Icrespin Journal Week 11

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• Thiuram Article

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Purpose

The purpose of this assignment is to better understand the paper that was given. It will begin the process of analyzing the info that was presented. By better understanding the paper, it will help for future data collections and analysis that occurs for a paper.

Definitions of Terms

1. Thiuram: "a synthetic sulphur-containing compound used as a fungicide and seed protectant" (Oxford Dictionary of Biochemistry and Molecular Biology)

2. Proteolysis: "the process whereby complex protein molecules, obtained from the diet, are broken down by digestive enzymes in the stomach and small intestine into their constituent amino" (Oxford Dictionary of Biochemistry and Molecular Biology)

3. Hybridization: "the process of an animal or plant breeding with an individual of another species" (Oxford Dictionary of Biochemistry and Molecular Biology)

4. YKL071W: "NADH-dependent aldehyde reductase" (Gene Ontology)

5. YCR102C: "Putative protein of unknown function" (Gene Ontology)

6. YLR303W: "O-acetyl homoserine-O-acetyl serine sulfhydrylase" (Gene Ontology)

7. YLL057C: "Fe(II)-dependent sulfonate/alpha-ketoglutarate dioxygenase" (Gene Ontology)

8. Sulfhydryl: "the chemical group −SH; it is found in the amino acid cysteine and in other molecules. Two sulfhydryls can be oxidized to a disulfide bond, −S−S−." (Oxford Dictionary of Biochemistry and Molecular Biology)

9. Mutagenicity: "Capable of causing mutations in an organism. Mutagenic substances may also be carcinogenic" (Oxford Dictionary of Biochemistry and Molecular Biology)

10. Detoxification: "The process by which harmful compounds, such as drugs and poisons, are converted to less toxic compounds in the body. Detoxification is an important function of the liver" (Oxford Dictionary of Biochemistry and Molecular Biology)

