Msamdars Week 11

Purpose

This week in our assignment we will read and analyze a scientific article and be able to explain the results to others. Furthermore, we will also be working in our teams for the first time and learn to navigate the team's dynamics.

Ten Definitions

retrograde pathway - Signals that travel from plastids to nuclei in plants and eukaryotic algae, and from mitochondria to nuclei in other eukaryotes, and tend to send messages regarding stress and environmental sensing (Lagarias et al., 2013)

septin ring - Septins are GTP-binding proteins in most eukaryotic cells (except in plant cells) and can form a protein complex in the shape of nonpolar filaments, filament bundles, rings, or cages (Douglas et al., 2005)

trehalose - A disaccharide made up of two α-glucose molecules, and serves as an energy source in certain fungi, bacteria, plants, and invertebrates (Biology Online, 2019)

immunoblot - (AKA Western Blot) A procedure in which proteins separated by electrophoresis in polyacrylamide gels are transferred (blotted) onto nitrocellulose or nylon membranes and identified by specific complexing with antibodies that are either pre-or post-tagged with a labelled secondary protein. (Biology Online, 2019)

triose phosphate - (AKA Glyceraldehyde phosphate) A phosphate ester of the 3-carbon sugar glyceraldehyde and has chemical formula: C3H7O6P (Biology Online, 2019)

chemostat - Apparatus for maintaining a bacterial population in the exponential phase of growth by regulating the input of a rate-limiting nutrient and the removal of medium and cells (Smith, 2000)

Cdc11 - One of a family of mitotic septins that aid in cell division by forming a ring structure at the septum (Biology Online, 2019)

RT-PCR - Reverse transcriptase polymerase chain reaction; reverse transcription PCR PCR in which the starting template is RNA, implying the need for an initial reverse transcriptase step to make a DNA template. Some thermostable polymerases have appreciable reverse transcriptase activity; however, it is more common to perform an explicit reverse transcription, inactivate the reverse transcriptase or purify the product, and proceed to a separate conventional PCR. Abbreviation ambiguous because also used sloppily for real-time PCR. (Lackie, 2007)

bud neck - The constriction between the mother cell and daughter cell (bud) in an organism that reproduces by budding.

isozyme - (AKA Isoenzyme) Enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (i.e. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (Biology Online, 2019)

Journal Article Outline

What is the main result presented in this paper?

The main result of the paper states that short-term potassium deprivation has an effect on the mRNA transcription rate of around a thousand genes. They see that in particular genes that control sulfur metabolism, trigger an oxidative response, and activate the retrograde pathway get affected. It also says that they observe a significant decrease in overall ribosome creation and translation, components like cyclins and protein kinases, and decrease in septin assembly.

What is the importance or significance of this work?

Given that a lack of potassium showed a significant decrease in gene expression of certain pathways, this opens up a new field of study to see how exactly the potassium cation regulates these pathways.

What were the limitations in previous studies that led them to perform this work?

This study had apparently never been done before. Thus, this study was necessitated to perform a complete transcriptomic analysis.

How did they treat the yeast cells (what experiment were they doing?)

The scientists grew the cells at 28C in translucent K+ free medium supplemented with KCl solution. Cells were then transferred to fresh media with KCl, or without K+. Samples of both were then taken at 20, 40, 60, and 120 min from four replicates.

What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?

The strains used were BY4741, BYT1, BYT2, BYT12, BY4741 rtg2, BY4741 rtg3, BY4741 fzo1, YNR055.1 YPC722 (5), YPC723, YPC724, W303-1A, DBY746. It was not told to us which ploidy each strain was. However, according to the SGD wiki, BY4741 is a haploid strain (Cherry et al., 2011).

What media did they grow them in? What temperature? What type of incubator? For how long?

The yeast were grown in translucent potassium-free YNB media at 28°C supplemented with 50 mM KCl. They did not specify an incubator, but did say the yeast was grown until the culture reached an optical density of 0.8.

What controls did they use?

They extracted a control from four replicates from the media supplemented with 50 mM KCl at every timepoints of the experiment.

How many replicates did they perform per treatment or timepoint?

They used four replicates of each experimental treatment and control at each timepoints of 0, 20, 40, 60, 120.

What method did they use to prepare the RNA, label it and hybridize it to the microarray?

They used the Ribo-Pure kit to extract the RNA, and didn't say how they labelled and hybridized it.

What mathematical/statistical method did they use to analyze the data?

They used GEPAS server to pre-process the microarray data, but made no mention of what mathematical approaches they used. For metabolite data, they used the DataAnalysis software and used standardized calibration curves to quantify the metabolites.

