Progress 01/03/22 to 01/02/24
Outputs
Target Audience:The target audience for my research has not changed much, simply different venues to share my work with and additional young scientists. This past year afforded me the opportunity to attend three conferences and share my research with the immunology and microbiology community. The first conference I attended was the Mississippi Academy of Sciences conference in Biloxi, MS on February 24th. This conference is asmall conference, but I was able to give an oral presentation on my work and also had the opportunity to judge the undergraduate poster session. This was a great mentoring opportunity and a way for me to interact with the local science community in Mississippi. Additionally, I was awarded the third best oral presentation in the Health Sciences division at this conference. The second conference was the American Association of Immunologists (AAI) conference in Washington, DC on May 12. This conference was a large conference with several days of talks, with multiple rooms running at a time, including a talk from Dr. Fauci. I was able to present a poster of my work among several hundred other poster presentations. This conference allowed me to network and meet several new acquaintances. I was also able to meet up with immunologists that I had met at previous meetings. This conference I learned so much about novel immunology topics as well. Shortly after the AAI meeting, I was able to attend the North American Comparative Immunology Workshop and present an oral presentation on my work for the second year in a row. This meeting was another great opportunity to network for my future career after graduation and meet new people to discuss my research with. I was also awarded a travel award for this meeting, so I was able to use my fellowship monies for other travel opportunities. Finally, I was able to mentor two great up-and-coming scientists in the lab throughout the summer of 2023. One student is a PhD student in the same program as myself, and rotated through the lab to learn some basic immunology techniques before finalizing her lab for her dissertation research. Additionally, I had a visiting undergraduate student for the summer. Both students learned many research techniques, including but not limited to cell culture, restriction site-mediated cloning, confocal microscopy, flow cytometry, primer design, RT-PCR, RT-qPCR, transient transfection of mammalian cells, western blotting, and DNA gel electrophoresis. The students also learned essential laboratory skills such as note-keeping, laboratory math, basic buffer chemistry, organization, attention to detail, data analysis, and asking good questions. The target audience for my research has been both the immunology community and future scientists in this area. Changes/Problems:There have been no additional changes from those reported in the last reporting period. I have continued to follow those changes in studying cellular localization of the duplicated TLR5 proteins in channel catfish and should have new data to report on that project soon. In this past reporting period, my department has gottenanother new chair. This has not affected my research much besides the extra meetings during the transitional period. Otherwise, my research has been on track and there have been minimal problems to report on this past year. What opportunities for training and professional development has the project provided?I have had the opportunity to mentor two students, teach dental students, and present my research, both of which are excellent professional development opportunities. In addition, I successfully defended my dissertation and accepted a job for after graduation. While I am not allowed to share many details, my new position will be a postdoctoral researcher at the USDA and has a potential start date in June. How have the results been disseminated to communities of interest?These results have been presented at three conferences the past year, and written up into one manuscript with possibly another manuscript or short communication on the way. The data generated from this project has been discussed with the target audience thoroughly and will continue to be discussed as we have provided a model that other scientists can use in their studies. While we have characterized TLR7 in channel catfish, there are many more TLRs left to characterize in this model and hopefully those future studies will be disseminated to the comparative immunology community too. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This year has been a year of progress. I was able to put together a manuscript on some of the TLR7 data generated from this fellowship. This manuscript has recently been submitted to the Developmental and Comparative Immunology journal and is under revision. I also completed and defended my dissertation within the past year and will take the skills learned through this fellowship to my postdoctoral position. The data published focus mainly on channel catfish TLR7 and the ligands imiquimod and resiquimod, both synthetic ssRNA agonists. In confirming that TLR7 in catfish recognizes both IMQ and RSQ, we also discovered that TLR7 in catfish does not respond to poly(I:C), a synthetic dsRNA analog. It has been shown in other teleosts that TLR7 can sometimes be a dual-acting receptor that responds to both ssRNA and dsRNA. This does not seem the case in channel catfish. We characterized much of the TLR7 signaling pathway in channel catfish as well in this manuscript. Additionally, we have been working with the duplicated TLR5 proteins in channel catfish. We first hypothesized that, unlike humans, TLR5 in channel catfish localizes internally within the cell and not on the cell surface. So far, we have shown colocalization with TLR5.1 and both early endosome marker Rab5a and lysosomal marker LAMP1. We have plans to study TLR5.2 alone as well as both TLR5.1 and TLR5.2 in co-culture to determine if both proteins are localized internally and from there, hopefully determine the ligands of each. In other teleosts, heterodimerization of both proteins is necessary for proper recognition and response to ligands. Mainly TLR5 has been shown to recognize bacterial flagellin. We hope to test TLR5.1 and TLR5.2 in co-culture with heat-killed Edwardsiella ictaluri, a flagellated bacteria that is detrimental to catfish farming.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2024
Citation:
Felch, K.L. (2023). Insights into Toll-like Receptor (TLR) Signaling in Channel Catfish, Ictalurus punctatus. Doctoral Dissertation, University of Mississippi Medical Center.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Felch K, Crider J, Bhattacharjee D, Huhn C, Wilson M, Bengt�n E. A model for assessment of channel catfish toll-like receptor (TLR) ligand potential in mammalian cells. (2024). Dev. And Comp. Immunol. Under review.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Felch K., Bengten E., and Wilson M. A cell reporter system to study TLR function in channel catfish. May 12, 2023. American Association of Immunologists Meeting, Washington D.C. Poster presentation.
