Ntesfaio Week 11
Contents
Purpose
Preparation to create my team's home page, Sulfiknights. Practice how to search an assigned primary literature. Prepare and give a journal club presentation.
10 Terms
proteome
the complement of proteins expressed in a cell, tissue, or organism by a genome https://www.merriam-webster.com/dictionary/proteome
glutathione biosynthesis
Protein involved in the synthesis of the tripeptide glutathione https://www.uniprot.org/keywords/KW-0317
glutathionylation
Protein which is posttranslationally modified by the attachment of a glutathione molecule by a disulfide bond. https://www.uniprot.org/keywords/KW-0318
Antimonite
antimonite refers to a salt of antimony(III), such as NaSb(OH)₄ and NaSbO₂, which can be prepared by reacting alkali with antimony trioxide, Sb₂O₃. https://en.wikipedia.org/wiki/Antimonite
thiol-reactive metals
Molecular Probes’ thiol-reactive reagents will react with thiol groups on proteins to give thioether-coupled products. tools.thermofisher.com/content/sfs/manuals/mp00003.pdf
glutamylcysteine
A precursor of glutathione formed by glutamate and cysteine ligase
https://en.wikipedia.org/wiki/Gamma-L-Glutamyl-L-cysteine
aquaglyceroporin
a aquaporin that transports water and glycerol
https://en.wiktionary.org/wiki/aquaglyceroporin
chelation
A bonding of ions and molecules to metal ions.
https://en.wikipedia.org/wiki/Chelation
Generalized additive model
Generalization from linear models. Focuses on interference of these linear models.
https://en.wikipedia.org/wiki/Generalized_additive_model
https://en.wikipedia.org/wiki/Generalized_additive_model
ubiquitination
"Kiss of death" as a protein is inactivated by attaching ubiquitin to it
https://www.medicinenet.com/script/main/art.asp?articlekey=26112
Outline of your journal article
1. What is the main result presented in this paper?
The purpose of this paper was to observe how Saccharomyces cerevisiae responded to arsenite. This was analyzed by quantitative transcriptome, proteome, and sulfur metabolite. Arsenite exposure can be toxic as it affects transcription in genes which is necessary for protein synthesis. Arsenic is highly toxic and is a risk to human health as well as the environment. Arsenic has been known to poison humansas epidemics of arsenic poisoning has occurred with contaminated drinking water. Chronic exposure can cause cardiovascular disease, neurological disorders, and liver injury. It is also associated with cancers of the skin, bladder, liver, and lung. As organisms because more exposed to arsenite there has been an evolution. Sulfur assimilation in yeasts in performed by having sulfate taken up and metabolized through a sulfate assimilation pathway. The sulfide goes through two possible pathways: the methyl cycle or the glutathione cycle. Glutathione (GSH) is a key factor in a cell's defense of metal toxicity. The metals used were sodium arsenite, cadmium chloride, and potassium antimonyl tartrate. Yeast strains were grown at 30 degrees Celsius on YNB mediums. RNA was isolated from the yeast cells that had been exposed to arsenite. For the proteome analysis yeast cells were exposed to arsenite for one hour. The yeast cells went through gel electrophoresis, protein extraction, and gel analysis. Most genes began responding to the arsenite in the first 15 minutes and peaked after 60 minutes. The lower concentration of 0.2mM triggered a faster transcriptional response that the higher concentration while the expression levels were larger at the higher concentrations. Proteome analysis had two-dimensional gel analysis that confirmed increased levels of proteins in the sulfur assimilation and GSH biosynthesis.
2. What is the importance or significance of this work?
Results showed that exposure to sodium arsenite of different concentrations yielded different effects. Cells exposed to 0.2 mM had a moderate effect on growth while those with 1 mM were severly affected.
3. What were the limitations in previous studies that led them to perform this work?
An understanding of how arsenic affects transcription of genes. Previous studies stated that Yap1p and Met4p control GSHI expression. This paper showed that the two also extend to most genes of the sulfur/GSH pathway.
4. How did they treat the yeast cells (what experiment were they doing?)
RNA was isolated from the yeast cells. cDNA was cleaned by vacuum-drying, and resuspension in hybridization buffer. The hybridization mix was incubated at 100 degree celsius for 2 minutes then 37 degrees celsius for 30 minutes. Hybridization was for 12-18 hours at 42 degrees celsius.
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
W303-1A RW124 CC849-1B RW104 YPDahl 166
6. What media did they grow them in? What temperature? What type of incubator? For how long?
Yeast strains were grown at 30 degrees celsius on YNB medium that were suplemented with autotrophic requirements. Glucose was used as a carbon source.
7. What controls did they use?
4 controls: Wildtype, met4, acr3, and acr3met4
8. How many replicates did they perform per treatment or timepoint? 4 replicates at 5 microM AS(III), 0.5mM AS(III), 5mM Sb(III), and 10 microM Cd(II)
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
RNA was isolated from the yeast cells that were both treated and untreated by arsenite. RNA was primed with a haxamer (invitrogen) and AB gene. It was labeled with Cy3-dUTP or Cy5-dUTP. The cDNA was resuspended in hybridization buffer. The hybridization max was heated at 100 degrees Celsius for 2 minutes and at 37 degrees Celsius for 30 minutes.
