Louisiana Research Day Program Book 2025

Biomedical Research: Section 1

Biomedical Research: Section 1

Sabeen Wazir, OMS-II; Anne B. Remorca, OMS-II; Trevor Morris, OMS-II; Erin Vasquez, OMS-II; Ariana Faraji, DO; Kasia Michalak, MSc; Lin Kang, PhD; Stephen DiGiuseppe, PhD; Pawel Michalak, PhD 1 VCOM- Louisiana 20 CRISPR-LIKE PROPERTIES OF EUKARYOTIC 45S RIBOSOMAL 45S RNA GENE CLUSTERS

David Kang 1 ; Jedidiah Lim 1 ; Victoria Lucas 1 ; Samreen Shah 1 ; Sherine Thomas 1 ; Meredith Gwin, PhD 2 ; Dara W. Frank, PhD 3 , Samir Gautam, MD, PhD 2 ; Melissa Lipsmeyer, PhD 1 ; Rebekah Morrow, PhD 4 ; K. Adam Morrow, PhD 1 ; Sarah Voth, PhD 1 1 Department of Cell Biology and Physiology, VCOM-Louisiana, Monroe, LA; 2 Section of Pulmonary, Critical Care, and Sleep Medicine, Yale School of Medicine, New Haven, CT; 3 Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI; 4 Department of Microbiology and Immunology, VCOM-Louisiana, Monroe, LA 21 PSEUDOMONAS INDUCED ARDS PROMOTES CARDIOMYOCYTE NECROSIS SECONDARY TO CYSTATIN-C DEPLETION

Context: 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. Interestingly, there are sequences within ITSs that are homologous to certain DNA viruses, such as herpesviruses in vertebrates. Preliminary data from the lab suggests that ITS

RNA fragments may play an extra ribosomal role in innate immune sensing that was previously overlooked. Hypothesis: We hypothesize that ITS regions contain ancient fragments of integrated virus-like sequences that are used by the host to stimulate the antiviral response against certain DNA viruses. by transfecting in small interfering RNA (siRNA) followed by infection with herpes simplex 1 virus (HSV-1) then measured viral titers by plaque assay and viral protein levels by immunoblotting. We also measured the average plaque sizes using GFP expressing HSV-1 following ITS2 depletion. Lastly, we transfected an overabundance of specific ITS2 RNA into NHDFs followed by HSV-1 infection and measured viral titers via plaque assay and measured viral protein levels by immunoblotting. Results: RNA sequencing confirmed depletion of ITS2 RNA using two specific siRNAs. Depletion of ITS2 RNA following infection Methods: We depleted specific ITS2 RNA in normal human dermal fibroblasts (NHDFs)

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. Conclusions: It was assumed that ITS spacer regions only serve as a precursor during rRNA maturation. However, our lab has identified integrated virus-like sequences that may serve immunostimulatory and antiviral effects against herpesvirus infection. While the exact pathways stimulated by ITS RNA have yet to be identified, these findings suggest a novel extra ribosomal function of ITS spacer regions and provide a basis for a new paradigm in understanding antiviral immunity.

Background: Infection-induced acute respiratory distress syndrome (ARDS) and pneumonia recurrently result from the pathogen, Pseudomonas aeruginosa. Virulent strains of P. aeruginosa employ a type III secretion system (T3SS) to inject cytotoxic exoenzymes directly into host cells. Exoenzyme Y (ExoY), a T3SS effector protein, is expressed in ~ 90% of clinical isolates. Cytotoxic ExoY induces a cascade of cellular disturbances which causes tau hyperphosphorylation, disrupts cytoskeletal integrity, and releases pathogenic tau into the extracellular space. Under physiological conditions, endothelial amyloids, including amyloid-beta (A β ) are cytoprotective and antimicrobial, thereby shielding cardiomyocytes from ischemic injury. Cytotoxic tau compromises the innate function of endothelial amyloids, suppresses antimicrobiocity, and may promote infection-induced cardiac microthrombi, a devastating complication of ARDS. Cystatin C (CysC), a ubiquitously expressed cysteine protease inhibitor, is fundamental in regulating amyloid homeostasis. Proteomic analyses of blood and urine samples from critically ill patients repeatedly demonstrated marked depletion of serum CysC through ARDS-associated inflammatory

proteinuria, yet the pathophysiological consequences of CysC depletion or remedy of this critical care phenomenon remain inadequately understood. Here, we sought to determine whether CysC depletion via inflammatory proteinuria promotes cardiomyocyte necrosis during ExoY induced pneumonia. Methods: Male and female wildtype C57BL/6J mice (10-12 weeks old) were infected intratracheally with either vehicle or 1 x 105 colony forming units (CFU) of bacteria in 40 µl of PBS. P. aeruginosa strain ExoY+ (secretes only ExoY in host cells) was used for infections. Weight, urine, and PT/INR were collected daily and survival was tracked. At 48 hours post-infection, surviving mice were sacrificed. Urine was collected via cystocentesis, blood was collected via cardiac puncture, and the brain, kidneys, heart, and lungs were fixed for histological analysis. CysC levels of the blood and urine were measured via ELISA. Amyloid and tau levels were assessed via immunoblotting and thioflavin T.

Results: TBD

Conclusions: TBD

30

31

2025 Research Recognition Day