Difference between revisions of "Mavila9 Week 12 Journal"

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==Purpose==
 +
The purpose of this project was to work as a team on [https://www.physiology.org/doi/full/10.1152/physiolgenomics.00236.2006 primary literature] to demonstrate our understanding of microarray data analysis.
 +
 +
==10 Words I Didn't Understand Before==
 +
#Acute promyelocytic leukemia - An aggressive type of acute myeloid leukemia in which there are too many immature blood-forming cells in the blood and bone marrow. It is usually marked by an exchange of parts of chromosomes 15 and 17. Also called APL and promyelocytic leukemia (NCI Dictionary of Cancer Terms, 2008).
 +
#Aquaglyceroporin - integral membrane proteins that are permeable to glycerol as well as water (Grosell, 2003).
 +
#Chelation - the combination of a metal ion with a chemical compound, forming a ring (biology-online.org dictionary, 2014).
 +
#Glutathionylation - the specific post-translational modification of protein cysteine residues by the addition of the tripeptide glutathione, the most abundant and important low-molecular-mass thiol within most cell types (Dalle-Donne, 2009).
 +
#Auxotrophic - any strain of microorganism (alga, bacterium, or fungus) that differs from the wild‐type by requiring a supply of one or more growth factors (Oxford Dictionary of Biochemistry and Molecular Biology).
 +
#Orthologue - a gene, protein, or biopolymeric sequence that is evolutionarily related to another by descent from a common ancestor, having diverged as a result of a speciation event (Oxford Dictionary of Biochemistry and Molecular Biology).
 +
#Dichotomous - a term that describes dividing into two parts or classifications (biology-online.org dictionary, 2014).
 +
#Antioxidant - any substance that inhibits oxidation, usually because it is preferentially oxidized itself (Lackie, 2007).
 +
#Glutathione - most abundant non-protein thiol, synthesized in the cytosol, that protects against oxidative stress, and regulates cell proliferation, immune response, apoptosis, and fibrogenesis (Lu, 2012)
 +
#Ubiquitination - formation of a highly efficient and selective isopeptide bond between a substrate lysine residue and the C-terminus of ubiquitin catalyzed by ubiquitin-protein ligase, or E3 (Pickart, 2001).
 +
 +
==Article Outline==
 +
# What is the main result presented in this paper?
 +
#* This paper's main result was that cells responded to cellular arsenite acquisition by stimulating sulfur assimilation or glutathione biosynthesis pathways through the control of transcription factors Yap1p and Met4p. These results are found by transcriptome, proteome, and sulfur metabolite profiling of ''Saccharomyces cerevisiae's'' response to arsenite.
 +
# What is the importance or significance of this work?
 +
#* This work provides further information about the impact of arsenite on the environmental and human health. Due to its causative and curative properties for disease a more full understanding of global and specific responses can help development of medical therapies.
 +
# What were the limitations in previous studies that led them to perform this work?
 +
#* Previous studies have not shown whether protein glutothionylation occurs in response to metals or not.
 +
# How did they treat the yeast cells (what experiment were they doing?)
 +
#* Metal sensitivity assays using sodium arsenite, cadmium chloride, and potassium antimonyl tartrate were done. The yeast cells were exposed to different concentrations of arsenite.
 +
# What strain(s) of yeast did they use?  Were the strain(s) haploid or diploid?
 +
#* The following yeast strains were used:
 +
#** W303-1A
 +
#** RW124
 +
#** CC849-1B
 +
#** RW104
 +
#** YPDAHL1166
 +
# What media did they grow them in?  What temperature? What type of incubator? For how long?
 +
#* The yeast was grown at 30 degrees Celsius in minimal YNB medium of 0.67% yeast nitrogen base, auxotrophic requirements, and 2% glucose or on SC medium.
 +
# What controls did they use?
 +
#* 18s rRNA was used for a control during Northern blot analysis
 +
#* A culture of yeast unexposed to arsenite was used as a control when comparing GSH synthesis following arsenite exposure
 +
# How many replicates did they perform per treatment or timepoint?
 +
#* The amount of replicates per treatment was not mentioned in the article.
 +
# What method did they use to prepare the RNA, label it and hybridize it to the microarray?
 +
#* The RNA was isolated, then 20 micrograms of RNA was primed with 3 micrograms of random hexamer and 3 micrograms of anchored oligo(dT)20 primer. Next the RNA was labeled using a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP in 30 microliters. The RNA was then hybridized with 32P-labeled PCR fragments of ''MET3'', ''MET25'', and ''MET14''.
 +
