Progress 05/01/23 to 04/30/24
Outputs
Target Audience:This project aims to foster collaborative partnerships to enhance research and education in Food, Agriculture, Natural Resources, and Human Sciences (FANH) at the Southern University Agricultural Research and Extension Center (SUAREC). By engaging faculty experts across diverse disciplines such as horticulture, analytical chemistry, toxicology, molecular biology, and partnering with USDA scientists, we seek to address multidisciplinary challenges comprehensively. Our primary objective is to disseminate research outcomes effectively to key stakeholders including USDA representatives, grape growers, and affiliated institutions, thereby amplifying the impact of our efforts. Specifically targeting grape growers from Louisiana and the Southeastern United States, our project endeavors to provide tailored solutions and insights relevant to their industry needs.The proposed objectives of this initiative will empower the Principal Investigator (PI) to showcase laboratory experiments planned with Air Liquid Interphase and using 3D culture systems, thereby enriching the educational and technical experience of students. Through hands-on participation in research activities, students will acquire valuable knowledge and practical skills crucial for pursuing careers in FANH sciences. This approach not only strengthens the academic foundation of students but also fosters their readiness to address real-world challenges in the nutraceutical and/or agricultural sector. Changes/Problems:It is imperative to propel the project forward, addressing critical components left incomplete due to the departure of Dr. Kambiranda, who relinquished his role as Principal Investigator (PI) and tenured professor at the close of 2022. Dr. Batra assumed leadership in 2023 and has demonstrated exceptional efficacy in advancing the project's objectives. What opportunities for training and professional development has the project provided?We are focused on preparing graduate and undergraduate students in the field and laboratory research by direct mentoring, engaging them in professional developmental tasks, graduate student thesis, teaching, research, and outreach activitiesat the Southern University Agricultural Research and Extension Center (SUAREC). How have the results been disseminated to communities of interest?The products of this project are being reportedvia university media,presentations at regional and national conferences and meetings, flyers, fact sheets, and posters. What do you plan to do during the next reporting period to accomplish the goals?The second No-Cost Extension (NCE) is imperative to propel the project forward, addressing critical components left incomplete due to the departure of Dr. Kambiranda, who relinquished his role as Principal Investigator (PI) and tenured professor at the close of 2022. Dr. Batra assumed leadership in 2023 and has demonstrated exceptional efficacy in advancing the project's objectives. Beyond diligently executing proposed experiments, we have introduced innovative methodologies, such as the Air Liquid Interface (ALI) Cell culture, a physiologically relevant model, and are at the forefront of implementing 3D culture (spheroid) in the laboratory. This second NCE phase promises not only the imminent submission of a manuscript currently in its final stages for submission but also an exploration into the impact of melatonin on A549 cells subjected to cigarette smoke extract (CSE) treatment under various physiologically relevant culture conditions for comparison with submerged cultures being currently conducted. The investigations will be conducted under both the Air Liquid Interface (ALI) culture system and a cutting-edge 3D culture system. Our commitment to advancing the research landscape is evident, and the extended timeframe ensures a comprehensive exploration of the effects of melatonin on cellular responses in varied culture environments, enriching the scientific contributions of the project.
Impacts
What was accomplished under these goals?
Several experiments were repeated and the results were confirmed and finalized for the manuscript which is ready for publication and is being internally reviewed by all the authors. Additionally,we haveintroduced innovative methodologies, such as the Air Liquid Interface (ALI) Cell culture, a physiologically relevant model, and we are at the forefront of implementing 3D culture (spheroid) technologyin the laboratory. While the ALI model has already been established,we are now testing the impact of CSE. All the setup for 3D spheroid culture is also complete and we are planning to start it inMay 2024. Our experiments during the next year will provide interesting information about the differences we encounter by using different culture model systems. We are planning to use the same proposedlung epithelial cells to keep a check on the number of variables so that the results can be more comparable. The exploration of the effects of melatonin on cellular responses induced by CSE under varied culture environmentswill significantly enrichour understandingof the project outcomes.
