Week 3 Individual Journal William P Fuchs

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Week 3 Journal Assignment

Purpose

To practice additional bioinformatics techniques through online tools like Biology Workbench and GenBank and to sequence several HIV and conduct an experiment on the relation between variants of HIV.

Methods

Part 2

  1. Find the Markham et al paper on [GenBank].
  2. Collect and save 4-6 of the archived sequences in the FASTA format.
  3. Download the sequences on the hard drive in the FASTA format.
  4. Re-open the file to confirm the FASTA configuration.

Part 3

  1. Acess the [Biology Workbench].
  2. Log in and create an account
  3. Once the created account is active browse the 5 toolsets and locate the nucleic acid tool.
  4. Select the command Add a new sequence and Run.
  5. Upload the FASTA data.
  6. Save the imported data for later use.
  7. Locate the ClustalW tool.
    • Highlight the tool and select Help to discover more information.
  8. Select the uploaded sequences and use ClustalW to run a multiple sequence alignment.
  9. Attempt to find correlations within the data.
  10. Analyze tree structure.

Results

Referring to the tree below (Figure A). There seems to be even divergence between the selected samples and there is little relation between the samples, resulting in the star-shaped output observed.

Activity Questions

  • Part 2
  1. What was the accession number of the sequence you chose?
    • AF089132.1
  2. Which subject of the study was that HIV sequence from?
    • It was collected from subject number 3
  3. Which section of the record contains information about who the HIV was collected from?
    • In the source section, subsection organism
  • Part 3
  1. Part 3 completion met through the analysis of the samples with the Biology workbench tool.

Data and Files

Figure A 351.CLUSTALW.1WPF.gif SequenceWPFbioworkbench.png Figure B

Conclusion

The data cultivated in the assignment yielded no clear relationship. The pertinence of this result is that the result was important for practice of new bioinformatic tools.

Preparation for Week 4 Journal Club

10 Unfamiliar Terms and Definitions

  1. chemostats
  2. coreceptor
  3. Seroconversion
  4. transversion
  5. Wilcoxon signed rank test
  6. Sanger method
  7. epitope
  8. monophyletic
  9. env gene
  10. Immunologic

Outline

Abstract

  • HIV-1 env sequences were studied in 15 patients and looked for for differences in CD4 T cell decline.
  • It was found that the incidence of divergence from the first seropositive visits were higher in progressors than in nonprogressors.
  • Viral progess in subjects with moderate to severe disease progression showed a selection towards nonsynonymous mutations.
  • Conversely, in non progressors with low viral loads had shown selection against the nonsynonymous mutations.
  • 10 subjects exhibited that a single variant predominated over the course of the study.
  • Summarily, the differences in the categorizations of the CD4-T cells fall in an interval of time show that not only numerical contrast in the subsequent

accumulation of mutations, but changes in the types of mutations that provide the best survivorship.

Introduction

  • HIV mutates very quickly.
  • In a unchanging environment the most fit virus would be minimally represented in the HIV gene pool.
    • There would be low occurrence of non-neutral mutations.
    • This style of mutational change is not limited to an HIV system.
  • Having a dynamic host environment causes a responsive change within HIV to create accommodations for itself.
  • HIV can be pressured to change by a differential display of coreceptors in the host immune system in CDT 4 cells.
  • The surviving HIV: Once the aforementioned selective forces such as an immune response only neutralize the most common viral variation, there would be a significant reduction in the viral population but a lack therein of a corresponding reduction in the HIV genetic diversity.
  • Limitations of prior studdies: Previous studies of HIV-1 genetic evolution evaluated only small sample sizes of HIV subjects and characterized HIV-1 genetic evolution, omitting direct examination of sequence patterns and subsequent analysis.
    • Also leaving out many time points in prior studies.
  • New Information: The attainment of increased genetic diversity is most commonly associated with the more rapid CD4 T cell loss over time.

Methods

The Subjects

  • 15 participants
    • All a part of the ALIVE study in Baltimore MD.
    • These individuals had reached HIV-1 seroconversion and had different levels of CD4T cells.
    • T-cell decline over the 4 year period of study

Sequence of the env Gene

  • Nested style PCR was employed to amplify a 285-bp region of the env gene.
    • extracted from peripheral blood mononuclear cells (PBMC)
      • Prior studies show that a majority of viral DNA is obtained from recently infected, unactivated PBMC in humans.
    • Both sessions of PCR were run for 2 minutes each and at a temperatue of at 95°, followed by 35 cycles of 94° for 30 seconds, 60° for another 30 seconds, and a final 72° for 45 seconds
    • Lastly, at the end of the 35 cycles the samples were held at 72° for 10 minutes then returning to the 4° temperature.
    • Cloned into pUC19
    • Sequenced by the Sanger chain termination method.
    • At this stage there were greater than 125 copies of viral DNA at the initial session of PCR amplification.
  • Likely that most to all of the clones generated were derived from a unique viral genomic template.

Generation of Phylogenetic Trees

  • Trees constructed from Mega program
  • The intrinsic relations were presented according to a color scheme.

Analysis

  • Nomenclature was employed to distinguish initial data and the various time points, cell count etc.
  • Various Figures and further computation of data points follows the remainder of the Methods.

Figure Analysis

  • Notes prepared for Class Discussion

Results/Discussion

  • CDT4 decline were inconsisstant among the 15 subjects
  • Scope of analysis focused on the viral env region around the third hypervariable (V3) domain.
    • this site is critical for the host–virus interactions.
  • Changes in HIV-1 sequences were measured in two ways
    • the genetic diversity upon each visit
    • divergence, i.e. the median percentage of nucleotides for each clone
  • The relation between diversity/divergence and the decline in CD4 T cell levels over the following year after a visit was included.
  • The subjects with the more genetically diverse viral clone genes experienced, collectively, a higher CD4 T cell decline in the subsequent year.

Left out Remaining Outline Questions

The paper addresses prior models of HIV progression by supporting the typical characteristics of HIV-immune system response relationships. (The higher the pressure the greater the variation).

Some limitations included the fact that they didn't physically conduct the study and were using bioinformatic techniques and relied on the data of others. They overcame this by conducting a very thorough and specific covering of HIV.

The results of the tables and figures showed phylogenetic trees, relationships of diversity and divergence, the differences in rapid/moderate progressors and nonprogressors and their corresponding relationships to divergence and diversity.

Future work:

  • Increase the amount of patients and to manipulate scenarios in which static or no-pressure environments can be observed.

Critique:

  • The tables and the figures were conducive only to a small audience and the lack of labeling made prompt comprehension difficult

Conclusion

N/A

Acknowledgements

  • Worked in class discussion with Matthew Allegretti.
  • While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

William P Fuchs 02:26, 20 September 2016 (EDT)

References

Markham, R.B., Wang, W.C., Weisstein, A.E., Wang, Z., Munoz, A., Templeton, A., Margolick, J., Vlahov, D., Quinn, T., Farzadegan, H., & Yu, X.F. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A. 95, 12568-12573. doi: 10.1073/pnas.95.21.12568 [PDF].

SDSC Biology Workbench see link: here

  • for ClustalW work

GenBank

  • Resource for the 4-6 HIV env gene sequences

Assignment for Week 3

  • Rubric/instructions for this assignment.

Other Work

Individual Journals

Class Journals

Assignments

Useful Links

  1. Bio Class Page
  2. BIOL368/F16:People
  3. Will Fuchs
  4. Link to LMU: http://www.lmu.edu/
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