|
Progress 01/03/22 to 01/02/23
Outputs
Target Audience:This reporting period provided me with many opportunities to share my research, and also interact with and teach the next generation of scientists. Beginning in March, I was invited to give a lecture in basic immunology to students in an introductory course at Tougaloo College. Tougaloo is a historic African American college, and the student body is largely made up of minority individuals. After the lecture, I also went back a few weeks later to teach an ELISA explorer laboratory that was very hands on for the students. In addition, I was asked to step in and teach UMMC medical students for an afternoon of POPS (patient-oriented problem solving) when a professor could not attend this session. Through these opportunities, I hope I was able to teach these students more about immunology, and maybe even inspire some of them to pursue research in their future careers. In addition, I learned that teaching could be a career goal of mine after I finish my postdoctoral research position. Moreover, this summer, funds from my fellowship allowed me to attend and present at the North American Comparative Immunology Workshop (NACI) held Banff, Alberta Canada June 5th - 8th. This conference was an amazing opportunity for me to share my research and progress on this project. Moreover, I also met many investigators who were looking for postdoctoral researchers, and many of them provided me with good feedback on my presentation. Several of them provided suggestions that would complement my project. For example, one suggestion was to examine if microplastics are potential ligands for any of the catfish TLRs. In addition, this past fall, October 27-29, this fellowship allowed me to also attend and present my research at the American Society of Microbiology (ASM) regional meeting in Shreveport, Louisiana. There, I again met many investigators that I would like to collaborate with in the future. Also, their feedback on my research progress was encouraging and positive. Combined, these meetings provided me the opportunity to network for potential postdoctoral research positions. I have also had the opportunity to mentor two undergraduate students one-on-one in the laboratory this past summer and fall. Over the summer, our volunteer who attends Rhodes College worked with me in the laboratory, helped me with several cell-based experiments. She also learned many benchtop skills and even helped us move in to our new laboratory. Currently, I am also mentoring a student worker that is working part-time in our catfish aquatic facility. He works on weekends, and it has been a pleasure to mentor him in how to care for our catfish and check the pumps and water flow. Changes/Problems: There have been no changes to the original course of study except to add in a little bit of emphasis on the interferon and cytokine production as well as cellular localization of the catfish TLRs after stimulation. Both of these two additional areas of interest are part of the TLR signaling complex and initiation of immune responses in the body. This information will complement the original intents of the project and provide even more information to be used in the future for the production of TLR-mediated vaccines for the catfish industry. We have encountered minor problems that have influenced the timeline of the plans for the fellowship. During this reporting period our department on campus was merged into another department. This would not have had a negative impact and actually would have provided more opportunities for collaboration and data generation if our laboratory space had remained on site and not have had to move as well. Several months of 2022 were dedicated to packing and moving laboratory equipment across campus to the new space and getting settled. This affected progress on the project by not allowing for bench work, experiments and data collection. In addition, once the laboratory supplies and equipment were moved, several reagents were ruined during transport which created problems and frustrations when I was able to get back to work in the laboratory. These problems have all been resolved and I am currently working hard to generate more data to make up for lost time. What opportunities for training and professional development has the project provided?This project has allowed me to learn how to perform confocal microscopy. Though I am still working on perfecting my experiments using confocal, I have now been taught how to operate the microscope that our department has and I have learned the general protocol for fixing, permeabilizing and staining confocal slides. This knowledge is immense and will greatly benefit me in the future and now with this current project. In addition, I have learned how to perform quantitative PCR and will soon learn to navigate publishing and responding to reviewers comments. How have the results been disseminated to communities of interest?These results have been disseminated to colleagues in the field through conference oral and poster presentations and will soon be published as open-access manuscripts and be incorporated into my dissertation. I have also shared my results with students through various teaching activies. What do you plan to do during the next reporting period to accomplish the goals?TLR5.1 will soon becloned into the fluorescent vector, pE2-crimson-N1 for use in confocal microscopy. In mammals, TLR5 recognizes flagellin, and this TLR is expressed on the cell surface of macrophages, dendritic cells, and epithelial cells that line the intestine. However, based on our phylogenetic analyses of catfish TLRs, TLR5.1 always groups with the endosomally-expressed TLRs with high bootstrap values whether using the TLR5.1 extracellular domain, the transmembrane region, or the intracellular TIR signaling domain. These same groups also occur when using full-length TLR sequences. If our confocal analyses data shows that TLR5.1 colocalizies with the endosome, this will be a novel finding. However, since flagellated bacteria can be either intracellular or extracellular, TLR5 may be localized to both the cell surface and the endosome. In addition, catfish TLR18 is the only TLR1 family member left to clone. Once this is done, I will then begin to study TLR heterodimerization patterns.After the first publication from this data, I will begin writing my dissertation based on this work as well.
