Desireegonzalez Week 3

Electronic Lab Notebook

Purpose

• The purpose of this assignment is to understand how to properly read a scientific research paper. This assignment will assist in determining the proper ways to interpret data presented by other researchers through the explanation of their studies in words and models (including but not limited to, graphical representations of data as figures).

Preparation For Journal Club 1

List of Biological Terms

1. Shine-Dalgarno Sequence: a small sequence of nucleotides upstream the translational start site of a prokaryotic mRNA molecule, that assists in binding of rRNA and bringing initiator codon to the mRNA (Biology Online 2014).
2. Trehalose: a disaccharide made from two alpha-glucose molecules, which functions as an energy source for some bacteria, fungi, plants, and invertebrates (Biology Online 2014).
3. Carbohydrate Metabolism: the breakdown of large carbohydrates into smaller units that can be used by the body as a form of energy (Biology Online 2014).
4. Oxidative Stress:a highly oxidized environment with in cells that forces cells into a highly activated state due to a loss of control on their regulatory systems (Biology Online 2014).
5. Ubiquitin: a small protein used for regulation in eukaryotes (Biology Online 2014).
6. Hyperosmotic: a scenario when the total amount of solutes in a solution is greater than that in another solution; often relatinf to an increase in osmotic pressure (Biology Online 2014).
7. Glycogen: a polymer of glucose, that is branched, and that is often produced in liver and muscle cells to serve as a secondary long-term energy storage in animal cells (Biology Online 2014).
8. Dendogram: a tree diagram showing relationships between similar sets of data using hierarchial clustering (S. 2016).
9. Hierarchical Clustering:the process of building a tree diagram to represent data where each node in the cluster tree contains a group of similar data (S. 2016).
10. Dithiothreitol (DTT): a reagent used to stabilize enzymes and other proteins by reducing disulfide bonds (Thermo Fischer Scientific).

References

Biology Online (2014, May 12) Carbohydrate Metabolism. Retrieved from https://www.biology-online.org/dictionary/Carbohydrate_metabolism on 06 February 2019.

Biology Online (2014, May 12) Glycogen. Retrieved from https://www.biology-online.org/dictionary/Glycogen on 06 February 2019.

Biology Online (2014, May 12) Hyperosmotic. Retrieved from https://www.biology-online.org/dictionary/Hyperosmotic on 06 February 2019.

Biology Online (2014, May 12) Oxidative Stress. Retrieved from https://www.biology-online.org/dictionary/Oxidative_stress on 06 February 2019.

Biology Online (2014, May 12) Shine-Dalgarno Sequence. Retrieved from https://www.biology-online.org/dictionary/Shine-dalgarno_sequence on 06 February 2019.

Biology Online (2014, May 12) Trehalose. Retrieved from https://www.biology-online.org/dictionary/Trehalose on 06 February 2019.

Biology Online (2014, May 12) Ubiquitin. Retrieved from https://www.biology-online.org/dictionary/Ubiquitin on 06 February 2019.

S. (2016, November 15). Hierarchical Clustering / Dendrogram: Simple Definition, Examples. Retrieved from https://www.statisticshowto.datasciencecentral.com/hierarchical-clustering/ on 06 February 2019.

Schade, B., Jansen, G., Whiteway, M., Entian, K. D., & Thomas, D. Y. (2004). Cold Adaptation in Budding Yeast. Molecular Biology of the Cell, 15(12), 5492-5502. doi:10.1091/mbc.e04-03-0167

Thermo Fischer Scientific (n.d.) DTT (dithiothreitol). Retrieved from https://www.thermofisher.com/order/catalog/product/R0861 on 06 February 2019.

Article Outline

Background Information

1. What is the importance or significance of this work?
   • Budding Yeast can serve as a great model organism to take a closer look at the mechanisms developed by cells in response to environmental stressors. It is important to understand adaptability since it is essential for all organisms who are exposed to rapid changes in environment. This experiment focuses on Budding Yeasts response to cold shock that occurs due to the decrease of temperature from 37 degrees C to 25 degrees C.
2. What were the limitations in previous studies that led them to perform this work?
   • A limitation that could have occurred in previous studies that led these researchers to perform this work include the need for better technology and the absence of a standard application for the measurement of transcriptional profiles, and the absence of a set of standard conditions used by many researchers when inducing stress on an organism.
3. How does this work compare with previous studies?
   • In comparison to other studies, this work also demonstrates the importance of RNA helicases and responses of membrane fluidity to adapt to colder temperatures.
4. What strain(s) of yeast did they use?
   • The researchers used the yeast strains BY4743 and BSY25. The experiment focused on S. cerevisiae wild-type, msn2, and msn4 mutant cells.
5. Were the strain(s) haploid or diploid?
   • The strains used in the research experiment were diploid.