Publications
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:One minority undergraduate student, Ms. Lauryn Langley, completed her honors college thesis work in viticulture research, and three undergraduate students: Ms. A Miracle Guidry, Ms. Destyne Howard, and Ms. Chastity M. Joseph gained experiential learning by participating in research on the melatonin study. Muscadine grapes harvested from the established vineyard were used to extract secondary metabolites, gene expression studies, and HPLC quantification of metabolites. Grape research was and will be presented at the ARD Symposium (April 2 - 5, 2022 in Atlanta, GA); LSU/SU Joint Conference (December 12-13, 2022 at the LSU Campus, Baton Rouge, LA);78th Joint Meeting of BKX and NIS, March 29-April 1, 2023; and American Society for Nutrition, July 2023. Also, the research was presented in the Orientation to the AG science and plant science courses. Plant science undergraduate students are always actively involved in the viticulture program activities that deal with vineyard management practices such as pruning, fertilization, sample collection, processing, and preparation for laboratory assays. The project's target audiences include grape growers, wineries, and grape breeders with specific interests in the research outcomes. Changes/Problems:On December 14, 2022 we requested and USDA/NIFA approved on January 5, 2023 change in project director from Dr. Devaiah Kambiranda who resigned from his position as project director (PD) and from the university to Dr. Sanjay Batra as the new PD. We requested on February 15, 2023 and USDA/NIFA approved the no-cost extension (NCE) on February 28, 2023 to extend the grant to April 30, 2024. What opportunities for training and professional development has the project provided?Ms. A Miracle Guidry, Ms. Destyne Howard, and Ms. Chastity M. Joseph gained experiential learning by participating in research on the melatonin study. Grape research was presented at the ARD Symposium (April 2 - 5, 2022 in Atlanta, GA), LSU/SU Joint Conference (December 12-13, 2022 at the LSU Campus, Baton Rouge, LA); and will be presented at the American Society for Nutrition, July 2023 by undergraduate and graduate students participating in the project related work. How have the results been disseminated to communities of interest?The work from the project has been disseminated to interested clientele in the garden workshop, national meeting/conferences, community events, and extension agents. Students presented their work on this project at the 1890 Association of Research Directors (ARD) Conference, April, 2022, LSU/SU Joint Annual Conference (December 12-13, 2022 at the LSU Campus, Baton Rouge, LA). What do you plan to do during the next reporting period to accomplish the goals?Train several undergraduate and graduate students for providing hands-on experience in research. From the data generated, the students will be presenting their research findings at regional and national conferences, preparing manuscripts, and making oral presentations at departmental and college meetings. Specifically, the studies willbe conducted to delineate the anti-inflammatory effects of melatonin-rich specific berry extracts identified after evaluating the melatonin content.
Impacts
What was accomplished under these goals?
Melatonin quantification from muscadine grapes: In the current study various varieties of muscadine grapes were analyzed for the content of melatonin. These included Nobel (row 1, plants 5 and 6), Nobel (row 5, plants 9 and 10), Carlos (row 7, plant 4), Carlos (row 7, plants 7 and 8), and Nobel (row 11, plant 6). On each plant, 16 clusters of grapes were sprayed with varying levels of melatonin, and the concentration ranged from 50 µM to 200 µM (repeated processing). The muscadine grape clusters were sprayed at three different ripening stages including before veraison, veraison, and after they were ripe. Before veraison: the berries have the slight color change, whereas in veraison the berries undergo color change and softening. Subsequently, around the final stages, the berries show 50% ripeness. Fully ripe berries were picked and tested for sugar content and weight of three fresh berries and the whole cluster was recorded. Finally, leaf samples were ground in liquid nitrogen, and melatonin was extracted