Matthew K. Oki Individual Journal 14: Difference between revisions

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*Other more recent studies have focused on the impact of this DTC on yeast glycolysis, but this study is investigating the overall physiology of S. cerevisiae.
*Other more recent studies have focused on the impact of this DTC on yeast glycolysis, but this study is investigating the overall physiology of S. cerevisiae.
===Methods and Results===
===Methods and Results===
*The prototrophic haploid yeast strain Saccharomyces cerevisiae CEN.PK113-7D (MATa) (33, 34) was used in this study
*The prototrophic haploid yeast strain Saccharomyces cerevisiae CEN.PK113-7D (MATa) was used in this study.
**Kept in an anaerobic controlled environment
**Kept in an anaerobic controlled environment
*Sequential batch reactors were used instead of single batches because of enhanced reproducibility of certain growth rate measurements in the SBRs.
*Sequential batch reactors were used instead of single batches because of enhanced reproducibility of certain growth rate measurements in the SBRs.
Line 121: Line 121:
**“Samples were taken during the fifth and/or sixth temperature cycle. To minimize disturbance, sampling volumes did not exceed 5% of the reactor volume during a single temperature cycle, and intervals of at least 3 h were maintained between sampling points”
**“Samples were taken during the fifth and/or sixth temperature cycle. To minimize disturbance, sampling volumes did not exceed 5% of the reactor volume during a single temperature cycle, and intervals of at least 3 h were maintained between sampling points”
*Analytical analysis methods included gas and biomass analysis, total cell concentration, cell size, and residual glucose and metabolite concentrations.
*Analytical analysis methods included gas and biomass analysis, total cell concentration, cell size, and residual glucose and metabolite concentrations.
Measured glucose consumption during DTC
*The rate of glucose consumption during DTC was calculated.
Determined the percentage of cells carrying a bud
*Determined the budding index or percentage of cells carrying a bud.
Cell cycle phase distribution analyzed
*The cell cycle phase distribution in the yeast population was analyzed.
“Samples for microarray analysis were taken during the fifth and sixth temperature cycles from two independent duplicate cultures”
“Samples for microarray analysis were taken during the fifth and sixth temperature cycles from two independent duplicate cultures”
“Sample points from the fifth and sixth temperature cycles were combined”
“Sample points from the fifth and sixth temperature cycles were combined”

Revision as of 13:12, 5 December 2016

Week 14 Individual Journals

Part 1 In-Class Assignment

  1. What database did you access? (link to the home page of the database)
  2. What is the purpose of the database?
    • "Provides for the first time detailed information on genes and associated genetic variants involved in digenic diseases, the simplest form of oligogenic inheritance."
    • http://dida.ibsquare.be/
  3. What biological information does it contain?
    • This database contains information on chromosome mutations. It shows the original allele, alternative allele, what kind of mutation it is, and what disease this is correlated with.
    • http://dida.ibsquare.be/browse/#
  4. What species are covered in the database?
  5. What biological questions can it be used to answer?
    • This database can be used to look into what types of mutations cause certain diseases. This database pools together certain diseases and relates that to what kind of mutation each one is exhibiting.
    • http://dida.ibsquare.be/statistics/
  6. What type (or types) of database is it (sequence, structure model organism, or specialty [what?]; primary or “meta”; curated electronically, manually [in-house], manually [community])?
    • Since anyone can submit data to this database, it is a primary database that is curated manually (in-house). "New submissions will only be accepted after careful manual inspection."
    • http://dida.ibsquare.be/submit/
  7. What individual or organization maintains the database?
  8. What is their funding source(s)?
  9. Is there a license agreement or any restrictions on access to the database?
  10. How often is the database updated? When was the last update?
    • There is no clear information given on how often the database is updated. However, it does have a yearly count of how many publications there were. This graph stops at 2015.
    • http://dida.ibsquare.be/references/
  11. Are there links to other databases?
  12. Can the information be downloaded? And in what file formats?
  13. Evaluate the “user-friendliness” of the database.
    • The website is well designed and organized.
    • The website informs the reader/user about what the database is about on the home page. It gives background on digenic diseases. There is also a help header that gives information on how to work finding and submitting data.
    • In order to run a specific query, you really need the name of the disease you are looking for information on. You can also sort data by which kind of mutation it is.