# What mathematical/statistical method did they use to analyze the data?
 +
#* A generalized additive model with a logic link was used to analyze whether SGD gene hits were spread equally among all genes or overrepresented among regulated genes
 +
#* SGD hits were obtained from searching for genes with the consensus motif present in the promoter and in at least 50% of the available promoters of other species
 +
# Are the data publicly available for download?  From which web site?
 +
#* The data is publicly available at the end of the paper on https://www.physiology.org/doi/full/10.1152/physiolgenomics.00236.2006
 +
# Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
 +
#* Figure 2A-B shows that within the first 30 minutes of exposure the pools of homocysteine, cystathionine, cysteine, gamma-glutamylcysteine, and GSH started to increase and continued to do so with time.
 +
#** x-axis shows time in hours; y-axis shows intracellular sulfur metabolite concentrations in micromolar.
 +
#* Figure 3 shows sulfate utilization in cells exposed to 0.1 micromolar As(III) and 0.2 micromolar As(III).
 +
#** x-axis shows exposure to molarity of As(III); y-axis shows amount of 35S in proteins and GSH in amount of radioactivity (cpm).
 +
#* Figure 4A shows a heat map of gene expression after exposure to As(III).
 +
#** x-axis shows amount of As(III) exposure in milimoles as well as time in hours; y-axis shows the gene beingobserved for expression changes; a third dimension shows upregulation or downregulation of gene expression.
 +
#** There is a trend of  ''met4'' and ''yap1'' causing downregulation of the observed genes after exposure to 0.2 milimolar As(III).
 +
#* Figure 4B shows a northern blot analysis of RNA extracted from wt, ''met4'', and ''yap1'' cells before and at various time points.
 +
#** x-axis shows time in minutes; y-axis shows observed RNA extract.
 +
#** There is a trend of ''met4'' and ''yap1'' having less RNA expression with time.
 +
#* Figure 5 shows how sulfur and GSH metabolism contribute to cellular As(III) tolerance.
 +
#** x-axis shows amount of metal exposure; y-axis shows the gene being expressed.
 +
#** There is a trend of inverse relationship between gene expression and amount of metal exposure.
 +
# How does this work compare with previous studies?
 +
#* This work adds to previous studies by confirming the effects of metal exposure on gene expression and suggesting that the presence of Met4 and Yap1 also play a role in controlling expression of other genes during exposure to As(III).
 +
# What are the important implications of this work?
 +
#* The important implications of this work are that Met4 and Yap1 are the major cotrollers of gene expression during presence of As(III).
 +
# What future directions should the authors take?
 +
#* The author should further investigate the specific molar amount of metal exposure necessary to inhibit cellular growth of particular cells to add to the knowledge of using metals for treating cancer patients.
 +
# 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 the paper did a good job at explaining their results and showing that there was significance in their findings. Their figures seemed to build off of each other to support their hypothesis.
 +
 +
==Files==
 +
 +
[[media:Sulfinights presentation mavila9.pptx | Sulfinights presentation mavila9]]
 +
 +
==Conclusion==
 +
In conclusion, this article demonstrated the how expression of several genes changed with exposure to As(III) and the role of Met4 and Yap1 presence on the changes of gene expression.
 +
 +
==References==
 +
(2006). auxotroph. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 12 Nov. 2019, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-1807
 +
 +
Biology Online, BWB Marketing, 12 May 2014, https://www.biology-online.org/dictionary
 +
 +
Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., & Milzani, A. (2009). Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends in biochemical sciences, 34(2), 85-96. doi: https://doi.org/10.1016/j.tibs.2008.11.002
 +
 +
Grosell, M., & Bury, N. R. (2003). Biochimica et Biophysica Acta (BBA)/Biomembranes: Preface. Biochimica et Biophysica Acta-Biomembranes, 1618(2). doi: https://doi.org/10.1016/j.bbamem.2015.10.004
 +
 +
Lackie, J. M., & Lackie, J. M. (Eds.). (2007). The dictionary of cell and molecular biology. Retrieved from https://ebookcentral.proquest.com
 +
 +
Lu, S. C. (2013). Glutathione synthesis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(5), 3143-3153. doi: https://doi.org/10.1016/j.bbagen.2012.09.008
 +
 +
“NCI Dictionary of Cancer Terms.” National Cancer Institute, 5 Apr. 2018, www.cancer.gov/publications/dictionaries/cancer-terms/.
 +
 +
Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annual review of biochemistry, 70(1), 503-533. doi: https://doi.org/10.1146/annurev.biochem.70.1.503