Are the data publicly available for download? From which web site?

Yes, the data is available at the Society of Applied Microbiology website.

Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.

1. Figure 1
   • This figure shows the results of clustering of the genes. One can see overall trends of genes tend to be of either repression or activation across all of the timepoints. One can see that there are no other trends, with clusters of genes increasing expression at one timepoints and decreasing expression at another timepoint.
2. Table 1
   • This table simply shows the number of genes activated and repressed at each of the timepoints of the experiments.
3. Figure 2
   • There are multiple parts to this figure.
     • A) is a figure of sulfur uptake, and Met/Cys metabolism, with the genes associated annotated.
     • B) shows graphs of mRNA level of a number of genes over time for both treatments. X-Axis: mRNA change (log-fold expression); Y-Axis: genes (by name). Measurements from microarray data.
     • C) shows graphs of intracellular levels of Met and Cys over time after transfer of cells to K+-free medium. X-Axis: Time (min); Y-Axis: Met/Cys concentration μM. Measurements from HPLC-mass spectrometry.
4. Figure 3
   • There are multiple parts to this figure.
     • A) shows a graph of the response of certain environmental-stress response genes over time. X-Axis: Time (min); Y-Axis: mRNA change (log-fold expression). Measurements from microarray data.
     • B) are images of stained cells that show the amount of ROS produced in each cell over the time of the experiment. This is shown by fluorescent dyes that activate upon exposure to ROS.
     • C) shows a graph of the concentration of oxidized and reduced forms of glutathione over over the course of the potassium-deprivation experiment in the cells. X-Axis: Time (min); Y-Axis: Glutathione concentration μM. Measurements from HPLC-mass spectrometry.
5. Figure 4
   • There are multiple parts to this figure.
     • A) shows a diagram of methylglyoxal generation/degradation and trehalose metabolism pathways.
     • B) shows graphs of the changes in expression levels of genes associated with methylglyoxal generation/degradation and trehalose metabolism. X-Axis: Time (min); Y-Axis: mRNA change (log-fold expression). Measurements from microarray data.
     • C) shows levels of methylglyoxal within the cell over the course of the potassium-deprivation experiment. X-Axis: Time (min); Y-Axis: Methylglyoxal concentration μM. Measurements from HPLC-mass spectrometry.
6. Figure 5
   • There are multiple parts to this figure.
     • A) shows the expression levels of certain genes controlling the retrograde response over time, accompanied by a picture of the results of RT-PCR on 3 genes, with a wild type and mutant. X-Axis: Time (min); Y-Axis: mRNA change (log-fold expression). Measurements from microarray data.
     • B) shows fluorescent microscopy images of mitochondrial morphology in wild type cells treated and untreated with KCl, and images of a mutant strain that act as a control.
     • C) shows concentrations of ammonium in wild type cells in K+-free medium and normal medium. X-Axis: Time (min); Y-Axis: NH4+ concentration nmol/mg. Measurement source unknown.
     • D) shows concentrations of ammonium in multiple mutant cells with different or knocked-down potassium transporters. X-Axis: Time (min); Y-Axis: NH4+ concentration nmol/mg. Measurement source unknown.
7. Figure 6
   • There are multiple parts to this figure.
     • A) shows changes in expression of genes that control cyclins. X-Axis: Time (min); Y-Axis: mRNA change (log-fold expression). Measurements from microarray data.
     • B) shows images of cyclin mRNA quantified by RT-PCR, with two treatments at two timepoints each.
     • C) shows images of a western blot of multiple cyclins at a variety of timepoints.
8. Figure 7
   • There are multiple parts to this figure.
     • A) shows changes in expression of genes related to bud neck and septin ring formation. X-Axis: Time (min); Y-Axis: mRNA change (log-fold expression). Measurements from microarray data.
     • B) shows fluorescent microscopy images of Cdc11-GFP in wild-type cells with and without K+ treatment.
     • C) shows images of a Western blot for Cdc11 at multiple timepoints in K+ free treatment and control, as well as in multiple forms, supernatant and pellet.
     • D) shows fluorescent microscopy images of wild type cells with Cdc11-GFP in them, treated with and without K+.
9. Table 2
   • This table contains a list of all of the strains used in the study, the genotype of each strain, and the source/reference for each strain.

How does this work compare with previous studies?

There have been no previous studies that are equivalent to this study. No genome-wide transcriptomic analysis has been carried out like this, with this kind of stressor. Thus, this research is novel and illuminates the effects of a lack of potassium on cells.

What are the important implications of this work?

