Niru Ancha

4-26-17

Objective: To make any last minute changes to our presentation and to present it to our peers.

Results: We found one citation which talked about the significance of finding terminator sequences. We presented our presentation to Dr. Adair and peers. We received good feedback and made the suggested changes.

Next Steps: We will present at the symposium on Friday.

Niru Ancha

4-24-17

Objective: To finish up independent project.

Tools: DNA master, ARNold

Results: We finished analyzing each predicted terminator. We checked each potential terminator on DNA master to see where it would most fit our criteria of a potential Rho-independent transcription terminator. Here are the final terminators identified from the Shrooms genome.

Also, here is the final abstract for our project.

Three bacteriophages isolated by using Arthrobacter sp. ATCC 21022 named Shrooms, Caterpillar, and Nubia were sequenced and annotated. We used a terminator identification software called ARNold to find probable Rho-independent transcription terminator sequences. ARNold identified 17 possible terminator sequences in Shrooms, 18 in Caterpillar, and 14 in Nubia. By using DNA Master to scan for the sequences in the whole genome of each phage, we looked for sequences that appeared within 100 bp from the end of the gene. ARNold identified 8 sequences in Shrooms, 12 sequences in Caterpillar, and 2 sequences in Nubia that fit our criteria of a potential Rho-Independent terminator sequence. In addition, many sequences that possessed higher ARNold scores were more likely to be potential terminator sequences. Finding terminator sequences in phage genomes can give insight into the promoter sequences in phage genomes. This can lead to new information about operons which can show how genes are regulated and their functions within the bacteriophage.

We are now in the process of working on our powerpoint presentation.

Conclusions: We will continue working on our powerpoint presentation and present it on Friday at the symposium.

Niru Ancha

The Food and Drug Administration is in place in order to ensure our health and safety. Some problems in the FDA approval process are the large amounts of funding necessary and the long time it takes for the approval process. Scientists strongly rely on larger businesses and investors to fund their work because it extremely costly to conduct research on phage. It is especially expensive to keep up with the quickly evolving phage and bacteria in the world. Additionally, the approval process can take upward of 10 years which a large time commitment. The progression of phage therapy was hindered because of these reasons. For example, the first time Sulakvelidze took IND do the FDA, he did not get the response he was looking for. The Food and Drug Administration had multiple follow up questions for Sulakvelidze. A few of their requests were for them to make sure lytic phages were used in Intralytix’s product, to start using a model that resembles a patient closer to the type that would be receiving the therapy, and to find the rate of mutations of each phage in Intralytix’s phage cocktail. Sulakvelide “decided it would require about 2 years and close to $1 million” to go through the FDA approval process again. Because of this, the project had to be shelved.

Furthermore, the process of phage therapy approval could have been improved. Wolcott was a scientist who got approved to move on to Phase II of his trial on multiple phages. The FDA required Wolcott and Intralytix to put each phage through a separate trial. This clinical trial was calculated to cost upward of $100 million. I agree with his proposition that the FDA should “approve the process, not the individual phages” like the flu vaccine. In this case, the methods would be approved and could be used to test different phages. Bacteria and phage evolve and mutate so quickly and this would be a practical way to keep up with them. Also, maybe if phages were more widely understood, there would be more support for them. Scientists could work on spreading the word and a shining a positive light on phages. This could lead to better results is the approval process because the idea of phage therapy would not seem so foreign.

Niru Ancha

4-19-17

Today, we worked on our independent project. We decided to look for Rho-independent transcription terminators in the three genomes we sequenced (Shrooms, Catepillar, and Nubia). Having more information on this will give us more information about promoter sequences in these genomes.

Julia, Daniel, and I decided to each take over a genome. I picked Shrooms.

Objective: To identify rho-indendent transcription terminators in the Shrooms genomes.

Tools: We used ARNOLD, a software that find probable terminators. Here is a link to the software. <http://rna.igmors.u-psud.fr/toolbox/arnold/>

Results:

Here are the suggested terminator sequences given by Arnold. 17 terminators were identified. Next, we used the DNA Scan option to see whether these terminators were at the end of genes. We are currently recording data on each probable terminator of each genome on and excel spreadsheet.

Conclusions and Later Steps: We will continue analyzing each probable terminators. Later, we will make conclusions about the terminators in each of the genomes.

Niru Ancha

4-10-17

Daniel, Julia, and I started planning our independent project topic today. Originally, we thought about looking at GC content differences between coding and non-coding regions. We wanted to see if GC content had anything to do with coding potential and if GC content could be used as a factor when annotating. After talking to Dr. Adair, we realized that this may not be a great idea because GC content is just mainly used for clustering genomes.