with ethanol. In this study, a total of 78 samples were quantitatively analyzed for the measurement of melatonin on AB SCIEX API 4000 mass spectrometer connected with a Waters Acquity UPLC. To begin with, the melatonin standard solution was dissolved in methanol for preparing the stock solution. The stock solution was diluted in methanol to obtain varying concentrations. Subsequently, samples were prepared for analysis by UPLC-MS/MS. Samples were analyzed with an AB SCIEX API 4000 mass spectrometry connected with a Waters Acquity Ultra Performance Liquid Chromatogram. The chromatographic condition included a Waters Acquity UPLC HSS T3 column coupled with a pre-column (VanGuard). The LC analysis had the following parameters: Mobile Phase A: Double distilled water (with 0.1% formic acid) and Mobile Phase B: Methanol (with 0.1% formic acid). The column temperature was set to 25°C. The sample chamber temperature was programmed for 4°C with a flow rate of 0.2 mL/min. While Mass Spectrometry parameters included: ESI positive mode; Ion source temperature: 350; IS: 4500 V; CAD: 7 psi; CUR: 40 psi; GS1: 30 psi; GS2: 40 psi; Q1 and Q3 parameters melatonin are as follows. Three transitions were used for the analysis. The first transition was used for quantification and the other transitions were used for qualitative analysis. Results indicated that melatonin concentration in muscadine grapes ranged between 0.063 ng (Fry seedless cv) and 30.416 ng (Magnolia cv). Anti-inflammatory studies related to Melatonin - Melatonin is a hormone derived from the essential amino acid tryptophan. This is involved in regulating the circadian rhythm and has been shown to have additional beneficial anti-oxidative, anti-cancer, anti-inflammatory, and immunomodulatory effects among others. Cigarette smoking and other nicotine-based products cause chronic lung inflammation. Despite improved therapeutic options, the prognosis of cigarette smoke (CS) induced inflammation and associated pathologies have remained to be a major health concern. Dietary supplements and related strategies are alluring in this regard because the implementation is relatively simple in low-income populations with limited resources. N-acetyl-5-methoxy-tryptamine (melatonin) is produced by the pineal gland and is critical in regulating the day night cycle. Owing to its anti-oxidative and anti-inflammatory properties, its role as a dietary supplement is extensively studied in various inflammatory models. The literature review suggests the oxidative stress induced by a variety of stimulants causes protein degradation, DNA damage, and cell death, thereby resulting in altered homeostasis. In this regard, the autophagy pathway is one of the essential degradation systems for proteins in vertebrates and has been documented to play critical roles in cellular homeostasis. Reports suggest that Reactive Oxygen Species (ROS) are sufficient to initiate autophagy and autophagy lysosomal flux in cells. This would imply that cells possess mechanisms like autophagy to alleviate oxidative stress-induced damage. There is a growing interest in studying the involvement of Danger Associated Molecular Patterns (DAMPs) in regulating the autophagy process in various pathological conditions. We, therefore, hypothesize that supplementation of melatonin can rescue CS-induced inflammation. In light of this, we cultured and treated human type II alveolar epithelial cells (A549) with melatonin prior to treatment with Cigarette Smoke Extract (CSE). Our results showed increased - production of cytokine/chemokine (IL-6, TNF-?, IL-8, CCL2 and CCL5); expression of DAMPs (HMGB1 &Hsp70); inflammatory mediators (NF-B, STAT3 and p38 MAPK); autophagy-related proteins (Beclin1 and LC3B); NADPH oxidase subunits (p22phox, p47 phox, p67 phox and gp91 phox); and antioxidants (MnSOD, Catalase & GPx), in CSE-challenged A549 cells. Interestingly, pretreatment with Melatonin reversed CSE-induced responses. Furthermore, melatonin mediated beneficial effects were similar to a well-known anti-oxidant, N-Acetyl cysteine (NAC- ROS inhibitor) and an Autophagy inhibitor (3MA). Overall, our findings demonstrate the important role of Melatonin in CSE-induced inflammatory responses. We used commercially available kits to determine the formation of autophagosomes and applied a machine-learning