Blog on the reflection concerning the Climate Choices Deliberation.

Did any of the Options appeal to you more than the others?

Did you hear or think of any new way of addressing the issues associated with the warming of the climate?

What are your thoughts on the use of Public Deliberation in the classroom or the community? Is this something you would like to facilitate?

All in all, I thought the public deliberation regarding climate change was actually very enjoyable. I think the option that appealed to me the most was the third option, which pushed towards increasing innovation to help climate change. I felt like the second option was the least desirable, as it focused on reacting to the effects of climate change, instead of trying to make a change towards our bad actions. Pushing for innovation and research to effect climate change, such as producing vehicles that are more emission efficient or creating more energy sufficient homes could greatly reduce our carbon footprint. I believe it is easier to make the changes we need through innovation, and give humans more desirable options for being better on the planet.

Our group discussed several options to address the issues associated with climate change. They all definitely had their pros and cons, with the majority of them being ways to incentivize people to be more eco-friendly. One of my favorite solutions was to offer a tax incentive to people who decided to be more “green”, possibly reducing taxes on cars or homes that were more environmentally friendly. This concept could also be applied to companies, even further reducing our impact on the environment. It is obviously impossible to completely force the entire population to become more environmentally friendly, but if there was a trend in society (or people just wanted to keep more money), we could see a trend towards a more environmentally friendly population.

I think the use of Public Deliberation in the classroom or the community is an effective way of allowing individuals to discuss a topic in a well facilitated manner, allowing for a better flow of ideas in a constructive manner. Especially in the community and in the world of academia, this could be used in such an effective manner to discuss topics with multiple ideas and individuals. I think this would be pretty enjoyable to facilitate, but honestly I am more interested in participating in these in different topics, especially in the world of science.

Rationale:

The purpose of today’s lab was to rewrite the final abstract as well as begin the final presentation. Also Jmol was used to try and represent endolysin complex with Glycine-Glycine bridges

Tools:

• Jmol
• Microsoft Word
• Microsoft Powerpoint

Procedure:

• Final abstract was rewritten using the notes left on the previous abstract submission.
• A general outline of the final presentation was made, with general ideas for slides put out onto each slide.
• Used Jmol to visually represent Glycine-Glycine fitting into the catalytic region of our M23 peptidase endolysin.

Results: 

Copy of the Final Abstract

Visual Representation of Glycine-Glycine bridges fitting in active site.

Conclusions:

With the final abstract written, we are able to continue with creating the presentation for the CURES Symposium. Also, the ability to represent Glycine bridges fitting into NapoleonB’s endolysin catalytic region gives us another method of comparison between M23 Peptidase and PGRP endolysins.

Next Steps:

Our next steps for this project are to finish the final presentation for the symposium as well as finalize the PGRP catalytic region.

One of the biggest problems in phage therapy has been in the approval process. Describe the trouble surrounding FDA approval and recommend some suggestions to improve the process of phage therapy approval.

Despite the promise phage therapy has, it has yet to become a practice used in medicine due to the constant overwhelmingly large obstacle that is FDA approval. The approval process is riddled with problems that make approval for phage therapy an incredibly tedious process. The largest obstacle and issue with the FDA approval process is the fact that the FDA wishes to see promising results regarding the use of phages against bacterium, but require scientist to jump through hoops with little to no resources or funding. This can best be seen The Forgotten Cure, when the FDA made completely unreasonable request towards Sulakvelidze and Intralytix when they requested Intralytix to “determine the rate at which each of the phages in Intralytix’s proposed VRE cocktail would mutate inside an experimental animal”. This request was deemed unreasonable by Sulakvelidze, and noted that “if they [FDA] persist in this, it will be very difficult for us to carry on”. These roadblocks intentionally put in place by the FDA can hinder or completely prevent phage therapy companies from continuing on with their research. This can cause approval for phage therapy to take an incredibly long time, as it wasn’t until 2006 that phages were approved by the FDA just for food usage. Unfortunately, the FDA is a government agency, and for phage therapy to be approved it must meet and overcome every rule and regulation set in place.