Experimental Procedure Used in the Research

1. How did they treat the yeast cells (what experiment were they doing?)
   • The researchers treated these yeast cells with a cold-shock experiment. They decreased the temperature to see how the yeast cells would respond to the stress of cold.
2. What media did they grow them in? What temperature? What type of incubator? For how long?
   • The media on which the cell cultures were grown was YPD medium; YPD medium is made of glucose, bactopeptone, and yeast extract. The experimental cultures were inocculated and grown over night in 50mL of media in a 250mL Erlenmeyer Flask that was being shaken at 170rpm. An incubator shaker was used until the overnight cultures were diluted. After the dilution of the cultures, a water bath shaker incubator was used.
3. What controls did they use?
   • The researchers carried out control microarrays (created with control hybridizations) to better determine the variability of the experimental factor in the experiment.
4. How many replicates did they perform per treatment or timepoint?
   • Two biological repeats were done for each of the experiments with the timepoints of 0, 2, and 12 hours.
   • Three biological repeats were done for each experiment with the timepoints of 30 and 60 minutes.
5. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
   • The researchers used the method of reverse transcription to properly label the RNA and have it ready for hybridization to the microarray.
   • The method of reverse transcription incorporated a Cy3- and Cy5-dCTP onto the mRNA to label it before using the resultant cDNA on yeast genomic DNA microarrays for hybridization.

Mathematical/ Statistical Methods Used in the Research

1. What mathematical/statistical method did they use to analyze the data?
   • The researchers in this experiment used the method of DNA microarrays to collect data. This data on the microarray slides was then analyzed by scanning using a ScanArray lite scanner. The resulting TIFF files were then quantified using the QuantArray software.
   • Microsoft Excel was used to normalize the data from the QuantArray software.
   • Each DNA spot underwent the following three quality control checks:
   1. Making sure the signal intensity was significantly greater than the background
   2. Making sure the signal intensity was within the dynamic range of the photomultiplier tube
   3. Making sure that the raw intensities of duplicate spots had to be within 50% of one another

Collected Data

1. Are the data publicly available for download?
   • The specific data from this experiment is not publicly available for download; the authors even refer to their data being unpublished on page 5499 of the scientific paper.
   • Although the specific data collected from this experiment is not publicly available for download, the other S. cerevisiae stress data they used for comparison in the paper is able to be downloaded publicly.
2. From which web site?
   • The supplemental stress data can be downloaded from the following websites.
     • Sahara Cold Expression Data
     • Genomic Expression Programs in the Response of Yeast Cells to Environmental Changes

Results Presented in Scientific Paper

1. What is the main result presented in this paper?
   • The main result presented in this paper is that S. cerevisiane has two expression patterns when looking at the transcriptional response to cold.
   • The two types of expression seen are early (ERS) and late (LSR) phase.
   • The earlier phase was seen to change fluidity of the membrane in response to colder temperatures. In addition to the membrane changes, the early phase also exemplifies destabilization of RNA secondary structures which are required for efficient protein translation.
   • The late phase was seen to involve the environmental stress response as a consequence of the altered cell state especially the decreased transport, reduced enzyme activity, and abundance of misfolded proteins.
   • Changes in response to the cold assist in cell survival and help it stay regulated in terms of its cellular processes.