approach to counting the fluorescent puncta in control, CSE-challenged, and melatonin + CSE-challenged cells. Our results from the machine learning approach showed melatonin-mediated rescue of CSE-induced autophagy. To advance our understanding in relation to protein homeostatic mechanisms, the effect of melatonin and berry extracts will be studied using CSE-regulated expression of inducible [ 1i (LMP2), 2i (MECL1), and 5i (LMP7)] 20S proteasome subunits. Importantly, the preparatory steps involving the substrate binding and commitment and gate (19S) opening of the 20S proteasome are equally critical in the ubiquitin-mediated degradation of proteins. In this regard, the regulation and role of regulatory subunits and deubiquitylating enzymes components of 19S gate/cap will be studied in CSE-exposed cells. While Rpn10 is associated with the degradation of proteins with single chains of K48-linked ubiquitin, Rpn13 has been shown to retard the degradation of various single-chain substrates (previous studies). Intriguingly, proteins with multiple short ubiquitin chains can be targeted more efficiently for degradation by proteasomes through the ubiquitin-like domain, when bound by Rpn13. On the contrary, deubiquitylating enzymes-UCHL-5 cleaves 'Lys-48'-linked polyubiquitin chains, while Rpn11 possesses isopeptidase activity in the proteasome. Our results demonstrate an increase in the transcription and translation of 19S subunits-Rpn10 and Rpn-13, but not Rpn-11 in CSE-challenged cells. We also observed an increase in NF-B activation and IBdegradation in CSE-challenged cells, the latter being a substrate for the Rpn13/UCH37 complex. Overall, our findings provide evidence about the possible role of 19S gate subunits in CSE-induced inflammation. A major finding from our studies was that pre-exposure to melatonin significantly rescued CSE-induced responses associated with 19S components.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
L. Langley, L. Stapleton, C. Sanders 1, J. Obuya, D. Kambiranda. Quantitative Analysis of Melatonin Biosynthesis Genes and Content in Muscadine Grapes. ARD Symposium, Atlanta, GA, April 2-6, 2022
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Obuya, J. O. Grapes for Fresh Fruit and Wine Production In Louisiana. LSU/SU Joint Symposium, Baton Rouge, LA, December 12-13, 2022
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
S. Thota, R. Begum, D. Muthyala, N. Bidarimath, J. Obuya, S. Batra. Melatonin rescues cigarette smoke extract0-induced inflammation and autophagy in lung epithelial cells. 78th Joint Meeting of BKX and NIS, March 29-April 1, 2023.
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:This project will maximize partnerships and collaborative efforts to strengthen FANH sciences research and education at SUAREC by the involvement of faculty experts participating in this multidisciplinary issue. This includes involvement of faculty experts in horticulture, analytical chemistry, toxicology, molecular biology, and USDA scientists. Largely our goal is to share our research findings with USDA, grape growers and collaborating institutions to expand this program. The target audience of the project are grape growers from Louisiana and the Southeatern United States. One graduate student is working on the project to determine the anti-inflammatory activity of berry extracts and melatonin. Two undergraduate students completed their honors thesis using the resources from the project. The research work was presented at conferences, plant science courses, and BAYOU 2021 virtual high school summer training program. The target audience of the project includes students, small farmers, and growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has trained several undergraduate students in laboratory and field research. Six students from the plant science program were trained in experimental design, literature review, metabolite extraction, processing of samples for HPLC, quantification of metabolites, and presenting data. One undergraduate student presented her work at the 1890 Association of Research Directors (ARD) Conference, April,2022. Twoundergraduate students completedtheir Honors thesis work from the project funds. One graduate student was employed in the project. Presentations Conference Proceedings Kambiranda D, Langley L, Stapleton L, Begum R, Thota S, Batra S, Obuya J, Screening muscadine grapes for melatonin content and investigating its anti-inflammatory activity.ARD conference, Atlanta, GA, March 30-April 3, 2022. Graduate student Thota S, Begum R, Kambiranda D, Batra S. Melatonin Mitigates Cigarette Smoke Extract-Induced Inflammation: Impact on Autophagy and Chaperones. SC-SOT meeting, San Diego, CA, March 27-30, 2022. Thota S, Begum R, Kambiranda D, Batra S. "Anti-Inflammatory Effects of Melatonin in Cigarette Smoke Extract-Induced Inflammation: In Vitro Study." 