I think the best way to improve the process of phage therapy is to loosen the reigns a little on the companies that are participating in this research. It is obvious that phage therapy, unfortunately, is relatively unknown and under-appreciated, meaning these companies have a hard enough time securing funding and the means necessary to do this type of research. In combination with reduced funding, the FDA putting up unnecessary roadblocks and obstacles can make it extremely difficult for these companies to succeed. This could be largely contributing to the lack of the fully achieved potential and promise phage therapy holds. I understand that the FDA is simply doing its duty, as it is trying to ensure phage therapy is completely free of any potential threats to public health, however the process of scientific inquiry is already a long and drawn out process as it is. The constant interference with the FDA and the lengthy approval process continues to slow down phage therapy to a crawl. With looser regulations, and increased support from the FDA and the public, I believe the process of phage therapy approval will not only go by much smoother, but also at an increased rate.

Rationale:

The purpose of today’s lab was to create an abstract for the powerpoint presentation as well as look at lysin A catalytic regions.

Tools:

• JMOL
• Word
• NCBI

Procedure:

• Drafted the abstract for the QTM
• Used JMOL to look at Lysin A structures to determine the possible catalytic region
• Researched the PGRP superfamily

Results:

The Written abstract

The Folded Lysin A Protein

Conclusions:

The lysin A protein has the same structurally conserved catalytic region as the annotated “endolysin” in NapoleonB. Both proteins contain the same beta sheet structure, while the lysin A is missing the alpha helices at the end of the molecule that NapoleonB’s endolysin possesses.

Next Steps:

Fold the PGRP proteins and determine catalytic region as well as any other lysin variation we find.

Rationale:

The purpose of today’s lab was to create a project outline for our presentation as well as compare phage endolysin proteins to determine whether the M23 peptidase domain is highly conserved.

Tools:

• Word
• PhagesDB
• BLASTp
• RaptorX

Procedure

• An outline that highlighted the rest of the independent project was created.
• After that, multiple Arthrobacter phage lysin proteins were compared using PhagesDB, BLASTp, and RaptorX to compare their sequences.

Results

The following outline was created:

In addition to this, the following comparisons were drawn between different phage holin and endolysin proteins

Conclusions:

After viewing a wide array of different clusters of Arthrobacter phages it was discovered that only AM phages have the annotated endolysin protein containing the specific “HLH” sequence we have been looking for. The M23 Peptidase domain is not as highly conserved as expected, with the majority of phages, such as cluster AN, having a PGRP domain within their lysin proteins. It was also noted that there is variation of annotation comments regarding these supposedly similar proteins ranging from “Lysin A” to “endolysin”. It is unclear what decides these variations.

Next Steps:

Continue looking for the “HLH” sequence in M23 Peptidase domains in the rest of the listed phages. Also determine the defining characteristics of the PGRP domain.

Rationale:

The purpose of today’s lab was to further our knowledge and ability of JMOL to better understand our protein structures.

Tools:

• JMOL
• JMOL Instruction Guide

Procedure:

• Began by familiarizing ourselves with the commands used in JMOL to produce effective results.
• Once completed, all Histidine, Tyrosines, and Aspartic acids were labeled in the protein with the colors red, blue, and yellow respectively.
• Following this, wireframe was turned on to allow us to see in which direction these residues faced, allowing us to visualize which residues interacted with each other best.
• Next, the group hypothesized which residues would best fit our catalytic region based off motility, orientation, and distance from the 4 histidines that are essentially bound in place.

Results:

These are the proposed catalytic regions found in 3 different phages from 3 different clusters (AM, FE, and FF)

NapoleonB Endolysin

Corgi Endolysin

Elesar Endolysin

Conclusions:

It can be concluded that these catalytic domains are conserved well throughout the M23 Peptidase family and Arthrobacter phages. The aspartic acid as well seems to be structurally conserved as every endolysin examined contained an acidic residue of aspartic acid on the neighboring beta sheet, which suggest it plays a role in the reaction.

Next Steps:

The next steps for this project are to use the knowledge of these endolysin proteins to help call function to NKF genes in different Arthrobacter phages.

Rationale:

The purpose of today’s lab was to analyze the RaptorX results generated for the phage holin and endolysin proteins and compare the different structures.

