Biology subjects

Kwan, E.

Publications and source records attributed to Kwan, E..

4 recordsLinked to original sources

bioRxiv2025

Peroxisome proliferator-activated receptor gamma (PPARG)-mediated myocardial salvage in acute myocardial infarction managed with left ventricular unloading and coronary reperfusion

Ischemic heart disease and acute myocardial infarction (AMI) is a leading cause of morbidity and mortality. Improvements have been made in coronary interventions to restore blood flow, but ischemia/reperfusion (I/R) injury significantly impacts clinical outcomes. We previously reported that activation of percutaneous mechanical unloading of the left ventricle (LV) with a transvalvular axial-flow device simultaneously with reperfusion improves myocardial salvage. However, the underlying mechanisms, potential adjuvant pharmacological interventions and the timing of the use of LV unloading as a cardioprotective approach in AMI are not well understood. This study investigated a) the mechanisms associated with improved myocardial salvage, b) a pharmacological intervention, and c) the timing of LV unloading. Following 90 minutes of ischemia, adult swine were subjected to reperfusion alone, simultaneous unloading with reperfusion, upfront unloading with delayed reperfusion, upfront reperfusion with delayed unloading, or reperfusion with concurrent use of esmolol and milrinone. Compared to controls, the simultaneous group had a 47% increase in myocardial salvage following AMI. This was associated with increased expression of neutrophil degranulation, macrophage activation, iNOS signaling, wound healing, and PPAR signaling. From these pathways, PPARG (peroxisome proliferator-activated receptor gamma) emerged as a potential cardioprotective gene that was uniquely overexpressed in the simultaneously unloaded and reperfused myocardium. Next, we showed PPARG agonism with rosiglitazone reduces mitochondrial oxygen demand in cardiomyocytes and in vivo, improves myocardial salvage following I/R injury in C57BL6/J mice. Thiazolidinediones (TZDs), such as rosiglitazone could be investigated as therapies combined with simultaneous LV unloading and coronary interventions to mitigate reperfusion injury. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/637726v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1c4d6e6org.highwire.dtl.DTLVardef@1f8d064org.highwire.dtl.DTLVardef@396eddorg.highwire.dtl.DTLVardef@9d932f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

rDNA copy number variation in yeast alters response to environmental conditions

Ribosomal DNA (rDNA) in eukaryotes is maintained in hundreds of copies with rDNA copy number varying greatly among individuals within a species. In the budding yeast Saccharomyces cerevisiae, the rDNA copy number across wild isolates ranges from 90 to 300 copies. Previous studies showed that 35 rDNA copies are sufficient for ribosome biogenesis in this yeast and enable wild-type-like growth in standard laboratory growth conditions. We addressed two major questions concerning rDNA copy number variation in this yeast: (1) What are the fitness consequences of rDNA copy number variation outside and within the natural range in standard laboratory growth conditions? (2) Do these fitness effects change in different growth conditions? We used growth competitions to compare the fitness effects of rDNA copy number variation in otherwise isogenic strains whose rDNA copy number ranged from 35 to 200. In standard growth conditions, we found that fitness gradually increases from 35 rDNA copies until reaching a plateau that spans from 98 to 160 rDNA copies, well within the natural range. However, rDNA copy number-dependent fitness differed across environments. Compared to standard growth conditions, strains with higher rDNA copy numbers showed improved fitness when grown at increased temperature or in glycerol media. Moreover, the gradual fitness increase with increasing rDNA copy number in standard growth conditions gave way to a markedly lower fitness of strains with copy numbers below the natural range in these two stress conditions. These results suggest that selective pressures drive rDNA copy number in this yeast to at least ~100 copies and that a higher number of copies buffers against environmental stress. The similarity of the S. cerevisiae rDNA copy number range to the ranges reported in C. elegans, D. melanogaster, and humans points to conserved selective pressures maintaining the range of natural rDNA copy number in these highly diverse species.

genetics↗

bioRxiv2021

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription. Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

genomics↗

bioRxiv2019

Phenotypic and genotypic consequences of CRISPR/Cas9 editing of the replication origins in the rDNA of Saccharomyces cerevisiae

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

genetics↗