEpC cells treated inflammatory cocktail resulted in significant increase in cytoplasmic ROS and 1:5 dilution of RCSC for 24 hours showed significant decrease in cytoplasmic ROS, which was identified by fluorescence microscopy. NCM 356 and CCD 841 CoN treated with RCSC showed a similar pattern. Flow cytometry analysis detected marked reduction in cell viability of NCM356 cells subjected to pro-inflammatory cytokine cocktail treatment. This effect was significantly reduced with RCSC treatment as indicated by Hoechst 33342 staining. Based on upregulated and downregulated following treatment with RCSC versus inflammatory control in NCM 356, we hypothesize that RCSC acts as a novel anti-inflammatory agent for the gastrointestinal tract, which is mediated through nuclear factor kappa beta (NF-Κβ), interleukin 2 (IL-2), and AKT dependent pathways. Blueberry extract showed significant anti-inflammatory effects on NCM 356 and CCD 841 CoN cells treated for 24 hours with the pro-inflammatory cytokine cocktail followed by 48 hours treatment with blueberry extract (0.5 mg/ml). The effects of blueberry extract were similar to that of RCSC treatment reducing cytoplasmic ROS and increasing cell viability compared to cells treated only with pro-inflammatory cocktail, based on fluorescent microscopy and flow cytometry analysis. Preliminary results suggest that RCSC has better anti-inflammatory effect than blueberry extract.
Publications
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Progress 02/01/14 to 01/31/15
Outputs
Target Audience: Two graduate students received training under this project. This will help their careers in biomedical research. Data generated during the reporting period will be used for research publication and presentation in a conference. We collaborated with two research groups in chemistry department and kinesiology and integrative physiology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Extensive training in cell biology. biochemistry and molecular biology was provided to two graduate students. Development of presentation and written communication skillsof students. How have the results been disseminated to communities of interest? Information about the project was communicated to Michigan Technological University marketing department which was published in TechToday. What do you plan to do during the next reporting period to accomplish the goals? We will test the efficacy of these treatments on an additional normal colon epithelial cell line, perform quantitative PCR, fluorescent microscopy and flow cytometry analysis. Further, high performance liquid chromatography (HPLC) fractions will be tested. If any HPLC fraction shows better antiinflammatory activity, isolation and characterization of active compounds will be performed.
Impacts
What was accomplished under these goals?
We evaluated anti-gastrointestinal inflammatory activity of rice callus culture. Normal-derived colon mucosa cell line (NCM 356) was treated for 24 hours with a pro-inflammatory cocktail containing TNF-α, IL-1β, IFNγ, and LPS. Following the 24-hour incubation, cells were treated with RCSC (1 mg/ml) for 72 hours. Quantitative polymerase chain reaction (qPCR) was performed on NCM356 for the RCSC treated samples using TaqMan® array (96-well) for human immune response and inflammation. Four genes encoding CACNB2, HLA-1, IL-1R1, and IL-10 were upregulated upon inflammation, and RCSC treatment reversed their expression, thereby suggesting a pathway regulated by RCSC to mitigate inflammation. IL-2 and CD80 were upregulated and CD38 was downregulated by RCSC treatment only which may not have a direct role in regulating inflammation. Fluorescent microscopy and flow cytometry showed free radical scavenging capabilities of RCSC based on cytoplasmic and nuclear ROS stains. We partially evaluated antiinflammatory effects of blueberry extract (1 mg/ml) based on fluorescent microscopy and flow cytometry. However, the results were inconclusive. Further research is needed to test the efficacy of these treatments on an additional normal colon epithelial cell line. RCSC fractions were obtained using high performance liquid chromatography (HPLC).
Publications
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