Search for: All records

Heart disease is a leading cause of mortality, with calcific aortic valve disease (CAVD) being the most prevalent subset. Being able to predict this disease in its early stages is important for monitoring patients before they need aortic valve replacement surgery. Thus, this study explored hydrodynamic, mechanical, and hemodynamic differences in healthy and very mildly calcified porcine small intestinal submucosa (PSIS) bioscaffold valves to determine any notable parameters between groups that could, possibly, be used for disease tracking purposes. Three valve groups were tested: raw PSIS as a control and two calcified groups that were seeded with human valvular interstitial and endothelial cells (VICs/VECs) and cultivated in calcifying media. These two calcified groups were cultured in either static or bioreactor-induced oscillatory flow conditions. Hydrodynamic assessments showed metrics were below thresholds associated for even mild calcification. Young’s modulus, however, was significantly higher in calcified valves when compared to raw PSIS, indicating the morphological changes to the tissue structure. Fluid–structure interaction (FSI) simulations agreed well with hydrodynamic results and, most notably, showed a significant increase in time-averaged wall shear stress (TAWSS) between raw and calcified groups. We conclude that tracking hemodynamics may be a viable biomarker for early-stage CAVD tracking. 

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3′-5′ exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.