Latest revision as of 00:53, 14 November 2019

Links

User Page

Template:mavila9

Assignment Page Individual Journal Entry Class Journal Entry
Week 1 Week 1 (User page) Shared Journal Week 1
Week 2 Mavila9 Week 2 Shared Journal Week 2
Week 3 Gene Page Week 3 Shared Journal Week 3
Week 4 Journal Entry Page Week 4 Shared Journal Week 4
Week 5 RNAct Database Page Week 5 Shared Journal Week 5
Week 6 Journal Entry Page Week 6 Shared Journal Week 6
Week 7 Journal Entry Page Week 7 Shared Journal Week 7
Week 8 Journal Entry Page Week 8 Shared Journal Week 8
Week 9 Journal Entry Page Week 9 Shared Journal Week 9
Week 10 Journal Entry Page Week 10 Shared Journal Week 10
Week 11 Sulfiknights Team Page Shared Journal Week 10
Journal Entry Page Week 11
Week 12/13 Journal Entry Page Week 12 Shared Journal Week 11
Week 12/13 Sulfiknights DA Week 12/13 Shared Journal Week 12
N/A Sulfiknights DA Week 14

Purpose

The purpose of this project was to work as a team on primary literature to demonstrate our understanding of microarray data analysis.

10 Words I Didn't Understand Before

  1. Acute promyelocytic leukemia - An aggressive type of acute myeloid leukemia in which there are too many immature blood-forming cells in the blood and bone marrow. It is usually marked by an exchange of parts of chromosomes 15 and 17. Also called APL and promyelocytic leukemia (NCI Dictionary of Cancer Terms, 2008).
  2. Aquaglyceroporin - integral membrane proteins that are permeable to glycerol as well as water (Grosell, 2003).
  3. Chelation - the combination of a metal ion with a chemical compound, forming a ring (biology-online.org dictionary, 2014).
  4. Glutathionylation - the specific post-translational modification of protein cysteine residues by the addition of the tripeptide glutathione, the most abundant and important low-molecular-mass thiol within most cell types (Dalle-Donne, 2009).
  5. Auxotrophic - any strain of microorganism (alga, bacterium, or fungus) that differs from the wild‐type by requiring a supply of one or more growth factors (Oxford Dictionary of Biochemistry and Molecular Biology).
  6. Orthologue - a gene, protein, or biopolymeric sequence that is evolutionarily related to another by descent from a common ancestor, having diverged as a result of a speciation event (Oxford Dictionary of Biochemistry and Molecular Biology).
  7. Dichotomous - a term that describes dividing into two parts or classifications (biology-online.org dictionary, 2014).
  8. Antioxidant - any substance that inhibits oxidation, usually because it is preferentially oxidized itself (Lackie, 2007).
  9. Glutathione - most abundant non-protein thiol, synthesized in the cytosol, that protects against oxidative stress, and regulates cell proliferation, immune response, apoptosis, and fibrogenesis (Lu, 2012)
  10. Ubiquitination - formation of a highly efficient and selective isopeptide bond between a substrate lysine residue and the C-terminus of ubiquitin catalyzed by ubiquitin-protein ligase, or E3 (Pickart, 2001).