Next, we looked into predicting terminator sequences. We looked found a couple articles about Rho-independent transcription terminator sequences. Here is the link to one of the articles. <https://genomebiology.biomedcentral.com/articles/10.1186/gb-2007-8-2-r22> This article was published in 2007 so we also looked on pubmed to find some more recent articles. We then found software from previous studies that predict terminator sequences. We decided to choose this topic for our independent project. We want to use this software to predict terminator sequences. Then, we plan to analyze nucleotide sequences of the genomes we sequenced in class to predict where the promotor sequences are. This will give us more insight in to topics like operons in the genomes Shrooms, Catepillar, and Nubia.

Niru Ancha

3-16-17

It has become clear to me that politics and events occurring in the world play a role in the progression of science and medicine. In the 1940s-50s, Russia was implementing a state health system. All resources and healthcare needs were provided for by the state. At this time, most Russian funding and resources were being prioritized for defense and weaponry. Because of this, drugs were “of inferior quality and vulnerable to…shortages” (pg. 56). Interestingly enough, alternative medicine became widely accepted in Russia because of this struggle. As a result, phage therapy became of more interest.

The L. Hirszfeld Institute in Poland worked to provide therapeutic phages for patients to beat antibiotic resistance. They conducted studies that lead to positive results due to phage therapy such as research about antibiotic-resistant septicemia.  Ludwik Hirszfeld and his family faced many struggles during this time. From escaping Nazi Germany to the death of his daughter due to anorexia nervosa, the second world war really did hinder Hirszfeld’s work.  Despite this series of unfortunate events, he was determined to discover the promising results of phage therapy. He continued to investigate phages until his passing. The Institute of Immunology and Experimental Therapy was created in his legacy. Both the Phage Therapy Center and the Eliava Institute are still open today.  The Eliava Institute seems to be a research based institute while the Phage Therapy Center focuses more on phage typing and using phages for antibiotic-resistant bacteria. I believe the Phage Therapy Center is a little more beneficial because it seems to be having more of a direct effect on people. Moreover, Eliava and Hirszfeld’s stories are prime examples of how politics affects scientific discovery.

The United States was more cautious that European countries to accept phage therapy due to many rules and regulations. After reading an article in the Washington post about the growth of antibiotic resistance, Carl Merril thought phage therapy could help. He was determined to find a phage that could get past the liver and the spleen. Along with Richard Carlton and Sankar Adhya, Merril tested mice by injecting them with phage lambda, one active against E. coli. The tried to isolate as many phages as possible by leaving the phages in the mice for multiple hours. After, they analyzed the phages by inserting E. coli into mice and treating them with the phages named Argo1 and Argo2. The success of this experiment and another one with mice lead to promising results of phage therapy as well as the combined use of phages and antibiotics.

After looking into various start-up companies such as Phage Therapy and Phage Biotics, I can clearly see that phage therapy has great potential. I believe phage therapy could be more widespread if more people are educated about the topic. With more education and research, I believe phage therapy could become more consistent in treatments all over the world. I am excited to look to the future to see how we incorporate phages into medicine and how politics and outside influences will play a role throughout this process.

Niru Ancha

3-13-17

Rationale of today’s work: To help Shrooms group members finish annotating Shrooms so we can finally submit the annotated genome. To help annotate the short genome of Stewie Griff.

Tools used and/or Methods:

Results:

Daniel and I checked all of the query and sequence numbers for Shrooms genes 50-100. We did this all on Phage Notes.

Next, I annotated gene 10 of StewieGriff. The results are below.

Phages DB

NCBI (had no conserved domains)

HHPred

DNA Master

Will be posted at another time. Phage Notes is not working for me.

Conclusions and Next Steps: Shrooms is on its way to being completely finished so it can be submitted soon. StewieGriff  will continue to be worked on next lab period.

Niru Ancha

3-1-17

Rationale of today’s work: To finish checking all start and stop codons as well as Glimmer and Gene mark calls. Also, I had the job to compile one DNA master file with just the gene functions in the notes box. 

Tools: Starterator, Phamerator, NCBI Blast, phagesDB Blast, HHpred, DNA Master, GeneMark

Results:

Daniel and I finished checking all the start and stop codons. Natalie, who is making the cover letter, wanted a record of every gene that was extended or shortened so we recorded all of these genes. Here are the results.

Furthermore, I made a DNA master file with the functions, if applicable, to each gene. This was done so there would be one easy file to analyze the gene functions from.

Conclusions and Next Steps: We finished out part in the peer review process. Other members of the Shrooms group are almost finished as well. A member is working on the cover letter as well as a final DNA master file with all of the gene annotation. The Shrooms groups is on its way to having a full annotation to submit.