Tools:

• RaptorX
• PubChem
• Jmol

Procedure:

• Initially began with looking at the RaptorX PDB images generated, however they did not give much information in regards to the catalytic regions especially as we had no information regarding specific residues.
• Tried switching to PubChem, and though we were able to spin the protein, there was still no information regarding the actual amino acids.
• Eventually, downloaded JMOL, which offered the information we were looking for, and we were able to find the Histidine, Leucine, Histidine region of the catalytic Domain.

Results:

Histidine, Leucine, Histidine Catalytic Region

Elesar Endolysin

NapoleonB Endolysin

Conclusions:

The supposed catalytic regions were found, however there is much about this program that is unknown and there is definitely a learning curve to it. The tyrosine and acidic residue locations are still unknown in the protein.

Next Steps:

Figuring out how to utilize JMOL effectively is number one priority as it has the ability to give incredible amounts of information regarding the protein structures above. The missing tyrosine and acidic residues still need to be identified in the protein.

Rationale:

The purpose of today’s lab was to continue researching our independent project topics to gather more information of the mechanisms behind phage holin and endolysin proteins.

Tools:

• PUBMED
• HHMI
• TMHMM

Procedure:

• Began by discussing previously discovered information and narrowing down a project title and worked on the QTM.
• Searched for articles discussing the peptidoglycan bridges in bacterial cell walls to find any information that suggests Arthrobacter has the same bridges as Staph. aureus.
• Discussed possible holin mechanisms given the presence of acidic residues placed outside the membrane.

Results:

• Noted a sequence of amino acids “PEPEK” that lies outside of the bacterial membrane called the periplasmic loop for the holin protein.
• No significant research could justify that Arthrobacter cell walls contain bridges similar to Staph. aureus.
• 

Conclusions:

It can be hypothesized that this region of amino acids is a key characteristic of phage holin and could possibly be used to determine function to phages with unknown holin genes. It is still unclear as to what mechanism stops the proton motive force, and very well could be caused by just the physical damage the holin protein inflicts on the bacterial plasma membrane.

Next Steps:

The next steps for this experiment is to finalize any research information on holins and endolysins and begin folding these proteins to determine any key characteristics that could help determine gene function in other phages.

Rationale:

The purpose of today’s lab was to practice running through poster presentations in preparation of URSA Scholar’s Week.

Tools:

• Poster

Procedure:

• Each presentation group took turns presenting the poster in front of the rest of the class.
• During each group presentation, questions were constantly being asked towards those presenting to test their knowledge on the subject and see if they were capable of handing being interrupted.

Results:

• For the most part it seemed that most of the groups had a general presentation outline for the poster, but there definitely were areas of knowledge or presentation etiquette that could be polished before the presentation day.

Conclusion:

The poster and for the most part the students were ready for presenting for URSA. There definitely was some explanation needed for some bits of information on the poster, but overall the presentations were good.

Next Steps:

Transition from URSA back to independent project primary literature research.

Rationale:

The purpose of today’s lab was to discuss the proposed research questions with our research leaders and begin reading through the literature to inform us about this project.

Tools:

• NCBI BLASTp
• PhagesDB
• PUBMED

Procedure:

• The group discussed with our research leader the potential of our proposed research question, and the methods concerning it.
• Once completed, the group began with BLASTp queries of both the holin and endolysin protein to determine any conserved domains that could be found in the proteins.
• After that, literature regarding the biochemical mechanisms of these proteins was sought after and read.

Results:

Both the holin and endolysin contain conserved domains

Few recent articles were found regarding the biochemical mechanisms behind the holin protein, but there was information that suggested the overall mechanism of interacting with the bacterial cell to promote lysis.

Conclusions:

The discovery of conserved domains can suggest that the mechanisms in which holins and endolysins interact with bacterial cell is conserved or highly similar across bacteriophages, so there must be some research regarding those mechanisms. It was discovered that holins initially begin as alpha helices and somehow manage to oligomerize into beta sheets to stop the proton motive force causing bacterial cell death.

Next Steps: 

The next steps for this experiment are to continue with researching the literature and fold the proteins to try and determine any similar characteristics across the holins and endolysins of different phages.