Part 2 Group Project

Biological Terms

  1. Sinusoidal: "Varying according to the regular undulating sine curve y = sin x."
  2. Diural: "Happening daily, or during the course of a day."
  3. Kinetics: "The study of the rates of chemical reactions or biological processes."
  4. Mesophilic: "describing organisms, especially bacteria, that grow best at temperatures of about 25–45°C."
  5. Succinate: "A salt of succinic (butanedioic) acid, HOOC(CH2)2COOH, a four-carbon fatty acid. Succinate occurs in living organisms as an intermediate in metabolism, especially in the Krebs cycle."
  6. Chemostat: "An apparatus in which a bacterial population can be maintained in the exponential phase of growth by regulating the input of a rate-limiting nutrient and the removal of exhausted medium and cells."
  7. bioreactors: "Reaction vessels used to grow large numbers of cells that synthesize a product of interest. Originally developed for fermentation to produce antibiotics but more recently used to grow large numbers of selected or genetically engineered cells (e.g. for production of monoclonal antibodies or recombinant proteins)."
  8. budding index: "a method of release of virus from a cell after replication has taken place: viral protein associates itself with an area of cell membrane, which forms a coat or envelope around the virus; some cellular proteins in the area of budding are replaced by virus-coded proteins"
  9. argon laser: "a type of laser that utilizes argon gas to produce a beam of intense light, used especially in eye surgery to treat disease of the retina" (e.g. diabetic retinopathy) or glaucoma (as in argon laser trabeculoplasty)
  10. Metabolites: "A product of metabolism, including intermediate and waste products, or something which takes part in the reactions"
  11. transcriptome: "The full complement of all types of RNA molecules in a cell. The study of the transcriptome gives insight into the real-time activity of an organism at the molecular level."
  12. Off-gas: "a gas that is given off, especially one emitted as the byproduct of a chemical process."
  13. Biogenesis: "The principle that a living organism can arise only from another living organism, a principle contrasting with concepts such as that of the spontaneous generation of living from non-living matter"
  14. Vacuolar protein: "any of numerous proteins that are involved in the vesicle‐mediated transfer of enzymes to the yeast vacuole. Many share similarity with proteins from higher eukaryotes."
  15. Concomitant: "at the same time: describing drugs that are administered together or symptoms that occur during the same period"
  16. Flow Cytometry: "the technique for counting or measuring some property of cells, or subcellular components, using a flow cytometer, often after labelling with a fluorescent marker"
  17. Upregulation: "An increase in the sensitivity of a cell to a chemical substance, such as a hormone, signal molecule, or drug, due to an increase in the density of cell-surface receptors for that molecule. The converse, downregulation, reduces the cell’s sensitivity"
  18. Trehalose: "Mushroom sugar, also called mycose, a disaccharide of glucose. Found in some fungi (Amanita spp.), manna, and some insects; hydrolysed by the intestinal enzyme trehalase"
  19. Expression Profiles: "The range of genes expressed at particular stages of cell development. The level and duration of expression of one or more genes in a particular cell or tissue (detected, e.g., via sample sequencing, serial analysis, or microarray‐based methods)"
  20. Principal component analysis: "Mathematical technique for condensing a metabolomic spectrum to a single point on a graph, permitting rapid comparison between different species, experimental and control groups, etc."
  21. Biosynthesis: "The production of a chemical substance by a living organism"
  22. Monod kinetics: "An unstructured model used to describe the correlation of substrate concentration with microbial growth kinetics"
  23. Acetyl coenzyme A: "a compound formed by the combination of an acetate molecule with coenzyme A. Acetylcoenzyme A has an important role in the Krebs cycle."
  24. Synthesis: "The combination of discrete items to form a new whole, such as the development of new ideas from existing ones, or the combination of separate elements to form a new complex product, synthetic chemical compound, or material"
  25. Adenine: "A purine derivative. It is one of the major component bases of nucleotides and the nucleic acids DNA and RNA."
  26. Endogenous: "A product or an activity that arises in the body or cell, in contrast to exogenous agents or stimuli that come from outside."
  27. Heterogenity: "not originating within the organism in question"
  28. biomass: The total mass of all the organisms of a given type and/or in a given area; for example, the world biomass of trees, or the biomass of elephants in the Serengeti National Park.
  29. prototrophic: any strain of a microorganism (alga, bacterium, or fungus) that does not require any substances in its nutrition additional to those required by the wild type
  30. dilution rate: the rate, F, at which existing medium is replaced with fresh medium in a continuous culture, divided by the volume, V, of the culture

Outline

Intro

  • Organisms undergo natural temperature cycles throughout a day and night cycle.
    • It is uncertain if organisms are able to adapt and acclimate within a 24 hour cycle, or if they deal with “continuous temperature shock”.
  • Saccharomyces cerevisiae were used in this experiment.
    • This mesophilic microorganism's optimal temperature is 33°C.
    • They are being studied because they are often used in sub-optimal temperatures in industrial applications, such as brewing and wine making.
    • Temperature ranges vary by strain, but this strain is able to grow in the range of 4°C and 40°C
  • While previous studies focused on different temperature analysis on heat and cold shock responses, this study used analysis of a continuous diurnal temperature cycle (DTC) to see if the responses can be predicted from steady-state analysis.
  • Other more recent studies have focused on the impact of this DTC on yeast glycolysis, but this study is investigating the overall physiology of S. cerevisiae.