Article Outline

  1. What is the main result presented in this paper?
    • This paper's main result was that cells responded to cellular arsenite acquisition by stimulating sulfur assimilation or glutathione biosynthesis pathways through the control of transcription factors Yap1p and Met4p. These results are found by transcriptome, proteome, and sulfur metabolite profiling of Saccharomyces cerevisiae's response to arsenite.
  2. What is the importance or significance of this work?
    • This work provides further information about the impact of arsenite on the environmental and human health. Due to its causative and curative properties for disease a more full understanding of global and specific responses can help development of medical therapies.
  3. What were the limitations in previous studies that led them to perform this work?
    • Previous studies have not shown whether protein glutothionylation occurs in response to metals or not.
  4. How did they treat the yeast cells (what experiment were they doing?)
    • Metal sensitivity assays using sodium arsenite, cadmium chloride, and potassium antimonyl tartrate were done. The yeast cells were exposed to different concentrations of arsenite.
  5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
    • The following yeast strains were used:
      • W303-1A
      • RW124
      • CC849-1B
      • RW104
      • YPDAHL1166
  6. What media did they grow them in? What temperature? What type of incubator? For how long?
    • The yeast was grown at 30 degrees Celsius in minimal YNB medium of 0.67% yeast nitrogen base, auxotrophic requirements, and 2% glucose or on SC medium.
  7. What controls did they use?
    • 18s rRNA was used for a control during Northern blot analysis
    • A culture of yeast unexposed to arsenite was used as a control when comparing GSH synthesis following arsenite exposure
  8. How many replicates did they perform per treatment or timepoint?
    • The amount of replicates per treatment was not mentioned in the article.
  9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
    • The RNA was isolated, then 20 micrograms of RNA was primed with 3 micrograms of random hexamer and 3 micrograms of anchored oligo(dT)20 primer. Next the RNA was labeled using a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP in 30 microliters. The RNA was then hybridized with 32P-labeled PCR fragments of MET3, MET25, and MET14.
  10. What mathematical/statistical method did they use to analyze the data?
    • A generalized additive model with a logic link was used to analyze whether SGD gene hits were spread equally among all genes or overrepresented among regulated genes
    • SGD hits were obtained from searching for genes with the consensus motif present in the promoter and in at least 50% of the available promoters of other species
  11. Are the data publicly available for download? From which web site?
  12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • Figure 2A-B shows that within the first 30 minutes of exposure the pools of homocysteine, cystathionine, cysteine, gamma-glutamylcysteine, and GSH started to increase and continued to do so with time.
      • x-axis shows time in hours; y-axis shows intracellular sulfur metabolite concentrations in micromolar.
    • Figure 3 shows sulfate utilization in cells exposed to 0.1 micromolar As(III) and 0.2 micromolar As(III).
      • x-axis shows exposure to molarity of As(III); y-axis shows amount of 35S in proteins and GSH in amount of radioactivity (cpm).
    • Figure 4A shows a heat map of gene expression after exposure to As(III).
      • x-axis shows amount of As(III) exposure in milimoles as well as time in hours; y-axis shows the gene beingobserved for expression changes; a third dimension shows upregulation or downregulation of gene expression.
      • There is a trend of met4 and yap1 causing downregulation of the observed genes after exposure to 0.2 milimolar As(III).
    • Figure 4B shows a northern blot analysis of RNA extracted from wt, met4, and yap1 cells before and at various time points.
      • x-axis shows time in minutes; y-axis shows observed RNA extract.
      • There is a trend of met4 and yap1 having less RNA expression with time.
    • Figure 5 shows how sulfur and GSH metabolism contribute to cellular As(III) tolerance.
      • x-axis shows amount of metal exposure; y-axis shows the gene being expressed.
      • There is a trend of inverse relationship between gene expression and amount of metal exposure.
  13. How does this work compare with previous studies?
    • This work adds to previous studies by confirming the effects of metal exposure on gene expression and suggesting that the presence of Met4 and Yap1 also play a role in controlling expression of other genes during exposure to As(III).
  14. What are the important implications of this work?
    • The important implications of this work are that Met4 and Yap1 are the major cotrollers of gene expression during presence of As(III).
  15. What future directions should the authors take?
    • The author should further investigate the specific molar amount of metal exposure necessary to inhibit cellular growth of particular cells to add to the knowledge of using metals for treating cancer patients.
  16. 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 the paper did a good job at explaining their results and showing that there was significance in their findings. Their figures seemed to build off of each other to support their hypothesis.

Files

Sulfinights presentation mavila9

Conclusion

In conclusion, this article demonstrated the how expression of several genes changed with exposure to As(III) and the role of Met4 and Yap1 presence on the changes of gene expression.

References

(2006). auxotroph. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 12 Nov. 2019, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-1807

Biology Online, BWB Marketing, 12 May 2014, https://www.biology-online.org/dictionary

Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., & Milzani, A. (2009). Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends in biochemical sciences, 34(2), 85-96. doi: https://doi.org/10.1016/j.tibs.2008.11.002

Grosell, M., & Bury, N. R. (2003). Biochimica et Biophysica Acta (BBA)/Biomembranes: Preface. Biochimica et Biophysica Acta-Biomembranes, 1618(2). doi: https://doi.org/10.1016/j.bbamem.2015.10.004

Lackie, J. M., & Lackie, J. M. (Eds.). (2007). The dictionary of cell and molecular biology. Retrieved from https://ebookcentral.proquest.com

Lu, S. C. (2013). Glutathione synthesis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(5), 3143-3153. doi: https://doi.org/10.1016/j.bbagen.2012.09.008

“NCI Dictionary of Cancer Terms.” National Cancer Institute, 5 Apr. 2018, www.cancer.gov/publications/dictionaries/cancer-terms/.

Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annual review of biochemistry, 70(1), 503-533. doi: https://doi.org/10.1146/annurev.biochem.70.1.503

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