Poster 97 - Investigating the Metabolic Effects of Hyaluronic Acid in Drosophila Using Advanced Multimodal Imaging

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Florence Z. Wang

Volunteer
UCSD Institute of of Engineering in Medicine, United States

Co-Author(s)

Sirasit Prayotamornkul (he/him/his)

Undergraduate Student Researcher
UCSD Shu Chien-Gene Lay Department of Bioengineering
San Diego, California, United States
Matthew Callahan (he/him/his)

Undergraduate Student Researcher
UCSD Shu Chien-Gene Lay Department of Bioengineering
Indio, California, United States
Zachary Z. Wang (he/him/his)

Student Mentor
University of California, San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

Florence Z. Wang

Volunteer
UCSD Institute of of Engineering in Medicine, United States

Co-Author(s)

Tanvi Movva (she/her/hers)

Volunteer
UCSD Institute of of Engineering in Medicine
San Diego, California, United States
DK

Derek Kang

Volunteer
UCSD Institute of of Engineering in Medicine, United States
SY

Sophia Yao

Volunteer
UCSD Institute of of Engineering in Medicine, United States
WZ

Will K. Zhou (he/him/his)

Volunteer
UCSD Institute of of Engineering in Medicine
Rancho Santa Fe, California, United States
YL

Yajuan Li

Postdoctoral Researcher
University of California, San Diego, United States

Primary Investigator(s)

LS

Linda Shi, PhD

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Last Author(s)

LS

Lingyan Shi

Advisor, Associate Professor, Postdoctoral Research Scientist
UCSD Shu Chien-Gene Lay Department of Bioengineering, United States

Introduction: Hyaluronic acid (HA) is highly prevalent in living systems, being found in joint fluids, skin dermis, and the vitreous body within eyes. Despite heavy research on HA’s cosmetic benefits, its metabolic effects have not been extensively studied, even when considering its potential to have considerable impacts. By feeding Drosophila with different polypeptide lengths of HA as well as deuterium oxide, carbon deuterium bonds could be formed to strengthen the use of Stimulated Raman Scattering (SRS) Microscopy, as these bonds isolates themselves from other biomolecular signals on the Raman spectrum. The results of this experiment show the strong points of Deuterium oxide-probed SRS and Two Photon Excitation Fluorescence (2PEF) microscopy to determine whether different molecular weights of HA have unique impacts on the metabolic states of different organs.

Materials and

Methods: Drosophila were used in the experiment as a test subject owing to their short lifespan, availability, and immense research history. Wild-type (w1118) Drosophila melanogaster were maintained on a cornmeal diet. Newly-eclosed flies were collected over a 24-hour period before being allowed to mate and reproduce for 48 hours. After that, they were shortly anesthetized with carbon dioxide and sorted into single sexes. Only female flies were studied in this experiment. They were maintained for 5 days on a 20% (v/v) deuterium oxide (D2O) sugar/yeast/agar diet either containing no HA (control), 2% (w/v) 8kDa HA, or 2% (w/v) 1430 kDa HA. This probing allows for the newly synthesized biomolecules to be identified by their carbon-deuterium (C-D) bonds as opposed to original molecules which contain only carbon-hydrogen (C-H) bonds. After the labeling period, the flies were sacrificed. Their fat body, gastrointestinal (GI) tract, and ovary were dissected and fixed in a 4% methanol-free paraformaldehyde solution diluted by 1× phosphate-buffered saline (PBS) for 30 minutes. These samples were subjected to SRS and 2PEF imaging within 24 hours of dissection and stored at 4˚C when not in use. Both microscopy techniques were performed without displacing the samples, allowing for a holistic analysis through multimodal imaging. SRS specifically allows the metabolism of an organism to be imaged to reveal crucial chemical information and special resolution without directly interfering with the cells. Image analysis was done through ImageJ, where unneeded background was subtracted and ratiometric images were generated for data quantification.

Results, Conclusions, and Discussions: In the analyzed images, it was discovered that although the ovaries and fat body did not show significance between groups, the data shows promising results in the GI tract. The main statistical significance appeared in the protein synthesis, de novo lipogenesis, and lipid saturation of the GI tract, meaning HA treatments have the most potential for future applications in said area. When exhibiting an impact, the results of the hyaluronic treatment differ based on which molecular weight of HA was used, as 8 kDA seems to enhance metabolic activity whereas 1430 kDA exhibits the opposite effect. Protein synthesis and lipid saturation in the control and 1430 kDA groups both have a p-value under 0.0001, suggesting a very high significance due to HA treatment. Meanwhile, lipogenesis in the control and 8 kDA groups have a p-value under 0.0001. Lipids seem to be most affected, since both test groups have a strong significance, while the protein and lipogenesis channels hold no significance for the 8kDA and 1430 kDA groups respectively. In terms of the test groups, the figure indicates that 1430 kDA HA seems to have the strongest influence compared to the 8 kDA which has a lesser significance in lipids. The results of the trial indicate that the gut of the HA-treated flies demonstrated significant alteration of metabolic activity as compared to other organs tested in this experiment. Further analysis is ongoing, but current findings indicate future research should be focused on the gut. Furthermore, it could be concluded that the 8 kDa and 1430 kDa strands have varying effects on the fly with different outcomes across channels. Overall, the experiment demonstrates that SRS and 2PEF microscopy are excellent in visualizing the metabolic effects of HA, while also warranting further investigation into effects from HA strands of other lengths, and expanding focus onto metabolic pathway channels not examined in this experiment.

Acknowledgements (Optional): We give special thanks to Dr Lingyan Shi and Dr. Shu Chien from UCSD Bioengineering, Dr. Lizhu Chen from CorDx Inc., in addition to Dr. Linda Shi for their expert guidance. This experiment was supported by a generous gift from Beckman Laser Institute Inc. to LS. The high school students (*) participated in the UCSD IEM OPALS internship.

References (Optional):