This study leaves a number of future studies available for study. We may see future studies into how potassium affects cell cycle regulation because it showed that cyclins are regulated by potassium. Furthermore, the retrograde response pathway and ammonium can also be targets of further study.

What future directions should the authors take?

I think that the authors should study the oxidative stress response and global environmental stress response, as these two were heavily touched on in the study. One may see that the authors studied the accumulation of methylglyoxal and how it affects the environmental stress response, and I would study that overall pathway.

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 think that this paper was very thorough with how they presented all their results. Every significant result that the authors presented, they supported with additional follow-up experiments. I think that they presented quite a bit of information, and provided a solid baseline study for a number of future studies. I do not think there were any major flaws.

Annotated Bibliography

Reference 1

1. The complete bibliographic reference in the APA style (see this link), with DOI.
2. The link to the abstract from PubMed.
3. The link to the full text of the article in PubMedCentral (if not available, write N/A).
4. The link to the full text of the article (HTML format) from the publisher web site.
5. The link to the full PDF version of the article from the publisher web site.
6. Who owns the rights to the article? Look at the first page of the PDF version of the article for the © symbol. Generally, either the journal/publisher or the authors will hold the copyright.
7. How is the article available to you?
   • Is the article available “Open Access” (look for the words “open access” or the “unlocked” icon on the article website or the first page of the PDF) If YES, stop here.
   • If the article is not Open Access, is it available for free after a certain period of time has elapsed? You would not find the words “open access” or the “unlocked” icon, but you would still be able to access the article. If YES, stop here.
   • Did LMU buy a subscription or pay a fee for your access to this article? You might see “Loyola Marymount University” or “LMU” on the article website. Alternately, a list of the journals that LMU pays for can be found at: http://sq4ya5rf2q.search.serialssolutions.com/ If YES, stop here.
   • Is the article behind a paywall or “subscription-only”? Your attempts to access it when on the LMU network have failed. In this case, if you needed the article, you would use the ILLIAD system to request it by logging in here: https://lmu.illiad.oclc.org/illiad/illiad.dll?Action=99. Note that you don’t need to actually request it for this assignment.
8. Is the article available online-only or both in print and online? Look on the journal website for a “subscription” link. If that page talks about subscribing to the print edition, then it is available in print. If not, it is available online-only.
9. Who is the publisher of the journal?
   • Is the publisher for-profit or non-profit?
   • Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
   • Does the publisher belong to the Open Access Publishers Association?
   • What country is the journal published in?
10. How Long has the journal been in operaion? (e.g., browse the archive for the earliest article published)
11. Are the articles in this journal peer-reviewed?
12. Provide a link to the scientific advisory board/editorial board of the journal.
13. What is the journal impact factor? (Look to see if it is provided on the journal home page.)
14. Is the article a review or primary research article?
15. Are the data associated with this article available? If so, provide a link to the dataset.

Reference 2

1. The complete bibliographic reference in the APA style (see this link), with DOI.
2. The link to the abstract from PubMed.
3. The link to the full text of the article in PubMedCentral (if not available, write N/A).
4. The link to the full text of the article (HTML format) from the publisher web site.
5. The link to the full PDF version of the article from the publisher web site.
6. Who owns the rights to the article? Look at the first page of the PDF version of the article for the © symbol. Generally, either the journal/publisher or the authors will hold the copyright.
7. How is the article available to you?
   • Is the article available “Open Access” (look for the words “open access” or the “unlocked” icon on the article website or the first page of the PDF) If YES, stop here.
   • If the article is not Open Access, is it available for free after a certain period of time has elapsed? You would not find the words “open access” or the “unlocked” icon, but you would still be able to access the article. If YES, stop here.
   • Did LMU buy a subscription or pay a fee for your access to this article? You might see “Loyola Marymount University” or “LMU” on the article website. Alternately, a list of the journals that LMU pays for can be found at: http://sq4ya5rf2q.search.serialssolutions.com/ If YES, stop here.
   • Is the article behind a paywall or “subscription-only”? Your attempts to access it when on the LMU network have failed. In this case, if you needed the article, you would use the ILLIAD system to request it by logging in here: https://lmu.illiad.oclc.org/illiad/illiad.dll?Action=99. Note that you don’t need to actually request it for this assignment.
8. Is the article available online-only or both in print and online? Look on the journal website for a “subscription” link. If that page talks about subscribing to the print edition, then it is available in print. If not, it is available online-only.
9. Who is the publisher of the journal?
   • Is the publisher for-profit or non-profit?
   • Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
   • Does the publisher belong to the Open Access Publishers Association?
   • What country is the journal published in?
10. How Long has the journal been in operaion? (e.g., browse the archive for the earliest article published)
11. Are the articles in this journal peer-reviewed?
12. Provide a link to the scientific advisory board/editorial board of the journal.
13. What is the journal impact factor? (Look to see if it is provided on the journal home page.)
14. Is the article a review or primary research article?
15. Are the data associated with this article available? If so, provide a link to the dataset.

