Ckaplan Week 12
Media:Hailey_Charlotte_Katie_Journal_Club_Presentation.pdf
- What is the main result presented in this paper?
The paper shows how Saccharomyces cerevisiae responds in the first 20 minutes to oxidative stress caused by cumene hydroperoxide (CHP). Using experiments with time controls and duplicate samples, the study finds new genes involved in this stress response and highlights differences in how cells react to CHP compared to hydrogen peroxide. The study used the data only up to t 20, highlighting how after 20 minutes, the yeast exhibited a stress response.
- What is the importance or significance of this work?
This research is important because it's the first to show how yeast quickly responds (within 0-6 minutes) to CHP-induced stress. It also uncovers the roles of Yap3, Yap5, and Yap7 in yeast's response to oxidative stress, which weren't known before. Understanding these processes better could help us grasp how cells deal with stress and how it relates to diseases.
- What were the limitations in previous studies that led them to perform this work?
Previous studies didn't use control groups and incubated the CHP. Because of these issues, it was hard to understand the data well. This study tried to fix these problems by keeping track of time, using enough samples, and controlling environmental factors tightly to get a better understanding of how cells respond early to CHP-induced stress.
- How did they treat the yeast cells (what experiment were they doing?)
CHP- cumene hydroperoxide.
- What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
Diploid- BY4743.
- What media did they grow them in? What temperature? What type of incubator? For how long?
2% (w/v) sucrose with uracil L-leucine, and L-histidine at 30ºC using BioFlo fermentors.
- What controls did they use?
Yeast strain BY4743 was used but was not exposed to CHP.
- How many replicates did they perform per treatment or timepoint?
3
- What method did they use to prepare the RNA, label it and hybridize it to the microarray?
The researchers used GeneChip® One-Cycle cDNA synthesis kit to amplify the RNA samples, as recommended by the manufacturer. After amplification, labeled targets were hybridized to Affymetrix GeneChip® Yeast Genome S98 arrays.
- What mathematical/statistical method did they use to analyze the data?
A 2 way ANOVA gene model was used to assess the significance of differences between transcripts across two time points.
- Are the data publicly available for download? From which web site?
Yes, the data is publicly available on Gene Expression Omnibus (GEO)database.
- Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
Figure 1- the data illustrate how yeast efficiently converts cumene hydroperoxide (CHP) into cumyl alcohol (COH) within the initial 20 minutes of exposure. This transformation indicates the effectiveness of yeast in processing CHP, with the majority of this conversion occurring due to yeast activity rather than spontaneous chemical reactions in the medium itself.
Figure 2 shows the researchers' exploration of yeast's response to CHP-induced oxidative stress over time. Control cultures without CHP were included. Significant alterations are observed in the control cultures at the 40-minute mark, prompting the researchers to focus their analysis on the first 20 minutes.
In Figure 3- the researchers identify each of the clusters: Cluster A exhibits a rapid surge in activity at 6 minutes, indicating an immediate defensive response to oxidative stress. In contrast, Cluster B shows a slower but sustained increase in activity starting at 12 minutes, suggesting a secondary wave of defense mechanisms kicking in. Cluster C displays a temporary decrease in activity, suggesting a redistribution of yeast resources to respond to oxidative stress defenses. Cluster D demonstrates a gradual and steady increase in activity commencing at the 20-minute mark, potentially reflecting long-term adjustments aimed at enhancing survival strategies against oxidative stress
Figure 4 illustrates the identification of gene induction or repression within 3 to 6 minutes of exposure. In Graph A, genes rapidly induced return to their normal mRNA levels by 12 minutes but continue to decrease afterward. In Graph B, genes rapidly repressed return to their normal mRNA levels by 12 minutes but continue to increase thereafter.
Figure 5 presents the expression of six YAP family genes (YAP1, 2, 3, 4, 5, and 7), which encode bZIP proteins acting as transcription factors. It indicates the time point at which each YAP gene's expression is induced. Remarkably, the study notes the first observation of YAP3, YAP5, and YAP7 being induced by oxidative stress, with their functions previously unknown.
Figure 6 displays the gene expression response of the genes belonging to three overlapping regulons: YAP1, SKN7, and MSN2/4. It demonstrates that there are more overlapping genes between MSN2/4 and YAP1 compared to any other category. In the visualization, red indicates induction, and green indicates repression. The logarithm of the ratio of median values divided by the median value at time zero quantifies the intensity.
In Figure 7, the data reveal the induction of redox protein-encoding genes upon exposure to CHP. These genes are crucial for maintaining the reduced state of the cytosol and are associated with the glutathione and thioredoxin systems.
Figure 8 is organized into four different "functions," with the lag time of transcriptional response of genes increasing from left to right. The squares represent CHP-treated cultures, while triangles represent control cultures.
Figure 9 shows proteasome proteolysis, which targets partially-oxidized proteins prone to aggregation. This process occurs later in the oxidative stress response, around the 20-minute mark. The transcription of genes encoding proteasome subunits is regulated by Rpn4, a transcription factor.
- What do the X and Y axes represent (if applicable)?
- How were the measurements made?
- What trends are shown by the plots and what conclusions can you draw from the data?
- How does this work compare with previous studies?
- What are the important implications of this work?
- What future directions should the authors take?
- Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any major flaws to the paper?
Assignment Pages
- Week 1
- Week 2
- Week 3
- Week 4
- Week 5
- Week 6
- Week 8
- Week 9
- Week 10
- Week 11
- Week 12
- Week 13
- Week 14
- Week 15
Individual Journal Entry Pages
- ckaplan Week 1
- ckaplan Week 2
- SIR2 Week 3
- AgeAnnoMO Week 4
- ckaplan Week 5
- ckaplan Week 6
- ckaplan Week 8
- ckaplan Week 9
- ckaplan Week 10
- ckaplan Week 11
- ckaplan Week 12
- ckaplan Week 13
- ckaplan Week 14
- ckaplan Week 15