2021 Southeast regional IDEA conference. San Juan, Puerto Rico, Nov 12-14, 2021 Undergraduate student Langley L, Stapleton L, Sanders C, Obuya J, Kambiranda D. Quantitative analysis of melatonin biosynthesis genes and content in muscadine grapes. ARD conference, Atlanta, GA, March 30-April 3, 2022. Undergraduate Students Honors College Thesis Langley L, Melatonin Profiling During Grape Berry Development and Ripening. Honors College, May 4, 2022, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA. Tiffany Poydras, Anti-Inflammatory Effects of Phytomelatonin on Cigarette Smoke Challenged Alveolar Epithelial Cells. Virtual Presentation May 5, 2021, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA Honor Thesis Citation Langley L, Melatonin Profiling During Grape Berry Development and Ripening. Honors college, Spring 2022, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA. Tiffany Poydras, Anti-Inflammatory Effects of Phytomelatonin on Cigarette Smoke Challenged Alveolar Epithelial Cells. Spring 2021, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA How have the results been disseminated to communities of interest?The work from the project has been disseminated to interested clientele in the fall garden workshop, national meeting, and community events. The work is also presented in the middle and high school recruitment events and the BAYOU high school summer training program.One student presented her work on this project at the 1890 Association of Research Directors (ARD) Conference, April,2022. What do you plan to do during the next reporting period to accomplish the goals?Muscadine cultivars will be treated with different concentrations of bio stimulators (0.5 - 100 µM) at the onset of veraison. Berry samples will be collected from different days post-treatment to measure the changes in flavonols, anthocyanins, and stilbenes. Changes in the total phenolics will be spectrophotometrically measured using the Folin-Ciocalteu method with gallic acid as the standard. Total anthocyanins and flavonoids will be measured using rutin as the standard. Total proanthocyanidin content will be measured using the vanillin assay with vanillin as the standard. Changes in anthocyanins will also be measured using the HPLC and individual anthocyanins will be quantified using known standards. Non-flavanoid phenolics and flavanols will also be quantified using HPLC using known external standards. Melatonin will be quantified from treated and untreated berries to determine the endogenous production and increase/decrease post-treatment. Total RNA will be extracted from the treated and control berries and key changes in the berry metabolism induced post-exposure to bio stimulators will be determined. The effect of bio-stimulator treated berry extracts on cigarette smoke extract challenged cells will be conducted to determine if an increase/decrease in phytochemicals can affect the translation of autophagy genes. The data will be compiled and the manuscript will be submitted to peer review journal and also presented at conferences, meetings, and recruitment events. Two undergraduate students and one graduate student will be recruited for the project to provide training in laboratory assays and techniques.
Impacts
What was accomplished under these goals?