Outline of Journal Article

1. What is the main result presented in this paper?
   • The main resuslt from this paper is that thiuram was used to expose to certain genes. Due to this exposure, it caused them to get altered. In order to get these results, a data microarray analysis occurred.
2. What is the importance or significance of this work?
   • The importance of this work is to better understand how different pesticides or any pesticide can cause an alteration to a gene. There are certain circumstances that may occur and this can help to see how genes are altered from these exposure. With this, it will hep understand different environmental stressors. In the end, it will help identify this toxin.
3. What were the limitations in previous studies that led them to perform this work?
   • The limitations included that it can be intoxicating in certain things, like in humans or other organisms. In addition, there wasn't any evidence that showed toxicity for thiuram using the data microarray. There were was studies done before, but not using this way of analysis.
4. How did they treat the yeast cells (what experiment were they doing?)
   • In order to treat the yeast cells, they cultured the four yeast cells. It was done in four different time intervals. Everything was measured in minutes. The exposed to a specific amount of thiuram and had them cultured between 15 minutes to 120 minutes.
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
   • The strain of yeast they used was Saccaromyces cerevisiae S288C. There is uncertainty as to whether it is a haploid or diploid. However, this strain was designed by Robert Mortimer in his biochemical studies. (Yeast Genome)
6. What media did they grow them in? What temperature? What type of incubator? For how long?
   • They used YPD medium for the media. It was incubated for 24 hours with the temperature at 25°C. The incubator that they used was the Biomek 2000 Laboratory Automation Workstation.
7. What controls did they use?
   • The control that was used was having a microarray that did not have thiuram injected. By having that, it helped to see how strong/fast the growth was in the cultures.
8. How many replicates did they perform per treatment or timepoint?
   • For each timepoint, it is unclear how many replicates they did. It just happened in different time intervals.
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
   • To prepare it, the hot-phenol method was used. The yeast cells were diluted and was cultures were done, it was centrifuged. After that, RNA was extracted.
10. What mathematical/statistical method did they use to analyze the data?
    • It doesn't state that they used a method. However, they used a yeast database called, MIPS (Munich Information Center for Protein Sequences).
11. Are the data publicly available for download? From which web site?
    • It is not publicly available to download, unless the user has special credentials to login for the website. In this case, the website was the publisher, ACS (American Chemical Society). If there are special credentials, such as paying for the access, then one can download content.
12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • What do the X and Y axes represent?
      • Figure 1
        • First graph has X representing time(hr) and Y representing OD650.
        • Second graph has X representing concentration of thiuram (μM) and the Y representing relative growth for 2 hrs (%).
      • Figure 2
        • Intensity of Cy5 for the Y-axis and Intensity of Cy3 for the X-axis.
      • Figure 3
        • The first one shows different time intervals without a X- or Y-axis.
        • The second one shows X-axis being number of induced genes and Y-axis showing different treatments.
      • Figure 4
        • The first one is a venn diagram of time intervals without X- or Y-axis.
        • The second one has X-axis representing number of repressed genes and Y-axis showing different treatments.
      • Table 1
        • It has the class name, which are the genes.
      • Table 2
        • It has the impact of thiuram on gene expression. It divides by description and two time intervals.
      • Table 3
        • The third one has induced genes with two time intervals.
      • Table 4
        • It talks about the effect of chemical treatment on the expression. It deals with the promoter activity.
    • How were the measurements made?
      • Figure 1
        • The measurement was made by using OD650 and comparing to the yeast cells that were inoculated in the YPD medium.
      • Figure 2
        • The signal intensities were measured with the two fluorescent indicators, which showed one being Cy3 and the other being Cy5. This was the comparison between the control and concentrations.
      • Figure 3
        • Figure 3 was measured based on classification of each gene for induced ones in the 30 and 120 minute.
      • Figure 4
        • This one had the classification of induced genes by thiuram treatment.
      • Table 1
        • It was about induced genes in the 30 minute and 120 minute treatments.
      • Table 2
        • It dealt with the impact of this toxicity to the gene expression based on different reactions.
      • Table 3
        • The induced genes was used in the DNA repair.
      • Table 4
        • This dealt with the effect of the chemical treatment. It used mRNA and the four genes.
    • What trends are shown by the plots and what conclusions can you draw from the data?
    • The trends that popped up was the gene expression and induced genes. There was a correlation that many of the previous studies talked about.
13. How does this work compare with previous studies?
    • It relates to previous studies that discusses environmental stressors. In addition, it talks a lot about the toxicity of thiuram.
14. What are the important implications of this work?
    • The important implication is that the repressed seems to be less important than the induced. This is significant to know because of the alterations that occurred.
15. What future directions should the authors take?
    • There should be more background about their own experience. The tables show be more organized as to what they are representing.
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?
    • The authors displayed on the necessary items needed in this research. It seemed very out of the place and super rushed when explaining these things. The major flaw is connecting it to other studies. It seems it was supported by other experiments. It was very confusing to follow and seemed like sometimes it didn't make sense. It didn't have enough background information.

Annotated Bibliography

1. Iwahashi, Y., Hosoda, H., Park, J. H., Lee, J. H., Suzuki, Y., Kitagawa, E., ... & Iwahashi, H. (2006). Mechanisms of patulin toxicity under conditions that inhibit yeast growth. Journal of agricultural and food chemistry, 54(5), 1936-1942. DOI: 10.1021/jf052264g
2. PubMed Abstract: https://www.ncbi.nlm.nih.gov/pubmed/16506856/
3. PubMed Central: N/A
4. Publisher Full Text (HTML): https://pubs.acs.org/doi/10.1021/jf052264g
5. Publisher Full Text (PDF): https://pubs.acs.org/doi/pdf/10.1021/jf052264g
6. Copyright: 2006 by the American Chemical Society (information found on the PDF version)
7. Article is not Open Access. LMU paid to download article.
8. Availability: in print and online
9. Publisher: American Chemical Society (scientific society). It is a non-profit, and it is not part of OAPA. It was published in the United States.
10. The journal's earliest date was in 2005.
11. Peer-reviewed articles: Yes
13. Impact factor:

References

Kitagawa, E., Takahashi, J., Momose, Y., & Iwahashi, H. (2002). Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray. Environmental science & technology, 36(18), 3908-3915. DOI: 10.1021/es015705v Oxford https://www.oxfordreference.com/search?q=detoxification&searchBtn=Search&isQuickSearch=true Gene Ontology. (2019). Gene Ontology. Retrieved November 13, 2019, from http://geneontology.org/ LMU BioDB 2019. (2019). Week 11. Retrieved November 13, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_11