Impacts
What was accomplished under these goals?
During this reporting period, I have made good progress on my project. Catfish TLR7 was successfully cloned into a fluorescent vector, pE2-crimson-N1, and will be used for confocal microscopy in the next two weeks. The goal of this study is to determine if catfish TLR7 localizes to the endosome or the cell surface. We predict based on the observed similarities of the catfish TLR7 transmembrane region to the human TLR7 transmembrane region that catfish TLR7 will be expressed on the endosome. In addition, we have examined interferon and cytokine production in catfish peripheral blood leukocytes (PBL) after they were stimulated with imiquimod, a TLR7 agonist. This work provided me with a co-author publication in Fish and Shellfish Immunology (2022), just before my project funding began. Also, this study included the full catfish interferon gene repertoire, and this information will allow us to screen interferon expression after TLR stimulation in our catfish clonal cell lines. We first created primers for these interferons, and used them to examine RNA expression in catfish cultured blood leukocytes after imiquimod stimulation. Initial results showed up regulation of IFNb, IFNc and IFNg. These results will be confirmed by real-time PCR. Once these few experiments are finished, I will submit a manuscript focused on TLR7 as a model for testing TLR ligands. In addition, I have cloned and expressed catfish TLR5.1 in the HEK-Blue reporter cells, and am currently testing different ligands. I am also using 5'-RACE to obtain the full length TLR26 sequence before cloning it into our expression vector. TLR26 is a TLR unique to catfish and of interest to me due to its novelty. The other TLRs that I have cloned are: TLR1, TLR2, TLR3, and TLR22. I am currently testing different ligands for TLR22, but I have not identified a positive ligand for this TLR yet. We arecurrently selecting for stable transfectants of three TLR constructs, TLR7-P3X-FLAG-CMV9, TLR5.1-P3X-FLAG-CMV9, and TLR22-P3X-FLAG-CMV9 for use in future studies. This project has many moving parts but is coming along quite well with several publications anticipated from the results. We have also been able to find several transcripts of known intermediate signaling molecules in the channel catfish. Transcripts that we have been able to locate so far include MyD88 and many different TRIF molecules which are both heavily involved in the signaling cascade after TLR activation in the mammalian models. We have also found an UNC93B1 molecule which in mammals is a chaperone protein specific for endosomal TLRs. This chaperone protein is involved in transporting the TLRs from the golgi complex to the endosome where it will then be expressed and recognize MAMPs specific for intracellular pathogens. From this initial data, we are currently writing the first publication directly related to this project.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Functional Characterization of Toll-Like Receptors (TLRs) in channel catfish, Ictalurus punctatus Felch, K., Crider, J., Bengt�n, E., Wilson, M. North American Comparative Immunology Workshop, Banff, Alberta, Canada, June 5-8, 2022
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Identification of Toll-like receptor ligands in channel catfish using a SEAP reporter model. Felch, K., Bengt�n, E., Wilson, M. South Central Branch of American Society of Microbiology Annual Meeting, Shreveport, LA, October 27-29, 2022.
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Identification of Toll-like receptor ligands in channel catfish using a SEAP reporter model. Felch, K., Bengt�n, E., Wilson, M. University of Mississippi Medical Center School of Graduate Studies Research Day, November 4, 2022.
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Functional Characterization of Toll-Like Receptors (TLRs) in channel catfish, Ictalurus punctatus. Kristianna Felch. University of Mississippi Medical Center Microbiology and Immunology Departmental Seminar, April 11, 2022.
|