UPLC-MS/MS The content of melatonin and the gene expression of melatonin biosynthesis pathway genes were studied during berry development and ripening (EL-34, EL-36, and EL-38). We further optimized the quantification and extraction protocol using UPLC/MS/MS to determine the content of melatonin in muscadine grapes. Two methods 80% methanol and methanol were used for extraction, dried and re-extracted with water/ethyl acetate twice. The dried residue was redissolved in methanol: water (75:25, v/v) before the analysis by UPLC-MS/MS. 10 μL of the sample was injected into the UPLC-MS/MS for analysis. Samples were analyzed with an AB SCIEX API 4000 mass spectrometry connected with a Waters Acquity Ultra Performance Liquid Chromatogram. Waters Acquity UPLC HSS T3 column (2.1×150mm 1.8 μm) coupled with a VanGuard precolumn (2.1×5mm 1.8 μm) with a mobile phase of (water with 0.1% formic acid) and (methanol with 0.1% formic acid) and flow rate of 0.2 mL/min was used. Three stages of berries were screened for melatonin content. Most of the berries tested had higher melatonin content in EL 34 and EL-36 stage compared to the ripe berries. Methanol resulted in higher melatonin content among the tested cultivars compared to 80% methanol extraction. Melatonin concentration ranged from 2- 4.5 ug/g FWin the methanol extracted berries and only 3-25 ng/g FWin the 80% methanol extracted berry samples respectively. The highest concentration (4.5 ug/g FW-1) was observed in the EL-34 stage of the Fry seedless muscadine cultivar. Anti-inflammatory Study Melatonin possesses additional properties including anti-oxidant, anti-cancer, anti-inflammatory, and immunomodulatory effects. We pre-treated 549 cells with melatonin (0.25 and 1.0 mM) to determine its anti-inflammatory property in our study model using cigarette smoke extract (CSE). Previous research has reported that exposure to CSE modulates the expression of antioxidant genes to combat the oxidative stress caused due to the production of reactive oxygen species (ROS). NADPH oxidase (NOX), a multiprotein complex, is responsible for superoxide generation and plays an important role in regulating inflammatory responses. We observed an increase in the mRNA levels of NOX subunits--neutrophil cytosolic factor 1 (NCF-1/p47phox), neutrophil cytosolic factor 2 (NCF-2/p67phox-), and gp91phox--in the CSE challenged cells as compared with the DMSO controls. Interestingly, pretreatment with melatonin reduced the expression of NOX subunits--neutrophil cytosolic factor 1 (NCF-1/p47phox). We did not observe any change in the NRF2 (nuclear factor erythroid-derived factor). Nuclear factor-kappa B (NF-κB) is an ancient protein transcription factor and is considered a regulator of innate immunity. The NF-κB signaling pathway links pathogenic signals and cellular danger signals thus organizing cellular resistance to invading pathogens. NF-κB target inflammation directly by increasing the production of inflammatory cytokines, and chemokines. STAT3 induces the expression of many cytokines, chemokines, and other mediators. With melatonin, we observed a decrease in the expression of NF-κB and STAT3 transcription factors which indicate a decrease in inflammation
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Minkyu Park, Daniel Vera, Devaiah Kambiranda, Pranavkumar Gajjar, Lance Cadle-Davidson, Violeta Tsolova, Islam El-Sharkawy. 2021. Chromosome-level genome sequence assembly and genome-wide association study of Muscadinia rotundifolia reveal the genetics of 12 berry-related traits. Hortic Res. 2022 Jan 18;9:uhab011
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kambiranda D, Langley L, Stapleton L, Begum R, Thota S, Batra S, Obuya J, Screening muscadine grapes for melatonin content and investigating its anti-inflammatory activity. ARD conference, Atlanta, GA, March 30-April 3, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Thota S, Begum R, Kambiranda D, Batra S. Melatonin Mitigates Cigarette Smoke Extract-Induced Inflammation: Impact on Autophagy and Chaperones. SC-SOT meeting, San Diego, CA, March 27-30, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Langley L, Stapleton L, Sanders C, Obuya J, Kambiranda D. Quantitative analysis of melatonin biosynthesis genes and content in muscadine grapes. ARD conference, Atlanta, GA, March 30-April 3, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Thota S, Begum R, Kambiranda D, Batra S. "Anti-Inflammatory Effects of Melatonin in Cigarette Smoke Extract-Induced Inflammation: In Vitro Study." 2021 Southeast regional IDEA conference. San Juan, Puerto Rico, Nov 12-14, 2021
- Type:
Other
Status:
Accepted
Year Published:
2022
Citation:
Langley L, Melatonin Profiling During Grape Berry Development and Ripening. Honors college, Spring 2022, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA.