10. What mathematical/statistical method did they use to analyze the data?
They used what is called a generalized additive model (GAM) to test if hits were spread equally among all genes.
11. Are the data publicly available for download? From which web site?
Yes, when googling this paper the data is included in the figures and can be downloaded as a PDF.
12. See question 13
13. What do the X and Y axes represent?
In Figure 2 the x axis shows metabolite concentrations and the y axis showed time in hours. Figure 3 x axis is the amount of 35S in proteins and GSH. With the y axis being the difference in control and the two arsine concentrations.
14. How were the measurements made?
The measurements were made based on timepoints, p value, or heat maps.
15. What trends are shown by the plots and what conclusions can you draw from the data?
Figure 1 outlines sulfur assimilation and GSH biosynthesis in response to arsenite. Figure 2 shows a kinetic response of sulfur metabolite in response to arsenite. Figure 3 shows sulfate's incorporation in arsenite exposed cells. Figure 4 shows a heat map of changes of genes in sulfur assimilation. Figure 5 shows sulfur/GSH metabolism contributes to arsenite tolerance.
16. How does this work compare with previous studies?
Haugen et. al previously reported on gene expression data that this paper corroborates to. It integrated phenotype expression and transcriptional profiling by mapping the data.
17. What are the important implications of this work?
Yap1p and Met4p control the response of arsenite tolerance acquisition. Metabolites accumulated over time and sulfur eventually would be metabolized.
18. What future directions should the authors take?
Observing the other genes that were stated to be affected by arsenite exposure.
19. 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 believe the figures were relevant and gave a clear explanation of the changes from the arsenite.
Powerpoint Slide
Annotated Bibliography
Create a bibliography of a minimum of 8-10 citations to primary research articles related to your assigned article (2 per group member). Each of the 8-10 references in your bibliography needs to have the following information (an example is given in the section below):
- The complete bibliographic reference in the APA style (see this link), with DOI.
Zhou, X., Arita, A., Ellen, T. P., Liu, X., Bai, J., Rooney, J. P., ... & Costa, M. (2009). A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity. Genomics, 94(5), 294-307.
DOI
https://doi.org/10.1016/j.ygeno.2009.07.003
- The link to the abstract from PubMed.
- The link to the full text of the article in PubMedCentral (if not available, write N/A).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763962/
- The link to the full text of the article (HTML format) from the publisher web site.
https://www.sciencedirect.com/science/article/pii/S088875430900158X
- The link to the full PDF version of the article from the publisher web site.
http://sq4ya5rf2q.search.serialssolutions.com/?V=1.0&sid=PubMed:LinkOut&pmid=19631266
- 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.
Copyright © 2009 Elsevier Inc. All rights reserved.
- How is the article available to you?
NIH Public ACCESS
- 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.
No
- 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.
Author manuscript; available in PMC on November 1, 2010
- 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.
online-only
- Who is the publisher of the journal?
Genomics
- Is the publisher for-profit or non-profit?
for-profit
- Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
for-profit publisher
- Does the publisher belong to the Open Access Publishers Association?
- What country is the journal published in?
- How Long has the journal been in operaion? (e.g., browse the archive for the earliest article published)
- Are the articles in this journal peer-reviewed?
- Provide a link to the scientific advisory board/editorial board of the journal.
- What is the journal impact factor? (Look to see if it is provided on the journal home page.)
- Is the article a review or primary research article?
- Are the data associated with this article available? If so, provide a link to the dataset.
You must use these three databases/tools to find the references that you include in your bibliography: PubMed, GoogleScholar, and Web of Science. Answer the following questions as part of your assignment:
- Use a keyword search for the first three databases/tools and answer the following:
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?
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?
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?
- Which advanced search functions were most useful to narrow down the search? How many results did you get?
Web of Science
- Which advanced search functions were most useful to narrow down the search? How many results did you get?
- 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?
THIS WILL BE COMPLETED TUESDAY NOVEMBER 19
Conclusion
The purpose of this week's assignment was to begin the annotated bibliography and create a powerpoint presentation of the article assigned.
Acknowledgments
My homework partners this week were User:Jnimmers (Quality Assurance), User:Imacarae (Data Analysis), User:Mavila9 (Data Analysis) , and User:Dmadere (Designer). We each split into different roles (as stated in parenthesis) with myself serving as a Quality Assurance representative and as project manager.
"Except for what is noted above, this individual journal entry was completed by me and not copied from another source."
Ntesfaio (talk) 12:50, 13 November 2019 (PST)
References
Thorsen, M., Lagniel, G., Kristiansson, E., Junot, C., Nerman, O., Labarre, J., & Tamás, M. J. (2007). Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite. Physiological genomics, 30(1), 35-43.