Explanation of Figures Found in Research Paper

1. Figure 1 A:
   • What do the X and Y axes represent?
     • The X-axis represents the hierarchical clusters formed with the data collected from the DNA microarrays. The Y-axis represents the course of time throughout the experiment.
   • How were the measurements made?
     • Ratios of transcript abundance were created by dividing the experimental value by the reference value.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • The trend shown by the data is a statistical significance (two-fold) in variation of the 634 genes plotted.
     • All the green genes (mainly depicted by A, B, and F) are down-regulated, while all the red genes (mainly depicted by E, D, and C) are up-regulated.
     • The similarities in time exposure to the cold are represented by a dendogram on the vertical side of the figure.
2. Figure 1 B:
   • What do the X and Y axes represent?
     • The X-axis represents the various classifications of ECR genes. The Y-axis represents the number of genes regulated.
   • How were the measurements made?
     • The measurements for this bar graph were created by identifying which ECR genes were induced greater than or equal to 2 fold. These induced genes were identified with the use of open reading frames.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • Based on the bar graph, it can be concluded that the largest number of early response up-regulated genes were represented as uncharacterized open reading frames.
3. Figure 1 C:
   • What do the X and Y axes represent?
     • The X-axis represents the various classifications of LCR genes. The Y-axis represents the number of genes regulated.
   • How were the measurements made?
     • The measurements for this bar graph were also created by identifying which LCR genes were induced. These induced genes were identified with the use of open reading frames.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • Based on the bar graph, it can be concluded that the largest number of late response down-regulated genes were represented as genes in protein synthesis or as uncharacterized open reading frames.
4. Figure 2:
   • What do the X and Y axes represent?
     • The X-axis represents the decrease in temperature (from 37 degrees C to 25 degrees C) over the time the experiment was run. The Y-axis depicts the amount of gene transcription.
   • How were the measurements made?
     • These measurements were made by comparing experimental transcription profiles with supplemental data of transcription profiles when undergoing various stress stimuli.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • The highest amount of fluorescent red genes occurred at the 2 hour mark. Until the time reached two hours, the genes had a correlation in transcriptional response that revealed more bright red in each time frame. The larger amount of red fluorescence can conclude that there was an increase in regulation (in order to adapt) to the longer time in the colder temperatures.
5. Figure 3 A:
   • What do the X and Y axes represent?
     • The X-axis represents the time and the given stress stimuli used. The Y-axis represents the ECR transcriptional responses compared to LCR genes on each of the given stimuli.
   • How were the measurements made?
     • These measurements were made by comparing experimental transcription profiles with supplemental data of transcription profiles as the chosen gene was placed under a specific environmental stressor condition.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • A larger number of the individual ESR genes were red with cold shock, in comparison to the other environmental stressors from the supplemental data. This was deemed statistically significant by the researchers; most likely because it suggested a change in the levels of gene regulation over time.
6. Figure 3 B:
   • What do the X and Y axes represent?
     • The X-axis represents the time and the given stress stimuli used. The Y-axis represents the LCR transcriptional responses compared to ECR genes on each of the given stimuli.
   • How were the measurements made?
     • These measurements were also made by comparing experimental transcription profiles with supplemental data of transcription profiles as the chosen gene was placed under a specific environmental stressor condition.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • A relatively similar number of the individual LCR genes were red with cold shock as they were green with cold shock. This was deemed not statistically significant by the researchers.
7. Figure 3 C:
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • The Venn diagrams depict the similarities between induced and repressed stress genes when comparing LCR with ESR and ECR with ESR.
     • It can be concluded that the LCR and ESR genes had the largest number of common genes with 111.
     • The LCR and ESR overlap was concluded to be statistically significant by the researchers due to the large number of common genes.
8. Figure 4:
   • What do the X and Y axes represent?
     • The X-axis represents the wild type at different temperatures and the wild type in contrast to the mutants msn2 and msn4.
   • How were the measurements made?
     • A DNA microarray was done to collect the experimental data. This experimental data was then used to create a gene expression regulation map that represented the expression ratio for each gene (calculated as the average from duplicate or triplicate experiments).
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • The solely wild type data expressed a red and green fluorescence after the 12 hour mark. This concludes that the change in temperature causes a change in the transcription regulation of the gene. The wild type and mutant data bar had only very little green fluorescence at the 12 hour mark. This concludes that a large percent of the genes (specifically 78% of the LCR genes) were unaffected by the mutation.
9. Figure 5:
   • What do the X and Y axes represent?
     • The X-axis represents the time passing and the type of gene (ECR or LCR) and whether or not it has a mutation or is wild type. The Y-axis are the concentrations of the reserve carbohydrates trehalose and glycogen.
   • How were the measurements made?
     • These measurements were collected after exposing the cells to cold shock. The collected data was then compared to the induction of gene data collected from the DNA microarrays.
   • What trends are shown by the plots and what conclusions can you draw from the data?
     • There is an increase in the accumulated levels of reserve carbohydrates in LCR genes as the time increases. This can conclude, along with the microarray data, that a larger amount of genes involved in reserve carbohydrate metabolism are induced as time passes.
10. Figure 6:
    • What do the X and Y axes represent?
      • The X-axis represents the given time and experimental change (i.e. use of environmental stress genes versus use of ribosomal genes). The Y-axis represents the transcriptional response to cold.
    • How were the measurements made?
      • The measurements were made by comparing the gene clusters of this experiment to the data collected by Sahara et al. Then the color scale was used to determine the similarities and differences between the two sets of data.
    • What trends are shown by the plots and what conclusions can you draw from the data?
      • Looking at the plots there is a larger amount of red fluorescence seen when using the ESR genes in comparison to the ribosomal genes. This can conclude that such large discrepancies can be due to differences in strain or experimental design.