- Type:
Other
Status:
Accepted
Year Published:
2021
Citation:
Tiffany Poydras, Anti-Inflammatory Effects of Phytomelatonin on Cigarette Smoke Challenged Alveolar Epithelial Cells. Virtual Presentation May 5, 2021, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:Two minority undergraduate students completed their Honors College thesis work using the resources from the project. The thesis work was presented to the committee for review and acceptance. One graduate student is working on the project to investigate the molecular mechanisms and anti-inflammatory activity of muscadine berry extracts. The research work was presented at the BAYOU 2020 High school summer training program, Orientation to the AG science and plant science courses. Students from the plant science program are actively involved in the vineyard, learning to grow grapes, pruning, fertilization, sample collection, processing, and laboratory assays. The target audience of the project isfarmers interested ingrowing grapes and consumers interested inidentifying muscadine grapes with higher secondary metabolites. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project personnel has trained undergraduate students currently enrolled in plant science program in DNA extraction, PCR, real-time PCR, enzyme assays, grapevine pruning, planting, and fertilization. Four undergraduate students are currently working on the project on RNA and metabolite extraction, PCR, real-time PCR, biochemical assays, and HPLC. The project activities and research has also helped the graduate students to present their work atthe regional and national conference. Two undergraduate students were mentored by the PI to completetheir Honors College thesis work. The projectfunds were also used to buy chemicals and supplies for undergraduate/graduate research work and training. Proceedings Begum R, Thota S, Kambiranda D, Batra S. Urolithins rescue e-cigarette vapor condensate induced distinct histone signatures-in vitro study. 2021 Proceedings of the 19 LBRN Annual Conference, February 12-13, Baton Rouge, Louisiana. Oral Presentations Begum R, Singh DP, Thota S, Kambiranda D, Batra S. Lipid raft mediated regulation of NADPH Oxidase: Vaping induced Inflammation. 2021 SOT Annual meeting and Tox Expo Virtual meeting, March 2021. Begum R, Thota S, Kambiranda D, Batra S. Urolithins rescue e-cigarette vapor condensate induced distinct histone signatures-in vitro study. 2021 Proceedings of the 19 LBRN Annual conference, February 12-13, Baton Rouge, Louisiana. Poster Presentation Begum R, Singh DP, Thota S, Kambiranda D, Batra S. Urolithins rescue e-cigarette condensate induced lipid raft-mediated inflammation in lung epithelial cells. 2020 SC-SOT Virtual Meeting, November 13, 2020, Mississippi State University, Starkville, MS. Undergraduate Students Honors College Thesis Presentation Naila L. McCraney, Examination of health benefits of Melatonin found in Muscadine Grapes and Wine. Virtual Zoom Meeting, April, 30, 2020, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA. Christopher Amos, Detection of Melatonin Biosynthesis pathway genes in Muscadine grapes. Virtual Zoom Meeting, December, 2, 2020, Dolores Margaret Richard Spikes Honors College, Southern University, Baton Rouge, LA. Thesis Citation Naila L. McCraney, Examination of health benefits of Melatonin found in Muscadine Grapes and Wine. May 2020. Christopher Amos, Detection of Melatonin Biosynthesis pathway genes in Muscadine grapes. December 2020. How have the results been disseminated to communities of interest?Data/information generated from this project have been disseminated via virtual conference meetings and introductory courses in plant and agricultural sciences. What do you plan to do during the next reporting period to accomplish the goals? Quantification of total phenolics, anthocyanin, flavonoids, stilbenes during berry development and ripening will be completed using biochemical and analytical methods. Application of bio-stimulator for eliciting secondary metabolites in muscadine grapes. Bio stimulator will be sprayed at different concentrations and optimum concentration for elicitation of secondary metaboliteswill be determined. HPLC method will be optimized for detection of secondary metabolites in different muscadine grapes in control and bio-stimulator treated berries. Human lung epithelial cells exposed to cigarette smoke will be treated with varying concentrations of grape berryextracts. Immunoblotting and real-time PCR will be performed to determine the changes in anti-inflammatory proteins/genes. Effect of bio-stimulator treated berry extracts on the translation of autophagy genes in cigarette smoke challenged cells will be determined. Data will be analyzed and the manuscript will be submitted to a peer-reviewed journal. Data will also be presented in conferences, seminars, recruitment events and disseminated via extension personnel. Undergraduate students will be provided hands-on experiential learning/training in various analytical techniques, sample preparation, quantification of metabolites, and data analysis.
Impacts
What was accomplished under these goals?
