Louisiana Via Research Day Book 2026

Biomedical Research: Section 1

Biomedical Research: Section 1

Hannah Khairandish, OMS-II; Rose Pwint, OMS-II; Kayla Griffith, OMS-II; Francheska Brzazgon, OMS-II; Kasia Michalak, MSc; Lin Kang, PhD; Stephen DiGiuseppe, PhD; Pawel Michalak, PhD VCOM-Louisiana 17 CRISPR-LIKE PROPERTIES OF EUKARYOTIC 45S RIBOSOMAL 45S RNA GENE CLUSTERS ARE CONSERVED ACROSS DISTINCT HSV-1 STRAINS

Jimmy T. Nguyen, OMS-III; Mujahid Hines, MD; Jamie Beckman, DO; Sarah Voth, PhD; Melissa Lipsmeyer, PhD; K. Adam Morrow, PhD VCOM-Louisiana 18 ASSESSING PROPHYLACTIC ANTIBIOTIC PROTECTION AGAINST PSEUDOMONAS AERUGINOSA INFECTED HUMAN CORNEAL ENDOTHELIAL CELLS

Background: Viruses lack their own ribosomes and have evolved strategies to usurp and repurpose host cell ribosomes to favor viral protein production. This challenge posed by viruses has resulted in virus-host coevolution, with ribosomes and ribosomal RNA (rRNA) gene clusters locked in genetic conflict with viral genomes. In addition to their tandem repeat redundancy, eukaryotic 45S rRNA gene clusters have several striking features reminiscent of bacterial and archaeal Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), such as palindromic organization of highly conserved sequences (18S, 5.8S, and 28S rDNA) separated by unique spacer regions called internal transcribed spacers 1 (ITS1) and 2 (ITS2). ITS sequences are highly dissimilar between species but tend to preserve RNA secondary structures that include a stem-loop. ITS regions are considered non-functional RNA located between structural rRNA that are transcribed as a precursor rRNA and degraded during the rRNA maturation process. However, previous work from our group demonstrated that ITS2 contains virus like sequences that restrict HSV-1 replication. Whether this antiviral activity is conserved across different HSV-1 strains remains unclear.

Hypothesis: We hypothesize that ITS2 mediated antiviral activity is conserved across distinct HSV-1 strains, including more aggressive laboratory variants, HSV-F. Method: Specific ITS2 RNA fragments were depleted in normal human dermal fibroblasts (NHDFs) using small interfering RNAs (siRNAs), followed by infection with herpes simplex virus type 1 (HSV-1). To expand and validate our previous findings, infections were performed using two distinct HSV-1 strains, including the laboratory strain HSV-1 (KOS) and the additional strain HSV-F. Viral titers were quantified by plaque assay, viral protein expression was assessed by immunoblotting, and average plaque sizes were measured using GFP-expressing HSV-1. In complementary experiments, NHDFs were transfected with an overabundance of specific ITS2 RNA prior to HSV-1 infection, followed by assessment of viral titers and protein levels. Results: RNA sequencing confirmed depletion of ITS2 RNA using two specific siRNAs. Depletion of ITS2 RNA following infection with HSV-1 showed a significant increase in

HSV-1 titers measured via plaque assay. The average plaque size was also increased when specific ITS2 RNA fragments were depleted during infection. We also observed increased viral protein levels of ICPO and ICP4 when specific ITS2 RNAs were depleted by siRNAs. Conversely, when we transfected into NHDFs an overabundance of specific ITS2 RNA followed by infection with HSV-1, and we observed a reduction in HSV-1 titers. These effects were reproducible across both HSV-1 strains tested, confirming and extending our prior observations. Conclusion: While ITS spacer regions were thought to serve only as precursors during rRNA maturation, these findings confirm that ITS mediated antiviral activity is conserved across distinct HSV-1 strains. This work validates and extends previous observations, supporting a strain-independent, CRISPR-like role for eukaryotic rRNA spacer regions in innate antiviral immunity.

Context: Endophthalmitis caused by Pseudomonas aeruginosa is a devastating complication of cataract surgery with poor visual prognosis if not treated rapidly. Although moxifloxacin is commonly administered intracamerally post-cataract surgery as prophylaxis to reduce endophthalmitis risk, it remains an off-label use. The high intracameral concentrations achieved clinically far exceed the FDA susceptibility breakpoint, supporting its clinical use for endophthalmitis prophylaxis. However, the safety profile of moxifloxacin at these therapeutic concentrations remains debatable, with potential dose-dependent cytotoxicity to corneal endothelial cells. This study evaluates the prophylactic efficacy of three commonly used ophthalmic antibiotics - azithromycin (a macrolide), moxifloxacin (a fluoroquinolone), and tobramycin (an aminoglycoside) - against P. aeruginosa (PAO1 strain) infection in immortalized human corneal endothelial cells (HCECs). Objective: To assess the prophylactic efficacy of azithromycin, moxifloxacin, and tobramycin against P. aeruginosa (PAO1 strain) infection in immortalized HCECs.

Methods: HCECs were cultured to confluence in 6-well plates. Dose-response studies were conducted 12-24 hours post-confluence. Prior to infection, cells were treated with either vehicle control or ascending doses of antibiotic for 2 hours. The dosages selected for this study should be based on achieving intracameral concentrations that exceed the FDA susceptibility breakpoints for P. aeruginosa while remaining within clinically safe ranges for corneal endothelial cells. The antibiotic concentrations include Azithromycin 2, 4, 8 mcg/mL, Moxifloxacin 0.5, 1, 2 mcg/mL, and Tobramycin 4, 8, 16 mcg/mL. After 2 hours, treatments were terminated, cells were rinsed and subsequently inoculated with PAO1 at a multiplicity of infection (MOI) of 20 1. Monolayer integrity was assessed via image capture at multiple time intervals post-infection using AmLite software and cell damage was quantified using Fiji ImageJ software by counting rounded (damaged) cells per image. Results: Moxifloxacin demonstrated dose dependent protection against PAO1 infection, with both the number and percentage of rounded cells decreasing with ascending dosages by ~ 22%. Azithromycin showed decreased

absolute numbers of rounded cells but increased percentages of rounded cells with higher dosages. Tobramycin showed more rounded cells than PBS control at most concentrations, with only the highest dosage (16 mcg/mL) demonstrating protective effects. Conclusion: PAO1 demonstrated susceptibility to moxifloxacin with dose-dependent protective effects, consistent with known P. aeruginosa susceptibility to fluoroquinolones. Variable resistance to azithromycin and tobramycin was observed, with tobramycin showing reduced efficacy compared to PBS vehicle except at the highest concentration. These findings align with established antimicrobial susceptibility patterns showing P. aeruginosa susceptibility to fluoroquinolones and aminoglycosides, and resistance to macrolides. Among the three antibiotics tested, moxifloxacin demonstrated the most promising protective effects for HCECs against P. aeruginosa strain PAO1, corroborating current clinical use of moxifloxacin. Further research is needed to comprehensively evaluate moxifloxacin toxicity to human corneal endothelial cells and establish the optimal concentration that balances antimicrobial efficacy with cellular safety.

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2026 Research Recognition Day