===Responses

1. PubMed
   • What original keyword(s) did you use? How many results did you get?
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
2. Google Scholar
   • What original keyword(s) did you use? How many results did you get?
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
3. Web of Science
   • What original keyword(s) did you use? How many results did you get?
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
4. Use the advanced search functions for each of these three databases/tools and answer the following:
5. PubMed
   • Which advanced search functions were most useful to narrow down the search? How many results did you get?
6. Google Scholar
   • Which advanced search functions were most useful to narrow down the search? How many results did you get?
7. Web of Science
   • Which advanced search functions were most useful to narrow down the search? How many results did you get?
8. Perform a prospective search on your article in the Web of Science and answer the following:
   • How many articles does this article cite?
   • How many articles cite this article?

• Reflect:
  1. What impact does the choice of keywords have on your results?
  2. What are the advantages and disadvantages of each of the three databases (Pubmed, Google Scholar, Web of Science)?

Data & Files

Journal Club Presentation

Conclusion

This week we read a paper, and critically analyzed the paper. Furthermore, we learned to use resources to better supplement our knowledge, and to increase our information literacy. We learned to analyze those resources and think critically about them as well. I learned quite a bit during this assignment, and was surprised to learn that the first genome-wide transcriptomic analysis on yeast deprived of such an important cation was only carried out a few years ago. I wonder what other analogous experiments have not been carried out, as a result of this?

Acknowledgments

This week my team members are in Skinny Genes which is composed of Jonar Cowan, Aby Mesfin, David Ramirez, Christina Dominguez, & myself. We worked in and out of class together.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Other Links

Links
Mihir Samdarshi User Page
Assignment Pages	Personal Journal Entries	Shared Journal Entries
Week 1	Journal Week 1	Class Journal Week 1
Week 2	Journal Week 2	Class Journal Week 2
Week 3	FAS2/YPL231W Week 3	Class Journal Week 3
Week 4	Journal Week 4	Class Journal Week 4
Week 5	Database - AmtDB	Class Journal Week 5
Week 6	Journal Week 6	Class Journal Week 6
Week 7	Journal Week 7	Class Journal Week 7
Week 8	Journal Week 8	Class Journal Week 8
Week 9	Journal Week 9	Class Journal Week 9
Assignment Pages	Personal Journal Entries
Week 10	Journal Week 10
Week 11	Journal Week 11
Week 12/13	Journal Week 12/13
Team Project Links
Skinny Genes Team Page

References

1. LMU BioDB 2019. (2019). Week 11. Retrieved November 7, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_11
2. MediaWiki (2019). Category: Help. Retrieved November 11, 2019, from https://www.mediawiki.org/wiki/Category:Help
3. Lagarias, J.C., Duanmu, D., Casero, D., Dent, R.M., Gallaher, S., Yang, W., Rockwell, N.C., Martin, S.S., Pellegrini, M., Niyogi, K.K., Merchant, S.S., Grossman, A.R. (2013). "Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival". Proceedings of the National Academy of Sciences of the United States of America. 110 (9): 3621–3626. doi:10.1073/pnas.1222375110.
4. Douglas, L. M., Alvarez, F. J., McCreary, C., & Konopka, J. B. (2005). "Septin function in yeast model systems and pathogenic fungi". Eukaryotic Cell. 4 (9): 1503–12.
5. Biology Online. (2019). Retrieved November 7, 2019 from https://biology-online.org
6. Smith, A. (2000). Oxford Dictionary of Biochemistry and Molecular Biology: Revised Edition. Oxford University Press.
7. Lackie, J. M. (Ed.). (2007). The dictionary of cell & molecular biology. Academic Press. Retrieved November 11, 2019 from http://ebookcentral.proquest.com/lib/lmu/detail.action?docID=311420.
8. Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S, AmiGO Hub, Web Presence Working Group. AmiGO: online access to ontology and annotation data. Bioinformatics. Jan 2009;25(2):288-289. Retrieved November 11, 2019 from http://amigo.geneontology.org/amigo/
9. Cherry, J. M., Hong, E. L., Amundsen, C., Balakrishnan, R., Binkley, G., Chan, E. T., ... & Fisk, D. G. (2011). Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Research, 40(D1), D700-D705.