Methods and Results

  • The prototrophic haploid yeast strain Saccharomyces cerevisiae CEN.PK113-7D (MATa) was used in this study.
    • Kept in an anaerobic controlled environment
  • Sequential batch reactors were used instead of single batches because of enhanced reproducibility of certain growth rate measurements in the SBRs.
    • After three volume cycles, (not sure what this means) with no significant changes in biomass, a sinusoidal temperature cycle began.
    • “Samples were taken during the fifth and/or sixth temperature cycle. To minimize disturbance, sampling volumes did not exceed 5% of the reactor volume during a single temperature cycle, and intervals of at least 3 h were maintained between sampling points”
  • Analytical analysis methods included gas and biomass analysis, total cell concentration, cell size, and residual glucose and metabolite concentrations.
  • The rate of glucose consumption during DTC was calculated.
  • Determined the budding index or percentage of cells carrying a bud.
  • The cell cycle phase distribution in the yeast population was analyzed.

“Samples for microarray analysis were taken during the fifth and sixth temperature cycles from two independent duplicate cultures” “Sample points from the fifth and sixth temperature cycles were combined” 

“All genes with a P value of 0.002 were considered to be significantly changed (1,102 genes)”

“Steady-state chemostat cultures at constant temperatures of 12°C and 30°C (independent duplicate cultures at both temperatures) were sampled for microarray analysis” 

“For each sampling point during DTC, an average expression profile was calculated based on the two biologically independent arrays.”

“The complete data set (17 arrays) was deposited at the Gene Expression Omnibus database (http://www .ncbi.nlm.nih.gov/geo) under accession number GSE55372.”

Results

CO2 production revealed a clear cyclic variation in fermentative activity CO2 decreased production as temperatures dropped “For both CO2 and residual glucose, the amplitude of this fluctuation decreased as yeast cells acclimated to the DTC and became steady and reproducible after three cycles” “residual glucose concentration was inversely correlated with temperature” in 5th and 6th cycle 

“glucose concentrations decreased much faster when the temperature increased after passing the temperature minimum than they increased as the temperature minimum was approached. This asymmetry was also visible in the off-gas CO2 profile”
“concentrations of extracellular metabolites (ethanol, glycerol, lactate, succinate, and pyruvate) (Fig. 3D to G) were largely unaffected by the cyclic temperature variation, with the notable exception of acetate, the concentration of which rhythmically varied by circa 70%”
“transcriptome analysis was performed, covering six time points during the 24-h temperature cycle”
“microarray analysis revealed major reprogramming of gene expression”

1102 genes showed transcription changes

Questions

  1. What is the main result presented in this paper?
  2. What is the importance or significance of this work?
  3. What were the limitations in previous studies that led them to perform this work?
  4. How did they treat the yeast cells (what experiment were they doing?)
  5. Draw a diagram or flow chart of the experimental design.
  6. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
  7. What media did they grow them in? What temperature? What type of incubator? For how long?
  8. What controls did they use?
  9. How many replicates did they perform per treatment or timepoint?
  10. What method did they use to prepare the RNA, label it and hybridize it to the microarray? (very brief description)
  11. What mathematical/statistical method did they use to analyze the data? (very brief description)
  12. Are the data publicly available for download? From which web site?
  13. Briefly state the result shown in each of the figures and tables.
  14. How does this work compare with previous studies?
  15. What are the important implications of this work?
  16. What future directions should the authors take?
  17. 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?

Presentation

Acknowledgements

  • I would like to thank my partners, Mia Huddleston, Matthew R Allegretti, and Colin Wikholm, for the assistance on this weeks project both in the understanding of our paper in class and completion of the powerpoint outside of class.
  • I would also like to thank Kam D. Dahlquist, Ph.D. for providing the instructions and information for this assignment both in class and on this document: BIOL368/F16:Week 14.
  • Even though I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.
  • Matthew K. Oki 14:29, 5 December 2016 (EST):

References

  1. BIOL368/F16:Week 14
  2. Hebly, M., de Ridder, D., de Hulster, E. A. F., de la Torre Cortes, P., Pronk, J. T., & Daran-Lapujade, P. (2014). Physiological and transcriptional responses of anaerobic chemostat cultures of Saccharomyces cerevisiae subjected to diurnal temperature cycles. Applied and Environmental Microbiology, 80(14), 4433-4449. doi: 10.1128/AEM.00785-14

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