Kvescio Week 3 Assignment

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Purpose

Purpose of this paper is to study the involvement of the CD4 T cell in HIV positive subjects. To address genetic variety among these subjects, and compare their progression of the virus. Also, purpose is to reinvestigate and improve on previous studies.

Biological Terms

  1. Seropositive: adjective (Medicine) giving a positive result in a test of blood serum, e.g., for the presence of a viru (Lindberg 2015).
  2. Chemostat: noun a system in which the chemical composition is kept at a controlled level, especially for the culture of microorganisms (Lindberg 2015).
  3. Cohort: a group of individuals of similar age within a population (Stansfield 2014).
  4. Seroconversion: Immunological jargon for having responded to immunization by producing antibody, becoming seropositive. (Lackie and Nation 2019).
  5. PBMC: Peripheral blood mononuclear cells. The mononuclear cells of the blood: monocytes and lymphocytes. (Lackie and Nation 2019).
  6. Sanger Chain Termination Method: a method in which free unprotonated amino groups react with 1‐fluoro‐2,4‐dinitrobenzene (FDNB). The dinitrophenylamino groups formed are stable to the acid used to hydrolyse the peptide bonds; the yellow arylated (DNP‐) amino acids so released can then be identified by chromatography and estimated spectrophotometrically (Cammack, Atwood, Campbell, Parish, Smith, Vella, Stirling 2008).
  7. Epidemiologically: adjective relating to the branch of medicine which deals with the incidence, distribution, and control of diseases: epidemiological studies of the effect of pesticides on humans (Stevenson and Lindberg 2015).
  8. Stratified: Showing distinct layers. Glacial till is often stratified through sorting and redeposition by meltwater (Mayhew 2015).
  9. Monophyletic: describing a number of individuals, species, etc., that have evolved from a single ancestral group (Martin 2015).
  10. Env gene: The retroviral gene that encodes the glycoproteins of the viral envelope. Since these viral proteins interact with host cell-surface molecules they determine host range, and rapid changes (as is the case with the HIV-1 env gene) make vaccine development very difficult (Lackie and Nation 2019).

Outline of Article

What is the importance or significance of this work?

  • The significance of this work is to investigate the evaluation of HIV, and genetic diversity among subjects who are infected with the virus. This work is important for studying how the virus mutates, and can help lead to better treatment.

What were the limitations in previous studies that led them to perform this work?

  • Previous studies were very limiting because they did not examine a large group of subjects. They also did not directly examine sequence patterns within the gene, and did not study the subjects for along enough time period.

How did they overcome these limitations?

  • Currently, HIV-1 virus is studied in a larger number of subjects, all with differing progression of the virus. They studied the subjects after seroconversion. The time period of observation often lasted for up to 4 years.

What is the main result presented in this paper? (Hint: look at the last sentence of the introduction and restate it in plain English.)

  • The main result is that the HIV virus progresses at different rates in different subjects. This paper reveals that the CD4 T cell decline, that is linked to HIV, is often in association with genetic diversity between subjects. In other words, there is a big difference in the genetics of people and their progression of the virus.

What were the methods used in the study?

  1. Study Population: chose a group of 15 people who were intravenous drug users, and also HIV positive. Studied these 15 subjects for six-month intervals.
  2. Sequencing of HIV-1 env Genes: Amplified the env gene from cells. Discover the env primers and nucleotide sequence. Both were run for 2 minutes at 95 degrees Celsius, followed by 35 cycles of 94 degrees for 30 seconds, 60 degrees for 30 seconds, and 72 degrees for 45 seconds.Afterwards, the samples were held at 72 degrees again, this time for 10 minutes. The sequences from the nested PCR were then cloned into pUC19 and sequenced by using the Sanger chain termination method. They were then screened for input viral DNA copy number. Result of this method indicated that it is likely that most or all of the clones generated were derived from a unique viral template.
  3. Plasma Viral Load: Reverse transcription occurred.
  4. Generation of Phylogenetic Trees: MEGA computer package was employed in order to generate phylogenetic trees.
  5. Correlation Analysis: In order to determine the correlation between genetic diversity and CD4 T cell count one year later. Analysis was stratified by whether the amount of CD4 T cell count divergence was in each of the categories.
  6. Determination of dS/dN Ratios: After the initial sequence for each person had been computed, they were compared with each subsequently observed strain. Differences were defined as either synonymous or nonsynonymous. Used the Jukes-Cantor correction. Values of dS and dN were averaged over all the strains observed at a certain visit.
  7. Examination of Source of Greater Initial Visit Diversity in Subjects 9 and 15: Three different phylogenetic trees were constructed in order to examine whether individuals may have been infected with two different viruses.
  8. Comparison of the Rate of Change of Divergence and Diversity: decline of CD4 T cell counts were compared by the use of the random effects models.

Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.

  • Figure 1 : In Rapid Progressors, the CD4 T cell counts seem to decline while diversity and divergence incline. Moderate Progressors had a similar result, however, not as extreme. In Non-Progressors, results were all different. In one subject (12), divergence and diversity stayed the same while CD4 T cell counts declined. In another subject (2), divergence and diversity increased, while CD4 T cell counts remained the same. In the last subject (13), CD4 T cell counts increased while divergence and diversity declined.
  • Table 1 : The results of this Table indicate that subjects with higher CD4 T cell counts, had a larger annual rate of cell decline. Rapid Progressors had a higher slope of change in intravisit nucleotide differences per clone per year, and slope of divergence.
  • Figure 2 A & B: Genetic diversity increased in all groups. Rapid Progressors had the largest mean slope/year of both intravisit genetic diversity and of % of nucleotides mutated from seroconversion virus.
  • Figure 3 : Figure represents the phylogenetic tree from subject 9. There is no dominant strain in the tree.
  • Figure 4 : All of the subjects selected have a specific pattern in their phylogenetic tree.

How do the results of this study compare to the results of previous studies (See Discussion).

  • Two recent studies have given contradictory results to the ones in this study. In this study, the subjects had higher levels of genetic diversity and divergence, while also having a greater decline in CD4 T cells. In another study done by McDonald et al., the diversity in the rapid Progresso's was less than observed in the slow progressors, during the second visit. In another study, by Wolinsky et al., there was less viral genetic diversity in two of the subjects with the most rapidly declining CD4 T cells, then those who's cells were slowly declining.

How do the results of this study support published HIV evolution models?

  • The association between CD4 T cells decline with increased diversity and divergence, is supported by the model of Nowak. Nowak proposed that increasing viral genetic diversity is associated with cell decline.

What are the important implications of this work?

  • This work implies that in most HIV positive individuals, the CD4 T cells decline as the diversity and divergence increase. This is important in studying the way in which the HIV virus evolves in different subjects, and how it mutates in order to fit into its environment. It is a very detrimental virus to many communities around the world, therefore, further research is important for all of society. This work will help scientists to study further and possibly create a better treatment for those infected.

What future directions should the authors take?

  • I believe that the authors should look into the results of a similar study, however, one that included HIV individuals who were not intravenous drug users. Drug use can also alter the body's response to certain viruses, and lower the immune system. It would be interesting to see a study in relation to those who were born HIV positive, and/or contracted it through sexual intercourse.

Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any limitations or major flaws to the paper?

  • I believe that the authors did a tremendous job in supporting their conclusions. Some of the data was a little bit more complicated to understand. It would have been nice if the Tables and Figures provided a detailed summary of the results underneath them. Their findings and relation to CD4 T cells, genetic divergence and diversity were well supported by all of the data, and discussed in the Results and Discussion.

Scientific Conclusion

  • In studying the evolution of HIV in the body, 15 subjects were selected and observed over a period of 4 years. Each of the subjects were intravenous drug users, and had different levels of progression of the virus. The results showed that the CD4 T cells declined when the genetic diversity and divergence increased. This was more evident for those who were Rapid Progressors, but the pattern was also shown in the other subjects. This study confirmed previous findings that have been shown in the model of Nowak. Nowak hypothesized that this is why the immune system is unable to control the HIV-1 infection. These findings are very crucial, due to the gravity of the virus. Hopefully this will lead to further research, and ultimately a better form of treatment for infected individuals.

Acknowledgments

  • I worked with Christina during class, and shared a google doc with information concerning Figure 2A
  • I asked Christina which source to use in order to find definitions for scientific terms.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Kvescio (talk) 21:29, 5 February 2020 (PST)

References

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