Molecular Studies in grapes In the current study, endogenous melatonin present in muscadine grapes and the gene expression pattern of melatonin biosynthesis genes were detected in leaf and berry tissues. In addition, the anti-inflammatory responses of melatonin in cigarette smoke exposed (CSE) lung epithelial cells were tested. Total RNA was extracted from muscadine berries and leaf samples. cDNA was constructed from total RNA. Semiquantitative and real-time PCR was performed using gene specific primers to measure the activity of melatonin pathway biosynthesis genes in berries and leaves. Berry samples included Florida Fry, Sugar pop, Supreme, Pride, Cowart, African Queen, Sweet Jenny, Fry, and four berry developmental stages of Carlos and Noble muscadine grapes. Relative gene expression was measured using an experimental control gene, glyceraldehyde 3-phosphate dehydrogenase. Data collected showed that different genes were expressed in muscadine grapes in berry stages and cultivars. Tryptophan 5 hydroxylase (T5H) gene expression was observed in eight muscadine cultivars. T5H gene expression was high in ripe berries compared to green berries. The SNAT gene that converts serotonin to melatonin in the final step was detected in most of the muscadine berries. Interestingly SNAT gene expression was more prominent in the ripe berries compared to green unripe berries indicating melatonin production is active in ripe berries. The expression of the melatonin 3-hydroxylase gene that converts melatonin to cyclic 3-hydroxymelatonin was active only in the ripe stages of the Noble and Carlos muscadine grape. HPLC HPLC analysis was optimized for quantifying the endogenous melatonin concentration in muscadine grape leaves and berries. The protocol was optimized using a combination of organic and aqueous solvents for leaves and berries. HPLC was performed using a gradient of acetonitrile and water with 0.1% phosphoric acid using a Kinetex® 5 µm C18 100 Å LC Column 50 x 4.6 mm. Calibration curves were generated using a standard melatonin concentration of 5-50 µg/ul. Our preliminary results reveal ~6-20µg/g of melatonin present in leaves and berries tested. We will further optimize the extraction procedure and purify our samples using Hypersep Spe columns to determine the concentration of endogenous melatonin in muscadine grapes. Anti-inflammatory activity of melatonin Our preliminary results showed a significant increase in the production of cytokines-IL-6, IL-1β, and chemokines IL-8, CCL5, and CCL-2 by A549 cells in response to cigarette smoke-challenge. We pre-treated 549 cells with melatonin (0.25 and 1.0 mM) to determine its anti-inflammatory property in our study model. Interestingly, we observed that pre-treatment with melatonin abrogated CS-induced production of cytokines and chemokines by A549 cells in a concentration-dependent manner. HMGB1 and HSP70 the Damage-associated molecular patterns (DAMP) biomarkers that serve as extracellular pro-inflammatory mediators that promote inflammation. We observed that pre-treatment with melatonin significantly reduced mRNA levels of both HMGB1 and HSP70 in CS-challenged A549 cells. In addition, to elucidate the role of melatonin in regulating CS-induced autophagy process, we first determined the expression levels of Beclin-1, LC3B, ATG5, and ATG16 in challenged cells. Our results showed a significant increase in autophagy-related genes in CS-challenged cells. Furthermore, pre-treatment with melatonin abrogated CS-induced Beclin-1 and LC3B transcription. Our results provide preliminary evidence that melatonin could reverse the CSE-induced autophagy mechanism in lung epithelial cells. Our findings will determine the interrelationship between endogenous or external MT treatment and the flavonoid/stilbenoid accumulation among different muscadine cultivars and the anti-inflammatory activity of muscadine grapes enriched with phyto-nutraceuticals.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Naila L. McCraney, Examination of health benefits of Melatonin found in Muscadine Grapes and Wine. https://www.subr.edu/assets/subr/2020-Spring---Summer-Commencement-Program.pdf Page 20
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Christopher Amos, Detection of Melatonin Biosynthesis pathway genes in Muscadine grapes. December 2020
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Melatonin alleviates cigarette smoke-induced inflammation (2021). Shilpa Thota, Rizwana Begum, Prathyusha Bagam, Abubakar Abdulkadir, Sanjay Batra and Devaiah Kambiranda. Toxicology
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