Implications and Future Directions of This Research

1. What are the important implications of this work?
   • This work with DNA microarrays and yeast cells is important to the scientific community because it can help increase the knowledge of the effects of various environmental stressors on a particular gene. The use of yeast is significant because it can act as a model organism to compare particular outcomes of a stressor on the cell regulation mechanisms of other organisms.
   • For this class in particular, this research is important since we will be using similar scientific experiments to collect data and also analyze it using DNA microarrays.
2. What future directions should the authors take?
   • The authors should focus on extending their research by publishing it and then expanding on experiments that will look into identifying the key regulatory mechanism that allows cells to adapt to the cold.

Evaluation of the Research Paper

1. 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?
   • I personally believe that the authors of the article did a mediocre job supporting their conclusions with the data they showed. The scientific paper itself was really hard to follow and understand. The reason that I believe the authors could have improved their supporting arguments, was that all the figures depicted used a red-green color scale to signify the data. No tables were attached to show that the interpretation of the results was not solely based on the color fluoresced by the DNA microarray. In addition I deemed it very wrong that the researchers were referencing their data although they never published it (like on the paragraph at the right hand of page 5499). I believe that the authors using a reference of data that was not published, was incorrectly used as support to aid in their conclusions. The data not being published makes the results harder to believe since they are not available for peer reviewing. I think that the only thing I would call a major flaw to this paper is the referencing of unpublished data. I think that this can be seen as a major flaw because it puts the study into jeopardy of undergoing data manipulation like that done by Dr. Potti in the video we watched for last weeks assignment.

Acknowledgements

I communicated with my homework partner, Fatima about how to answer some of the assignment questions and we also spoke about how to interpret the figure that we were assigned. I also spoke with another classmate Ava about specific questions that were being asked to be explained on the article outline.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Desireegonzalez (talk) 21:21, 6 February 2019 (PST)

Below are the links to all the Assignments and Journal Entries of the Spring 2019 Semester.

User Page: user:desireegonzalez

Template Page: template:desireegonzalez

Weekly Assignment Pages:

• Overall Weekly Assignment Schedule

1. Week 1
2. Week 2
3. Week 3
4. Week 4
5. Week 5
6. Week 6
7. Week 7
8. Week 9
9. Week 10
10. Week 11
11. Week 12
12. Week 15

Individual Journal Entry Pages:

1. desireegonzalez Week 1
2. desireegonzalez Week 2
3. desireegonzalez Week 3
4. desireegonzalez Week 4/5
5. desireegonzalez Week 6
6. desireegonzalez Week 7
7. desireegonzalez Week 9
8. desireegonzalez Week 10
9. desireegonzalez Week 11
10. desireegonzalez Week 12
11. desireegonzalez Week 15

Shared Journal Pages:

1. Shared Journal Week 1
2. Shared Journal Week 2
3. Shared Journal Week 3
4. Shared Journal Week 4
5. Shared Journal Week 5
6. Shared Journal Week 6
7. Shared Journal Week 7
8. Shared Journal Week 9
9. Shared Journal Week 10
10. Shared Journal Week 11
11. Shared Journal Week 12
12. Shared Journal Week 14/15